1. FIELD OF THE INVENTION
[001] Compositions and methods for detecting Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus (RSV) are provided.
In particular, SARS-CoV-2, influenza, and RSV markers and panels of markers useful in the
5 detection of SARS-CoV-2, influenza, and RSV virus are provided.
2. BACKGROUND
[002] On December 31 2019, an outbreak of respiratory illness of unknown etiology
was reported to the World Health Organization (WHO). A novel coronavirus (2019-nCoV)
10 was identified, which has resulted in thousands of confirmed human infections in multiple
provinces throughout the world. Cases of severe illness and some deaths have been reported.
The International Committee for Taxonomy of Viruses (ICTV) renamed the virus SARSCoV-2, which is short for “Severe Acute Respiratory Syndrome Coronavirus 2.” The World
Health Organization has named the disease caused by the SARS-CoV-2 as coronavirus disease
15 2019 (COVID-19). COVID-19 is associated with a variety of clinical outcomes, including
asymptomatic infection, mild upper respiratory infection, severe lower respiratory disease
including pneumonia and respiratory failure, and in some cases, death. According to the
Center for Disease Control and Prevention (U.S. CDC), as of June 2020, it was known that
patients with COVID-19 exhibit a wide range of symptoms – ranging from mild symptoms to
20 severe illness. Symptoms, including but not limited to fever or chills, cough, shortness of
breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or
smell, sore throat, congestion or runny nose, nausea or vomiting, and/or diarrhea, may appear
2-14 days after exposure to the virus.
[003] Influenza, or the flu, is a contagious viral infection of the respiratory tract.
25 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.
30 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
3
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.
5 In addition to seasonal flu, a novel H1N1 strain was identified in humans in the United States
in early 2009.
[004] Respiratory syncytial virus (RSV), a member of the Pneumoviridae family
(formerly 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 immune10 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. By the age of two, most children have
already been infected by RSV, but because only weak immunity develops, both children and
15 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
20 infections, however, also occur at other times of the year, although rarely.
[005] Active surveillance programs in conjunction with infection control precautions
are important components for preventing transmission of SARS-CoV-2, influenza, and RSV.
The use of assays providing rapid results to identify patients infected with these infections is
also an important factor for effective control, proper choice of treatment, and prevention of
25 widespread outbreaks.
[006] 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
30 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, swine origin H3N2).
[007] 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
4
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.
[008] This constant antigenic drift and antigenic shift makes it difficult to maintain
5 influenza detection assays from season to season. Additionally, there is a need for a nextgeneration test to assist global efforts in the fight against the spread of COVID-19, especially
during future respiratory virus seasons. There is a need for a robust SARS-CoV-2, influenza,
and RSV detection assay that will remain accurate even as the viral genomes undergo genetic
drift. Patients with COVID-19, Flu, and RSV have overlapping clinical presentations, but
10 fundamentally different treatment and management pathways. Infection with the viruses is
often associated with fever and other systemic manifestations that may be coupled with severe
outcomes, especially in the elderly. There is thus a need and demand for a test that can deliver
qualitative detection and differentiation of SARS-CoV-2, Flu A, Flu B, and RSV from a single
patient sample. Furthermore, obtaining results in a short amount of time is beneficial.
15 3. SUMMARY
[009] In some instances, the following non-limiting embodiments are provided:
Embodiment 1. A method of detecting the presence or absence of influenza A,
influenza B, RSV, and SARS-CoV-2 in a biological sample from a subject comprising:
a) contacting a biological sample from the subject with sets of primers that detect an
20 influenza A gene, an influenza B gene, a RSV gene, and a SARS-CoV-2 gene;
b) conducting one or more polymerase chain reaction (PCR); and
c) detecting an amplicon that is produced by the PCR.
Embodiment 2. A method of determining whether a subject has influenza, RSV, and/or
COVID-19 comprising detecting the presence or absence of at least one gene selected from
25 influenza A or influenza B, RSV, and SARS-CoV-2 in a sample from the subject comprising:
a) contacting a biological sample from the subject with sets of primers that detect an
influenza A gene, an influenza B gene, an RSV gene, and a SARS-CoV-2 gene;
b) conducting a polymerase chain reaction (PCR); and
c) detecting an amplicon that is produced by the PCR.
30 Embodiment 3. The method of embodiment 1 or embodiment 2,
a) wherein the set of primers that detects the presence or absence of influenza A
comprises at least one of:
i) a set of primers that detects influenza A PB2 selected from:
5
1) a forward and reverse primer for detecting a sequence of the influenza PB2 gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 1, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1; and
5 3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 17, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 18;
ii)a set of primers that detects influenza A PA selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PA gene;
10 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 2, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 2; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 20, and a reverse primer comprising a sequence that
15 is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 21;
iii)a set of primers that detects influenza A MP selected from:
1) a forward and reverse primer for detecting a sequence of the influenza A MP gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 3, and a reverse primer comprising a sequence that is
20 at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 3; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 23, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 24;
iv)a set of primers that detects avian influenza MP selected from:
25 1) a forward and reverse primer for detecting a sequence of the avian influenza MP gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 4, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 4; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
30 contiguous nucleotides of SEQ ID NO: 26, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 27;
b) wherein the set of primers that detects the presence or absence of influenza B
comprises at least one of:
i)a set of primers that detects influenza B MP selected from:
6
1) a forward and reverse primer for detecting a sequence of the influenza B MP gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 6, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 6; and
5 3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 32, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 33;
ii)a set of primers that detects influenza B NS selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B NS gene;
10 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 7, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 7; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 35, and a reverse primer comprising a sequence that
15 is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 36;
c) wherein the set of primers that detects the presence or absence of RSV comprises at
least one of:
i)a set of primers that detects RSV A selected from:
1) a forward and reverse primer for detecting a sequence of the RSV A gene; and
20 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 38, and a reverse primer comprising a sequence that
is at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 39; and
ii)a set of primers that detects RSV B selected from:
1) a forward and reverse primer for detecting a sequence of the RSV B gene; and
25 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 41, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 42; and
d) wherein the set of primers that detects the presence or absence of SARS-CoV-2
comprises at least one of:
30 i)a set of primers that detects SARS-CoV-2 E selected from:
1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 E gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 44, and a reverse primer comprising a sequence that
is at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 44; and
7
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 48, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 49; and
ii)a set of primers that detects SARS-CoV-2 N2 selected from:
5 1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 N2 gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 45, and a reverse primer comprising a sequence that
is at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 45; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
10 contiguous nucleotides of SEQ ID NO: 51, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 52.
Embodiment 4. The method of embodiment 1 or embodiment 2,
a) wherein the set of primers that detects the presence or absence of influenza A
comprises at least one of:
15 i)a set of primers that detects influenza A PB2 selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PB2 gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 1, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1; and
20 3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 17, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 18;
ii)a set of primers that detects influenza A PA selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PA gene;
25 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 2, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 2; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 20, and a reverse primer comprising a sequence that
30 is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 21;
iii)a set of primers that detects influenza A MP selected from:
1) a forward and reverse primer for detecting a sequence of the influenza A MP gene;
8
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 3, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 3; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
5 contiguous nucleotides of SEQ ID NO: 23, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 24;
iv)a set of primers that detects avian influenza MP selected from:
1) a forward and reverse primer for detecting a sequence of the avian influenza MP gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
10 contiguous nucleotides of SEQ ID NO: 4, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 4; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 26, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 27;
15 b) wherein the set of primers that detects the presence or absence of influenza B
comprises at least one of:
i)a set of primers that detects influenza B MP selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B MP gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
20 contiguous nucleotides of SEQ ID NO: 6, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 6; and
3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 32, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 33;
25 ii)a set of primers that detects influenza B NS selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B NS gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 7, and a reverse primer comprising a sequence that is
at least 85% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 7; and
30 3) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 35, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 36;
c) wherein the set of primers that detects the presence or absence of RSV comprises at
least one of:
9
i)a set of primers that detects RSV A selected from:
1) at least one forward and at least one reverse primer for detecting a sequence of the
RSV A gene; and
2) at least one forward primer comprising a sequence that is at least 85% identical to at
5 least 15 contiguous nucleotides of SEQ ID NO: 38 and/or SEQ ID NO: 67, and at least one
reverse primer comprising a sequence that is at least 85% complementary to at least 15
contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 68, and/or SEQ ID NO: 69; and
ii)a set of primers that detects RSV B selected from:
1) a forward and reverse primer for detecting a sequence of the RSV B gene; and
10 2) a forward primer comprising a sequence that is at least 85% identical to at least 15
contiguous nucleotides of SEQ ID NO: 41, and a reverse primer comprising a sequence that
is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID NO: 42; and
d) wherein the set of primers that detects the presence or absence of SARS-CoV-2
comprises at least one of:
15 i)a set of primers that detects SARS-CoV-2 E selected from:

WE CLAIM:
1. A method of detecting the presence or absence of influenza A, influenza B, RSV,
and SARS-CoV-2 in a biological sample from a subject comprising:
a) contacting a biological sample from the subject with sets of primers that detect
5 an influenza A gene, an influenza B gene, a RSV gene, and a SARS-CoV-2 gene;
b) conducting one or more polymerase chain reaction (PCR); and
c) detecting an amplicon that is produced by the PCR.
2. A method of determining whether a subject has influenza, RSV, and/or COVID10 19 comprising detecting the presence or absence of at least one gene selected from
influenza A or influenza B, RSV, and SARS-CoV-2 in a sample from the subject
comprising:
a) contacting a biological sample from the subject with sets of primers that detect
an influenza A gene, an influenza B gene, an RSV gene, and a SARS-CoV-2 gene;
15 b) conducting a polymerase chain reaction (PCR); and
c) detecting an amplicon that is produced by the PCR.
3. The method of claim 1 or claim 2,
a) wherein the set of primers that detects the presence or absence of influenza A
20 comprises at least one of:
i)a set of primers that detects influenza A PB2 selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PB2 gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 1, and a reverse primer comprising a
25 sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 1; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 17, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
30 NO: 18;
ii)a set of primers that detects influenza A PA selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PA gene;
118
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 2, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 2; and
5 3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 20, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 21;
iii)a set of primers that detects influenza A MP selected from:
10 1) a forward and reverse primer for detecting a sequence of the influenza A MP
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 3, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
15 SEQ ID NO: 3; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 23, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 24;
20 iv)a set of primers that detects avian influenza MP selected from:
1) a forward and reverse primer for detecting a sequence of the avian influenza MP
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 4, and a reverse primer comprising a
25 sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 4; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 26, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
30 NO: 27;
b) wherein the set of primers that detects the presence or absence of influenza B
comprises at least one of:
i)a set of primers that detects influenza B MP selected from:
119
1) a forward and reverse primer for detecting a sequence of the influenza B MP
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 6, and a reverse primer comprising a
5 sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 6; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 32, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
10 NO: 33;
ii)a set of primers that detects influenza B NS selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B NS
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 15 contiguous nucleotides of SEQ ID NO: 7, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 7; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 35, and a reverse primer comprising a
20 sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 36;
c) wherein the set of primers that detects the presence or absence of RSV comprises
at least one of:
i)a set of primers that detects RSV A selected from:
25 1) a forward and reverse primer for detecting a sequence of the RSV A gene; and
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 38, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 39; and
30 ii)a set of primers that detects RSV B selected from:
1) a forward and reverse primer for detecting a sequence of the RSV B gene; and
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 41, and a reverse primer comprising a
120
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 42; and
d) wherein the set of primers that detects the presence or absence of SARS-CoV-2
comprises at least one of:
5 i)a set of primers that detects SARS-CoV-2 E selected from:
1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 E
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 44, and a reverse primer comprising a
10 sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 44; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 48, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
15 NO: 49; and
ii)a set of primers that detects SARS-CoV-2 N2 selected from:
1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 N2
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
20 15 contiguous nucleotides of SEQ ID NO: 45, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 45; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 51, and a reverse primer comprising a
25 sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 52.
4. The method of claim 1 or claim 2,
a) wherein the set of primers that detects the presence or absence of influenza A
30 comprises at least one of:
i) a set of primers that detects influenza A PB2 selected from:
1) a forward and reverse primer for detecting a sequence of the influenza PB2 gene;
121
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 1, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 1; and
5 3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 17, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 18;
ii)a set of primers that detects influenza A PA selected from:
10 1) a forward and reverse primer for detecting a sequence of the influenza PA gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 2, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 2; and
15 3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 20, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 21;
iii)a set of primers that detects influenza A MP selected from:
20 1) a forward and reverse primer for detecting a sequence of the influenza A MP
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 3, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
25 SEQ ID NO: 3; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 23, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 24;
30 iv)a set of primers that detects avian influenza MP selected from:
1) a forward and reverse primer for detecting a sequence of the avian influenza MP
gene;
122
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 4, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 4; and
5 3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 26, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 27;
b) wherein the set of primers that detects the presence or absence of influenza B
10 comprises at least one of:
i)a set of primers that detects influenza B MP selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B MP
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 15 contiguous nucleotides of SEQ ID NO: 6, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 6; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 32, and a reverse primer comprising a
20 sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 33;
ii)a set of primers that detects influenza B NS selected from:
1) a forward and reverse primer for detecting a sequence of the influenza B NS
gene;
25 2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 7, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 7; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
30 15 contiguous nucleotides of SEQ ID NO: 35, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 36;
123
c) wherein the set of primers that detects the presence or absence of RSV comprises
at least one of:
i)a set of primers that detects RSV A selected from:
1) at least one forward and at least one reverse primer for detecting a sequence of
5 the RSV A gene; and
2) at least one forward primer comprising a sequence that is at least 85% identical
to at least 15 contiguous nucleotides of SEQ ID NO: 38 and/or SEQ ID NO: 67, and at
least one reverse primer comprising a sequence that is at least 85% complementary to
at least 15 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 68, and/or SEQ ID
10 NO: 69; and
ii)a set of primers that detects RSV B selected from:
1) a forward and reverse primer for detecting a sequence of the RSV B gene; and
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 41, and a reverse primer comprising a
15 sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 42; and
d) wherein the set of primers that detects the presence or absence of SARS-CoV-2
comprises at least one of:
i)a set of primers that detects SARS-CoV-2 E selected from:
20 1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 E
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 44, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
25 SEQ ID NO: 44;
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 70, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 71; and
30 4) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 48, and a reverse primer comprising a
sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 49;
124
ii)a set of primers that detects SARS-CoV-2 N2 selected from:
1) a forward and reverse primer for detecting a sequence of the SARS-CoV-2 N2
gene;
2) a forward primer comprising a sequence that is at least 85% identical to at least
5 15 contiguous nucleotides of SEQ ID NO: 45, and a reverse primer comprising a
sequence that is at least 85% complementary to at least 15 contiguous nucleotides of
SEQ ID NO: 45; and
3) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 73 or SEQ ID NO: 51, and a reverse primer
10 comprising a sequence that is at least 85% identical to at least 15 contiguous nucleotides
of SEQ ID NO: 52;
iii)a set of primers that detects SARS-CoV-2 RdRP selected from:
1) a forward and at least one reverse primer for detecting a sequence of the SARSCoV-2 RdRP gene;
15 2) a forward primer comprising a sequence that is at least 85% identical to at least
15 contiguous nucleotides of SEQ ID NO: 76, and at least one reverse primer comprising
a sequence that is at least 85% identical to at least 15 contiguous nucleotides of SEQ ID
NO: 61 and/or SEQ ID NO: 78.
20 5. The method of claim 1 or claim 2,