JP2007000040A - Method for detecting b-type hepatitis virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting B-type hepatitis virus rapidly and supersensitively detecting B-type hepatitis virus comprising disease virus regardless of its genetic type for diagnosis of infection with the B-type hepatitis virus. <P>SOLUTION: An oligonucleotide primer is specifically hybritized with optional base sequence designed based on an S-area base sequence of the B-type hepatitis virus; a nucleic-acid amplification method uses the primer; a diagnostic method of infection with the B-type hepatitis virus detects nucleic-acid amplification; and a kit diagnoses infection with the B-type hepatitis virus. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting hepatitis B virus (hereinafter sometimes referred to as “HBV”), and more specifically, an oligonucleotide primer for detecting hepatitis B virus, and hepatitis B using the primer. The present invention relates to a virus detection method, a hepatitis B virus infection diagnosis method, and a kit for diagnosing hepatitis B virus infection.

  Hepatitis B is caused by HBV and is not only acute hepatitis but also a cause of chronic hepatitis, cirrhosis, and even liver cancer. The number of infected people reaches approximately 400 million worldwide. Estimated.

  Diagnosis of hepatitis B infection is mainly performed by the presence or absence of viral antigens or antibodies present in human serum. A nucleic acid detection method for measuring HBV DNA is also used as a high sensitivity method. Recently, both Amplicor ™ HBV monitor testing and Taqman ™ technology for real-time detection have been used to detect and quantify HBV DNA in a more sensitive PCR method.

  In blood for blood transfusion, the PCR method, which is a nucleic acid detection method, is used for blood screening at the time of donation. However, even with these virus tests, there is a serious social problem that slipping through occurs and infection by blood transfusion occurs.

  Therefore, a test method that can detect hepatitis B virus quickly and with high sensitivity has been desired.

Publication No. 03-43100 Publication 2000-201698

  As a result of diligent research to solve the above problems, the present inventors have found oligonucleotide primers that hybridize with base sequences specific for HBV (A-H) of all known genotypes. The present invention was completed by finding that only hepatitis B virus can be detected with high sensitivity by amplifying a base sequence specific to hepatitis B virus by LAMP (loop-mediated isothermal amplification) method.

That is, the present invention provides the following (1) to (8).
(1) An oligonucleotide primer designed from an arbitrary base sequence selected from base sequences 304 to 526 of the S region of hepatitis B virus represented by SEQ ID NO: 1, or a base sequence complementary thereto .
(2) The oligonucleotide primer according to (1), comprising an oligonucleotide selected from the following (a) to (c).
(A) an oligonucleotide comprising at least 15 consecutive bases selected from the base sequences represented by SEQ ID NOs: 2 to 9 or a base sequence complementary thereto;
(B) An oligonucleotide that can hybridize with the oligonucleotide according to (a) under stringent conditions.
(C) An oligonucleotide having a primer function, comprising a base sequence in which one to several bases are substituted, deleted, inserted or added among the oligonucleotides described in (a) or (b).
(3) Select a base sequence region of F3c, F2c, F1c from the 3 ′ end side on the target nucleic acid of the S region of hepatitis B virus, and select a base sequence region of B3, B2, B1 from the 5 ′ end side, (1) to (1), wherein the complementary base sequences are F3, F2, F1, and B3c, B2c, B1c. 2) The oligonucleotide primer as described.
(A) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(B) A base sequence having the F3 region of the target nucleic acid.
(C) A base sequence having the B2 region of the target nucleic acid on the 3 ′ end side and the B1c region of the target nucleic acid on the 5 ′ end side.
(D) A base sequence having a B3 region of the target nucleic acid.
(4) A base sequence specific to hepatitis B virus can be amplified and consists of a base sequence selected from the following (a) to (b) from the 5 ′ end to the 3 ′ end (1) The oligonucleotide primer according to (3).
(A) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'.
(B) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3'.
(5) A method for detecting hepatitis B virus, comprising performing an amplification reaction of a target nucleic acid region of hepatitis B virus using the oligonucleotide primers according to (1) to (4).
(6) The method for detecting hepatitis B virus according to (5), wherein the amplification reaction of the target nucleic acid region of hepatitis B virus is the LAMP method.
(7) Influenza characterized by diagnosing the presence or absence of hepatitis B virus infection by detecting amplification of the target nucleic acid region of hepatitis B virus using the oligonucleotide primers described in (1) to (4) Diagnosis method.
(8) A kit comprising the oligonucleotide primer according to (1) to (4), for diagnosing infection with hepatitis B virus.

  According to the present invention, an oligonucleotide primer that selectively hybridizes with a base sequence specific to hepatitis B virus is prepared, and a base sequence specific to hepatitis B virus is amplified by the LAMP method. All known genotypes of HBV (A-H) can be detected with high sensitivity and speed.

  Samples used in the present invention include specimens derived from living organisms of humans or other animals suspected of being infected with hepatitis B virus, such as blood, serum, plasma, spinal fluid, semen, sputum, gargle, saliva, urine Stool, tissue, etc. In addition, cells used for infection experiments, culture solutions thereof, specimens containing viruses isolated from biological specimens or cultured cells, and the like can also be used as samples. These samples may be subjected to pretreatment such as separation, extraction, concentration and purification.

  Amplification of such nucleic acids is a new nucleic acid amplification method developed by Natomi et al. That does not require temperature control, which is indispensable for the PCR method: loop-mediated isothermal amplification method called LAMP method (International Publication No. 00/28082 pamphlet) To be achieved. This method anneals its 3 'end to the template nucleotide to serve as the starting point for complementary strand synthesis, and by combining the primer that anneals with the loop formed at this time, isothermal complementary strand synthesis reaction is performed. This is a possible nucleic acid amplification method. The LAMP method is a highly specific nucleic acid amplification method that uses four primers that recognize at least six regions.

  Oligonucleotide primers used in the LAMP method have a total of 6 regions of the base sequence of the template nucleic acid, that is, the regions of F3c, F2c, F1c from the 3 ′ end and the bases of B3, B2, B1 from the 5 ′ end. These are at least four kinds of primers for recognizing sequences, and are referred to as inner primer F and B and outer primer F and B, respectively. The complementary sequences of F1c, F2c, and F3c are referred to as F1, F2, and F3, respectively, and the complementary strands of B1, B2, and B3 are referred to as B1c, B2c, and B3c, respectively. The inner primer is a nucleic acid synthesis reaction product that recognizes “a specific nucleotide sequence region” on the target base sequence and has a base sequence that gives a synthesis origin at the 3 ′ end, and at the same time, uses this primer as the origin. An oligonucleotide having a base sequence complementary to an arbitrary region at the 5 ′ end. Here, a primer including “a base sequence selected from F2” and “a base sequence selected from F1c” is an inner primer F (hereinafter abbreviated as FIP), and “a base sequence selected from B2” and “B1c A primer containing “a more selected base sequence” is referred to as an inner primer B (hereinafter abbreviated as BIP). On the other hand, an outer primer is an oligonucleotide having a base sequence that recognizes “a specific nucleotide sequence region existing on the 3 ′ end side of a“ specific nucleotide sequence region ”” on the target base sequence and provides a starting point for synthesis. is there. Here, a primer containing “a base sequence selected from F3” is an outer primer F (hereinafter abbreviated as F3), and a primer containing “a base sequence selected from B3” is an outer primer B (hereinafter abbreviated as B3). Call it. Here, F in each primer is a primer display that complementarily binds to the sense strand of the target base sequence and provides a starting point for synthesis, while B is complementary to the antisense strand of the target base sequence. The primer display provides a starting point for synthesis. Here, the length of the oligonucleotide used as a primer is 10 bases or more, preferably 15 bases or more, and may be either chemically synthesized or natural. Each primer may be a single oligonucleotide, It may be a mixture of oligonucleotides.

  In the LAMP method, in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used. The loop primer (Loop Primer) is a primer having a base sequence complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure. When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (WO 02/24902 pamphlet). The base sequence of the loop primer may be selected from the base sequence of the target gene or its complementary strand as long as it is complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure described above. The base sequence may be used. Further, one or two kinds of loop primers may be used.

  Oligonucleotides can be produced by known methods, for example, chemically synthesized. Alternatively, a natural nucleic acid can be cleaved with a restriction enzyme or the like, modified so as to have a desired base sequence, or ligated. Specifically, it can be synthesized using an oligonucleotide synthesizer or the like. A known production method can also be used as a method for synthesizing an oligonucleotide having a base sequence in which one to several bases are substituted, deleted, inserted or added. For example, it is possible to synthesize such oligonucleotides by site-directed mutagenesis, gene homologous recombination, primer extension, or PCR alone or in appropriate combination.

As the “stringent hybridization conditions” in the present specification, generally known conditions can be selected. As stringent conditions, for example, 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhart's solution, 10% dextran sulfate, and 20 μg / ml After overnight hybridization at 42 ° C. in a solution containing DNA of the above, first wash in 2 × SSC · 0.1% SDS at room temperature, then 0.1 × SSC · 0.1% at about 65 ° C. A condition that secondary washing is performed with SDS can be given.

As a result of intensive studies on the base sequence of a LAMP primer that can rapidly amplify a base sequence specific to hepatitis B virus and its combination, the present inventors have found that the base sequence of the S region of hepatitis B virus (SEQ ID NO: 1). The following one set was selected as a primer set.
(Primer set)
FIP: 5'-GATAAAACGCCGCAGACACATCCCCAACCTCTTGTCCTCCAA-3 '(SEQ ID NO: 10)
BIP: 5'-CCTGCTGCTATGCCTCATCTTCTGACAAACGGGCAACATACCTT-3 '(SEQ ID NO: 11)
F3: 5′-CCAAAATTCGCAGTCCCCAA-3 ′ (SEQ ID NO: 4)
B3: 5′-GCAGGTTTTGCATGGTCC-3 ′ (SEQ ID NO: 8)
LF: 5′-CAGCGATAACCAGGACAA-3 ′ (SEQ ID NO: 5)
LB: 5′-TGTTGGTTCTTCTGGA-3 ′ (SEQ ID NO: 9)

  The enzyme used in nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Examples of such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase (large fragment).

  Enzymes and reverse transcriptases used in nucleic acid synthesis may be purified from viruses or bacteria, or may be prepared by genetic recombination techniques. These enzymes may be modified by fragmentation or amino acid substitution.

  A known technique can be applied to the detection of the nucleic acid amplification product after the LAMP reaction. For example, it can be detected using a labeled oligonucleotide that specifically recognizes the amplified base sequence or a fluorescent intercalator method (Japanese Patent Laid-Open No. 2001-242169), and the reaction solution after completion of the reaction is used as it is. It can be easily detected even by agarose gel electrophoresis. In agarose gel electrophoresis, a large number of bands with different base lengths are detected in a ladder form from the LAMP amplification product. Further, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate ions as a by-product react with coexisting magnesium ions to become magnesium pyrophosphate, and the reaction solution becomes cloudy enough to be confirmed with the naked eye. Therefore, the nucleic acid amplification reaction is confirmed by confirming the white turbidity with a measuring instrument capable of optically observing an increase in turbidity over time after the reaction is completed or during the reaction, for example, a change in absorbance at 400 nm using a normal spectrophotometer Can also be detected (WO 01/83817 pamphlet).

  Various reagents necessary for detecting nucleic acid amplification using the primer of the present invention can be packaged in advance to form a kit. Specifically, various oligonucleotides necessary as a primer or loop primer of the present invention, four types of dNTPs serving as a substrate for nucleic acid synthesis, a DNA polymerase that performs nucleic acid synthesis, an enzyme having reverse transcription activity, and suitable for an enzymatic reaction Buffers and salts that give conditions, protective agents that stabilize enzymes and templates, and reagents necessary for detection of reaction products as necessary are provided as kits.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1: Confirmation of primer reactivity In order to detect all HBV genotypes (A to H) with the same sensitivity, Genbank shared S of genotypes based on a total of 29 samples including 8 types of genotypes. Regions were selected and primers were made. The reactivity of the primer was confirmed by the following method. The composition of the reaction solution for performing nucleic acid amplification by the LAMP method is as follows. In addition, the synthesis | combination of the primer used the QIAGEN company and used what was purified by HPLC (high performance liquid chromatography).
20 mM Tris-HCl pH 8.8
10 mM KCl
8 mM MgSO ₄
1.4 mM dNTPs
10 mM (NH ₄ ) ₂ SO ₄
0.8M Betaine (SIGMA)
0.1% Tween20
1.6μM FIP
1.6μM BIP
0.2 μM F3
0.2 μM B3
0.8μM LF
0.8μM LB
Bst DNA polymerase 16U (NEB)

A dilution series of 10 to 10 ⁶ copies of HBV plasmid DNA (produced by Eiken Chemical) was added to the reaction solution, and a LAMP reaction was performed at 60 ° C. for 60 minutes. The reaction was detected in real time using a real-time turbidity measuring apparatus LA-200 (Eiken Chemical). FIG. 1 is a graph showing the results of detection sensitivity when HBV plasmid DNA is used. As shown in FIG. 1, amplification of up to 10 copies of HBV plasmid DNA was confirmed for this primer set.

Example 2: Confirmation of amplification product The LAMP product amplified with the above primer set was confirmed by electrophoresis and restriction enzyme FokI. FIG. 2 is a diagram showing the results of electrophoresis. As is clear from lane 2 in FIG. 2, a ladder pattern peculiar to the LAMP product was confirmed. In addition, digestion was confirmed in the sample treated with FokI (lane 3). From the above results, it was revealed that the target sequence was specifically amplified.

Example 3: Evaluation of primers (specificity test)
A total of 8 types of commercially available positive specimens for each HBV genotype (Genotype A, B, C, D, E, F, G, H) were diluted with normal human serum to prepare samples. As sample concentrations, these specimens were quantified in advance with an Amplicor ™ HBV monitor (Roche) and diluted to 30, 100, and 1000 copies / mL with human normal serum, respectively. Details of each specimen are as follows. Genotype A (Code Number: 536550, ProMedDx), Genotype B (Code Number: 339347, ProMedDx), Genotype C (Code Number: 536863, ProMedDx), Genotype D (Code Number: 536461, ProMedDx), Genotype E (Code Number: 44858, teragenix), Genotype F (Code Number: 27485, teragenix), Genotype G (Code Number: 61827, teragenix), Genotype H (Code Number: 78621, teragenix).

  In order to isolate HBV-DNA in serum, it was extracted by the following method. Add 300 μL of lysis reagent [5.76 M guanidine thiocyanate, 194 mM DTT, 10 mM Tris-HCl (pH 8.8)] to 100 μL of HBV positive specimen, leave it at room temperature for 5 minutes, and then incubate at 95 ° C. for 10 minutes. did. Next, it was rapidly cooled on ice for 3 minutes, 1 μL of Pellet Paint (Novagen) and 400 μL of isopropanol were added and mixed, and centrifuged at 15000 rpm for 15 minutes. After removing the supernatant, an appropriate amount of 70% ethanol was added, mixed, and centrifuged at 15000 rpm for 10 minutes. Finally, the supernatant was removed, left at room temperature for 5 minutes, and dissolved with 5 μL of sterile water. All of this solution was brought into the LAMP reaction.

  From Table 1, it was confirmed that 100% of all genotype specimens could be detected up to 100 copies / mL, and the detection sensitivity was constant between genotypes.

Example 4: Evaluation of primers (sensitivity test)
A sample obtained by diluting WHO standard HBV DNA 97/746 (NIBSC), which is an international standard of WHO, with human normal serum was used. Extraction and LAMP reaction were the same as in Example 3.

  From Table 2, 100% of the virus concentration could be detected up to 15 IU / mL.

Example 5: Sensitivity comparison Three sets of commercially available HBV Sero-conversion panels (Impath-BCP) (Catalog No. 6277, 6290, 6291) were used to detect other detection methods [Amplicor HBV Monitor (Roche), PCR, Sensitivity comparison with PRISM HBsAg test (Abott) was performed. In addition, the Amplicor HBV monitor requested a measurement request from SRL, and PCR and PRISM HBsAg test were attached with the measurement results from Impath-BCP.

  The result by the LAMP method was represented by ○ or ×. As shown in Tables 3 to 5, the LAMP method had the highest detection sensitivity, and the window period was shortened. In summary, the detection sensitivity was higher in the order of LAMP method> Amplicor HBV monitor> PCR> PRISM HBsAg test.

It is a graph showing the result of a sensitivity test. 10 to 10 ⁶ represent the copy number of plasmid DNA per assay. It is a figure showing the result of electrophoresis. Lane 1 is a 100 bp ladder marker, lane 2 is a sample of the LAMP product, and lane 3 is a sample obtained by treating the LAMP product with FokI.

Claims (8)

  An oligonucleotide primer designed from an arbitrary nucleotide sequence selected from nucleotide sequences 304 to 526 of the S region of hepatitis B virus represented by SEQ ID NO: 1, or a complementary nucleotide sequence thereto. The oligonucleotide primer according to claim 1, comprising an oligonucleotide selected from the following (a) to (c).
(A) an oligonucleotide comprising at least 15 consecutive bases selected from the base sequences represented by SEQ ID NOs: 2 to 9 or a base sequence complementary thereto;
(B) An oligonucleotide that can hybridize with the oligonucleotide according to (a) under stringent conditions.
(C) An oligonucleotide having a primer function, comprising a base sequence in which one to several bases are substituted, deleted, inserted or added among the oligonucleotides described in (a) or (b). Select base sequence regions F3c, F2c, and F1c from the 3 ′ end side on the target nucleic acid of the S region of hepatitis B virus, and select base sequence regions B3, B2, and B1 from the 5 ′ end side. 3. The oligo according to claim 1, comprising a base sequence selected from the following (a) to (d) when the base sequence is F3, F2, F1, and B3c, B2c, B1c: Nucleotide primer.
(A) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(B) A base sequence having the F3 region of the target nucleic acid.
(C) A base sequence having the B2 region of the target nucleic acid on the 3 ′ end side and the B1c region of the target nucleic acid on the 5 ′ end side.
(D) A base sequence having a B3 region of the target nucleic acid. A base sequence specific to hepatitis B virus can be amplified, and consists of a base sequence selected from the following (a) to (b) from the 5 'end to the 3' end: The described oligonucleotide primer.
(A) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'.
(B) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3'.   A method for detecting hepatitis B virus, comprising performing an amplification reaction of a target nucleic acid region of hepatitis B virus using the oligonucleotide primer according to claim 1.   6. The method for detecting hepatitis B virus according to claim 5, wherein the amplification reaction of the target nucleic acid region of hepatitis B virus is the LAMP method.   Hepatitis B virus infection characterized by diagnosing the presence or absence of hepatitis B virus infection by detecting the amplification of the target nucleic acid region of hepatitis B virus using the oligonucleotide primer according to claims 1 to 4. Diagnosis method.   A kit comprising the oligonucleotide primer according to claim 1 for diagnosing hepatitis B virus infection.

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