JP2005270031A - Method for discriminating subtype of genotype a of hepatitis b virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and accurately discriminating the subtype Aa and Ae of the genotype A of a hepatitis B virus. <P>SOLUTION: The subtype Aa and Ae of the genotype A of a hepatitis B virus can be discriminated by examining whether the nt1812 of the hepatitis B virus gene is c or not, or t or not and/or nt1809 is g or not or t or not. In addition to the procedure, the subtype Aa and Ae of the genotype A of a hepatitis B virus can be discriminated by further examining whether the nt1984 of the hepatitis B virus gene is a or not. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for determining whether the subtype of genotype A of hepatitis B virus is Aa or Ae, and a primer used therefor.

  Hepatitis B virus (HBV) is one of the most important causes of acute and chronic liver diseases such as fulminant hepatitis, cirrhosis and hepatocellular carcinoma (HCC) in the world, especially in Asia and African countries. Eight types of HBV classified as A to H (HBV / A to HBV / H) are identified by differences between genotypes exceeding 8% of the total base sequence of about 3200 nucleotides (nt) (non- Patent Documents 2, 18, 19, and 22). These genotypes have different geographical distributions, virological characteristics and clinical symptoms (Non-patent Documents 15 and 17). In addition, virological characteristics may differ between HBV isolates of the same genotype.

  Recently, two subtypes of genotype B of HBV, namely Ba (a means Asia) and Bj (j means Japan) have been reported in Asian countries (Non-patent Document 24), and HBV / Clinical differences are being recognized between patients infected with Ba and patients infected with HBV / Bj (Non-patent Documents 1 and 25).

  Similarly, a subtype of genotype A has also been reported and is called A 'in sub-Saharan Africa (Non-patent Document 4). By phylogenetic analysis of both the pre-S2 / S region (NPL 4) and the complete genome (NPL 13), numerous HBV isolates from South Africa are clustered with this subtype A '. I understood it. This subgroup was also found in HBV isolates from Malawi (Non-patent Document 26). Genotype A, which is mostly subtype A 'in African countries, has a low serum concentration of HBV DNA and a low frequency of hepatitis Be antigen (HBeAg) in the serum (Non-patent Documents 11 and 12). Africa has a high ability to cause HCC. On the other hand, HBV carriers widely distributed in European countries and the United States infected with isolates belonging to the rest of genotype A sustained remission after seroconversion during long-term follow-up compared to other groups, The rate of death associated with liver disease is low (Non-Patent Documents 6, 21, 29).

  USA, Japan, India, South Africa HBV 40 cases 421 bases (HBV; nt 1631-2051) was determined, existing sequences were added, systematically analyzed (6-parameter, neighbor joining method), bootstrap Strains divided by law were classified as HBV / Aa and HBV / Ae, respectively. The basic sequence of HBV genotype A is described in Non-Patent Document 30 (GenBank Accession No. AB014370) (SEQ ID NO: 1 in the sequence listing), and T1809, T1812, T1862, H1888, A1951, C1978, G1984, A1990. , T1996, A1999, G2017 SNPs are Aa. Therefore, whether the HBV genotype A subtype is Aa or Ae can be reliably determined by determining the entire nucleotide sequence of at least the region of nt 1631-2051. However, determining the entire base sequence of this region is time-consuming and costly, and in clinical examinations that determine subtypes within a predetermined time for a large number of specimens, a method that can more easily determine the subtype is desired. .

Akuta, N., F. Suzuki, M. Kobayashi, A. Tsubota, Y. Suzuki, T. Hosaka, T. Someya, S. Saitoh, Y. Arase, K. Ikeda, and H. Kumada. 2003. The influence of hepatitis B virus genotype on the development of lamivudine resistance during long-term treatment. J Hepatol 38: 315-21. 2.Arauz-Ruiz, P., H. Norder, B. H. Robertson, and L. O. Magnius. 2002. Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol 83: 2059-73. Botha, J. F., M. J. Ritchie, G M. Dusheiko, H. W. Mouton, and M. C. Kew. 1984. Hepatitis B virus carrier state in black children in Ovamboland: role of perinatal and horizontal infection. Lancet 1: 1210-2. Bowyer, SM, L. van Staden, MC Kew, and J. G Sim. 1997.A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa.J Gen Virol 78 (Pt 7): 1719-29 . Dusheiko, G. M., S. M. Bowyer, A. Paterson, E. Song, A. Dibisceglie, and M. C. Kew. 1985. Clinical and serological events accompanying changes in hepatitis B viral replication: case reports.Liver 5: 77-83. Erhardt, A., U. Reineke, D. Blondin, WH Gerlich, O. Adams, T. Heintges, C. Niederau, and D. Haussinger. 2000. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B Hepatology 31: 716-25. Fattovich, G`, G Giustina, SW Schalm, S. Hadziyannis, J. Sanchez-Tapias, P. Almasio, E. Christensen, K. Krogsgaard, F. Degos, M. Carneiro de Moura, and et al. 1995. Occurrence of hepatocellular carcinoma and decompensation in western European patients with cirrhosis type B. The EUROHEP Study Group on Hepatitis B Virus and Cirrhosis. Hepatology 21: 77-82. Felsentein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-91. Gojobori, T., K. Ishii, and M. Nei.1982.Estimation of average number of nucleotide substitutions when the rate of substitution varies with nucleotide.J Mol Evol 18: 414-23. Kew, M. C., and P. Macerollo. 1988. Effect of age on the etiologic role of the hepatitis B virus in hepatocellular carcinoma in blacks.Gastroenterology 94: 439-42. Kramvis, A., S. Bukofzer, M. C. Kew, and E. Song. 1997. Nucleic acid sequence analysis of the precore region of hepatitis B virus from sera of southern African black adult carriers of the virus.Hepatology 25: 235-40. Kramvis, A., M. C. Kew, and S. Bukofzer. 1998. Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma. J Hepatol 28: 132-41. Kramvis, A., L. Weitzmann, W. K. Owiredu, and M. C. Kew. 2002. Analysis of the complete genome of subgroup A 'hepatitis B virus isolates from South Africa.J Gen Virol 83: 835-9. Lee, W. M. 1997. Hepatitis B virus infection. N Engl J Med 337: 1733-45. Lindh, M., A. S. Andersson, and A. Gusdal. 1997. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus--large-scale analysis using a new genotyping method.J Infect Dis 175: 1285-93. Lok, A. S., E. J. Heathcote, and J. H. Hoofnagle. 2001. Management of hepatitis B: 2000--summary of a workshop. Gastroenterology 120: 1828-53. Magnius, L. 0., and H. Norder. 1995.Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 38: 24-34. Norder, H., A. M. Courouce, and L. O. Magnius. 1994. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology 198: 489-503. Okamoto, H., F. Tsuda, H. Sakugawa, RI Sastrosoewignjo, M. Imai, Y. Miyakawa, and M. Mayumi. 1988. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 69 (Pt 10): 2575-83. Saitou, N., and M. Nei. 1987. The neighbor joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-25. Sanchez-Tapias, JM, J. Costa, A. Mas, M. Bruguera, and J. Rodes. 2002. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients.Gastroenterology 123: 1848- 56. Stuyver, L., S. De Gendt, C. Van Geyt, F. Zoulim, M. Fried, RF Schinazi, and R. Rossau. 2000. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness.J Gen Virol 81: 67-74. Sugauchi, F, M. Mizokami, E. Orito, T. Ohno, H. Kato, S. Suzuki, Y. Kimura, R. Ueda, LA Butterworth, and WC Cooksley. 2001. A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness.J Gen Virol 82: 883-92. Sugauchi, F., E. Orito, T. Ichida, H. Kato, H. Sakugawa, S. Kakumu, T. Ishida, A. Chutaputti, CL Lai, R. Ueda, Y. Miyakawa, and M. Mizokami. 2002 Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene.J Virol 76: 5985-92. Sugauchi, F., E. Orito, T. Ichida, H. Kato, H. Sakugawa, S. Kakumu, T. Ishida, A. Chutaputti, CL Lai, R. G Gish, R. Ueda, Y. Miyakawa, and M. Mizokami. 2003. Epidemiologic and virologic characteristics of hepatitis B virus genotype B having the recombination with genotype C. Gastroenterology 124: 925-32. Sugauchi, F., E. Orito, H. Kato, S. Suzuki, S. Kawakita, Y. Sakamoto, K. Fukushima, T. Akiba, N. Yoshihara, R. Ueda, and M. Mizokami. 2003. Genotype, serotype, and phylogenetic characterization of the complete genome sequence of hepatitis B virus isolates from Malawian chronic carriers of the virus.J Med Virol 69: 33-40. Usuda, S., H. Okamoto, H. Iwanari, K. Baba, F. Tsuda, Y. Miyakawa, and M. Mayumi. 1999. Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product. J Virol Methods 80: 97-112. Usuda, S., H. Okamoto, T. Tanaka, K. Kidd-Ljunggren, PV Holland, Y. Miyakawa, and M. Mayumi. 2000. Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product. J Virol Methods 87: 81-9. Zhang, X., F. Zoulim, F. Habersetzer, S. Xiong, and C. Trepo. 1996. Analysis of hepatitis B virus genotypes and pre-core region variability during interferon treatment of HBe antigen negative chronic hepatitis B. J Med Virol 48: 8-16. Takahashi, K., Akahane, Y., Hino, K., Ohta, Y. and Mishiro, S. Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates. Arch. Virol. 143 (12), 2313-2326 (1998)

  An object of the present invention is to provide a method capable of easily and accurately discriminating whether the hepatitis B virus genotype A subtype is Aa or Ae.

  As a result of diligent research, the inventors of the present invention have found that in the subtype Ae of genotype A of hepatitis B virus, in most cases, nt1809 is g and nt1812 is c, whereas in subtype Aa, most cases Nt1809 was found to be t and nt1812 was found to be t, and the present invention was completed by conceiving that it was possible to easily determine whether the subtype was Aa or Ae. Furthermore, in addition to this, it was also found that whether or not the subtype is Aa or Ae can be accurately determined by examining whether nt1984 is a or not.

  That is, the present invention examines whether nt1812 of the hepatitis B virus gene is c or t and / or whether nt1809 is g or t. A method for discriminating whether the hepatitis B virus genotype A subtype is Aa or Ae is provided. In addition to the method of the present invention described above, the present invention further comprises examining whether nt1984 of the hepatitis B virus gene is a or not, wherein the hepatitis B virus genotype A subtype is Aa and Ae A method is provided for determining which one of the two.

  According to the present invention, it is possible to easily and accurately determine whether the hepatitis B virus genotype A subtype is Aa or Ae.

  As specifically described in the Examples below, hepatitis B virus genotype A subtype Ae is almost always nt1809 g and nt1812 c, whereas subtype Aa is mostly In this case, nt1809 is t and nt1812 is t. The present invention is based on this new finding. The identification of the position of the base in the HBV genotype A gene has already been established. The base sequence that serves as a base for specifying the position is described in, for example, Non-Patent Document 30 (GenBank Accession No. AB014370) (SEQ ID NO: 1 in the Sequence Listing). In the present invention, the positions of nt1812 and nt1809 and nt1984 are also in accordance with a standard established in this field, in which the first base of this sequence is nt1.

  Whether nt1812 is c or t can be examined by determining the base sequence of the region containing this site. In addition, it is possible to set a primer so that nt1812 is at the 3 'end, perform PCR using that primer as one primer, and check whether amplification occurs. This is preferable. As is well known, if a primer has a mismatch at the 3 ′ end, no further extension of the strand will proceed. The primer may be a sense primer or an antisense primer, but is preferably a sense primer so that nt1984 in the amplified fragment can be examined as described in detail later. The primer can be set so that amplification occurs in the case of HBV / Ae, or can be set so that amplification occurs in the case of HBV / Aa. However, as shown in Fig. 1-1 (a), in HBV / Aa, there is a SNP between nt1809 and nt1812, so that amplification occurs in HBV / Ae where such SNP does not exist. It is preferable to set it because it can be more accurately determined using one type of sense primer.

  In HBV / Ae, nt1809 is g, whereas in HBV / Aa, when nt1812 is t, nt1809 is also t, so whether nt1809 is g or not It is possible to discriminate between HBV / Aa and HBV / Ae by examining the above. Whether nt1809 is g or t can be examined by determining the base sequence of the region containing this site, as in the case of nt1812. In addition, the primer can be set so that nt1809 is at the 3 'end, PCR can be performed using that primer as one primer, and whether or not amplification occurs is examined. This is preferable. The primer may be a sense primer or an antisense primer, but is preferably a sense primer so that nt1984 in the amplified fragment can be examined as described in detail later. The primer can be set so that amplification occurs in the case of HBV / Ae, or can be set so that amplification occurs in the case of HBV / Aa.

  For type discrimination, either nt1812 or nt1809 SNPs may be used, but since these sites are close to each other, it is preferable to use both sites simultaneously in order to improve accuracy. And, as described above, it is preferable to set the sense primer so that amplification occurs in HBV / Ae.As a result, as specifically described in the examples below, nt1812 is the 3 ′ end, and HBV / Ae Most preferably, PCR is performed using a sense primer having a partial sequence. The partial sequence of this region of HBV / Ae is described in FIGS. 1-1 and 1-2, and is naturally described in SEQ ID NO: 1 in which the entire sequence is described. The size of the primer is not particularly limited, but is usually about 15 to 50 bp, preferably about 20 to 35 bp.

  When type discrimination is performed using a sense primer, the antisense primer is preferably set downstream of nt1984 so that nt1984 in the amplified fragment can be examined as will be described later. In the following examples, the antisense primer is set in the region of nt2072-nt2052, but is not limited to this, as long as amplification occurs in both HBV / Ae and HBV / Aa, other regions are used. It may be set.

  Amplification by PCR may be performed in one step, but in order to further increase the sensitivity, it can be obtained by amplifying a wider region by PCR in the first step as specifically described in the Examples below. Using the amplified product as a template, the region contained therein may be amplified by the second stage PCR (nested PCR). In this case, both primers used for the second stage PCR may be different from the primers used for the first stage PCR, or only one primer was used, and the other was used for the first stage PCR. The same primers may be used (hemi-nested PCR, see examples below). Furthermore, for the sake of accuracy, as specifically described in the examples below, if HBV / A is used, PCR is performed separately using a primer that causes amplification even if the subtype is Aa or Ae, and the specimen is HBV / A. You may confirm that it is A. Examples of such a primer include a method using a sense primer having nt1808 at the 3 ′ end as used in the following examples, but is not limited thereto, and HBV / Aa and As long as the regions of HBV / Ae have substantially the same sequence, other regions may be set. As long as there is no mismatch at the 3 'end of the primer, amplification occurs even if there is a mismatch of about 1 base in the middle, so it is also possible to set the primer in a region where there is a SNP of about 1 base in the middle. is there.

  By the above method, HBV / Aa and HBV / Ae can be distinguished almost accurately, but as shown in FIGS. 1-1 and 1-2, nt1809 and nt1812 are HBV in HBV / Aa. There is a small number (one example in Figs. 1-1 and 1-2) of the same base as / Ae. For this reason, the above method cannot perform type discrimination completely accurately. Therefore, in order to complete the accuracy, it is preferable to further check whether nt1984 is a or not. It is possible to check whether nt1984 is a or not by checking the nucleotide sequence of this part, but in HBV / Ae, nt1984 to nt1989 are agatct, which is the restriction enzyme BglII site. It can also be determined by RFLP using BglII, which is preferred because it is simpler. In this case, it is preferable to perform RFLP on the amplified fragment amplified by PCR as described above, that is, to perform PCR-RFLP because it is simple and highly sensitive.

  PCR-RFLP itself is a well-known technique. That is, the amplification product containing the site to be examined (namely, nt1984) is digested with a predetermined restriction enzyme (namely, BglII), and the size of the digested DNA fragment is examined by electrophoresis or the like. The number and size of the fragments that are generated differ depending on whether or not cleavage by the restriction enzyme has occurred. Based on this, it is possible to know whether or not cleavage has occurred, and thus whether or not the base at the target position is a. it can. That is, to examine whether nt1984 is a or not, the region containing nt1984 is amplified by PCR, the amplified product is digested with BglII, and the digest is subjected to electrophoresis. When cleavage by BglII does not occur, that is, in the case of subtype Aa, the amplified fragment is not cleaved, so that one fragment having the size of the amplified fragment is detected. On the other hand, when cleavage by BglII occurs, that is, in the case of subtype Ae, the amplified fragment is cleaved, so that two fragments of the cleaved size are detected.

  The amplification region is set so that the two fragments produced when cleavage occurs can be clearly detected by electrophoresis, preferably the smaller fragment is 1 bp or more, more preferably 20 bp or more. Further, the size difference between the two generated fragments is preferably set to 1 bp or more, more preferably 20 bp or more so that it can be clearly detected by electrophoresis. There is no particular upper limit on the size of the amplification region, but if it is too large, it takes time and cost for PCR, and there are no advantages, so it is preferably 1000 bp or less.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Materials and methods
(1) Serum samples HBV / A subtype specific combined with RFLP analysis using 40 HBV / A positive serum samples (10 samples each from Japan, USA, India and South Africa) collected from carriers in 4 countries PCR specificity and sensitivity were tested.

  To actually test this method, 333 sera from HBsAg positive paid donors collected by ProMedDx Inc. (California, USA) were used. Of the 333 samples, 139 samples belonged to HBV / A. Excluding HCV- and HIV-infected specimens and specimens of unknown patient ethnicity, a total of 109 people from 38 African-Americans, 16 Asians, 24 Whites and 31 Latinos Samples were obtained and used for testing. For control, 32 HBV / A isolates from black HBV carriers from South Africa were used.

(2) Serological measurement of HBV genotype
A commercially available kit (HBV Genotypes EIA) that uses five types of monoclonal antibodies (mAbs) corresponding to epitopes in the pre-S2 region (Non-patent Documents 27 and 28), designated as b, m, k, s, and u. (Product name), manufactured by Institute of Immunology), and serologically confirmed that the genotype of these specimens was HBV / A.

(3) Detection and cloning of HBV DNA
DNA was extracted from 100 μL of each serum using a QIAamp DNA Blood Mini Kit (trade name, manufactured by Qiagen Inc., Hilden, Germany). X and the precore / core region in the HBV genome sequence were amplified by a known method (Non-patent Document 23). That is, the first stage PCR of hemi-nested PCR is performed using HB7F: 5′-gag acc acc gtg aac gcc ca-3 ′ [nt1611-1630] and HBIR-2: 5′-ata ggg gca ttg gtc as sense primers. Using t-3 '[nt2314-2229], after denaturation at 96 ° C for 5 minutes, 94 ° C, 1 minute denaturation step, 60 ° C, 1 minute annealing step and 72 ° C, 1 minute extension step (final) The cycle consisting of only a further 5 minutes was repeated 40 times in a 96-hole cycler (GeneAmp 9600 (trade name), Perkin-Elmer Cetus, Norwalk, USA). The second stage PCR uses the same sense primer (HB7F) as the first stage, and HB7R-2: 5'-cct gag tgc tgt atg gtg agg-3 '[nt2072-2052] as the antisense primer. 35 cycles were performed under the same conditions as the first stage. Amplified products were analyzed by 3% agarose gel electrophoresis, stained with ethidium bromide and observed under ultraviolet light.

  In order to confirm the specificity of amplification, the base sequence of the PCR product was directly determined using Prism Big Dye (trade name, Applied Biosystems, Foster City, Calif., USA) in an ABI 3100 DNA automatic sequencer. For positive control of HBV / Aa and HBV / Ae isolates, the second stage PCR product was cloned into a TOPO TA cloning vector (trade name, manufactured by InVirogen Corp, Carlsbad, Calif.). The plasmid was amplified in E. coli DH5α cells (TaKaRa Shuzo, Tokyo) and purified by the Qiagen method.

(4) Molecular evolution analysis
A phylogenetic tree was created to discriminate HBV / A subtype. 24 sequences of 10 HBV / Ae and 6 HBV / Aa isolates obtained from DDBJ / EMBL / GenBank DNA database were obtained from 421 bases (nt1631-2051) of 40 isolates amplified in this example. Aligned and analyzed. Using the computer program Hepatitis Virus Database Server (http://hvd.med.nagoya-cu.ac.jp/index.html), the number of base substitutions per site and the genetic distance between isolates were determined using a 6-parameter method ( Based on these values, a neighbor-joining (NJ) tree was created based on these values (Non-patent document 20) Bootstrap resampling test to confirm the reliability of the phylogenetic tree (Non-patent document 8) was performed 1000 times.

(5) HBV / A subtype-specific PCR combined with RFLP analysis
Newly developed HBV / A subtype discrimination analysis based on HBV / A subtype specific PCR combined with RFLP analysis after serological determination of genotype using HBV Genotypes EIA (trade name) Was applied to the HBV / A isolate. The first step of hemi-nested PCR uses sense primer HB7F and antisense primer HB7R-2, denaturation at 96 ° C. for 5 minutes, followed by denaturation step at 94 ° C. for 1 minute, annealing step at 60 ° C. for 1 minute, and 72 A cycle consisting of a 1 minute extension step at 5 ° C. (only 5 minutes for the final cycle) was repeated 40 times in a 96-hole cycler. The second stage PCR uses the primer set of set 1 or set 2 and uses a denaturation step at 94 ° C. for 45 seconds, an annealing step at 62 ° C. for 45 seconds, and an extension step at 72 ° C. for 45 seconds (only 5 cycles for the final cycle). Cycle) was repeated 40 times. Primer set 1 is a sense primer HBxA: 5′-att ggt ctg cgc acc a-3 ′ [nt1793-1803] (nt1793-1803) (FIG. 1) for confirming that it is HBV / A set in the stored X region. -1 (a)) and antisense primer HB7R-2. Primer set 2 is an HBV / Ae-specific sense primer having an HBV / Ae-specific sequence at the 3 ′ end, HBxAe: 5′-att ggt ctg cgc acc agg ac-3 ′ [nt1793-1812] (FIG. 1-1 (a)) and antisense primer HB7R-2. To discriminate exceptional isolates, another subtype-specific restriction site (FIGS. 1-2 (b)) was utilized to combine PCR analysis with RFLP analysis using BglII. For digestion with restriction enzymes, 5 μL of the 282 base pair (bp) amplified fragment amplified in set 2 was used, and this was digested with 10 units of restriction enzyme BglII (New England BioLabs, Massachusetts, USA) for 3 hours. This was done by digestion. The digested PCR product was electrophoresed on a 3% agarose gel and stained with ethidium bromide. The RFLP pattern was examined under ultraviolet light. The amplification product derived from HBV / Ae was cleaved into two fragments (221 bp and 61 bp), and the amplification product derived from HBV / Aa was not cleaved.

(6) Statistical analysis HBV / A subtype differences between ethnic groups were evaluated using chi-square test with Yates correction. When the p value was less than 0.05, it was judged that there was a significant difference.

2. result
(1) Molecular evolution analysis of HBV / A 40 specimens from 4 countries and 24 sequences including HBV / B to HBV / H obtained from DDBJ / EMBL / GenBank DNA database were aligned, and HBV X / A phylogenetic tree in the pre-core / core region was created (Figure 2). All 20 HBV / A isolates from the United States and Japan were confirmed to be HBV / Ae, and all 20 isolates from India and South Africa had a high bootstrap value. Classified as Aa.

(2) Alignment of HBV / A isolates Figure 1-1 and Figure 1-2 show the primer subtype-specific region (Figure 1-1 (a)) and the BglII RFLP site (Figure 1-2 ( In the core region corresponding to b)), 56 sequences of the above-described sequences are aligned with 4 short sequences from Gambia or India. Almost all HBV / Ae isolates had a pattern in which nt1809 was g and nt1812 was c (g1809 / c1812). On the other hand, the HBV / Aa isolate was t1809 / t1812. Using these two types of SNPs, two sets of hemi-nested PCR were developed with the sense primer set to distinguish HBV / Ae and HBV / Aa. While looking up available sequences from the database, I noticed an exception. A few HBV / Aa isolates have g1809 / c1812 and were therefore initially classified as HBV / Ae. To discriminate between these exceptional isolates, we developed an RFLP analysis using BglII in different regions of the HBV genome, including SNPs that can discriminate between HBV / Aa and HBV / Ae. Since the restriction enzyme BglII recognizes the base sequence a / gatct (nt1984-1989) (FIG. 1-2 (b)), the HBV / Ae isolate was cleaved into a 221 bp fragment and a 61 bp fragment. The isolate was not cleaved (282 bp fragment). In addition, atypical HBV / Aa, a1984, was found in the database. However, they are excluded by the previous PCR because of their base sequence pattern (t1809 or a1809 / t1812).

(3) Sensitivity of HBV / A subtype-specific PCR combined with RFLP analysis Sensitivity to distinguish HBV / Aa and HBV / Ae in newly developed PCR analysis, HBV / Ae clone and HBV with known copy number Evaluation was performed using a 10-fold reduced dilution series containing both / Aa clones. Subtype-specific PCR using primers HBxAe is of ¹⁰² copies per 1 ml, can be detected isolates having g1809 / c1812, HBV / Aa isolates having t1809 / t1812 were not amplified. Meanwhile, PCR using primers HBxA can detect both HBV / Ae and HBV / Aa, the detection limit was 10 ² copies per 1 ml.

(4) Strategy for HBV / A subtype discrimination based on HBV / A subtype specific PCR combined with RFLP analysis Figure 3 (a) shows HBV / A based on subtype specific PCR followed by RFLP analysis. The final strategy for subtype discrimination of A is shown. A combination of two sets of second stage PCR using the sense primer HBxAe named “HBV / Ae specific primer” or the sense primer HBxA named “HBV / A confirmation primer” and RFLP analysis using BglII, / Ae and HBV / Aa could be clearly distinguished. That is, when the amplification product is detected using any set of PCR primers, and the amplified fragment is not cleaved in RFLP (FIG. 3 (b)), the isolate becomes HBV / Aa. Classified. On the other hand, when any set of PCR primers was used, the amplification product was detected, and when the amplified fragment was cleaved with BglII in RFLP, the isolate belonged to HBV / Ae. Amplification occurred only when primer HBxA was used, and HBV / Aa was also recognized when amplification did not occur when HBxAe was used (FIG. 3 (b)).

(5) Application of HBV / A subtype discrimination to samples from the United States To confirm the practicality of this analytical method, the serum of 109 paid donors in the United States, known to be HBV / A by ELISA The subtype discrimination analysis was performed on the HBV isolate obtained from the above. Of the 109 samples, 79 samples were classified as HBV / Ae, and 30 samples were classified as HBV / Aa. As shown in FIG. 4 (a), 84% of isolates from African-Americans, 83% of isolates from white races, and 74% of isolates from Latinos are HBV / Ae, while 75% of isolates from Asians were HBV / Aa. This suggests that the frequency of HBV / Aa in Asians is significantly higher than in the other groups. As a control test, subtype Aa is known to be the main type (Non-Patent Documents 4 and 13), and the above subtype discrimination method was applied to 32 HBV / A sera from South Africa. It was confirmed that 94% of the HBV isolates belong to HBV / Aa (FIG. 4 (b)), and findings from the previous experiments (Non-Patent Documents 4 and 13) were confirmed.

  The method of the present invention is a method that can easily and accurately determine whether the subtype of HBV genotype A is Aa or Ae, and is useful for the determination. Since subtypes Aa and Ae have different clinical symptoms as described above, determining whether subtype is Aa or Ae is useful for treating liver disease and preventing progression. Also, since the subtypes Aa and Ae have different regional distributions, it is useful for identifying the infection route to determine whether the subtype is Aa or Ae.

FIG. 3 is a view showing a base sequence (a) in the vicinity of nt1812 and a setting region (a) of a sense primer that are examined in the method of the present invention, divided into HBV / Ae and HBV / Aa. FIG. 3 is a view showing a base sequence (b) and a BglII site (b) in the vicinity of nt1894 examined by the method of the present invention, divided into HBV / Ae and HBV / Aa. It is a figure which shows the phylogenetic tree of HBV genotype A. Figure (a) for explaining a preferred embodiment of the present invention and a type discrimination method therefor, and an electrophoresis photograph showing the electrophoresis pattern of fragments after BglII digestion for each subtype in PCR-RFLP using BglII (b) ). It is a figure which shows the frequency of HBV / Aa and HBV / Ae in each race.

Claims (7)

  Hepatitis B virus comprising examining whether nt1812 of the hepatitis B virus gene is c or t and / or whether nt1809 is g or t To determine whether the subtype of genotype A is Aa or Ae.   The method according to claim 1, wherein the amplification is carried out by PCR using a sense primer set so that the 3 'end is nt1812 or nt1809.   The method according to claim 2, comprising examining whether nt1812 is c by using a primer having a 3 'end of nt1812, and a subtype Ae partial sequence.   The method according to any one of claims 1 to 3, further comprising examining whether nt1984 of the hepatitis B virus gene is a.   The method according to claim 4, wherein whether or not nt1984 is a is examined by RFLP using BglII.   The method according to claim 5, wherein the primer according to claim 2 or 3 is used to amplify a fragment to a site downstream of nt1989 by PCR, and then the obtained amplified fragment is subjected to RFLP using BglII. The primer for subtype discrimination | determination of the genotype A of hepatitis B virus described in Claim 2 or 3.