JPH11127860A - Highly sensitive detection of hepatitis b virus resistant to lamivudine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To highly sensitively detect a hepatitis B virus resistant to lamivudine or the like by rapidly and highly sensitively judge the mutation of the virus in simple operations by a polymerase chain reaction(PCR) method comprising specific two step processes. SOLUTION: This method for highly sensitively detecting a hepatitis B virus resistant to lamivudine comprises proliferating a constant region of the DNA of the virus in the first step process of a PCR method (nested PCR) comprising two step processes, treating the obtained proliferation product with a restriction enzyme capable of cleaving only a proliferation product originated from the wild strain of the virus, performing the proliferation of the viral DNA with a primer in the second step process of the PCR method, treating the obtained proliferation product with a restriction enzyme capable of cleaving only a proliferation product originated from the mutant of the virus, and subsequently examining the pattern cleaved with the restriction enzyme to detect the mutation of the virus. The primer is designed by introducing a mismatched base so that a site to be recognized with the restriction enzyme is produced only in a proliferation product originated from the mutant of the virus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting a mutation in a virus with high sensitivity, and more particularly to a method for detecting a lamivudine-resistant hepatitis B virus with high sensitivity and a primer used therefor.

[0002]

2. Description of the Related Art Lamivudine is a hepatitis B virus (HB).
V) and HIV are found to be effective as replication inhibitors, and are used for the treatment of chronic hepatitis B, acquired human immunodeficiency syndrome, and the like. However, long-term administration of lamivudine causes drug resistance, and recently, in patients who received immunosuppressive drugs after liver transplantation, cases in which lamivudine administration caused lamivudine resistance were reported. Is a problem.

[0003] Lamivudine resistance in hepatitis B virus is known to be caused by mutation of the amino acid sequence YMDD motif (tyrosine-methionine-aspartic acid-aspartic acid) present in the active center of the viral DNA polymerase to YIDD or YVDD. ing.
These mutations correspond to the nucleotide sequence AT encoding methionine.
This is because G was mutated to AT T , G TG or AT C. To determine the presence or absence of these mutations and to detect lamivudine-resistant hepatitis B virus, a method of determining the base sequence of viral DNA has been used so far.

[0004] However, such a method is not suitable for processing a large number of samples due to the time and labor required for cloning and sequencing. Moreover, it is difficult to detect the mutant strain with high sensitivity when the amount is small, and it is also possible to accurately grasp the progress of the appearance of the lamivudine-resistant hepatitis B virus in the patient and the return to the wild strain. Did not.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for determining a mutation of a virus quickly and with high sensitivity by a simple operation, and in particular, a method for detecting a lamivudine-resistant hepatitis B virus with high sensitivity. , And reagents used for the same. Another object of the present invention is to provide a virus mutation detection method for accurately grasping the course of a patient in which a resistant strain has appeared.

[0006]

Means for Solving the Problems The present inventors can easily and efficiently detect a mutant strain by devising a two-step PCR method (nested PCR method) even when the mutant strain contains only a trace amount. By combining the method and the two detection methods, a method was found in which the course in a patient in which a mutant strain appeared could be accurately observed. Furthermore, when a region containing a mutation site involved in lamivudine resistance of hepatitis B virus is amplified by the polymerase chain reaction method (PCR method), detection of lamivudine-resistant hepatitis B virus can be performed by using specific primers. They have found that they can be performed simply and accurately, and have completed the present invention.

That is, the gist of the present invention is a method for detecting a virus mutation with high sensitivity using a two-step polymerase chain reaction method comprising the following steps (1) to (5).

(1) A certain region of the viral DNA is amplified by the first-stage polymerase chain reaction method.

(2) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from a wild strain of the virus.

(3) The amplification of the viral DNA by the polymerase chain reaction method in the second step is carried out by using a primer designed by introducing a mismatch base so that a restriction enzyme recognition site is generated only in an amplification product derived from a virus mutant. Do it.

(4) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from the virus mutant.

(5) Virus mutations are detected by examining the pattern of cleavage by restriction enzymes.

[0013] The present invention also relates to the above method.
In the first-stage polymerase chain reaction method, the present invention also relates to a method of performing amplification using a primer designed by introducing a mismatched base such that a restriction enzyme recognition site is generated only in an amplification product derived from a wild strain of a virus.

These methods can be used for the detection of lamivudine-resistant hepatitis B virus, and specifically, the mutation of the YMDD motif in the DNA polymerase of hepatitis B virus to YIDD or YVDD.

A preferred embodiment of the present invention is a method for detecting lamivudine-resistant hepatitis B virus with high sensitivity using a two-step polymerase chain reaction method comprising the following steps (1) to (5).

(1 ') A certain region including the region encoding the DNA polymerase YMDD motif in the hepatitis B virus genomic DNA is amplified by the first stage polymerase chain reaction method.

(2 ') The obtained amplification product is treated with a restriction enzyme Fo capable of cleaving only an amplification product derived from a wild-type hepatitis B virus.
Process with kI.

(3 ') The amplification of the viral DNA by the polymerase chain reaction method of the second step is carried out by using only the amplification product derived from lamivudine-resistant hepatitis B virus as a restriction enzyme.
This is carried out using a primer having the nucleotide sequence shown in SEQ ID NO: 3 or 9 designed by introducing a mismatch base so as to generate a recognition site for SspI or EcoRV.

(4 ′) The obtained amplification product is treated with a restriction enzyme SspI or EcoRV capable of cleaving only the amplification product derived from lamivudine-resistant hepatitis B virus.

(5 ') Lamivudine-resistant hepatitis B virus is detected by examining the pattern of cleavage by restriction enzymes.

The method comprises the following steps (1) to (5):
Using the polymerase chain reaction method of the stage,
The present invention also relates to a method for detecting lamivudine-resistant hepatitis B virus with high sensitivity.

(1 ") A first region of the polymerase chain reaction method is used to amplify a certain region including the region encoding the DNA polymerase YMDD motif in the hepatitis B virus genomic DNA. The reaction is carried out using a primer having a base sequence shown in SEQ ID NO: 7 designed by introducing a mismatch base so as to generate a recognition site for restriction enzyme Ppul0I only in the product.

(2 ") The obtained amplification product is treated with a restriction enzyme Pp capable of cleaving only an amplification product derived from a wild-type hepatitis B virus strain.
Process with ul0I.

(3 ") Amplification of the viral DNA by the polymerase chain reaction method of the second stage is carried out only with the amplification product derived from the lamivudine-resistant hepatitis B virus.
This is performed using a primer having the nucleotide sequence shown in SEQ ID NO: 8 designed by introducing a mismatched base so as to generate an ApaLI recognition site.

(4 ") The obtained amplification product is treated with a restriction enzyme ApaLI capable of cleaving only the amplification product derived from lamivudine-resistant hepatitis B virus.

(5 ") Lamivudine-resistant hepatitis B virus is detected by examining the pattern of cleavage by restriction enzymes.

Furthermore, the present invention is a method for detecting a mutation of a virus, which combines the following two detection methods.

(A) The detection method comprising the steps (1) to (5), and (B) In the above detection method, the step (1) is directly carried out without the restriction enzyme treatment in the step (2). A method of performing the polymerase chain reaction method of (3).

This method can be used for detecting lamivudine-resistant hepatitis B virus.

Further, the present invention provides a method wherein the restriction enzyme SspI or EcE is used only for an amplification product derived from lamivudine-resistant hepatitis B virus.
Using a primer having the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 designed by introducing a mismatch base so as to generate an oRV recognition site, hepatitis B virus DN
A certain region of the sequence encoding the DNA polymerase in A is amplified, and the obtained amplification product is treated with a restriction enzyme SspI or EcoRV capable of cutting only the amplification product derived from lamivudine-resistant hepatitis B virus, and the cleavage pattern is changed. The present invention also relates to a method of detecting a lamivudine-resistant hepatitis B virus by examining the virus.

Furthermore, the present invention also relates to primers having the nucleotide sequences shown in SEQ ID NOs: 3, 7, 8 and 9.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The mutation detection method of the present invention will be described in detail below.

A method for detecting a virus mutation with high sensitivity using a two-step polymerase chain reaction method comprises the following procedures.

(1) A certain region of the viral DNA is amplified by the first-stage polymerase chain reaction method.

(2) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from the wild strain of the virus.

(3) The amplification of the viral DNA by the polymerase chain reaction method in the second step is performed by using a primer designed by introducing a mismatch base so that a restriction enzyme recognition site is generated only in the amplification product derived from the virus mutant. Do it.

(4) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from the virus mutant.

(5) Virus mutation is detected by examining the pattern of cleavage by restriction enzymes.

The case where this method is used for detecting lamivudine-resistant hepatitis B virus will be described.

The gene structure of hepatitis B virus is as shown in FIG. 1, and a part (411-895) of the pol portion encoding DNA polymerase is enlarged and shown on the right. 736-
The base sequence of 747 is a region encoding the amino acid sequence YMDD motif present in the active center of DNA polymerase. This YMDD motif is YIDD or YVD
Changes to D, that is, lamivudine resistance caused by methionine YMDD motif is changed to an isoleucine or valine, which is the base sequence ATG is AT T or AT C (YIDD mutant) methionine, G
This is due to mutation to TG (YVDD mutant) (see FIG. 2).

First, among the YIDD mutant strains, the base sequence A
Detection of a mutated strain of the case by the mutation of the TG → AT T, the first
A certain region including the YMDD motif region is amplified by the first-stage PCR (abbreviated as 1st PCR). For example, primer
An amplification product of 240 bp is obtained using BF108 (SEQ ID NO: 1) and BR112 (SEQ ID NO: 2) (see FIG. 1).

The 1st PCR amplification product thus obtained is treated with the restriction enzyme FokI. As shown in FIG.
Has a recognition site of FokI, ATG is a mutant strain → AT T
This recognition site does not exist due to the mutation of Therefore, the amplification product from the wild-type strain by digestion with FokI is 100 bp to some extent.
And the amplified product derived from the mutant remains unchanged.

Next, the second stage PCR (abbreviated as 2nd PCR) is performed on the 1st PCR amplification product treated with the restriction enzyme.
In this amplification, a primer having a nucleotide sequence in which a mismatch base is introduced near the mutation site so as to provide a restriction enzyme recognition site specific to the mutation is used. Specifically, FIG.
As shown in the upper row, only the restriction enzyme Ss
Amplification is carried out using a mismatch primer YNSspI (SEQ ID NO: 3) in which the 736th base T is changed to A so as to generate a pI recognition site, and using BR109 (SEQ ID NO: 4) as the other primer.

The G → T mutation in the YIDD mutant and the mismatched base A create a recognition site for the restriction enzyme SspI only in the amplification product derived from the mutant, whereas the wild-type strain has
No recognition site for SspI occurs. Therefore, after digestion of the amplification product with SspI, analysis by electrophoresis or the like makes it possible to discriminate between the mutant strain and the wild strain.

D treated with a specific restriction enzyme according to the mutation
The procedure of separating and detecting NA under appropriate separation conditions such as electrophoresis can be performed according to a conventional method. For example, after electrophoresis using 4% agarose gel, staining with ethidium bromide, UV irradiation, and band detection are performed. I do.

The DNA fragments separated and detected are examined for the presence or absence of cleavage by the respective restriction enzymes used. Since a restriction enzyme that cuts only the mutant strain is used, if only the wild-type strain is present in the sample, it is not cut by the restriction enzyme,
The amplified original band is detected, and when only the mutant is present, a band cut by the restriction enzyme is given. If wild and mutant strains are mixed,
Cut and uncut bands are detected.

When the above-mentioned YNSspI and BR109 were used as a primer and amplified, a band obtained by treating with the restriction enzyme SspI was only 160 bp, all of the wild strain and 132 b
In the case of p alone, all of the mutant strains alone, and when both bands are observed, it is determined that the wild strain and the mutant strain are mixed. If no band appears, it may be HBV-DNA (-) or a small amount of a wild-type strain was present.

[0048] When the ATG at YIDD mutants are mutated to AT C, was amplified using primers having the nucleotide sequence of SEQ ID NO: 9 instead of YNSspI as primers containing mismatched base recognition restriction enzyme EcoRV By creating a site, the presence of the mutant can be determined.

[0049] In the YVDD mutant, FokI
In the first PCR, a restriction enzyme recognition site is introduced only into an amplification product derived from a wild-type strain using a primer containing a mismatched base. For example, in the first PCR, as shown in FIG. 2, PCR is performed using a mismatch primer TMPpul0I (SEQ ID NO: 7) in which the base G is changed to C and a primer BF105 (SEQ ID NO: 5). When the obtained amplification product is digested with Ppul0I that can cut only the wild strain, only the wild strain is cut to some extent, and the mutant strain remains as it is. Then, in the 2nd PCR, as shown in FIG.
By amplifying TMApaLI (SEQ ID NO: 8) changed to C as a mismatch primer, a recognition site for the restriction enzyme ApaLI is introduced only into the amplification product derived from the mutant strain. Therefore, when the band obtained by treating the 2nd PCR amplification product with ApaLI is only 189 bp, all bands are wild strains,
In the case of only 163 bp, all are mutant strains, and when both bands are observed, it is determined that the wild strain and the mutant strain are mixed.

As described above, if the product amplified by 1st PCR is treated with a restriction enzyme that cuts only the amplification product derived from the wild-type strain, amplification of the wild-type strain can be suppressed to some extent. Effective for detection.

Next, a method combining two types of mutation detection methods using two-step PCR will be described. This method
(A) After treating the 1st PCR amplification product with a restriction enzyme that cuts only the product derived from the wild-type strain, use a primer that generates a restriction enzyme recognition site only in the amplification product derived from the mutant strain.
nd PCR, the above-described mutation detection method, and (B) 1st P
Without performing restriction enzyme treatment of CR amplification products, 2nd PCR
The method comprises using a primer that generates a restriction enzyme recognition site only in the amplification product derived from the mutant strain.

In the case of the above (B), the same level of amplification is applied to both the wild strain and the mutant strain, so that it is easy to track the wild strain and to determine the timing of the return from the mutant strain to the wild strain. Useful for Therefore, by analyzing the virus by both the methods (A) and (B), it is possible to accurately grasp the change in the virus.

[0053]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to the following examples.

Extraction method of hepatitis B virus genomic DNA Hepatitis B virus genomic DNA is extracted from 300 μl of hepatitis B patient serum as follows. The serum used is
It is freshly separated or stored at -80 ° C. 20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1
% Proteinase K (manufactured by MERK) with sodium dodecyl sulfate to a final concentration of 0.8 mg / ml.
Incubate at 4 ° C for 4 hours. Thereafter, the mixture was treated three times with an equal amount of a phenol / chloroform solution (a solution of phenol: chloroform = 4: 1 saturated with 1 × TE buffer) and once with chloroform, and the aqueous phase was diluted with a 1/10 volume of 3M 3M.
The extracted DNA is precipitated by adding sodium acetate, 20 μg of glycogen, and 2.5 times the amount of ethanol, followed by purification.
After a 70% ethanol wash, the resulting pellet is dissolved in 50 μl of 1 × TE buffer.

A method for detecting a YIDD mutant strain and a YVDD mutant strain using the above extracted DNA is described below.

In the following experiments, in order to increase the specificity of the PCR, Tag DNA polymerase was added to the antibody (Taq Start
^TM Antibody, CLONTEC)
Perform a start. A solution obtained by mixing equal amounts of 5 units / μl Gene Taq (manufactured by Wako Pure Chemical Industries) and 7 μM Taq Start ^™ Antibody and incubating at room temperature for 5 minutes was used.

[0057]Detection of hepatitis B virus YIDD mutant
(1) YIDD by two-step PCR using the extracted DNA
Mutant (ATG → ATT2)
The different methods are described below.

(A) In the case of treatment with the restriction enzyme Fok I: Extract D into an autoclaved 0.5 ml PCR tube.
2 μl of NA, 10 × Gene Taq Universal Buffer (Gen T
2.5 μl of dNTP mixture (attached to Gene Taq)
1 μl each of the primers BF108 (SEQ ID NO: 1) and BR112 (SEQ ID NO: 2) adjusted to 2 μl and 0.1 μg / μl respectively, and further to Gene Taq and Ta described above.
q Add 0.3 µl of Start ^™ Antibody solution, make up to 25 µl with sterile distilled water, add liquid paraffin, and start the PCR cycle. PCR conditions were 94 ° C,
After 4 minutes incubation, 94 ° C, 1 minute; 58 ° C,
35 cycles of 1.5 minutes at 72 ° C for 1 minute
(1st PC)
R).

After the 1st PCR, the cells are treated with the restriction enzyme FokI.
That is, 10 mM Tris-HCl,
pH 7.5, 10 mM MgCl ₂ , 1 mM DDT, 50 mM NaCl, 0.01%
Add 2.5 U of Fok I (manufactured by Takara Shuzo Co., Ltd.) with BSA, and make up to 10 μl with sterile distilled water.
Incubate for at least an hour.

The 2nd PCR is performed in the same manner as the 1st PCR, but the primers used are YNSspI (SEQ ID NO: 3) and BR109 (SEQ ID NO: 4), and the amount brought in from the previous step is 1 μl.

After the 2nd PCR, digestion is performed with the restriction enzyme SspI.
That is, 10 mM Tris was added to 5 μl of the 2nd PCR product.
-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM DDT, 100 mM NaCl
After adding 3 U of SspI (manufactured by Takara Shuzo Co., Ltd.) and making up to 10 μl with sterile distilled water, incubation is carried out at 37 ° C. for 1 hour or more.

Thereafter, electrophoresis is performed on a 4% agarose gel, and a photograph is taken after ethidium bromide staining.

The determination is carried out as follows: if no band appears, it is possible that HBV-DNA (-) or a small amount of a wild type strain is present. If the band appears only at a height of 160 bp, the wild type is determined. Only the strain, if the band has a height of only 132 bp, only the mutant strain, and if both bands are recognized, it is determined that the wild strain and the mutant strain coexist.

Amplification of a wild strain can be suppressed to some extent by digestion with the restriction enzyme FokI, which is effective for detecting a trace amount of a mutant strain. In preliminary experiments using plasmids, 1/100
The amount of mutant was detectable (however, wild strain, 10
In the range of ⁵ -10 ³ copies / PCR).

The primers used are as follows.

BF108 (SEQ ID NO: 1): 5′-AGTCCGTTTCTCCTGGCTCA-3 ′ BR112 (SEQ ID NO: 2): 5′-TTCCGTCGACATATCCCATGAAGTT
AAGGGA-3 'YNSspI (SEQ ID NO: 3): 5'-TTTCCCCCACTGTTTGGCTTTCAGT
AATAT-3 'BR109 (SEQ ID NO: 4): 5'-AAGGGAGTAGCCCCAACGTT -3' (B) When not treated with the restriction enzyme Fok I In the above method (A), the 1st PCR amplification product was not treated with the restriction enzyme. Proceed directly to 2nd PCR. However, 2nd from 1st PCR
The volume to be carried into the nd PCR is 0.5 μl.

The determination is carried out as follows: if no band appears, HBV-DNA (-); if the band appears only at a height of 160 bp, only the wild-type strain, and the band has a height of 132 bp. If only the mutant strain is found, it is determined that the wild strain and the mutant strain are mixed if both are found. It is easier to track the wild strain than in the case (A).

[0068]Detection of hepatitis B virus YIDD mutant
(2) (A) YIDD mutant (ATG → ATCDue to mutations)
Detection was performed in the 2nd PCR of the above method (1).
Primer 5'-TTTCCCCCACTGTTTGGC in place of mer YNSspI
Using the primer of TTTCAGTGATAT-3 '(SEQ ID NO: 9)
Using EcoRV as the restriction enzyme to be used after 2nd PCR.
Other than the above.

(B) The 2nd PCR is performed directly without performing the step of digesting the 1st PCR amplification product with the restriction enzyme Ppul0I.

The determination is carried out as follows: if no band appears, there is a possibility that HBV-DNA (-) or a small amount of a wild-type strain is used (when the 1st PCR amplification product is treated with a restriction enzyme).
If the band appears only at the height of 160 bp, it is determined that only the wild strain is present, if the band is only 132 bp high, only the mutant strain is present, and if both bands are recognized, the wild strain and the mutant strain are mixed.

Detection of Hepatitis B Virus YVDD Mutant (A) The same operation as in Experimental Example 1 is performed, but the primers and restriction enzymes used are different. In the first PCR, primers BF105 (SEQ ID NO: 5) and TMPpul0I (SEQ ID NO: 7)
To introduce a Ppul0I recognition site only into the 1st PCR amplification product of the wild type. The 1st PCR amplification product was digested with Ppul0I, and then the 2nd PCR was digested with primers BF111 (SEQ ID NO: 6) and TM
Amplification was performed using ApaLI (SEQ ID NO: 8)
Digest with ApaL.

The primers used are as follows.

BF105 (SEQ ID NO: 5): 5'-TCCTGCTGCTATGCCTCATC-3 'BF111 (SEQ ID NO: 6): 5'-TCGGACGGAAACTGCACTTG-3' TMPpul0I (SEQ ID NO: 7): 5'-CAGACTTGGCCCCCAATACCACA
TCATGCA -3 'TMApaLI (SEQ ID NO: 8): 5'-CAGACTTGGCCCCCAATACCACATC
GTGCA-3 '(B) 2nd PCR is performed directly without performing the step of digesting the 1st PCR amplification product with the restriction enzyme Ppul0I.

The determination is carried out as follows: If no band appears, there is a possibility that HBV-DNA (-) or a small amount of a wild-type strain is used (when the 1st PCR amplification product is treated with a restriction enzyme).
If the band appears only at a height of 189 bp, it is determined that only the wild strain is present, if the band is only 163 bp high, only the mutant strain is present, and if both bands are observed, the wild strain and the mutant strain are mixed.

[0075]

Example 1 A wild type strain and a mutant strain were mixed in the amounts shown at the top of each figure in FIG. 4, and the method for detecting the YIDD mutant strain (1) was performed.
YIDD mutants were detected according to the methods (A) and (B). Wild strains and mutant strains can be easily determined from the difference in migration after digestion with restriction enzymes.

FIG. 4 shows bands obtained when the 1st PCR product was not digested with FokI and when it was digested. When digested with FokI, the mutant can be detected even in a 1/100 amount of the mutant strain, which is effective for high-sensitivity detection of the mutant strain. Also, Fo
When digestion is not performed with kI, the band derived from the wild strain appears stronger than when digested, and it is easy to confirm the presence of the wild strain.

[0077]

Example 2 Changes in hepatitis B virus when lamivudine was administered to patients with chronic hepatitis B were examined over 2 years and 6 months. FIG. 5 shows the case where the administration of lamivudine was continued, and FIG. 6 shows the case where the administration of lamivudine was stopped in one year.

The amount of HBV-DNAR was measured by a solution hybridization method, and ALT (alanine aminotransferase) was measured.
Was measured by a biochemical automatic analyzer. Hepatitis B virus genomic DNA extracted from serum at each collection time indicated by numbers 1 to 21 and 1 to 16 in the graph,
(A) and (B) of the method (1) for detecting the YIDD mutant strain
The YIDD mutant strain was detected using the method described above.

From FIG. 5 and FIG. 6, the appearance time of the lamivudine-resistant hepatitis B virus and the time of returning to the wild strain can be determined. It is clear that the growth of the mutant strain continued in patients who continued lamivudine administration (FIG. 5), and that the patients who stopped administration returned to the wild strain (Example 6).

[0080]

According to the present invention, there is provided a method for detecting even a small amount of a mutant strain with high sensitivity by applying a two-step PCR method to the determination of a virus mutation.
In addition, it is possible to easily make a determination in a short time. In addition, by combining the two types of two-step PCR methods, it is easy to accurately follow the course in a patient in which a mutant strain has appeared. The virus mutation detection method of the present invention can be used for detecting lamivudine-resistant hepatitis B virus.

[0081]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGTCCGTTTC TCCTGGCTCA 20 SEQ ID NO: 2 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTCCGTCGAC ATATCCCATG AAGTTAAGGG A 31 SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands : Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTTCCCCCAC TGTTTGGCTT TCAGTAATAT 30 SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AAGGGAGTAG CCCCAACGTT
20 SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCCTGCTGCT ATGCCTCATC 20 SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCGGACGGAA ACTGCACTTG 20 SEQ ID NO: 7 Sequence length: 30 Sequence type: Number of nucleic acid strand: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CAGACTTGGC CCCCAATACC ACATCATGCA 30 SEQ ID NO: 8 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear sequence Type: Other nucleic acid Synthetic DNA sequence CAGACTTGGC CCCCAATACC ACATCGTGCA 30 SEQ ID NO: 9 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTTCCCCCAC TGTTTGGCTT TCAGTGATAT 30

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of hepatitis B virus DNA.

FIG. 2 is a diagram showing a method for detecting lamivudine-resistant hepatitis B virus.

FIG. 3 shows a method for detecting lamivudine-resistant hepatitis B virus.

FIG. 4 is a diagram showing the results of Example 1.

FIG. 5 is a diagram showing the results when lamivudine administration in Example 2 was continued.

FIG. 6 is a diagram showing the results when administration of lamivudine in Example 2 was stopped.

Claims (11)

[Claims] 1. A method for detecting a virus mutation with high sensitivity using a two-step polymerase chain reaction method comprising the following steps (1) to (5). (1) A certain region of the viral DNA is amplified by the first-stage polymerase chain reaction method. (2) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from the wild strain of the virus. (3) Amplification of the viral DNA by the polymerase chain reaction method in the second step is performed using primers designed by introducing mismatched bases such that a restriction enzyme recognition site is generated only in an amplification product derived from a virus mutant. (4) The obtained amplification product is treated with a restriction enzyme capable of cutting only the amplification product derived from the virus mutant. (5) Detect viral mutations by examining the pattern of cleavage by restriction enzymes. 2. In the first step of the polymerase chain reaction method of the step (1), a primer designed by introducing a mismatch base so that a restriction enzyme recognition site is generated only in an amplification product derived from a wild strain of a virus is used. The method according to claim 1, wherein the amplification is carried out. 3. The method according to claim 1, which is used for detecting lamivudine-resistant hepatitis B virus. 4. The lamivudine-resistant hepatitis B virus is detected by determining a mutation of the YMDD motif to YIDD in the hepatitis B virus DNA polymerase.
The described method. 5. The lamivudine-resistant hepatitis B virus is detected by determining the mutation of the YMDD motif to YVDD in the hepatitis B virus DNA polymerase.
The described method. 6. The method according to claim 4, wherein the lamivudine-resistant hepatitis B virus is detected with high sensitivity using a two-step polymerase chain reaction method comprising the following steps (1) to (5). (1) A certain region including a region encoding a DNA polymerase YMDD motif in hepatitis B virus genomic DNA is amplified by the first-stage polymerase chain reaction method. (2) The obtained amplification product is treated with a restriction enzyme FokI capable of cutting only the amplification product derived from the wild-type hepatitis B virus strain. (3) The amplification of the viral DNA by the polymerase chain reaction method of the second step was performed using the restriction enzyme SspI or Ec only for the amplified product derived from lamivudine-resistant hepatitis B virus.
This is performed using a primer having the nucleotide sequence shown in SEQ ID NO: 3 or 9 designed by introducing a mismatched base so as to generate an oRV recognition site. (4) The obtained amplification product is treated with a restriction enzyme SspI or EcoRV that can cut only the amplification product derived from lamivudine-resistant hepatitis B virus. (5) Lamivudine-resistant hepatitis B virus is detected by examining the pattern of cleavage by restriction enzymes. 7. The method according to claim 5, wherein the lamivudine-resistant hepatitis B virus is detected with high sensitivity using a two-step polymerase chain reaction method comprising the following steps (1) to (5). (1) The DNA in hepatitis B virus genomic DNA was determined by the first-stage polymerase chain reaction method.
A certain region including a region encoding a polymerase YMDD motif is amplified, and this amplification is shown in SEQ ID NO: 7 designed by introducing a mismatch base to generate a recognition site of a restriction enzyme Ppul0I only in an amplification product derived from a wild strain of a virus. This is performed using a primer having a base sequence. (2) The obtained amplification product is treated with a restriction enzyme Ppul0I capable of cutting only an amplification product derived from a wild-type hepatitis B virus strain. (3) A sequence designed by introducing a mismatch base such that the amplification site of the restriction enzyme ApaLI is generated only in the amplification product derived from the lamivudine-resistant hepatitis B virus in the amplification of the viral DNA by the polymerase chain reaction method in the second step. This is performed using a primer having the nucleotide sequence shown in No. 8. (4) The obtained amplification product is treated with a restriction enzyme ApaLI capable of cleaving only the amplification product derived from lamivudine-resistant hepatitis B virus. (5) Lamivudine-resistant hepatitis B virus is detected by examining the pattern of cleavage by restriction enzymes. 8. A method for detecting a mutation of a virus, comprising a combination of the following two detection methods. (A) The detection method according to claim 1 or 2, and (B) the detection method according to claim 1 or 2,
Directly from step (1) without the restriction enzyme treatment of (2)
A method of performing the polymerase chain reaction method of (3). 9. The method according to claim 8, which is used for detecting lamivudine-resistant hepatitis B virus. 10. The nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 designed by introducing a mismatched base such that a recognition site of a restriction enzyme SspI or EcoRV is generated only in an amplification product derived from lamivudine-resistant hepatitis B virus. DNA in hepatitis B virus DNA using primers
Amplifying a certain region of the sequence encoding A polymerase;
Next, a method of detecting the lamivudine-resistant hepatitis B virus by treating the obtained amplification product with a restriction enzyme SspI or EcoRV capable of cleaving only the amplification product derived from the lamivudine-resistant hepatitis B virus and examining the cleavage pattern. 11. A primer having the nucleotide sequence shown in SEQ ID NOs: 3, 7, 8, and 9.