[DESCRIPTION]

[Invention Title]

A METHOD FOR DETECTING HBV USING REAL TIME PCR
[Technical Field]

The present invention relates to the detection of
hepatitis B virus gene. More particularly, the present
invention relates to a pair of primers complementary to HBV
gene, a composition for the detection of HBV comprising a
pair of primers and a probe complementary to HBV gene, a HBV
detection kit comprising the composition, and a method for
detecting HBV gene.

[Background Art]
Hepatitis B Virus (hereinbelow, referred to as HBV) is
known as a major cause of viral hepatitis, and an estimated
2 billion people are infected with HBV worldwide. Of these
infected people, approximately 350 million people are
chronic carriers, and a majority of the people are at
substantially increased risk of developing liver cirrhosis,
and hepatocellular carcinoma. The HBV envelope gene
consists of the preS region composed of preS1 and preS2, and
the S region. After transcription of the entire envelope
gene, three envelope proteins (L protein, M protein and S
protein) are synthesized by alternate translation at each of
three regions. The L protein, for which translation is
initiated at the preS1 region, consists of approximately 400
amino acids containing all of the preS1 domain, preS2 domain
and S domain. The M protein, for which translation is
initiated at the preS2 region, consists of approximately 281
amino acids containing the preS2 domain and S domain. The S
protein consists of approximately 226 amino acids containing
the S domain only, and is the most abundantly expressed to
be the main component of virus particles. The S domains of
the envelope protein bind to each other to form a particle
as S antigen. The preS1 and preS2 domains contained in the
M protein and L protein are located on the outside of the
particle, and serve as a preS antigen to induce high immune
responses.
Diagnosis of HBV infection can be performed by
detecting an antibody against HBV that is present in a
sample, detecting an HBV antigen, or detecting an HBV gene.
Frequently, the method of HBV antigen detection is to
detect its absence or presence in blood using HBV Surface
Antigen (HBsAg) or HBV e antigen (HBeAg) as a diagnostic
marker. However, early diagnosis of HBV infection can be
hindered depending on the life cycle of the virus. Reduction
or absence of HBsAg or HBeAg in the blood of an HBV-infected
patient is also an obstacle to an accurate diagnosis of HBV
in a patient during treatment.
HBV gene detection is performed by a nucleic acid
amplification technique (NPT) or a DNA probe method. At
present, these methods are widely employed in clinical
practice. In particular, PCR is a method to selectively
amplify a low amount of a target gene to a detectable level,
and thus it is widely used. However, there is a problem in
that the DNA extraction procedures lead to an inevitable
loss of DNA. For example, even though a sufficient amount
of the target DNA is contained in the sample as a template
for amplification, the suitable amount of DNA for
amplification could not be recovered due to the loss during
the extraction process, leading to a reduction in the
amplification efficiency of the template. Therefore, it is
hard to sufficiently amplify the target DNA and detect the
target DNA in the sample. Consequently, the technique
produces a false negative result, and thus it is difficult
to ensure accurate results. Even though using the same
sample, variations in DNA yields during the extraction
process may produce different results, leading to a lack of
reproducibility. That is, the DNA amplification technique
has problems in that it requires the DNA extraction process
to cause the loss of trace target DNA in the sample, whereby
the detection sensitivity is reduced. In addition, when
amplification inhibitors (e.g., heparin, surfactant, protein
denaturants, organic solvent, etc.) are contained in the
sample liquid, the amplification efficiency is also reduced
to lower the detection sensitivity.
Further, a region having very little genetic variation,
such as the S protein, the pre-Core region or the envelope
protein, is useful for the HBV detection by PCR.
Nevertheless, these regions also have a low probability of
variation such as mutation. If genetic variation in the
base sequence occurs, the amplification procedures using a
PCR primer and/or probe are failed. Consequently, a falsenegative
result is obtained or the detection sensitivity is
reduced due to reduced amplification efficiency. Thus, it
is difficult to ensure accurate quantification.
[Disclosure]

[Technical Problem]
The present inventors have made many efforts to solve
these problems. They have developed a method of accurately
and rapidly detecting and quantifying HBV gene using a trace
amount of target HBV gene contained in a sample, and a
method for simultaneous detection and quantification of two
regions of HBV gene having very little genetic variation
such as mutation. According to these methods, even though
genetic variation via mutation occurs in one region of the
gene, the other region of the gene can be detected, so as to
achieve accurate diagnosis of patients with suspected HBV
infection with high sensitivity and specificity, thereby
completing the present invention.
[Technical Solution]
It is an object of the present invention to provide a
pair of primers complementary to HBV gene, which have base
sequences represented by SEQ ID NOs. 1 and 2, or SEQ ID NOs.
3 and 4.
It is another object of the present invention to
provide a composition for the detection of HBV gene,
comprising a pair of primers having base sequences of SEQ ID
NOs. 1 and 2, a pair of primers having base sequences of SEQ
ID NOs. 3 and 4, and probes having base sequences
represented by SEQ ID NOs. 7 and 8.

It is still another object of the present invention to
provide an HBV detection kit comprising the composition for
the detection of HBV gene.
It is still another object of the present invention to
provide a method for detecting HBV gene using the
composition for the detection of HBV gene.
[Description of Drawings]
FIG. 1 is the result showing that a positive detection
and quantification can be made without competitive
inhibition of the internal control for the HBV-specific
primer and probe;
FIG. 2 shows a linearity of HBV quantification
standard;
FIG. 3 shows the result of PCR, in which the
amplification of HBV S-protein and pre-core region was
performed separately; and
FIG. 4 shows the result of PCR, in which the
amplification of HBV S-protein and the pre-core region was
performed simultaneously. In this case, there was also no
competitive inhibition in the detection and no significant
difference in the quantification.
[Advantageous Effects]
As described above, when the detection and
quantification of HBV infection is performed by the Real-
Time PCR according to the present invention, the sensitivity
and specificity equal to or greater than that of the known
methods can be obtained.
[Best Mode]
Accordingly, in accordance with one aspect, the present
invention relates to a pair of primers complementary to HBV
gene. Preferably, the primers are a pair of primers having
the base sequences represented by SEQ ID NOs. 1 and 2 or SEQ
ID NOs. 3 and 4.
As used herein, the term “primer” means a short nucleic
acid sequence having the free 3’ hydroxyl group, which forms
a base-pair with a complementary template and serves as a
starting point for template strand replication. The primer
is able to initiate DNA synthesis in the presence of four
different nucleoside triphosphates and an agent for
polymerization in an appropriate buffer and at a suitable
temperature. In accordance with the object of the present
invention, the primer is a primer that specifically
amplifies the HBV S protein or HBV pre-Core region.
Therefore, the primer of the present invention is composed
of a pair of forward and reverse primers, which are
complementary to the gene and have base sequences of 7 to 50,
preferably 10 to 30. In particular, the HBV S gene region
can be specifically amplified by a pair of primers having
the base sequences of SEQ ID NOs. 1 and 2, and the HBV pre-
Core region can be specifically amplified by a pair of
primers having the base sequences of SEQ ID NOs. 3 and 4.
The primer of the present invention may be chemically
synthesized by the phosphoramidite solid support method or
other well-known methods. These nucleic acid sequences may
also be modified using many means known in the art. Nonlimiting
examples of such modifications include methylation,
capsulation, replacement of one or more native nucleotides
with analogues thereof, and inter-nucleotide modifications,
for example, modifications to uncharged conjugates (e.g.,
methyl phosphonate, phosphotriester, phosphoroamidate,
carbamate, etc.) or charged conjugates (e.g.,
phosphorothioate, phosphorodithioate, etc.). Nucleic acids
may contain one or more additionally covalent-bonded
residues, which are exemplified by proteins (e.g., nucleases,
toxins, antibodies, signal peptides, poly-L-lysine, etc.),
intercalating agents (e.g., acridine, proralene, etc.),
chelating agents (e.g., metals, radioactive metals, iron,
oxidative metals, etc.), and alkylating agents. The nucleic
acid sequences of the present invention may also be altered
using a label capable of directly or indirectly supplying a
detectable signal. Examples of the label include
radioisotopes, fluorescent molecules and biotin.
When PCR is performed using the primers having the base
sequences of SEQ ID NOs. 1 and 2, and SEQ ID NOs. 3 and 4
provided by the present invention, each HBV genotype can be
detected with high specificity and sensitivity. Herein, the
term ‘sensitivity’ means the proportion of people with a
disease who tested positive, namely, the ability of a test
to recognize the presence of a particular disease in a
patient. The term ‘specificity’ means the proportion of
those free of the disease who tested negative, namely, the
ability of a test to recognize healthy individuals.
Therefore, the primers can be used in the diagnosis of
HBV infection, analysis of infected HBV genotype,
epidemiological analysis of HBV, and evaluation of efficacy
and safety of HBV vaccine.
In accordance with still another embodiment, the
present invention relates to a composition for the detection

of HBV gene, comprising a pair of primers having the base
sequences of SEQ ID NOs. 1 and 2, a pair of primers having
the base sequences of SEQ ID NOs. 3 and 4, and probes having
the base sequences of SEQ ID NOs. 7 and 8.
Specifically, the present invention provides a
composition for the detection of HBV, comprising novel
primers specific to the HBV S protein region, which consist
of a sense primer having the base sequence of SEQ ID NO. 1
and/or an antisense primer having the base sequence of SEQ
ID NO. 2; and a probe having the base sequence of SEQ ID NO.
7. Further, the present invention provides a composition
for the detection of HBV, comprising novel primers specific
to the HBV pre-Core region, which consist of a sense primer
having the base sequence of SEQ ID NO. 3 and/or an antisense
primer having the base sequence of SEQ ID NO. 4; and a probe
having the base sequence of SEQ ID NO. 8.
Further, the present invention provides a composition
for the detection of an internal control, comprising novel
primers that are specific to a gene being not present in
human and HBV gene and serves as an internal control to
examine a false negative reaction, in which the primers
consist of a sense primer having the base sequence of SEQ ID
NO. 5 and/or an antisense primer having the base sequence of
SEQ ID NO. 6; and a probe having the base sequence of SEQ ID
NO. 9.
In the present invention, the detection of HBV in blood
can be preferably achieved with higher sensitivity and
specificity by using the S protein-specific primer and probe
together with the pre-Core-specific primer and probe,
compared to the technique using a primer and probe specific
to base sequence of a single gene. The specific sequences
of the primers are described in the following Table 1.
[Table 1]
Name Primer base sequence Length SEQ ID No.
BSFOR GTR TCT GCG GCG TTY TAT CA 20 1
BSREV GGA CAA ACG GGC AAC ATA CC 20 2
BCFOR AAA TGC CCC TAT CYT ATC AAC ACT 24 3
BCREV CCG AGA TTK AGA TCT TCT GCG A 22 4
ICFOR CGA TGG CCC TGT CCT TTT ACA TTT A 25 5
ICREV CTT TTC GTT GGG ATC TTT GAT TAA A 25 6
As used herein, the term “HBV (Hepatitis B virus)” is a
kind of virus, and causes hepatitis B that is commonly
transmitted by blood transfusion. It is largely divided
into acute and chronic hepatitis. The symptoms of acute
hepatitis B are similar to those of hepatitis A or C. Most
people have no symptoms, or some people experience flu-like
symptoms, but recover without any treatment. The symptoms
of chronic hepatitis B are also similar to those of
hepatitis C, and include fatigue, fever, vomiting, muscle
and joint pain. The symptoms continue for long periods of
time, increasing the risk of development of hepatic
cirrhosis and hepatoma. Therefore, continuous examination
is required. PCR is generally used for diagnosis of
hepatitis B, but the known PCR diagnostic methods are
problematic in that the methods amplify and detect a single
region, and thus mutation in the region generates low
sensitivity and specificity, thereby providing false
positive and false negative results. In the diagnostic
method of the present invention, the S protein-specific
primer and probe are used together with the pre-Core regionspecific
primer and probe, and their amplification products
are used in a single detection step, thereby providing
convenience, high specificity and sensitivity, and improved
accuracy of quantitative determination.
In the preferred embodiment, the composition of the
present invention further comprises a probe sequence to

detect the nucleic acids that are amplified by the primer
sequences.
As used herein, the term “probe” refers to an
oligonucleotide, whether occurring naturally or produced
synthetically, recombinantly or by PCR amplification, which
is capable of hybridizing to another oligonucleotide of
interest. A probe may be single-stranded or double-stranded.
Probes are useful in the detection, identification and
isolation of particular gene sequences. In the preferred
embodiment, it is contemplated that any probe used in the
present invention will be labeled with any “reporter
molecule,” so that is detectable in any detection system
including fluorescent, radioactive, and luminescent systems.
It is not intended that the present invention is limited to
any particular detection system or label. Preferably, the
probe of the present invention is a probe represented by SEQ
ID NO. 7 or 8. In the preferred Example of the present
invention, it was found that the primer and probe were used
to detect the presence and absence of HBV with high
sensitivity and specificity and there was no crossreactivity.
In one preferred embodiment, the probe of the present
invention may be a probe having fluorescent molecules at its

5’ and 3’ ends. In one preferred embodiment, the probe
contains a fluorescent reporter and a quencher at its 5’ and
3’ ends, respectively, in which they may show interference
with each other. Therefore, when the probes bind to the S
protein or pre-Core region present in the sample, the
generation of fluorescence signals is restricted. Upon
performing polymerase chain reaction, the probe is
decomposed, and the fluorescent reporter at 5’end is
released away from the quencher at 3’ end, thereby generates
fluorescence signals. The presence of HBV in the sample can
be detected by the fluorescence signals.
Any fluorescent molecules that are typically used in
the related art can be labeled at 5’ end, exemplified by 6-
carboxyfluorescein, hexachloro-6-carboxyfluorescein,
tetrachloro-6-carboxyfluorescein, FAM (5-carboxy
fluorescein), HEX (2’,4’,5’,7’-tetrachloro-6-carboxy-4,7-
dichlorofluorescein) and Cy5 (cyanine-5), but are not
limited thereto.
Any fluorescent molecules that are typically used in
the related art can be also labeled at 3’ end without
limitation, exemplified by 6-carboxytetramethyl-rhodamine,
TAMRA (5-Carboxytetramethylrhodamine), and BHQ 1, 2 and 3
(black hole quencher 1, 2, 3), but are not limited thereto.
- 15 -
In the present invention, base sequences of the probes
of SEQ ID NOs. 7 to 9 are described in the following Table 2.
[Table 2]
Name 5’
Probe base sequence (5’-
>3’)
3’ Length
SEQ ID
No.
BSFAM FAM
CAT CCT GCT GCT ATG CCT
CAT CTT C
TAMRA 25 7
BCFAM FAM
CCC CTA GAA GAA AGA ACT
CCC TCG CC
TAMRA 26 8
ICCy5 Cy5
ATT ACC TGT CCA CAC AAT
CTG CCC TT
BHQ3 26 9
The HBV-specific primer and probe of the present
invention are able to produce the PCR product having a
length of 100 bp, which is suitable for Real Time PCR. In
particular, the probe specific to S-protein and pre-Core is
useful for PCR quantification analysis by taqman assay or
molecular beacon assay.
The present inventors have examined the risk of false
positive and false negative results by base sequence
analysis. They found that the primer and probe of the
present invention is a primer base sequence capable of
amplifying HBV gene only, and shows a 100% positive
predictive value in the test using Genotype Panel
((PHD201(M)) provided by BBI, indicating very low risk of
false positive and false negative results. In addition,
their quantitative results were evaluated using Genotype
Panel. As a result, there was no significant difference in
the quantitative results between panels (Table 5).
Accordingly, the primers of SEQ ID NOs. 1 and 2, and the
probes of SEQ ID NOs. 3 and 4 and SEQ ID NOs. 7 and 8 are
used to perform the detection and quantitation of HBV in
blood with high reliability.
In the preferred embodiment, the present invention may
include additional primer and probe sequences as an internal
control to minimize the false positive and false negative
results. The internal control group serves as a marker for
direct comparison to prevent false positive and false
negative results.
Preferably, the internal control group may be a GFP
(Green Fluorescence Protein) derived from a plant. More
preferably, the GFP primer sequence may include the primers
represented by SEQ ID NOs. 5 and 6 (see Table 1).
- 17 -
The primer and probe as the internal control group for
prevention of false negative results are a primer and probe
that are specific to GFP (Green Fluorescence Protein) gene
derived from a plant, and do not inhibit Real Time PCR of
the HBV-specific primer and probe, and separately form PCR
products. Particularly, in the detection of a low level of
HBV, the formation of HBV PCR product is not inhibited by
the PCR product of the internal control, whereby the false
negative results can be effectively prevented, reliability
of quantitation result is improved, and the quantitation is
also available at a lower detection level. Meanwhile, in
the detection of a high level of HBV, formation of the PCR
products of the internal control is not inhibited by the PCR
products of HBV gene, and thus the test reliability is
effectively maintained to reduce the possibility of retest
due to inhibition of internal control reaction, thereby
providing convenience and high efficiency, as well as
allowing quantitative determination in a wide range of
levels. The probe base sequence of internal control is SEQ
ID NO. 9, as described in Table 2.
In one preferred embodiment, the composition of the
present invention may be a composition for the detection and

quantification of HBV, which further comprises a buffer
solution, DNA polymerase, dNTP and distilled water. In
addition, the buffer solution, DNA polymerase, dNTP and
distilled water may be any solutions and enzymes that are
typically used in the related art, without limitation.
Preferably, the composition for the detection of HBV may
have the composition of Table 3.
Preferably, the additional enzyme may be HS-Taq. HSTaq
is a modified form of Taq DNA Polymerase, which is
activated by heat treatment. A chemical moiety is attached
to the enzyme at the active site, which renders the enzyme
inactive at room temperature. Thus, during amplification,
misprimed primers are not extended. The result is higher
specificity and greater yields, when compared to standard
DNA polymerases.
The specific composition of the primer, probe, HS-Taq
and buffer solution of the present invention is described in
the following Table 3.
[Table 3]
Addition Added amount (μl)
Primer 100 μM BSFOR 0.1
100 μM BSREV 0.1
100 μM BCFOR 0.1
100 μM BCREV 0.1
100 μM ICFOR 0.1
100 μM ICREV 0.1
100 μM BSFAM 0.02
Probe 100 μM BCFAM 0.02
100 μM ICCy5 0.02
2U HS prime Taq
premix
4.845
1M MGCl2 0.004
100mM dUNTP 0.025
PCR reactant
2000 U/mL UDG 0.125
Extracted DNA 5
Milli-Q Water 4.34
Total 20
In the present invention, the target amplification
reaction may be preferably PCR, and the PCR conditions are
described in the following Table 4.
[Table 4]
Reaction Conditions of Real Time PCR
Temperature/Time Cycle

50°C/2 min 1
95°C/10 min 1
95°C/15 sec
57°C/60 sec
72°C/20 sec
40
In still another embodiment, the present invention
relates to a method for detecting HBV gene, comprising the
steps of obtaining a sample from a subject; and detecting
HBV gene in the biological sample using the composition for
the detection of HBV.
The term “biological sample”, as used herein, includes
tissues, cells, whole blood, serum, plasma, saliva, sputum,
cerebrospinal fluid or urine obtained from the individuals
with HBV infection, suspected of having HBV infection or
vaccinated with HBV vaccine, but is not limited thereto. A
sample obtained from the blood of an individual suspected of
having HBV infection is preferred.
A method for identifying the presence and genotype of
HBV is particularly not limited as long as it employs the
aforementioned primers. Examples of such methods include
direct identification of HBV DNA using a primer of a
specific strand as a probe, Southern blotting, dot blotting,
and FISH (filter in situ hybridization). Alternative
methods include a method based on amplifying HBV DNA using a
pair of primers, genotype-specific polymerase chain reaction
(hereinbelow, referred to as PCR), and general-primer PCR.
PCR is more preferred, and Real Time PCR is most preferred.
As used herein, the term “polymerase chain reaction
(PCR)” is a representative nucleic acid amplification
technique (NAT), which enzymatically amplifies a specific
DNA region of interest in vitro. The PCR method, which was
developed in 1985 by Mullis et al., can amplify any segment
of a DNA molecule if its boundary sequences are known. PCR
basically consists of three major steps: denaturation,
annealing and extension. A specific DNA sequence is
amplified while these three steps are repeated. In the
first step (denaturation) of PCR, a double-stranded template
DNA is denatured into two single strands. In the second
step (annealing), primers anneal with the two kinds of
single-stranded DNA, in which a sequence desired to be
amplified is interposed between the primer binding regions.
In the third step, a heat-resistant DNA polymerase extends
the primers and synthesizes the complementary strand. This
cycle is repeated 25 to 30 times.
Preferably, the composition according to the present

invention is used to amplify DNA extracted from the clinical
specimen, and the products are subjected to real time PCR.
Such real time PCR analysis may be performed by any
commercially available Real-time PCR reactor, exemplified by
SLAN real time PCR detection system (LG Life Science, Korea),
LightCyclerTM (Roche, Germany), ABI PRISMTM 7000/7700
(Applied Biosystems, USA), iCyclerTM (Bio-Rad, USA), Rotor-
GeneTM(Corbett, Australia), and OpticonTM (PharmaTech, USA),
but is not limited thereto.
Primers are the most important factor determining the
reliability of PCR results. Some primer sequences can give
rise to non-specific amplification, leading to false results.
In this regard, the present invention provides reliable
primer pairs. The performance of PCR with the primer pairs
of the present invention enables accurate detection of HBV
genotypes and sensitive quantitative analysis of very small
amounts. Also, when PCR is carried out with the primer
pairs of the present invention, consistent results are
obtained upon repeated PCR performance. That is, since the
primer pairs of the present invention are highly valid and
reliable, the results obtained with the present primer pairs
are highly reliable.
In still another embodiment, the present invention
relates to a kit for the detection and quantification of HBV,
comprising the composition for the detection of HBV.
Preferably, the kit can be manufactured by including
the primer and probe having the sequences of the present
invention, and the internal control primer and probe
sequences. The kit may include a buffer, KCl, MgCl2, and
dNTP for HBV detection and quantification. More preferably,
the kit having the composition of Table 3 is used to
manufacture a kit for the amplification and detection of HBV.
In the preferred Example of the present invention, a kit
having the above composition was manufactured, thereby
detecting and quantifying HBV with high sensitivity and
specificity.
[Mode for Invention]
Hereinafter, the present invention will be described in
more detail with reference to Examples. However, these
examples are for illustrative purposes only and are not
intended to limit the scope of the present invention.
Example 1. Construction of Primer and Probe
The HBV-specific primer and probe used in the present

invention were primer sequences that can amplify only HBV
virus gene, confirmed by analyzing the DNA sequence in
GenBank (www.ncbi.nlm.nih.gov) managed by National Center
for Biotechnology Information (NCBI) of U.S. National
Institutes of Health (NIH) using a DNAsis program from
Hitachi Software, sequencing the DNA sequence, and then
analyzing the DNA sequence again with BLAST
(www.ncbi.nlm.nih.gov/BLAST/).
After obtaining the base sequence, the plant-derived
gene specific primer and probe that did not competitively
inhibit the HBV-specific primer and probe in the composition
for HBV detection were determined by using HS-Taq.
Example 2. Synthesis of Primer
The primers analyzed in Example 1 were synthesized by
Metabion (Germany) using a method such as “Synthesis of
Oligonucleotide” described in a paragraph 10.42 of Molecular
cloning 3rd (Sambrook and Rusell, Cold Spring Harbor
Laboratory Press, New York, USA, 2001).
Example 3. Extraction of HBV RNA from Clinical Specimen
Serum or EDTA-Plasma was separated from the blood of
patients with suspected HBV infection, and extracted using a

silica membrane-based spin column such as QIAamp MineElute
Virus Spin Column (Qiagen).
Example 4. Multiplex Real-Time PCR Analysis Using the
Primer and Probe
A PCR reaction solution was prepared according to the
compositions in Table 3, and PCR was performed under the
conditions of Table 4 in a SLAN real time PCR detection
system (LG Life Science, Korea). The reaction products were
measured in real-time, and the results were analyzed using a
SLAN 8.0 program after completing the reaction.
Of the HBV DNA quantification methods using PCR, Cobas
Amplicor HBV MonitorTM test (Roche Diagnostics Systems,
Meylan, France) has high sensitivity, but a narrow dynamic
range of 2.0×102-2.0×105 copies/mL. Thus, to measure within
the linear range of accurate quantification, a sample
dilution is required. In addition, a bDNA method is a DNA
quantification method based on signal amplification, in
which a viral gene present in the serum of a patient is
captured using each viral DNA-specific probe and then the
captured gene is reacted with a probe and amplifier to
quantify DNA. This method has been used in diagnosis of
virus infection and evaluation of therapeutic effect for

several years. In the present invention, the serum HBV DNA
levels of chronic HBV-infected patients in Korea were
measured by a real-time PCR which was invented by the
present inventors, and compared with Cobas Amplicor HBV
MonitorTM test and bDNA method to evaluate its validity.
As shown in Table 5 and FIG. 3, the positive predictive
value was found to be 100% in the test using the Genotype
Panel ((PHD201(M)) provided by BBI, indicating very low risk
of false positive and false negative results. In addition,
FIG. 1 suggests that a positive detection and quantification
can be made without competitive inhibition of the internal
control for the HBV-specific primer and probe.
[Table 5]
Member
ID#
Origin Genotypes %Identity GI#
Roche Cobas
Amplicor
Monitor
(copies/mL)
Bayer bDNA
(copies/mL)
AdvanSure
HBV QPCR
(copies/mL)
PHD201-01 US Negative N/A N/A < 300 < 2000 < 58.2
94 416174A
PHD201-02 US A
94 416076A
1.9 X 104 4.8 X 104 3.2 X 104
97 416074A
PHD201-03 US A
95 416076A
1.5 X 104 4.2 X 104 3.6 X 104
PHD201-04 E Asia B 99 416078B 1.9 X 104 8.0 X 103 4.7 X 103
98 452622C
97 416080C
98 452641C
PHD201-05 E Asia C
98 416082C
1.7 X 104 1.9 X 104 5.6 X 103
94 416086D
PHD201-06
Middle
East
D
95 416084D
1.6 X 104 9.5 X 103 3.2 X 103
99 416074A
PHD201-07 US A
97 416076A
1.1 X 104 1.4 X 104 6.5 X 103
PHD201-08 Africa E 99 X75657 2.4 X 104 1.8 X 104 2.5 X 104
PHD201-09
South
America
F 99 X69798 1.3 X 104 1.5 X 104 1.3 X 104
Example 6. Test on HBV detection limit of the kit of
the present invention
The kit of the present invention was used to test its
detection limit for HBV infection. The test results are
shown in the following Table 6.
[Table 6]
Test of Detection Limit
IU/mL # Tested # Detected % Detected
10 24 24 100
6 24 24 100
5 24 24 100
3 24 23 96
2 24 22 92
1 24 20 83
Probit 95% Hit rate
2.6 IU/mL (95% confidence limits of 1.6
~ 10.4 IU/mL)
To determine the lower limit of detection of HBV, an
international standard for HBV, NIBSC 97/746 was applied to
compensate the matrix effect for normalization, and then DNA
was extracted from 6 different concentrations of HBV sera
diluted with Drug Free Serum (Bio-rad), and quantified by
AdvanSure HBV Real-Time PCR, followed by Probit analysis and
calculation of 95% detection limit. As a result, the 95%
detection limit was 2.6 IU/mL.
OPA10026-Sequence listing
<110> LG LIFE SCIENCES LTD.
<120> A METHOD FOR DETECTING HBV USING REAL TIME PCR
<130> OPA10026
<150> KR10-2009-0035638
<151> 2009-04-23
<160> 9
<170> KopatentIn 1.71
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for amplication of HBV S protein
<400> 1
gtrtctgcgg cgttytatca 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Reverse primer for amplication of HBV S protein region
<400> 2
ggacaaacgg gcaacatacc 20
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for amplication of HBV pre-Core region
<400> 3
aaatgcccct atcytatcaa cact 24
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Reverse primer for amplication of HBV pre-Core region
<400> 4
ccgagattka gatcttctgc ga 22
Page 1
OPA10026-Sequence listing
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for amplication of GFP
<400> 5
cgatggccct gtccttttac attta 25
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Reverse primer for amplication of GFP
<400> 6
cttttcgttg ggatctttga ttaaa 25
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> probe for HBV S protein region
<400> 7
catcctgctg ctatgcctca tcttc 25
<210> 8
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> probe for HBV pre-Core region
<400> 8
cccctagaag aaagaactcc ctcgcc 26
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> probe for GFP
Page 2
OPA10026-Sequence listing
<400> 9
attacctgtc cacacaatct gccctt 26
Page 3
- 29 -
[CLAIMS]
[Claim 1]
A pair of primers complementary to hepatitis B virus
(HBV) gene, which have the base sequences represented by SEQ
ID NOs. 1 and 2, and SEQ ID NOs. 3 and 4.
[Claim 2]
A composition for the detection of HBV gene, comprising
a pair of primers having the base sequences of SEQ ID NOs. 1
and 2, a pair of primers having the base sequences of SEQ ID
NOs. 3 and 4, and probes having the base sequences of SEQ ID
NOs. 7 and 8.
[Claim 3]
The composition according to claim 2, wherein the
composition further comprises a pair of primers having the
base sequences of SEQ ID NOs. 5 and 6 and a probe having the
base sequence of SEQ ID NO. 9 as an internal control, and
shows no competitive inhibition.
[Claim 4]
The composition according to claim 2, further
comprising HS-Taq, a buffer solution, dNTP or distilled
water.
[Claim 5]
The composition according to claim 4, comprising each
- 30 -
100 μM of the primers of SEQ ID NOs. 1 to 4, each 100 μM of
the probes of SEQ ID NOs. 7 and 8, 2.5U HS-Taq, and 2 mM
dUTP.
[Claim 6]
The composition according to claim 2, wherein the probe
contains fluorescent molecules at 5’ and 3’-ends.
[Claim 7]
The composition according to claim 6, wherein the
fluorescent molecule at 5’-end is any one selected from the
group consisting of 6-carboxyfluorescein, hexachloro-6-
carboxyfluorescein, tetrachloro-6-carboxyfluorescein, FAM
(5-carboxy fluorescein), HEX (2’,4’,5’,7’-tetrachloro-6-
carboxy-4,7-dichlorofluorescein), and Cy5 (cyanine-5).
[Claim 8]
The composition according to claim 6, wherein the
fluorescent molecule at 3’-end is any one selected from the
group consisting of 6-carboxytetramethyl-rhodamine, TAMRA
(5-Carboxytetramethylrhodamine), and BHQ 1, 2 and 3 (black
hole quencher 1, 2, 3).
[Claim 9]
A kit for the detection of HBV, comprising the
composition of any one of claims 1 to 8.
[Claim 10]
- 31 -
A method for detecting HBV gene, comprising the steps
of obtaining a sample from a subject; and detecting HBV gene
in the biological sample using the composition of any one of
claims 1 to 8.
[Claim 11]
The method according to claim 10, wherein the detection
is performed by polymerase chain reaction.
[Claim 12]
The method according to claim 10, wherein the sample is
blood.
Dated this 14th Day of April, 2010

OMANA RAMAKRISHNAN

OF K & S PARTNERS
AGENT FOR THE APPLICANT