1. FIELD OF THE INVENTION
[001] Compositions and methods for detecting influenza are provided. In
particular, influenza markers and panels of markers useful in the detection of influenza are
provided.
2. BACKGROUND
[002] Influenza, or the flu, is a contagious viral infection of the respiratory tract.
Transmission of influenza is primarily airborne (i.e., coughing or sneezing); the peak of
transmission usually occurs in the winter months. Symptoms commonly include fever,
chills, headache, muscle aches, malaise, cough, and sinus congestion. Gastrointestinal
symptoms (i.e., nausea, vomiting, or diarrhea) may also occur, primarily in children, but are
less common in adults. Symptoms generally appear within two days of exposure to an
infected person. Pneumonia may develop as a complication of influenza infection, causing
increased morbidity and mortality in pediatric, elderly, and immunocompromised
populations. Influenza viruses are classified into types A, B, and C, the former two of
which cause most human infections. Influenza A is the most common type of influenza
virus in humans, and is generally responsible for seasonal flu epidemics and occasionally
for pandemics. Influenza A viruses can also infect animals such as birds, pigs, and horses.
Infections with influenza B virus are generally restricted to humans and are less frequent
causes of epidemics. Influenza A viruses are further divided into subtypes on the basis of
two surface proteins: hemagglutinin (H) and neuraminidase (N). Seasonal flu is normally
caused by subtypes HI, H2, H3, and Nl and N2. In addition to seasonal flu, a novel H1N1
strain was identified in humans in the United States in early 2009.
[003] Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family
consisting of two strains (subgroups A and B), is also the cause of a contagious disease that
afflicts primarily infants and the elderly who are immune-compromised, e.g., chronic lung
or heart disease or undergoing treatment for conditions that reduces the strength of their
immune system. The virus can live for hours on countertops and toys and cause both upper
respiratory infections, such as colds, and lower respiratory infections manifesting as
bronchiolitis and pneumonia.4 By the age of two, most children have already been infected
by RSV, but because only weak immunity develops, both children and adults can become
reinfected. Symptoms usually appear four to six days after infection. The disease is typically
self-limiting, lasting about one to two weeks in infants. In adults, the infection lasts about
five days and presents with symptoms consistent with a cold, such as rhinorrhea, fatigue,
headache, and fever. The RSV season overlaps with influenza season somewhat as
infections begin to rise during the fall and continue through early spring. RSV infections,
however, also occur at other times of the year, although rarely.
[004] Active surveillance programs in conjunction with infection control
precautions are important components for preventing transmission of influenza and RSV.
The use of assays providing rapid results to identify patients infected with these seasonal
infections is also an important factor for effective control, proper choice of treatment, and
prevention of widespread outbreaks.
[005] The genome of influenza viruses comprises eight RNA segments of 0.9-2.3
kb that together span approximately 13.5 kb and encode 11 proteins. These 8 segments
designated PB2, PB1, PA, HA, NP, NA, MP and NS are under constant selective pressure
which leads to rapid sequence changes (antigenic drift). In addition to changes on the
sequence level Influenza A has the ability to exchange whole segments with other Influenza
A viruses (antigenic shift). This process leads to the emergence of pandemic influenza
strains (i.e. Influenza A HlNlpdm09, swing origin H3N2).
[006] The two proteins, hemagglutinin (HA) and neuraminidase (NA) determine
the subtypes (H and N, respectively) of Influenza A virus. There are 16 H subtypes and 9 N
subtypes. The H1N1 and H3N2 subtypes cause the vast majority of influenza infections in
humans. Influenza B virus has a similar structure of RNA segments; however the Flu B
viruses do not have subtypes.
[007] This constant antigenic drift and antigenic shift makes it difficult to maintain
influenza detection assays from season to season. There remains a need for a robust
influenza detection assay that will remain accurate even as the influenza genome undergoes
genetic drift.
3. SUMMARY
[008] In some embodiments, methods of detecting the presence or absence of
influenza in a sample from a subject are provided. In some embodiments, a method
comprises detecting the presence or absence of at least one influenza gene selected from
polymerase acidic (PA) and polymerase basic 2 (PB2) in the sample.
[009] In some embodiments, methods of determining whether a subject has
influenza are provided. In some embodiments, a method comprises detecting the presence
or absence of at least one influenza gene selected from a polymerase acidic (PA) gene and a
polymerase basic 2 (PB2) gene in a sample from the subject.
[0010] In some embodiments, a method comprises detecting the presence of absence
of a PA gene. In some embodiments, a method comprises detecting the presence of absence
of a PB2 gene. In some embodiments, a method comprises detecting the presence of
absence of a PA gene and a PB2 gene. In some embodiments, the PA gene and/or PB2 gene
is an influenza A gene. In some embodiments, the sequence of the PA gene is at least 95%
identical to the sequence of SEQ ID NO: 2. In some embodiments, the sequence of the PB2
gene is at least 95% identical to the sequence of SEQ ID NO: 1.
[001 1] In some embodiments, the method further comprises detecting the presence
or absence of at least one influenza matrix protein (MP) gene. In some embodiments, the
method comprises detecting the presence or absence of an influenza A MP gene. In some
embodiments, the method comprises detecting the presence or absence of an influenza B
MP gene. In some embodiments, the method comprises detecting the presence or absence
of an influenza A MP gene and an influenza B MP gene. In some embodiments, the method
comprises detecting the presence or absence of an avian influenza MP gene. In some
embodiments, the avian influenza MP gene is a hemagglutinin (H) 5 or H7 subtype. In
some embodiments, the sequence of the influenza A MP gene is at least 95% identical to the
sequence of SEQ ID NO: 3 or 4. In some embodiments, the sequence of the influenza B
MP gene is at least 95% identical to the sequence of SEQ ID NO: 6.
[0012] In some embodiments, the method further comprises detecting the presence
or absence of at least one influenza nonstructural (NS) gene. In some embodiments, the
method comprises detecting the presence or absence of an influenza B NS gene. In some
embodiments, the sequence of the influenza B NS gene is at least 95% identical to the
sequence of SEQ ID NO: 7.
[0013] In some embodiments, the method further comprises detecting the presence
or absence of at least one influenza hemagglutinin (HA) gene. In some embodiments, the
method comprises detecting the presence or absence of an influenza A HA gene. In some
embodiments, the method comprises detecting the presence or absence of an avian influenza
HA gene. In some embodiments, the avian influenza is an H7 subtype. In some
embodiments, the sequence of the influenza HA gene is at least 95% identical to the
sequence of SEQ ID NO: 5.
[0014] In some embodiments, the method comprises detecting the presence or
absence of an influenza A PA gene, an influenza A PB2 gene, an influenza A MP gene, an
avian influenza MP gene, and an avian influenza HA gene. In some embodiments, the
sequence of the influenza A PA gene is at least 95% identical to SEQ ID NO: 2, the
sequence of the influenza A PB2 gene is at least 95% identical to SEQ ID NO: 1, the
sequence of the influenza A MP gene is at least 95% identical to SEQ ID NO: 3, the
sequence of the avian influenza MP gene is at least 95% identical to SEQ ID NO: 4, and the
sequence of the avian influenza HA gene is at least 95% identical to SEQ ID NO: 5.
[0015] In some embodiments, the method comprises detecting the presence or
absence of an influenza B MP gene and an influenza B NS gene. In some embodiments, the
sequence of the influenza B MP gene is at least 95% identical to SEQ ID NO: 6 and the
sequence of the influenza B NS gene is at least 95% identical to SEQ ID NO: 7.
[0016] In some embodiments, detection of the presence of any one of the influenza
genes indicates the presence of influenza in the sample. In some embodiments, the method
distinguishes between influenza A and influenza B. In some embodiments, the method does
not distinguish between influenza A and influenza B.
[0017] In some embodiments, the method comprises detecting the presence or
absence of respiratory syncytial virus (RSV) in a sample from the subject. In some
embodiments, the method comprises detecting the presence or absence of RSV A. In some
embodiments, the method comprises detecting the presence or absence of RSV B. In some
embodiments, the method comprises detecting the presence or absence of RSV A and RSV
B.
[0018] In some embodiments, wherein the subject has one or more symptoms of
influenza. In some embodiments, the subject has one or more symptoms selected from
fever, chills, cough, sore throat, runny nose, nasal congestion, muscle ache, headache,
fatigue, vomiting, and diarrhea.
[0019] In some embodiments, the method comprises detecting an exogenous control.
In some embodiments, the exogenous control is a sample processing control. In some
embodiments, the exogenous control comprises an RNA sequence that is not expected to be
present in the sample. In some embodiments, the exogenous control is an Armored® RNA.
[0020] In some embodiments, the method comprises PCR. In some embodiments,
the method comprises quantitative PCR. In some embodiments, the PCR reaction takes less
than 2 hours from an initial denaturation step through a final extension step.
[0021] In some embodiments, the method comprises contacting nucleic acids from
the sample with a first primer pair for detecting the influenza PA gene. In some
embodiments, the first primer pair comprises a first primer and a second primer, wherein the
first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 2, and wherein the
second primer comprises a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 2. In some embodiments, the first primer pair comprises a first primer and a second
primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 20, and wherein the second primer comprises a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19,
or at least 20 contiguous nucleotides of SEQ ID NO: 21. In some embodiments, the first
primer has the sequence of SEQ ID NO: 20 and the second primer has the sequence of SEQ
ID NO: 21.
[0022] In some embodiments, the method comprises contacting nucleic acids from
the sample with a second primer pair for detecting the influenza PB2 gene. In some
embodiments, the second primer pair comprises a third primer and a fourth primer, wherein
the third primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to
at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22,
at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1, and wherein
the fourth primer comprises a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 1. In some embodiments, the second primer pair comprises a third primer and a fourth
primer, wherein the third primer comprises a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleotides of SEQ ID NO: 17, and wherein the fourth primer comprises a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO:
18. In some embodiments, the third primer has the sequence of SEQ ID NO: 17 and the
fourth primer has the sequence of SEQ ID NO: 18.
[0023] In some embodiments, the method comprises contacting nucleic acids from
the sample with at least one additional primer pair, wherein each of the additional primer
pairs is for detecting a different influenza gene selected from an influenza A MP gene, an
avian influenza MP gene, and an avian influenza HA gene. In some embodiments, each
additional primer pair comprises a fifth primer and a sixth primer independently selected
from: (a) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3,
and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 3; (b) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4,
and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4; (c) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 5,
and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 5; (d) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 23,
and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 24; (e) a fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 26,
and a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 27; and (f) a fifth primer comprising a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 29, and a sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 30.
[0024] In some embodiments, the method comprises contacting nucleic acids from
the sample with at least one additional primer pair, wherein each of the additional primer
pairs is for detecting a different influenza gene selected from an influenza B MP gene and
an influenza B NS gene. In some embodiments, each additional primer pair comprises a
seventh primer and an eighth primer independently selected from: (a) a seventh primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6, and an eighth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6; (b) a seventh primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7, and an eighth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7; (c) a seventh primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 32, and an eighth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 33; and (d) a seventh
primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 35, and an eighth
primer comprising a sequence that is at least 90%, at least 95%, or 100% complementary to
at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22,
at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 36.
[0025] In some embodiments, the method comprises contacting nucleic acids from
the sample with at least one additional primer pair, wherein each of the additional primer
pairs is for detecting RSV A or RSV B. In some embodiments, each additional primer pair
comprises a ninth primer and a tenth primer independently selected from: (a) a ninth primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 38, and a tenth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 39; and (b) a ninth
primer comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 41, and a tenth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 42.
[0026] In some embodiments, the method comprises contacting nucleic acids from
the sample with primer pairs for detecting an influenza A PA gene, an influenza A PB2
gene, an influenza A MP gene, an avian influenza MP gene, and an avian influenza HA
gene. In some embodiments, the method further comprises contacting nucleic acids from
the sample with primer pairs for detecting RSV A and RSV B. In some embodiments, the
method comprises contacting nucleic acids from the sample with a control primer pair for
detecting an exogenous control. In some embodiments, each primer pair produces an
amplicon that is 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300
nucleotides long, 50 to 200 nucleotides long, or 50 to 150 nucleotides long.
[0027] In some embodiments, the method comprises forming an amplicon from each
primer pair when the target of the primer pair is present. In some embodiments, the method
comprises forming at least one amplicon selected from an influenza A PA amplicon, an
influenza A PB2 amplicon. In some embodiments, the influenza A PA amplicon has the
sequence of SEQ ID NO: 9 and the influenza A PB2 amplicon has the sequence of SEQ ID
NO: 8.
[0028] In some embodiments, the method comprises forming at least one amplicon
selected from an influenza A MP amplicon, an avian influenza MP amplicon, and an avian
influenza HA amplicon. In some embodiments, the influenza A MP amplicon has the
sequence of SEQ ID NO: 10, the avian influenza MP amplicon has the sequence of SEQ ID
NO: 11, and the avian influenza HA amplicon has the sequence of SEQ ID NO: 12.
[0029] In some embodiments, the method further comprises forming an influenza B
MP amplicon and/or an influenza B NS amplicon. In some embodiments, the influenza B
MP amplicon has the sequence of SEQ ID NO: 13 and the influenza B NS amplicon has the
sequence of SEQ ID NO: 14. In some embodiments, the method further comprises forming
an RSV A amplicon and/or an RSV B amplicon. In some embodiments, the RSV A
amplicon has the sequence of SEQ ID NO: 15 and the RSV B amplicon has the sequence of
SEQ ID NO: 16.
[0030] In some embodiments, the method comprises contacting the amplicons with
at least one probe selected from an influenza A PA probe and an influenza A PB2 probe. In
some embodiments, the influenza PA probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 2, and the influenza PB2 probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1. In some
embodiments, the influenza PA probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 19, and the influenza PB2 probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 22.
[0031] In some embodiments, the method comprises contacting the amplicons with
at least one probe selected from an influenza A MP probe, an avian influenza MP probe, and
an avian influenza HA probe. In some embodiments, the influenza MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and the avian
influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 4, and the avian influenza HA probe comprises a sequence that is at least
90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 5. In some embodiments, the influenza MP probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 25, and the
avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 28, and the avian influenza HA probe comprises a sequence that is at least
90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 31.
[0032] In some embodiments, the method comprises contacting the amplicons with
at least one probe selected from an influenza B MP probe and an influenza B NS probe. In
some embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 6, and the influenza B NS probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7. In some
embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 34, and the influenza B NS probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 37.
[0033] In some embodiments, the method comprises contacting the amplicons with
at least one probe selected from an RSV A probe and an RSV B probe. In some
embodiments, the RSV A probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 15, and the influenza B NS probe comprises a sequence that is
at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 40, and the RSV B probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 43.
[0034] In some embodiments, each probe comprises a detectable label. In some
embodiments, the each probe comprises a fluorescent dye and a quencher molecule. In
some embodiments, the influenza A probes and the influenza B probes comprise detectable
labels that are detectably different. In some embodiments, the influenza A probes and the
influenza B probes comprise detectable labels that are not detectably different. In some
embodiments, each probe consists of 15 to 30 nucleotides.
[0035] In some embodiments, the method comprises forming an exogenous control
amplicon. In some embodiments, the method comprises contacting the exogenous control
amplicon with a control probe capable of selectively hybridizing with the exogenous control
amplicon.
[0036] In some embodiments, the method comprises detecting the presence of
absence of at least one influenza A subtype and at least one influenza B subtype and an
exogenous control in a single multiplex reaction. In some embodiments, the at least one
influenza A subtype includes at least one avian influenza. In some embodiments, the
method comprises detecting RSV A and/or RSV B in the same multiplex reaction.
[0037] In some embodiments, the sample is selected from a nasopharyngeal swab
sample, a nasal aspirate sample, and a nasal wash sample.
[0038] In some embodiments, compositions are provided. In some embodiments, a
composition comprises a first primer pair for detecting an influenza PA gene. In some
embodiments, the first primer pair comprises a first primer and a second primer, wherein the
first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 2, and wherein the
second primer comprises a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 2. In some embodiments, the first primer pair comprises a first primer and a second
primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 20, and wherein the second primer comprises a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19,
or at least 20 contiguous nucleotides of SEQ ID NO: 21. In some embodiments, the first
primer has the sequence of SEQ ID NO: 20 and the second primer has the sequence of SEQ
ID NO: 21.
[0039] In some embodiments, a composition comprising a second primer pair for
detecting an influenza PB2 gene is provided. In some embodiments, the second primer pair
comprises a third primer and a fourth primer, wherein the third primer comprises a sequence
that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 1, and wherein the fourth primer comprises a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the
second primer pair comprises a third primer and a fourth primer, wherein the third primer
comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID
NO: 17, and wherein the fourth primer comprises a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19,
or at least 20 contiguous nucleotides of SEQ ID NO: 18. In some embodiments, the third
primer has the sequence of SEQ ID NO: 17 and the fourth primer has the sequence of SEQ
ID NO: 18.
[0040] In some embodiments, a composition comprises at least one additional
primer pair, wherein each of the additional primer pairs is for detecting a different influenza
gene selected from an influenza A MP gene, an avian influenza MP gene, and an avian
influenza HA gene. In some embodiments, each additional primer pair comprises a fifth
primer and a sixth primer independently selected from: (a) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 3, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 3; (b) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 4, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 4; (c) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 5, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 5; (d) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 23, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 24; (e) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 26, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 27; and (f) a fifth primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 29, and a sixth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 30.
[0041] In some embodiments, the composition further comprises at least one
additional primer pair, wherein each of the additional primer pairs is for detecting a different
influenza gene selected from an influenza B MP gene and an influenza B NS gene. In some
embodiments, each additional primer pair comprises a seventh primer and an eighth primer
independently selected from: (a) a seventh primer comprising a sequence that is at least
90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 6, and an eighth primer comprising a sequence that is at least
90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 6; (b) a seventh primer comprising a sequence that is
at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 7, and an eighth primer comprising a sequence that
is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 7; (c) a seventh primer comprising a sequence that is
at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 32, and an eighth primer comprising a sequence that
is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 33; and (d) a seventh primer comprising a sequence
that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 35, and an eighth primer comprising a sequence that
is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 36.
[0042] In some embodiments, a composition further comprises at least one
additional primer pair, wherein each of the additional primer pairs is for detecting RSV A or
RSV B. In some embodiments, each additional primer pair comprises a ninth primer and a
tenth primer independently selected from: (a) a ninth primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 38, and a tenth primer comprising a sequence that is
at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 39; and (b) a ninth primer comprising a sequence
that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 41, and a tenth primer comprising a sequence that is
at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 42.
[0043] In some embodiments, a composition comprises primer pairs for detecting an
influenza A PA gene, an influenza A PB2 gene, an influenza A MP gene, an avian influenza
MP gene, and an avian influenza HA gene. In some embodiments, a composition further
comprises primer pairs for detecting RSV A and RSV B. In some embodiments, a
composition further comprises a primer pair for detecting an exogenous control. In some
embodiments, the exogenous control is a sample processing control.
[0044] In some embodiments, a composition comprises at least one probe selected
from an influenza A PA probe and an influenza A PB2 probe. In some embodiments, the
influenza PA probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 2, and the influenza PB2 probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the influenza PA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 19, and the
influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 22.
[0045] In some embodiments, a composition further comprises at least one probe
selected from an influenza A MP probe, an avian influenza MP probe, and an avian
influenza HA probe. In some embodiments, the influenza MP probe comprises a sequence
that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and the avian influenza MP
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4, and the avian influenza HA probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 5. In some embodiments, the influenza MP probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 25, and the
avian influenza MP probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 28, and the avian influenza HA probe comprises a sequence that is at least
90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 31.
[0046] In some embodiments, the composition further comprises at least one probe
selected from an influenza B MP probe and an influenza B NS probe. In some
embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 6, and the influenza B NS probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7. In some
embodiments, the influenza B MP probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 34, and the influenza B NS probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 37.
[0047] In some embodiments, the composition further comprises at least one probe
selected from an RSV A probe and an RSV B probe. In some embodiments, the RSV A
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 15, and the influenza B NS probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 40, and the
RSV B probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 43.
[0048] In some embodiments, the composition further comprises a probe for
detecting an exogenous control.
[0049] In some embodiments, each probe comprises a detectable label. In some
embodiments, each probe comprises a fluorescent dye and a quencher molecule. In some
embodiments, each probe consists of 15 to 30 nucleotides.
[0050] In some embodiments, the composition is a lyophilized composition. In
some embodiments, the composition is in solution. In some embodiments, the composition
comprises nucleic acids from a sample from a subject being tested for the presence of
absence of influenza. In some embodiments, the sample is selected from a nasopharyngeal
swab sample, a nasal aspirate sample, and a nasal wash sample.
[005 1] In some embodiments, kits are provided. In some embodiments, a kit
comprises a composition described herein. In some embodiments, the kit further comprises
an exogenous control. In some embodiments, the exogenous control is an Armored® RNA.
In some embodiments, the kit comprises dNTPs and/or a thermostable polymerase. In some
embodiments, the kit comprises a reverse transcriptase.
[0052] In some embodiments, an oligonucleotide consisting of a sequence selected
from SEQ ID NOs: 17 to 43 is provided. In some embodiments, the oligonucleotide
comprises at least one modified nucleotide. In some embodiments, the oligonucleotide
comprises a detectable label. In some embodiments, the oligonucleotide comprises a
fluorescent dye and a quencher molecule. In some embodiments, the oligonucleotide is a
fluorescence resonance energy transfer (FRET) probe.
[0053] In some embodiments, a composition comprising a first primer consisting of
the sequence of SEQ ID NO: 17 and a second primer consisting of the sequence of SEQ ID
NO: 18, wherein the first primer and the second primer each comprises at least one modified
nucleotide is provided. In some embodiments, the composition comprises a probe
consisting of the sequence of SEQ ID NO: 19, wherein the probe comprises at least one
modified nucleotide and/or a detectable label.
[0054] In some embodiments, a composition comprising a first primer consisting of
the sequence of SEQ ID NO: 20 and a second primer consisting of the sequence of SEQ ID
NO: 21, wherein the first primer and the second primer each comprises at least one modified
nucleotide is provided. In some embodiments, the composition comprises a probe
consisting of the sequence of SEQ ID NO: 22, wherein the probe comprises at least one
modified nucleotide and/or a detectable label. In some embodiments, the probe is a
fluorescence resonance energy transfer (FRET) probe. In some embodiments, the probe
comprises at least one modified nucleotide. In some embodiments, the composition is a
lyophilized composition. In some embodiments, the composition is in solution. In some
embodiments, the composition comprises nucleic acids of a sample from a subject.
4. DETAILED DESCRIPTION
4.1. Definitions
[0055] To facilitate an understanding of the present invention, a number of terms
and phrases are defined below:
[0056] As used herein, the terms "detect", "detecting" or "detection" may describe
either the general act of discovering or discerning or the specific observation of a detectably
labeled composition.
[0057] As used herein, the term "detectably different" refers to a set of labels (such
as dyes) that can be detected and distinguished simultaneously.
[0058] As used herein, the terms "patient" and "subject" are used interchangeably to
refer to a human. In some embodiments, the methods described herein may be used on
samples from non-human animals.
[0059] As used herein, the terms "oligonucleotide," "polynucleotide," "nucleic acid
molecule," and the like, refer to nucleic acid-containing molecules, including but not limited
to, DNA or RNA. The term encompasses sequences that include any of the known base
analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-
methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil,
dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-
methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-
2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and
2,6-diaminopurine.
[0060] As used herein, the term "oligonucleotide," refers to a single-stranded
polynucleotide having fewer than 500 nucleotides. In some embodiments, an
oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides
long. Oligonucleotides may be referred to by their length, for example, a 24 residue
oligonucleotide may be referred to as a "24-mer."
[0061] As used herein, the term "complementary" to a target RNA (or target region
thereof), and the percentage of "complementarity" of the probe sequence to that of the target
RNA sequence is the percentage "identity" to the sequence of target RNA or to the reverse
complement of the sequence of the target RNA. In determining the degree of
"complementarity" between probes used in the compositions described herein (or regions
thereof) and a target RNA, such as those disclosed herein, the degree of "complementarity"
is expressed as the percentage identity between the sequence of the probe (or region thereof)
and sequence of the target RNA or the reverse complement of the sequence of the target
RNA that best aligns therewith. The percentage is calculated by counting the number of
aligned bases that are identical as between the 2 sequences, dividing by the total number of
contiguous nucleotides in the probe, and multiplying by 100.
1. FIELD OF THE INVENTION
[001] Compositions and methods for detecting influenza are provided. In
particular, influenza markers and panels of markers useful in the detection of
influenza are provided.
2. BACKGROUND
[002] Influenza, or the flu, is a contagious viral infection of the
respiratory tract. Transmission of influenza is primarily airborne (i.e., coughing or
sneezing); the peak of transmission usually occurs in the winter months.
Symptoms commonly include fever, chills, headache, muscle aches, malaise,
cough, and sinus congestion. Gastrointestinal symptoms (i.e., nausea, vomiting, or
diarrhea) may also occur, primarily in children, but are less common in adults.
Symptoms generally appear within two days of exposure to an infected person.
Pneumonia may develop as a complication of influenza infection, causing
increased morbidity and mortality in pediatric, elderly, and immunocompromised
populations. Influenza viruses are classified into types A, B, and C, the former
two of which cause most human infections. Influenza A is the most common type
of influenza virus in humans, and is generally responsible for seasonal flu
epidemics and occasionally for pandemics. Influenza A viruses can also infect
animals such as birds, pigs, and horses. Infections with influenza B virus are
generally restricted to humans and are less frequent causes of epidemics.
Influenza A viruses are further divided into subtypes on the basis of two surface
proteins: hemagglutinin (H) and neuraminidase (N). Seasonal flu is normally
caused by subtypes H1, H2, H3, and N1 and N2. In addition to seasonal flu, a
novel H1N1 strain was identified in humans in the United States in early 2009.
[003] Respiratory syncytial virus (RSV), a member of the
Paramyxoviridae family consisting of two strains (subgroups A and B), is also the
cause of a contagious disease that afflicts primarily infants and the elderly who are
immune-compromised, e.g., chronic lung or heart disease or undergoing treatment
for conditions that reduces the strength of their immune system. The virus can live
for hours on countertops and toys and cause both upper respiratory infections,
such as colds, and lower respiratory infections manifesting as bronchiolitis and
pneumonia.4 By the age of two, most children have already been infected by
RSV, but because only weak immunity develops, both children and adults can
become reinfected. Symptoms usually appear four to six days after infection. The
disease is typically self-limiting, lasting about one to two weeks in infants. In
adults, the infection lasts about five days and presents with symptoms consistent
with a cold, such as rhinorrhea, fatigue, headache, and fever. The RSV season
overlaps with influenza season somewhat as infections begin to rise during the fall
3
and continue through early spring. RSV infections, however, also occur at other
times of the year, although rarely.
[004] Active surveillance programs in conjunction with infection control
precautions are important components for preventing transmission of influenza
and RSV. The use of assays providing rapid results to identify patients infected
with these seasonal infections is also an important factor for effective control,
proper choice of treatment, and prevention of widespread outbreaks.
[005] The genome of influenza viruses comprises eight RNA segments of
0.9-2.3 kb that together span approximately 13.5 kb and encode 11 proteins.
These 8 segments designated PB2, PB1, PA, HA, NP, NA, MP and NS are under
constant selective pressure which leads to rapid sequence changes (antigenic
drift). In addition to changes on the sequence level Influenza A has the ability to
exchange whole segments with other Influenza A viruses (antigenic shift). This
process leads to the emergence of pandemic influenza strains (i.e. Influenza A
H1N1pdm09, swing origin H3N2).
[006] The two proteins, hemagglutinin (HA) and neuraminidase (NA)
determine the subtypes (H and N, respectively) of Influenza A virus. There are 16
H subtypes and 9 N subtypes. The H1N1 and H3N2 subtypes cause the vast
majority of influenza infections in humans. Influenza B virus has a similar
structure of RNA segments; however the Flu B viruses do not have subtypes.
[007] This constant antigenic drift and antigenic shift makes it difficult to
maintain influenza detection assays from season to season. There remains a need
for a robust influenza detection assay that will remain accurate even as the
influenza genome undergoes genetic drift.
3. SUMMARY
[008] In some embodiments, methods of detecting the presence or
absence of influenza in a sample from a subject are provided. In some
embodiments, a method comprises detecting the presence or absence of at least
one influenza gene selected from polymerase acidic (PA) and polymerase basic 2
(PB2) in the sample.
[009] In some embodiments, methods of determining whether a subject
has influenza are provided. In some embodiments, a method comprises detecting
the presence or absence of at least one influenza gene selected from a polymerase
acidic (PA) gene and a polymerase basic 2 (PB2) gene in a sample from the
subject.
[0010] In some embodiments, a method comprises detecting the presence
of absence of a PA gene. In some embodiments, a method comprises detecting
the presence of absence of a PB2 gene. In some embodiments, a method
4
comprises detecting the presence of absence of a PA gene and a PB2 gene. In
some embodiments, the PA gene and/or PB2 gene is an influenza A gene. In
some embodiments, the sequence of the PA gene is at least 95% identical to the
sequence of SEQ ID NO: 2. In some embodiments, the sequence of the PB2 gene
is at least 95% identical to the sequence of SEQ ID NO: 1.
[0011] In some embodiments, the method further comprises detecting the
presence or absence of at least one influenza matrix protein (MP) gene. In some
embodiments, the method comprises detecting the presence or absence of an
influenza A MP gene. In some embodiments, the method comprises detecting the
presence or absence of an influenza B MP gene. In some embodiments, the
method comprises detecting the presence or absence of an influenza A MP gene
and an influenza B MP gene. In some embodiments, the method comprises
detecting the presence or absence of an avian influenza MP gene. In some
embodiments, the avian influenza MP gene is a hemagglutinin (H) 5 or H7
subtype. In some embodiments, the sequence of the influenza A MP gene is at
least 95% identical to the sequence of SEQ ID NO: 3 or 4. In some embodiments,
the sequence of the influenza B MP gene is at least 95% identical to the sequence
of SEQ ID NO: 6.
[0012] In some embodiments, the method further comprises detecting the
presence or absence of at least one influenza nonstructural (NS) gene. In some
embodiments, the method comprises detecting the presence or absence of an
influenza B NS gene. In some embodiments, the sequence of the influenza B NS
gene is at least 95% identical to the sequence of SEQ ID NO: 7.
[0013] In some embodiments, the method further comprises detecting the
presence or absence of at least one influenza hemagglutinin (HA) gene. In some
embodiments, the method comprises detecting the presence or absence of an
influenza A HA gene. In some embodiments, the method comprises detecting the
presence or absence of an avian influenza HA gene. In some embodiments, the
avian influenza is an H7 subtype. In some embodiments, the sequence of the
influenza HA gene is at least 95% identical to the sequence of SEQ ID NO: 5.
[0014] In some embodiments, the method comprises detecting the
presence or absence of an influenza A PA gene, an influenza A PB2 gene, an
influenza A MP gene, an avian influenza MP gene, and an avian influenza HA
gene. In some embodiments, the sequence of the influenza A PA gene is at least
95% identical to SEQ ID NO: 2, the sequence of the influenza A PB2 gene is at
least 95% identical to SEQ ID NO: 1, the sequence of the influenza A MP gene is
at least 95% identical to SEQ ID NO: 3, the sequence of the avian influenza MP
5
gene is at least 95% identical to SEQ ID NO: 4, and the sequence of the avian
influenza HA gene is at least 95% identical to SEQ ID NO: 5.
[0015] In some embodiments, the method comprises detecting the
presence or absence of an influenza B MP gene and an influenza B NS gene. In
some embodiments, the sequence of the influenza B MP gene is at least 95%
identical to SEQ ID NO: 6 and the sequence of the influenza B NS gene is at least
95% identical to SEQ ID NO: 7.
[0016] In some embodiments, detection of the presence of any one of the
influenza genes indicates the presence of influenza in the sample. In some
embodiments, the method distinguishes between influenza A and influenza B. In
some embodiments, the method does not distinguish between influenza A and
influenza B.
[0017] In some embodiments, the method comprises detecting the
presence or absence of respiratory syncytial virus (RSV) in a sample from the
subject. In some embodiments, the method comprises detecting the presence or
absence of RSV A. In some embodiments, the method comprises detecting the
presence or absence of RSV B. In some embodiments, the method comprises
detecting the presence or absence of RSV A and RSV B.
[0018] In some embodiments, wherein the subject has one or more
symptoms of influenza. In some embodiments, the subject has one or more
symptoms selected from fever, chills, cough, sore throat, runny nose, nasal
congestion, muscle ache, headache, fatigue, vomiting, and diarrhea.
[0019] In some embodiments, the method comprises detecting an
exogenous control. In some embodiments, the exogenous control is a sample
processing control. In some embodiments, the exogenous control comprises an
RNA sequence that is not expected to be present in the sample. In some
embodiments, the exogenous control is an Armored® RNA.
[0020] In some embodiments, the method comprises PCR. In some
embodiments, the method comprises quantitative PCR. In some embodiments, the
PCR reaction takes less than 2 hours from an initial denaturation step through a
final extension step.
[0021] In some embodiments, the method comprises contacting nucleic
acids from the sample with a first primer pair for detecting the influenza PA gene.
In some embodiments, the first primer pair comprises a first primer and a second
primer, wherein the first primer comprises a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 2, and wherein the second primer
6
comprises a sequence that is at least 90%, at least 95%, or 100% complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 2. In some embodiments, the first primer pair comprises a first primer and a
second primer, wherein the first primer comprises a sequence that is at least 90%,
at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 20, and wherein the second primer
comprises a sequence that is at least 90%, at least 95%, or 100% complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20
contiguous nucleotides of SEQ ID NO: 21. In some embodiments, the first primer
has the sequence of SEQ ID NO: 20 and the second primer has the sequence of
SEQ ID NO: 21.
[0022] In some embodiments, the method comprises contacting nucleic
acids from the sample with a second primer pair for detecting the influenza PB2
gene. In some embodiments, the second primer pair comprises a third primer and
a fourth primer, wherein the third primer comprises a sequence that is at least
90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 1, and wherein the fourth primer
comprises a sequence that is at least 90%, at least 95%, or 100% complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 1. In some embodiments, the second primer pair comprises a third primer
and a fourth primer, wherein the third primer comprises a sequence that is at least
90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least
18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 17, and
wherein the fourth primer comprises a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least
19, or at least 20 contiguous nucleotides of SEQ ID NO: 18. In some
embodiments, the third primer has the sequence of SEQ ID NO: 17 and the fourth
primer has the sequence of SEQ ID NO: 18.
[0023] In some embodiments, the method comprises contacting nucleic
acids from the sample with at least one additional primer pair, wherein each of the
additional primer pairs is for detecting a different influenza gene selected from an
influenza A MP gene, an avian influenza MP gene, and an avian influenza HA
gene. In some embodiments, each additional primer pair comprises a fifth primer
and a sixth primer independently selected from: (a) a fifth primer comprising a
7
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 3; (b) a fifth primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 4, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4; (c) a
fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 5, and a sixth primer comprising a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 5; (d) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 23, and
a sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 24; (e) a fifth primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 26, and a sixth primer comprising
a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:
27; and (f) a fifth primer comprising a sequence that is at least 90%, at least 95%,
or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 29, and a sixth primer comprising a sequence that is at
least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least
8
17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 30.
[0024] In some embodiments, the method comprises contacting nucleic
acids from the sample with at least one additional primer pair, wherein each of the
additional primer pairs is for detecting a different influenza gene selected from an
influenza B MP gene and an influenza B NS gene. In some embodiments, each
additional primer pair comprises a seventh primer and an eighth primer
independently selected from: (a) a seventh primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 6, and an eighth primer
comprising a sequence that is at least 90%, at least 95%, or 100% complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 6; (b) a seventh primer comprising a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 7, and an eighth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7; (c) a
seventh primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 32, and an eighth primer comprising a sequence that is at least 90%,
at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 33; and (d) a seventh primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 35, and an eighth primer comprising a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 36.
[0025] In some embodiments, the method comprises contacting nucleic
acids from the sample with at least one additional primer pair, wherein each of the
additional primer pairs is for detecting RSV A or RSV B. In some embodiments,
9
each additional primer pair comprises a ninth primer and a tenth primer
independently selected from: (a) a ninth primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 38, and a tenth primer comprising
a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:
39; and (b) a ninth primer comprising a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 41, and a tenth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 42.
[0026] In some embodiments, the method comprises contacting nucleic
acids from the sample with primer pairs for detecting an influenza A PA gene, an
influenza A PB2 gene, an influenza A MP gene, an avian influenza MP gene, and
an avian influenza HA gene. In some embodiments, the method further comprises
contacting nucleic acids from the sample with primer pairs for detecting RSV A
and RSV B. In some embodiments, the method comprises contacting nucleic
acids from the sample with a control primer pair for detecting an exogenous
control. In some embodiments, each primer pair produces an amplicon that is 50
to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50
to 200 nucleotides long, or 50 to 150 nucleotides long.
[0027] In some embodiments, the method comprises forming an amplicon
from each primer pair when the target of the primer pair is present. In some
embodiments, the method comprises forming at least one amplicon selected from
an influenza A PA amplicon, an influenza A PB2 amplicon. In some
embodiments, the influenza A PA amplicon has the sequence of SEQ ID NO: 9
and the influenza A PB2 amplicon has the sequence of SEQ ID NO: 8.
[0028] In some embodiments, the method comprises forming at least one
amplicon selected from an influenza A MP amplicon, an avian influenza MP
amplicon, and an avian influenza HA amplicon. In some embodiments, the
influenza A MP amplicon has the sequence of SEQ ID NO: 10, the avian
influenza MP amplicon has the sequence of SEQ ID NO: 11, and the avian
influenza HA amplicon has the sequence of SEQ ID NO: 12.
10
[0029] In some embodiments, the method further comprises forming an
influenza B MP amplicon and/or an influenza B NS amplicon. In some
embodiments, the influenza B MP amplicon has the sequence of SEQ ID NO: 13
and the influenza B NS amplicon has the sequence of SEQ ID NO: 14. In some
embodiments, the method further comprises forming an RSV A amplicon and/or
an RSV B amplicon. In some embodiments, the RSV A amplicon has the
sequence of SEQ ID NO: 15 and the RSV B amplicon has the sequence of SEQ
ID NO: 16.
[0030] In some embodiments, the method comprises contacting the
amplicons with at least one probe selected from an influenza A PA probe and an
influenza A PB2 probe. In some embodiments, the influenza PA probe comprises
a sequence that is at least 90%, at least 95%, or 100% identical or complementary
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 2, and the influenza PB2 probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 1. In some
embodiments, the influenza PA probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 19, and the influenza
PB2 probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 22.
[0031] In some embodiments, the method comprises contacting the
amplicons with at least one probe selected from an influenza A MP probe, an
avian influenza MP probe, and an avian influenza HA probe. In some
embodiments, the influenza MP probe comprises a sequence that is at least 90%,
at least 95%, or 100% identical or complementary to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and the avian
influenza MP probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 4, and the avian influenza HA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
11
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 5. In some embodiments, the influenza MP probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 25, and the avian influenza MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to
at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 28, and the avian influenza HA probe comprises a sequence that is at least
90%, at least 95%, or 100% identical or complementary to at least 15, at least 16,
at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 31.
[0032] In some embodiments, the method comprises contacting the
amplicons with at least one probe selected from an influenza B MP probe and an
influenza B NS probe. In some embodiments, the influenza B MP probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 6, and the influenza B NS probe comprises a sequence
that is at least 90%, at least 95%, or 100% identical or complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7.
In some embodiments, the influenza B MP probe comprises a sequence that is at
least 90%, at least 95%, or 100% identical or complementary to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 34, and
the influenza B NS probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 37.
[0033] In some embodiments, the method comprises contacting the
amplicons with at least one probe selected from an RSV A probe and an RSV B
probe. In some embodiments, the RSV A probe comprises a sequence that is at
least 90%, at least 95%, or 100% identical or complementary to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 15, and
12
the influenza B NS probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 40, and the RSV B probe comprises a sequence that is
at least 90%, at least 95%, or 100% identical or complementary to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 43.
[0034] In some embodiments, each probe comprises a detectable label. In
some embodiments, the each probe comprises a fluorescent dye and a quencher
molecule. In some embodiments, the influenza A probes and the influenza B
probes comprise detectable labels that are detectably different. In some
embodiments, the influenza A probes and the influenza B probes comprise
detectable labels that are not detectably different. In some embodiments, each
probe consists of 15 to 30 nucleotides.
[0035] In some embodiments, the method comprises forming an
exogenous control amplicon. In some embodiments, the method comprises
contacting the exogenous control amplicon with a control probe capable of
selectively hybridizing with the exogenous control amplicon.
[0036] In some embodiments, the method comprises detecting the
presence of absence of at least one influenza A subtype and at least one influenza
B subtype and an exogenous control in a single multiplex reaction. In some
embodiments, the at least one influenza A subtype includes at least one avian
influenza. In some embodiments, the method comprises detecting RSV A and/or
RSV B in the same multiplex reaction.
[0037] In some embodiments, the sample is selected from a
nasopharyngeal swab sample, a nasal aspirate sample, and a nasal wash sample.
[0038] In some embodiments, compositions are provided. In some
embodiments, a composition comprises a first primer pair for detecting an
influenza PA gene. In some embodiments, the first primer pair comprises a first
primer and a second primer, wherein the first primer comprises a sequence that is
at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17,
at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 2, and wherein the second
primer comprises a sequence that is at least 90%, at least 95%, or 100%
13
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 2. In some embodiments, the first primer pair
comprises a first primer and a second primer, wherein the first primer comprises a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 20, and
wherein the second primer comprises a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least
19, or at least 20 contiguous nucleotides of SEQ ID NO: 21. In some
embodiments, the first primer has the sequence of SEQ ID NO: 20 and the second
primer has the sequence of SEQ ID NO: 21.
[0039] In some embodiments, a composition comprising a second primer
pair for detecting an influenza PB2 gene is provided. In some embodiments, the
second primer pair comprises a third primer and a fourth primer, wherein the third
primer comprises a sequence that is at least 90%, at least 95%, or 100% identical
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 1, and wherein the fourth primer comprises a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the second
primer pair comprises a third primer and a fourth primer, wherein the third primer
comprises a sequence that is at least 90%, at least 95%, or 100% identical to at
least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous
nucleotides of SEQ ID NO: 17, and wherein the fourth primer comprises a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides
of SEQ ID NO: 18. In some embodiments, the third primer has the sequence of
SEQ ID NO: 17 and the fourth primer has the sequence of SEQ ID NO: 18.
[0040] In some embodiments, a composition comprises at least one
additional primer pair, wherein each of the additional primer pairs is for detecting
a different influenza gene selected from an influenza A MP gene, an avian
influenza MP gene, and an avian influenza HA gene. In some embodiments, each
additional primer pair comprises a fifth primer and a sixth primer independently
selected from: (a) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
14
contiguous nucleotides of SEQ ID NO: 3, and a sixth primer comprising a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3; (b) a
fifth primer comprising a sequence that is at least 90%, at least 95%, or 100%
identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 4, and a sixth primer comprising a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 4; (c) a fifth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 5, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 5; (d) a fifth primer comprising a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 23, and a sixth primer comprising
a sequence that is at least 90%, at least 95%, or 100% complementary to at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO:
24; (e) a fifth primer comprising a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 26, and a sixth primer comprising a sequence that is at
least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 27; and (f) a fifth primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 29, and a sixth primer comprising a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 30.
15
[0041] In some embodiments, the composition further comprises at least
one additional primer pair, wherein each of the additional primer pairs is for
detecting a different influenza gene selected from an influenza B MP gene and an
influenza B NS gene. In some embodiments, each additional primer pair
comprises a seventh primer and an eighth primer independently selected from: (a)
a seventh primer comprising a sequence that is at least 90%, at least 95%, or
100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 6, and an eighth primer comprising a sequence that is
at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6; (b) a seventh
primer comprising a sequence that is at least 90%, at least 95%, or 100% identical
to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 7, and an eighth primer comprising a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 7; (c) a seventh primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 32, and
an eighth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 33; and (d) a seventh primer comprising a sequence
that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 35, and an eighth
primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 36.
[0042] In some embodiments, a composition further comprises at least one
additional primer pair, wherein each of the additional primer pairs is for detecting
RSV A or RSV B. In some embodiments, each additional primer pair comprises a
ninth primer and a tenth primer independently selected from: (a) a ninth primer
comprising a sequence that is at least 90%, at least 95%, or 100% identical to at
16
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 38, and a tenth primer comprising a sequence that is at least 90%, at least
95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 39; and (b) a ninth primer comprising a
sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 41, and
a tenth primer comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 42.
[0043] In some embodiments, a composition comprises primer pairs for
detecting an influenza A PA gene, an influenza A PB2 gene, an influenza A MP
gene, an avian influenza MP gene, and an avian influenza HA gene. In some
embodiments, a composition further comprises primer pairs for detecting RSV A
and RSV B. In some embodiments, a composition further comprises a primer pair
for detecting an exogenous control. In some embodiments, the exogenous control
is a sample processing control.
[0044] In some embodiments, a composition comprises at least one probe
selected from an influenza A PA probe and an influenza A PB2 probe. In some
embodiments, the influenza PA probe comprises a sequence that is at least 90%, at
least 95%, or 100% identical or complementary to at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 2, and the influenza PB2
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 1. In some embodiments, the influenza PA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 19, and the influenza PB2 probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to
at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 22.
17
[0045] In some embodiments, a composition further comprises at least one
probe selected from an influenza A MP probe, an avian influenza MP probe, and
an avian influenza HA probe. In some embodiments, the influenza MP probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 3, and the avian influenza MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to
at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4, and the avian influenza HA probe comprises a sequence that is at least
90%, at least 95%, or 100% identical or complementary to at least 15, at least 16,
at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 5. In some
embodiments, the influenza MP probe comprises a sequence that is at least 90%,
at least 95%, or 100% identical or complementary to at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 25, and the avian
influenza MP probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 28, and the avian influenza HA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 31.
[0046] In some embodiments, the composition further comprises at least
one probe selected from an influenza B MP probe and an influenza B NS probe.
In some embodiments, the influenza B MP probe comprises a sequence that is at
least 90%, at least 95%, or 100% identical or complementary to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6, and
the influenza B NS probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 7. In some embodiments, the influenza
B MP probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at
18
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 34, and the influenza B NS probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 37.
[0047] In some embodiments, the composition further comprises at least
one probe selected from an RSV A probe and an RSV B probe. In some
embodiments, the RSV A probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 15, and the influenza B NS probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 16. In some embodiments, the RSV A probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 40, and the RSV B probe comprises a sequence that is
at least 90%, at least 95%, or 100% identical or complementary to at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 43.
[0048] In some embodiments, the composition further comprises a probe
for detecting an exogenous control.
[0049] In some embodiments, each probe comprises a detectable label. In
some embodiments, each probe comprises a fluorescent dye and a quencher
molecule. In some embodiments, each probe consists of 15 to 30 nucleotides.
[0050] In some embodiments, the composition is a lyophilized
composition. In some embodiments, the composition is in solution. In some
embodiments, the composition comprises nucleic acids from a sample from a
subject being tested for the presence of absence of influenza. In some
embodiments, the sample is selected from a nasopharyngeal swab sample, a nasal
aspirate sample, and a nasal wash sample.
[0051] In some embodiments, kits are provided. In some embodiments, a
kit comprises a composition described herein. In some embodiments, the kit
further comprises an exogenous control. In some embodiments, the exogenous
control is an Armored® RNA. In some embodiments, the kit comprises dNTPs
19
and/or a thermostable polymerase. In some embodiments, the kit comprises a
reverse transcriptase.
[0052] In some embodiments, an oligonucleotide consisting of a sequence
selected from SEQ ID NOs: 17 to 43 is provided. In some embodiments, the
oligonucleotide comprises at least one modified nucleotide. In some
embodiments, the oligonucleotide comprises a detectable label. In some
embodiments, the oligonucleotide comprises a fluorescent dye and a quencher
molecule. In some embodiments, the oligonucleotide is a fluorescence resonance
energy transfer (FRET) probe.
[0053] In some embodiments, a composition comprising a first primer
consisting of the sequence of SEQ ID NO: 17 and a second primer consisting of
the sequence of SEQ ID NO: 18, wherein the first primer and the second primer
each comprises at least one modified nucleotide is provided. In some
embodiments, the composition comprises a probe consisting of the sequence of
SEQ ID NO: 19, wherein the probe comprises at least one modified nucleotide
and/or a detectable label.
[0054] In some embodiments, a composition comprising a first primer
consisting of the sequence of SEQ ID NO: 20 and a second primer consisting of
the sequence of SEQ ID NO: 21, wherein the first primer and the second primer
each comprises at least one modified nucleotide is provided. In some
embodiments, the composition comprises a probe consisting of the sequence of
SEQ ID NO: 22, wherein the probe comprises at least one modified nucleotide
and/or a detectable label. In some embodiments, the probe is a fluorescence
resonance energy transfer (FRET) probe. In some embodiments, the probe
comprises at least one modified nucleotide. In some embodiments, the
composition is a lyophilized composition. In some embodiments, the composition
is in solution. In some embodiments, the composition comprises nucleic acids of
a sample from a subject.
4. DETAILED DESCRIPTION
4.1. Definitions
[0055] To facilitate an understanding of the present invention, a number of
terms and phrases are defined below:
[0056] As used herein, the terms “detect”, “detecting” or “detection” may
describe either the general act of discovering or discerning or the specific
observation of a detectably labeled composition.
[0057] As used herein, the term “detectably different” refers to a set of
labels (such as dyes) that can be detected and distinguished simultaneously.
20
[0058] As used herein, the terms “patient” and “subject” are used
interchangeably to refer to a human. In some embodiments, the methods
described herein may be used on samples from non-human animals.
[0059] As used herein, the terms “oligonucleotide,” “polynucleotide,”
“nucleic acid molecule,” and the like, refer to nucleic acid-containing molecules,
including but not limited to, DNA or RNA. The term encompasses sequences that
include any of the known base analogs of DNA and RNA including, but not
limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil,
dihydrouracil, inosine, N6-isopentenyladenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, Nuracil-
5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil,
queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0060] As used herein, the term “oligonucleotide,” refers to a singlestranded
polynucleotide having fewer than 500 nucleotides. In some
embodiments, an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to
75, or 12 to 50 nucleotides long. Oligonucleotides may be referred to by their
length, for example, a 24 residue oligonucleotide may be referred to as a “24-
mer.”
[0061] As used herein, the term “complementary” to a target RNA (or
target region thereof), and the percentage of “complementarity” of the probe
sequence to that of the target RNA sequence is the percentage “identity” to the
sequence of target RNA or to the reverse complement of the sequence of the
target RNA. In determining the degree of “complementarity” between probes
used in the compositions described herein (or regions thereof) and a target RNA,
such as those disclosed herein, the degree of “complementarity” is expressed as
the percentage identity between the sequence of the probe (or region thereof) and
sequence of the target RNA or the reverse complement of the sequence of the
target RNA that best aligns therewith. The percentage is calculated by counting
the number of aligned bases that are identical as between the 2 sequences,
21
dividing by the total number of contiguous nucleotides in the probe, and
multiplying by 100. When the term “complementary” is used, the subject
oligonucleotide is at least 90% complementary to the target molecule, unless
indicated otherwise. In some embodiments, the subject oligonucleotide is at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% complementary to the target molecule.
[0062] A “primer” or “probe” as used herein, refers to an oligonucleotide
that comprises a region that is complementary to a sequence of at least 8
contiguous nucleotides of a target nucleic acid molecule, such as DNA (e.g., a
target gene) or an mRNA (or a DNA reverse-transcribed from an mRNA). In
some embodiments, a primer or probe comprises a region that is complementary
to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least
14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least
21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least
28, at least 29, or at least 30 contiguous nucleotides of a target molecule. When a
primer or probe comprises a region that is “complementary to at least x
contiguous nucleotides of a target molecule,” the primer or probe is at least 95%
complementary to at least x contiguous nucleotides of the target molecule. In
some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%,
at least 99%, or 100% complementary to the target molecule.
[0063] The term “nucleic acid amplification,” encompasses any means
by which at least a part of at least one target nucleic acid is reproduced, typically
in a template-dependent manner, including without limitation, a broad range of
techniques for amplifying nucleic acid sequences, either linearly or exponentially.
Exemplary means for performing an amplifying step include polymerase chain
reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR),
multiplex ligation-dependent probe amplification (MLPA), ligation followed by
Q-replicase amplification, primer extension, strand displacement amplification
(SDA), hyperbranched strand displacement amplification, multiple displacement
amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step
multiplexed amplifications, rolling circle amplification (RCA), and the like,
including multiplex versions and combinations thereof, for example but not
limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR,
LCR/PCR, PCR/LCR (also known as combined chain reaction--CCR), digital
amplification, and the like. Descriptions of such techniques can be found in,
among other sources, Ausbel et al.; PCR Primer: A Laboratory Manual,
Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book,
Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The
22
Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J.
(2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.;4(1):41-7, U.S. Pat.
No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No. WO
97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics,
29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al.,
PCR Protocols: A Guide to Methods and Applications, Academic Press (1990);
Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection
28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex
Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium
on Human Identification, 1995 (available on the world wide web at:
promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction
Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl.
Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-
2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc
Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11
(1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf.
Dis. 2:18- (2002); Lage et al., Genome Res. 2003 Feb.;13(2):294-307, and
Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol
Diagn. 2002 Nov.;2(6):542-8., Cook et al., J Microbiol Methods. 2003
May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.;12(1):21-7,
U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, PCT
Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.
[0064] In some embodiments, amplification comprises at least one
cycle of the sequential procedures of: annealing at least one primer with
complementary or substantially complementary sequences in at least one target
nucleic acid; synthesizing at least one strand of nucleotides in a templatedependent
manner using a polymerase; and denaturing the newly-formed nucleic
acid duplex to separate the strands. The cycle may or may not be repeated.
Amplification can comprise thermocycling or can be performed isothermally.
[0065] Unless otherwise indicated, the term “hybridize” is used herein
refer to “specific hybridization” which is the binding, duplexing, or hybridizing of
a nucleic acid molecule preferentially to a particular nucleotide sequence, in some
embodiments, under stringent conditions. The term “stringent conditions” refers
to conditions under which a probe will hybridize preferentially to its target
sequence, and to a lesser extent to, or not at all to, other sequences. A “stringent
hybridization” and “stringent hybridization wash conditions” in the context of
nucleic acid hybridization (e.g., as in array, Southern, or Northern hybridization)
are sequence-dependent and are different under different environmental
23
parameters. An extensive guide to the hybridization of nucleic acids is found in,
e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of
principles of hybridization and the strategy of nucleic acid probe assays,”
Elsevier, NY (“Tijssen”). Generally, highly stringent hybridization and wash
conditions for filter hybridizations are selected to be about 5 C. lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent conditions are selected to be equal to the Tm for a particular probe.
Dependency of hybridization stringency on buffer composition, temperature, and
probe length are well known to those of skill in the art (see, e.g., Sambrook and
Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY).
[0066] A “sample,” as used herein, includes various nasal samples, such as
nasopharyngeal swab samples, nasal aspirate samples, nasal wash samples, and
other types of human samples. In some embodiments, a nasal sample comprises a
buffer, such as a preservative. Further nonlimiting exemplary samples include
nasal swabs, oropharyngeal swabs, throat swabs, bronchoalveolar lavage samples,
bronchial aspirates, bronchial washes, endotracheal aspirates, endotracheal
washes, tracheal aspirates, nasal secretion samples, mucus samples, sputum
samples, and lung tissue samples. In some embodiments, the sample comprises a
buffer, such as a preservative.
[0067] An “endogenous control,” as used herein refers to a moiety that is
naturally present in the sample to be used for detection. In some embodiments, an
endogenous control is a “sample adequacy control” (SAC), which may be used to
determine whether there was sufficient sample used in the assay, or whether the
sample comprised sufficient biological material, such as cells. In some
embodiments, an endogenous control is an RNA (such as an mRNA, tRNA,
ribosomal RNA, etc.), such as a human RNA. Nonlimiting exemplary
endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA,
TUBB mRNA, and UPK1a mRNA. In some embodiments, an endogenous
control, such as an SAC, is selected that can be detected in the same manner as the
target RNA is detected and, in some embodiments, simultaneously with the target
RNA.
[0068] An “exogenous control,” as used herein, refers to a moiety that is
added to a sample or to an assay, such as a “sample processing control” (SPC). In
some embodiments, an exogenous control is included with the assay reagents. An
24
exogenous control is typically selected that is not expected to be present in the
sample to be used for detection, or is present at very low levels in the sample such
that the amount of the moiety naturally present in the sample is either undetectable
or is detectable at a much lower level than the amount added to the sample as an
exogenous control. In some embodiments, an exogenous control comprises a
nucleotide sequence that is not expected to be present in the sample type used for
detection of the target RNA. In some embodiments, an exogenous control
comprises a nucleotide sequence that is not known to be present in the species
from whom the sample is taken. In some embodiments, an exogenous control
comprises a nucleotide sequence from a different species than the subject from
whom the sample was taken. In some embodiments, an exogenous control
comprises a nucleotide sequence that is not known to be present in any species. In
some embodiments, an exogenous control is selected that can be detected in the
same manner as the target RNA is detected and, in some embodiments,
simultaneously with the target RNA. In some embodiments, the exogenous
control is an RNA. In some such embodiments, the exogenous control is an
Armored RNA®, which comprises RNA packaged in a bacteriophage protective
coat. See, e.g., WalkerPeach et al., Clin. Chem. 45:12: 2079-2085 (1999).
[0069] In the sequences herein, “U” and “T” are used interchangeably,
such that both letters indicate a uracil or thymine at that position. One skilled in
the art will understand from the context and/or intended use whether a uracil or
thymine is intended and/or should be used at that position in the sequence. For
example, one skilled in the art would understand that native RNA molecules
typically include uracil, while native DNA molecules typically include thymine.
Thus, where an RNA sequence includes “T”, one skilled in the art would
understand that that position in the native RNA is likely a uracil.
[0070] In the present disclosure, “a sequence selected from” encompasses
both “one sequence selected from” and “one or more sequences selected from.”
Thus, when “a sequence selected from” is used, it is to be understood that one, or
more than one, of the listed sequences may be chosen.
[0071] In the present disclosure, a method that comprises detecting a “a
set of influenza A (fluA) markers consisting of…” involves detection of only the
fluA markers of the set, and not any further fluA markers. The method may
comprise additional components or steps, however, such as for detecting fluB,
respiratory syncytial virus (RSV), and/or endogenous and/or exogenous controls.
Similarly, a method or composition that comprises “a set of influenza A (fluA)
marker primer pairs” and/or “a set of fluA marker probes” can include primer
pairs and/or probes for only the fluA markers of the set, and not for any other fluA
25
markers. The method or composition may comprise additional components,
however, such as one or more fluB primer pairs, RSV primer pairs, endogenous
control primer pairs and/or exogenous control primer pairs.
[0072]
4.2. Detecting Influenza A
[0073] The present inventors have developed a more sensitive assay for
detecting Influenza A. In some embodiments, the assay comprises detecting the
Flu A polymerase basic 2 (PB2) gene and/or the Flu A polymerase acidic (PA)
gene. In some embodiments, the assay comprises detecting PA and/or PB2 in
addition to the Flu A matrix protein (MP) gene. The present assay relies on the
polymerase chain reaction (PCR), and can be carried out in a substantially
automated manner using a commercially available nucleic acid amplification
system. Exemplary nonlimiting nucleic acid amplification systems that can be
used to carry out the methods of the invention include the GeneXpert® system, a
GeneXpert® Infinity system, and a Smartcycler System (Cepheid, Sunnyvale,
CA). The present assay can be completed in under 3 hours, and in some
embodiments, under 2 hours, using an automated system, for example, the
GeneXpert® system.
4.2.1. General methods
[0074] Compositions and methods for detecting Flu A are provided. In
some embodiments, the method comprises detecting the Flu A PB2 gene and/or
PA gene. In some embodiments, the method comprises detecting the Flu A PB2
gene and PA gene. In some embodiments, the method comprises detecting the Flu
A PB2 gene and/or PA gene, and also detecting the Flu A MP gene. In some
embodiments, the method comprises detecting one or more of avian Flu (such as
Flu A 2 and/or Flu A 3), Flu B, RSV A, and RSV B.
[0075] In some embodiments, a method of detecting Flu A in a subject
comprises detecting the presence of the Flu A PB2 gene and/or PA gene in a
sample from the subject. In some embodiments, the method comprises detecting
the Flu A PB2 gene and PA gene in a sample from the subject. In some
embodiments, the sample is selected from a nasopharyngeal swab sample, a nasal
aspirate sample, and a nasal wash sample.
[0076] In some embodiments, a method of detecting Flu A further
comprises detecting at least one endogenous control, such as a sample adequacy
control (SAC). In some embodiments, a method of detecting Flu A further
comprises detecting at least one exogenous control, such as a sample processing
control (SPC). In some embodiments, the SPC is Armored® RNA.
26
[0077] In some embodiments, a method of detecting Flu A comprises
detecting the Flu A PB2 gene and/or PA gene in a sample. In some embodiments,
a method of detecting Flu A further comprises detecting a sample processing
control (SPC), such as an Armored® RNA.
[0078] In the present disclosure, the terms “target RNA” and “target gene”
are used interchangeably to refer to the Flu A PB2 gene and Flu A PA gene, and
also to other Flu and RSV genes, as well as to exogenous and/or endogenous
controls. Thus, it is to be understood that when a discussion is presented in terms
of a target gene, that discussion is specifically intended to encompass the Flu A
PB2 gene and Flu A PA gene, other Flu and RSV genes, any endogenous
control(s) (e.g., SAC), and any exogenous control(s) (e.g., SPC).
[0079] In some embodiments, the presence of the Flu A PB2 gene and/or
Flu A PA gene is detected in a nasal sample. In some embodiments, the target
gene is detected in a nasal aspirate sample or a nasal wash sample. In some
embodiments, a target gene is detected in a sample to which a buffer (such as a
preservative) has been added. In some embodiments, the presence of the Flu A
PB2 gene and/or Flu A PA gene is detected in a nasopharyngeal swab sample. In
some embodiments, the target gene is detected in an nasopharyngeal swab sample
that has been placed in a buffer (such as a preservative).
[0080] In some embodiments, detection of the Flu A PB2 gene and/or Flu
A PA gene in a sample from a subject indicates the presence of Flu A in the
subject. In some embodiments, detection of the Flu A PB2 gene and/or Flu A PA
gene in a sample from a subject indicates the presence of Flu A 1 in the subject.
In some embodiments, the detecting is done quantitatively. In other
embodiments, the detecting is done qualitatively. In some embodiments,
detecting a target gene comprises forming a complex comprising a polynucleotide
and a nucleic acid selected from a target gene, a cDNA reverse transcribed from a
target gene, a DNA amplicon of a target gene, and a complement of a target gene.
In some embodiments, detecting a target gene comprises RT-PCR. In some
embodiments, detecting a target gene comprises quantitative RT-PCR or real-time
RT-PCR. In some embodiments, a sample adequacy control (SAC) and/or a
sample processing control (SPC) is detected in the same assay as the target gene.
In some embodiments, if the Flu A PB2 gene or Flu A PA gene is detected, Flu A
is considered to be detected even if the SPC is not detected in the assay. In some
embodiments, if the Flu A PB2 gene and Flu A PA gene are not detected, Flu A is
considered to be not detected only if the SPC is detected in the assay.
[0081] In some embodiments, the presence of the Flu A PB2 gene and/or
Flu A PA gene can be measured in samples collected at one or more times from a
27
subject to monitor treatment for Flu in the subject. In some embodiments, the
present assay may be used as part of routine and/or preventative healthcare for a
subject. In some embodiments, the present assay may be used seasonally as part
of routine and/or preventative healthcare for a subject. In some embodiments, the
present assay may be used as part of routine and/or preventative healthcare for
subjects who are at particular risk from influenza, such as immunocompromised
and elderly subjects.
[0082] In some embodiments, a sample to be tested is a nasal aspirate
sample or nasal wash sample, or is derived from a nasal aspirate sample or nasal
wash sample. In some embodiments, a buffer (such as a preservative) is added to
the nasal aspirate sample or nasal wash sample. In some embodiments, the buffer
is added to the nasal aspirate sample or nasal wash sample 5 minutes, within 10
minutes, within 30 minutes, within 1 hour, or within 2 hours of sample collection.
[0083] In some embodiments, a sample to be tested is a nasopharyngeal
swab sample. In some embodiments, the swab is placed in a buffer. In some
embodiments, the swab is immediately placed in the buffer. In some
embodiments, the swab is placed in the buffer within 5 minutes, within 10
minutes, within 30 minutes, within 1 hour, or within 2 hours of sample collection.
[0084] In some embodiments, less than 5 ml, less than 4 ml, less than 3
ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of sample or buffered
sample are used in the present methods. In some embodiments, 0.1 ml to 1 ml of
sample or buffered sample is used in the present methods.
[0085] In some embodiments, the sample to be tested is another bodily
fluid, such as saliva, nasal swabs, oropharyngeal swabs, throat swabs,
bronchoalveolar lavage samples, bronchial aspirates, bronchial washes,
endotracheal aspirates, endotracheal washes, tracheal aspirates, nasal secretion
samples, mucus samples, sputum samples, lung tissue samples, etc.
[0086] The clinical sample to be tested is, in some embodiments, fresh
(i.e., never frozen). In other embodiments, the sample is a frozen specimen. In
some embodiments, the sample is a tissue sample, such as a formalin-fixed
paraffin embedded sample. In some embodiments, the sample is a liquid cytology
sample.
[0087] In some embodiments, the sample to be tested is obtained from an
individual who has one or more symptoms of influenza infection. Nonlimiting
exemplary symptoms of influenza include fever, chills, cough, sore throat, runny
nose, nasal congestion, muscle ache, headache, fatigue, vomiting, diarrhea, and
combinations of any of those symptoms. In some embodiments, the sample to be
tested is obtained from an individual who has previously been diagnosed with
28
influenza. In some such embodiments, the individual is monitored for recurrence
of influenza.
[0088] In some embodiments, methods described herein can be used for
routine screening of healthy individuals with no risk factors. In some
embodiments, methods described herein are used to screen asymptomatic
individuals, for example, during routine or preventative care. In some
embodiments, methods described herein are used to screen women who are
pregnant or who are attempting to become pregnant.
[0089] In some embodiments, the methods described herein can be used to
assess the effectiveness of a treatment for influenza infection in a patient.
[0090] In some embodiments, use of the polymerase acidic (PA) gene
and/or polymerase basic 2 (PB2) gene for detecting Flu A is provided. In some
embodiments, use of the PA gene and/or PB2 gene for detecting Flu A is
provided. In some embodiments, use of the PA gene and/or PB2 gene, and
optionally, one or more genes selected from the matrix protein (MP) gene and
nonstructural protein (NP) gene for detecting Flu A is provided. In some
embodiments, use of the PA gene, PB2 gene, MP gene, and NP gene for detecting
Flu A and Flu B is provided.
[0091] In any of the embodiments described herein, the polymerase acidic
(PA) gene and/or polymerase basic 2 (PB2) gene may be detected in the same
assay reaction as a sample processing control (SPC).
[0092] In some embodiments, a method of facilitating detection of Flu A
infection in a subject is provided. Such methods comprise detecting the presence
or absence of the Flu A PB2 gene and/or Flu A PA gene in a sample from the
subject. In some embodiments, information concerning the presence or absence
of the Flu A PB2 gene and/or Flu A PA gene in the sample from the subject is
communicated to a medical practitioner. A “medical practitioner,” as used herein,
refers to an individual or entity that diagnoses and/or treats patients, such as a
hospital, a clinic, a physician’s office, a physician, a nurse, or an agent of any of
the aforementioned entities and individuals. In some embodiments, detecting the
presence or absence of the Flu A PB2 gene and/or Flu A PA gene is carried out at
a laboratory that has received the subject’s sample from the medical practitioner
or agent of the medical practitioner. The laboratory carries out the detection by
any method, including those described herein, and then communicates the results
to the medical practitioner. A result is “communicated,” as used herein, when it is
provided by any means to the medical practitioner. In some embodiments, such
communication may be oral or written, may be by telephone, in person, by e-mail,
by mail or other courier, or may be made by directly depositing the information
29
into, e.g., a database accessible by the medical practitioner, including databases
not controlled by the medical practitioner. In some embodiments, the information
is maintained in electronic form. In some embodiments, the information can be
stored in a memory or other computer readable medium, such as RAM, ROM,
EEPROM, flash memory, computer chips, digital video discs (DVD), compact
discs (CDs), hard disk drives (HDD), magnetic tape, etc.
[0093] In some embodiments, methods of detecting Flu A are provided.
In some embodiments, methods of diagnosing Flu A infection are provided. In
some embodiments, the method comprises obtaining a sample from a subject and
providing the sample to a laboratory for detection of the Flu A PB2 gene and/or
Flu A PA gene in the sample. In some embodiments, the method further
comprises receiving a communication from the laboratory that indicates the
presence or absence of the Flu A PB2 gene and/or Flu A PA gene in the sample.
A “laboratory,” as used herein, is any facility that detects the target gene in a
sample by any method, including the methods described herein, and
communicates the result to a medical practitioner. In some embodiments, a
laboratory is under the control of a medical practitioner. In some embodiments, a
laboratory is not under the control of the medical practitioner.
[0094] When a laboratory communicates the result of detecting the
presence or absence of the Flu A PB2 gene and/or Flu A PA gene to a medical
practitioner, in some embodiments, the laboratory indicates whether or not the Flu
A PB2 gene and/or Flu A PA gene was detected in the sample. In some
embodiments, the laboratory indicates whether the sample comprises Flu A, by
indicating, for example, “Flu positive” or “Flu negative” or “Flu present” or “Flu
absent,” and the like.
[0095] As used herein, when a method relates to detecting Flu A,
determining the presence of Flu A, monitoring for Flu A, and/or diagnosing Flu A
infection, the method includes activities in which the steps of the method are
carried out, but the result is negative for the presence of Flu A. That is, detecting,
determining, monitoring, and diagnosing Flu A or Flu A infection include
instances of carrying out the methods that result in either positive or negative
results.
[0096] In some embodiments, at least one endogenous control (e.g., an
SAC) and/or at least one exogenous control (e.g., an SPC) are detected
simultaneously with the Flu A PB2 gene and/or Flu A PA gene in a single
reaction. In some embodiments, at least one exogenous control (e.g., an SPC) is
detected simultaneously with the Flu A PB2 gene and/or Flu A PA gene in a
single reaction.
30
[0097] In any of the embodiments described herein, the Flu A PB2 gene
and/or Flu A PA gene may be detected along with one or more additional Flu
genes, including but not limited to, Flu A 1 MP, Flu A 2 MP, Flu A 3
haemagglutinin (HA), and Flu B MP. In any of the embodiments described
herein, the Flu A PB2 gene and/or Flu A PA gene may be detected along with one
or more additional Flu genes such as those listed above, and RSV, such as RSV A
and/or RSV B.
4.2.2. Exemplary controls
[0098] In some embodiments, an assay described herein comprises
detecting the Flu A PB2 gene and/or Flu A PA gene and at least one endogenous
control. In some embodiments, the endogenous control is a sample adequacy
control (SAC). In some such embodiments, if neither the Flu A PB2 gene nor Flu
A PA gene is detected in a sample, and the SAC is also not detected in the sample,
the assay result is considered “invalid” because the sample may have been
insufficient. While not intending to be bound by any particular theory, an
insufficient sample may be too dilute, contain too little cellular material, contain
an assay inhibitor, etc. In some embodiments, the failure to detect an SAC may
indicate that the assay reaction failed. In some embodiments, an endogenous
control is an RNA (such as an mRNA, tRNA, ribosomal RNA, etc.). Nonlimiting
exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH
mRNA, TUBB mRNA, and UPK1a mRNA.
[0099] In some embodiments, an assay described herein comprises
detecting the Flu A PB2 gene and/or Flu A PA gene and at least one exogenous
control. In some embodiments, the exogenous control is a sample processing
control (SPC). In some such embodiments, if the PB2 gene and/or the PA gene is
not detected in a sample, and the SPC is also not detected in the sample, the assay
result is considered “invalid” because there may have been an error in sample
processing, including but not limited to, failure of the assay. Nonlimiting
exemplary errors in sample processing include, inadequate sample processing, the
presence of an assay inhibitor, the presence of a nuclease (such as an RNase),
compromised reagents, etc. In some embodiments, an exogenous control (such as
an SPC) is added to a sample. In some embodiments, an exogenous control (such
as an SPC) is added during performance of an assay, such as with one or more
buffers or reagents. In some embodiments, when a GeneXpert® system is to be
used, the SPC is included in the GeneXpert® cartridge. In some embodiments, an
exogenous control (such as an SPC) is an Armored RNA®, which is protected by a
bacteriophage coat.
31
[00100] In some embodiments, an endogenous control and/or an exogenous
control is detected contemporaneously, such as in the same assay, as detection of
the PA gene and/or PB2 gene. In some embodiments, an assay comprises
reagents for detecting the PA gene and/or PB2 gene and an exogenous control
simultaneously in the same assay reaction. In some such embodiments, for
example, an assay reaction comprises a primer set for amplifying the PA gene
and/or a primer set for amplifying the PB2 gene, and, a primer set for amplifying
an exogenous control, and labeled probes for detecting the amplification products
(such as, for example, TaqMan® probes).
4.2.3. Exemplary sample preparation
4.2.3.1. Exemplary buffers
[00101] In some embodiments, a buffer is added to the sample. In some
embodiments, the buffer is added within one hour, two hours, three hours, or six
hours of the time the sample was collected. In some embodiments, a buffer is
added to the sample within one hour, two hours, three hours, or six hours before
the sample is analyzed by the methods described herein.
[00102] In some embodiments, a swab sample is placed in a buffer. In
some embodiments, the swab sample is placed in the buffer within one hour, two
hours, three hours, or six hours of the time the swab sample was collected. In
some embodiments, the swab sample is placed in a buffer within one hour, two
hours, three hours, or six hours before the sample is analyzed by the methods
described herein.
[00103] Non-limiting exemplary commercial buffers include the viral
transport medium provided with the GeneXpert® Nasal Pharyngeal Collection Kit
(Cepheid, Sunnyvale, CA); universal transport medium (UTM™, Copan,
Murrieta, CA); universal viral transport medium (UVT, BD, Franklin Lakes, NJ);
M4, M$RT, M5, and M6 (Thermo Scientific). Further nonlimiting exemplary
buffers include liquid Amies medium, PBS/0.5% BSA, PBS/0.5% gelatin, Bartel
BiraTrans™ medium, EMEM, PBS, EMEM/1% BSA, sucrose phosphate,
Trypticase™ soy broth (with or without 0.5% gelatin or 0.5% BSA), modified
Stuart’s medium, veal infusion broth (with or without 0.5% BSA), and saline.
4.2.3.2. Exemplary RNA preparation
[00104] Target RNA can be prepared by any appropriate method. Total
RNA can be isolated by any method, including, but not limited to, the protocols
set forth in Wilkinson, M. (1988) Nucl. Acids Res. 16(22):10,933; and Wilkinson,
M. (1988) Nucl. Acids Res. 16(22): 10934, or by using commercially-available
32
kits or reagents, such as the TRIzol® reagent (Invitrogen), Total RNA Extraction
Kit (iNtRON Biotechnology), Total RNA Purification Kit (Norgen Biotek Corp.),
RNAqueous™ (Ambion), MagMAX™ (Ambion), RecoverAll™ (Ambion),
RNAeasy (Qiagen), etc.
[00105] In some embodiments, RNA levels are measured in a sample in
which RNA has not first been purified from the cells. In some such embodiments,
the cells are subject to a lysis step to release the RNA. Nonlimiting exemplary
lysis methods include sonication (for example, for 2-15 seconds, 8-18 μm at 36
kHz); chemical lysis, for example, using a detergent; and various commercially
available lysis reagents (such as RNAeasy lysis buffer, Qiagen). In some
embodiments, RNA levels are measured in a sample in which RNA has been
isolated.
[00106] In some embodiments, RNA is modified before a target RNA is
detected. In some embodiments, all of the RNA in the sample is modified. In
some embodiments, just the particular target RNAs to be analyzed are modified,
e.g., in a sequence-specific manner. In some embodiments, RNA is reverse
transcribed. In some such embodiments, RNA is reverse transcribed using
MMLV reverse transcriptase. Nonlimiting exemplary conditions for reverse
transcribing RNA using MMLV reverse transcriptase include incubation from 5 to
20 minutes at 40°C to 50°C.
[00107] When a target RNA is reverse transcribed, a DNA complement
of the target RNA is formed. In some embodiments, the complement of a target
RNA is detected rather than a target RNA itself (or a DNA copy of the RNA
itself). Thus, when the methods discussed herein indicate that a target RNA is
detected, or the level of a target RNA is determined, such detection or
determination may be carried out on a complement of a target RNA instead of, or
in addition to, the target RNA itself. In some embodiments, when the
complement of a target RNA is detected rather than the target RNA, a
polynucleotide for detection is used that is complementary to the complement of
the target RNA. In some such embodiments, a polynucleotide for detection
comprises at least a portion that is identical in sequence to the target RNA,
although it may contain thymidine in place of uridine, and/or comprise other
modified nucleotides.
4.2.4. Exemplary analytical methods
[00108] As described above, methods are presented for detecting influenza,
and optionally, respiratory syncytial virus (RSV). The methods comprise
detecting the presence of the Flu A polymerase basic 2 (PB2) gene and/or
polymerase acidic (PA) gene in a sample from a subject. In some embodiments,
33
the method further comprises detecting one or more additional target genes
selected from Flu A 1 matrix protein (MP) gene, Flu A 2 matrix protein (MP)
gene, Flu A 3 haemagglutinin (HA) gene, Flu B nonstructural protein (NS) gene,
RSV A genome, or RSV B genome, and optionally, at least one exogenous control
(such as an SPC). In some embodiments, detection of one or more genes selected
from Flu A polymerase basic 2 (PB2) gene, polymerase acidic (PA) gene, Flu A 1
matrix protein (MP) gene, Flu A 2 matrix protein (MP) gene, Flu A 3
haemagglutinin (HA) gene, and Flu B nonstructural protein (NS) gene indicates
the presence of influenza, even if the endogenous control and/or exogenous
control is not detected in the assay. In some embodiments, detection of RSV A or
RSV B indicates the presence of RSV, even if the endogenous control and/or
exogenous control is not detected in the assay. In some embodiments, if none of
the flu target genes (such as the Flu A polymerase basic 2 (PB2) gene, polymerase
acidic (PA) gene, Flu A 1 matrix protein (MP) gene, Flu A 2 matrix protein (MP)
gene, Flu A 3 haemagglutinin (HA) gene, and Flu B nonstructural protein (NS)
gene) is detected, the result is considered to be negative for influenza only if the
control detected.
[00109] Any analytical procedure capable of permitting specific detection
of a target gene may be used in the methods herein presented. Exemplary
nonlimiting analytical procedures include, but are not limited to, nucleic acid
amplification methods, PCR methods, isothermal amplification methods, and
other analytical detection methods known to those skilled in the art.
[00110] In some embodiments, the method of detecting a target gene, such
as the Flu A 1 matrix protein (MP) gene or Flu A 2 matrix protein (MP) gene,
comprises amplifying the gene and/or a complement thereof. Such amplification
can be accomplished by any method. Exemplary methods include, but are not
limited to, isothermal amplification, real time RT-PCR, endpoint RT-PCR, and
amplification using T7 polymerase from a T7 promoter annealed to a DNA, such
as provided by the SenseAmp Plus™ Kit available at Implen, Germany.
[00111] When a target gene is amplified, in some embodiments, an
amplicon of the target gene is formed. An amplicon may be single stranded or
double-stranded. In some embodiments, when an amplicon is single-stranded, the
sequence of the amplicon is related to the target gene in either the sense or
antisense orientation. In some embodiments, an amplicon of a target gene is
detected rather than the target gene itself. Thus, when the methods discussed
herein indicate that a target gene is detected, such detection may be carried out on
an amplicon of the target gene instead of, or in addition to, the target gene itself.
In some embodiments, when the amplicon of the target gene is detected rather
34
than the target gene, a polynucleotide for detection is used that is complementary
to the complement of the target gene. In some embodiments, when the amplicon
of the target gene is detected rather than the target gene, a polynucleotide for
detection is used that is complementary to the target gene. Further, in some
embodiments, multiple polynucleotides for detection may be used, and some
polynucleotides may be complementary to the target gene and some
polynucleotides may be complementary to the complement of the target gene.
[00112] In some embodiments, the method of detecting a target gene, such as
the Flu A 1 matrix protein (MP) gene or Flu A 2 matrix protein (MP) gene,
comprises PCR, as described below. In some embodiments, detecting one or
more target genes comprises real-time monitoring of a PCR reaction, which can
be accomplished by any method. Such methods include, but are not limited to, the
use of TaqMan®, molecular beacons, or Scorpion probes (i.e., energy transfer
(ET) probes, such as FRET probes) and the use of intercalating dyes, such as
SYBR green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc.
[00113] Nonlimiting exemplary conditions for amplifying a cDNA that has
been reverse transcribed from the target RNA are as follows. An exemplary cycle
comprises an initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes,
followed by cycling that comprises denaturation at 90°C to 100°C for 1 to 10
seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40
seconds. A further exemplary cycle comprises 20 seconds at 94°C, followed by
up to 3 cycles of 1 second at 95°C, 35 seconds at 62°C, 20 cycles of 1 second at
95°C, 20 seconds at 62°C, and 14 cycles of 1 second at 95°C, 35 seconds at 62°C.
In some embodiments, for the first cycle following the initial denaturation step,
the cycle denaturation step is omitted. In some embodiments, Taq polymerase is
used for amplification. In some embodiments, the cycle is carried out at least 10
times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at
least 35 times, at least 40 times, or at least 45 times. In some embodiments, Taq is
used with a hot start function. In some embodiments, the amplification reaction
occurs in a GeneXpert® cartridge, and amplification of the target genes and an
exogenous control occurs in the same reaction. In some embodiments, detection
of the target genes occurs in less than 3 hours, less than 2.5 hours, less than 2
hours, less than 1 hour, or less than 30 minutes from initial denaturation through
the last extension.
[00114] In some embodiments, detection of a target gene comprises
forming a complex comprising a polynucleotide that is complementary to a target
gene or to a complement thereof, and a nucleic acid selected from the target gene,
a DNA amplicon of the target gene, and a complement of the target gene. Thus,
35
in some embodiments, the polynucleotide forms a complex with a target gene. In
some embodiments, the polynucleotide forms a complex with a complement of
the target RNA, such as a cDNA that has been reverse transcribed from the target
RNA. In some embodiments, the polynucleotide forms a complex with a DNA
amplicon of the target gene. When a double-stranded DNA amplicon is part of a
complex, as used herein, the complex may comprise one or both strands of the
DNA amplicon. Thus, in some embodiments, a complex comprises only one
strand of the DNA amplicon. In some embodiments, a complex is a triplex and
comprises the polynucleotide and both strands of the DNA amplicon. In some
embodiments, the complex is formed by hybridization between the polynucleotide
and the target gene, complement of the target gene, or DNA amplicon of the target
gene. The polynucleotide, in some embodiments, is a primer or probe.
[00115] In some embodiments, a method comprises detecting the complex.
In some embodiments, the complex does not have to be associated at the time of
detection. That is, in some embodiments, a complex is formed, the complex is
then dissociated or destroyed in some manner, and components from the complex
are detected. An example of such a system is a TaqMan® assay. In some
embodiments, when the polynucleotide is a primer, detection of the complex may
comprise amplification of the target gene, a complement of the target gene, or a
DNA amplicon of the target gene.
[00116] In some embodiments the analytical method used for detecting at
least one target gene in the methods set forth herein includes real-time quantitative
PCR. In some embodiments, the analytical method used for detecting at least one
target gene includes the use of a TaqMan® probe. The assay uses energy transfer
(“ET”), such as fluorescence resonance energy transfer (“FRET”), to detect and
quantitate the synthesized PCR product. Typically, the TaqMan® probe comprises
a fluorescent dye molecule coupled to the 5’-end and a quencher molecule
coupled to the 3’-end, such that the dye and the quencher are in close proximity,
allowing the quencher to suppress the fluorescence signal of the dye via FRET.

WE CLAIM:
1. A composition comprising a first primer pair for detecting an influenza PA
gene.
2. The composition of claim 1, wherein the first primer pair comprises a first
primer and a second primer, wherein the first primer comprises a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 2, and wherein the second primer
comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 2.
3. The composition of claim 1, wherein the first primer pair comprises a first
primer and a second primer, wherein the first primer comprises a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 20, and wherein the second primer
comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous
nucleotides of SEQ ID NO: 21.
4. The composition of claim 1, wherein the first primer has the sequence of SEQ
ID NO: 20 and the second primer has the sequence of SEQ ID NO: 21.
5. A composition comprising a second primer pair for detecting an influenza
PB2 gene.
6. The composition of claim 5, wherein the second primer pair comprises a third
primer and a fourth primer, wherein the third primer comprises a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 1, and wherein the fourth primer
comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1.
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7. The composition of claim 5, wherein the second primer pair comprises a third
primer and a fourth primer, wherein the third primer comprises a sequence that is at
least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 17, and
wherein the fourth primer comprises a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, or
at least 20 contiguous nucleotides of SEQ ID NO: 18.
8. The composition of claim 5, wherein the third primer has the sequence of
SEQ ID NO: 17 and the fourth primer has the sequence of SEQ ID NO: 18.
9. A composition comprising a first primer pair of any one of claims 951 to 984
and a second primer pair of any one of claims 5 to 8.
10. The composition of any one of claims 1 to 9, further comprising at least one
additional primer pair, wherein each of the additional primer pairs is for detecting a
different influenza gene selected from an influenza A MP gene, an avian influenza
MP gene, and an avian influenza HA gene.
11. The composition of claim 10, wherein each additional primer pair comprises a
fifth primer and a sixth primer independently selected from:
a) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 3;
b) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 4;
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c) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 5, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 5;
d) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 23, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 24;
e) a fifth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 26, and a
sixth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 27; and
f) a fifth primer comprising a sequence that is at least 90%, at least 95%,
or 100% identical to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 29, and a sixth primer
comprising a sequence that is at least 90%, at least 95%, or 100%
complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least
25 contiguous nucleotides of SEQ ID NO: 30.
12. The composition of any one of claims 1 to 11, further comprising at least one
additional primer pair, wherein each of the additional primer pairs is for detecting a
different influenza gene selected from an influenza B MP gene and an influenza B NS
gene.
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13. The composition of claim 12, wherein each additional primer pair comprises a
seventh primer and an eighth primer independently selected from:
a) a seventh primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6, and an
eighth primer comprising a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 6;
b) a seventh primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 7, and an
eighth primer comprising a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 7;
c) a seventh primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 32, and an
eighth primer comprising a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 33; and
d) a seventh primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 35, and an
eighth primer comprising a sequence that is at least 90%, at least 95%,
or 100% complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 36.
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14. The composition of any one of claims 1 to 13, further comprising at least one
additional primer pair, wherein each of the additional primer pairs is for detecting
RSV A or RSV B.
15. The composition of claim 14, wherein each additional primer pair comprises a
ninth primer and a tenth primer independently selected from:
a) a ninth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 38, and a
tenth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 39; and
b) a ninth primer comprising a sequence that is at least 90%, at
least 95%, or 100% identical to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 41, and a
tenth primer comprising a sequence that is at least 90%, at least 95%, or
100% complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 42.
16. The composition of any one of claims 1 to 15, further comprising primer pairs
for detecting an influenza A PA gene, an influenza A PB2 gene, an influenza A MP
gene, an avian influenza MP gene, and an avian influenza HA gene.
17. The composition of any one of claims 1 to 16, further comprising primer pairs
for detecting RSV A and RSV B.
18. The composition of any one of claims 1 to 17, further comprising a primer
pair for detecting an exogenous control.
19. The composition of claim 18, wherein the exogenous control is a sample
processing control.
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20. The composition of any one of claims 1 to 19, further comprising at least one
probe selected from an influenza A PA probe and an influenza A PB2 probe.
21. The composition of claim 20, wherein the influenza PA probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 2, and
the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 1.
22. The composition of claim 20, wherein the influenza PA probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 19,
and the influenza PB2 probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 22.
23. The composition of any one of claims 20 to 22, further comprising at least one
probe selected from an influenza A MP probe, an avian influenza MP probe, and an
avian influenza HA probe.
24. The composition of claim 23, wherein the influenza MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 3, and
the avian influenza MP probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 4, and the avian influenza HA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
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complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 5.
25. The composition of claim 23, wherein the influenza MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 25,
and the avian influenza MP probe comprises a sequence that is at least 90%, at least
95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at
least 25 contiguous nucleotides of SEQ ID NO: 28, and the avian influenza HA probe
comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides
of SEQ ID NO: 31.
26. The composition of any one of claims 1 to 25, wherein composition further
comprises at least one probe selected from an influenza B MP probe and an influenza
B NS probe.
27. The composition of claim 26, wherein the influenza B MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 6, and
the influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 7.
28. The composition of claim 26, wherein the influenza B MP probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 34,
and the influenza B NS probe comprises a sequence that is at least 90%, at least 95%,
or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18,
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at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 37.
29. The composition of any one of claims 1 to 28, further comprising at least one
probe selected from an RSV A probe and an RSV B probe.
30. The composition of claim 29, wherein the RSV A probe comprises a sequence
that is at least 90%, at least 95%, or 100% identical or complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 15, and the
influenza B NS probe comprises a sequence that is at least 90%, at least 95%, or
100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 16.
31. The composition of claim 29, wherein the RSV A probe comprises a sequence
that is at least 90%, at least 95%, or 100% identical or complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 40, and the
RSV B probe comprises a sequence that is at least 90%, at least 95%, or 100%
identical or complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 43.
32. The composition of any one of claims 1 to 31, further comprising a probe for
detecting an exogenous control.
33. The composition of any one of claims 20 to 32, wherein each probe comprises
a detectable label.
34. The composition of claim 33, wherein the each probe comprises a fluorescent
dye and a quencher molecule.
35. The composition of any one of claims 20 to 34, wherein each probe consists
of 15 to 30 nucleotides.
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36. The composition of any one of claims 1 to 35, wherein the composition is a
lyophilized composition.
37. The composition of any one of claims 1 to 35, wherein the composition is in
solution.
38. The composition of claim 37, wherein the composition comprises nucleic
acids from a sample from a subject being tested for the presence of absence of
influenza.
39. The composition of claim 38, wherein the sample is selected from a
nasopharyngeal swab sample, a nasal aspirate sample, and a nasal wash sample.
40. A kit comprising a composition of any one of claims 1 to 37.
41. The kit of claim 40, wherein the kit further comprises an exogenous control.
42. The kit of claim 41, wherein the exogenous control is an Armored® RNA.
43. The kit of any one of claims 40 to 42, wherein the kit comprises dNTPs and/or
a thermostable polymerase.
44. The kit of any one of claims 40 to 43, wherein the kit comprises a reverse
transcriptase.
45. An oligonucleotide consisting of a sequence selected from SEQ ID NOs: 17 to
43, wherein the oligonucleotide comprises at least one modified nucleotide.
46. The oligonucleotide of claim 45, wherein the oligonucleotide comprises a
detectable label.
47. The oligonucleotide of claim 46, wherein the oligonucleotide comprises a
fluorescent dye and a quencher molecule.
48. The oligonucleotide of claim 47, which is a fluorescence resonance energy
transfer (FRET) probe.
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49. A composition comprising a first primer consisting of the sequence of SEQ ID
NO: 17 and a second primer consisting of the sequence of SEQ ID NO: 18, wherein
the first primer and the second primer each comprises at least one modified
nucleotide.
50. The composition of claim 49, wherein the composition comprises a probe
consisting of the sequence of SEQ ID NO: 19, wherein the probe comprises at least
one modified nucleotide and/or a detectable label.
51. A composition comprising a first primer consisting of the sequence of SEQ ID
NO: 20 and a second primer consisting of the sequence of SEQ ID NO: 21, wherein
the first primer and the second primer each comprises at least one modified
nucleotide.
52. The composition of claim 51, wherein the composition comprises a probe
consisting of the sequence of SEQ ID NO: 22, wherein the probe comprises at least
one modified nucleotide and/or a detectable label.
53. The composition of claim 50 or 52, wherein the probe is a fluorescence
resonance energy transfer (FRET) probe.
54. The composition of any one of claims 50, 52, and 53, wherein the probe
comprises at least one modified nucleotide.
55. The composition of any one of claims 49 to 54, wherein the composition is a
lyophilized composition.
56. The composition of any one of claims 49 to 54, wherein the composition is in
solution.
57. The composition of claim 56, wherein the composition comprises nucleic
acids of a sample from a subject.