特异性引物聚合酶链反应乙型肝炎病毒基因分型法的建立及应用

目的应用型特异性引物聚合酶链反应法（PCR）进行乙型肝炎病毒基因分型并分析该法的可靠性。方法应用型特异性引物PCR和INNO-LiPA分别对深圳、长春、北京152份HBV DNA阳性慢性乙型肝炎患者的血清标本进行了基因型分型，对该两种分型法不一致的血清标本再进行S区基因测序分型，以确定该两法的可靠性。结果型特异性引物PCR和INNO-LiPA的总符合率为86．8％（132／152），不一致率为13．2％（20／152）。型特异性引物PCR检测到81份（53．3％）B型；58份（38．2％）C型；13份（8．5％）B＋C型混合感染，未检出其他基因型或混合感染的基因型。INNO-LiPA检测到74份（48．7％）B型；61份（40．1％）C型；5份（3．3％）B＋C型混合感染；另检出3份（2．0％）A＋B型混合感染；1份（0．7％）B＋E型混合感染；1份（0．7％）C型与D型，1份（0．7％）D型感染，3份（2．0％）B／C／D型及3份未能分型。20份两法分型不一致的标本中，6份无剩余血清，对其余14份进行了S区基因测序分型，结果型特异性引物PCR与S区基因测序分型法的符合率为71．4％（10／14），而INNO-LiPA与S区测序法的符合率仅为7．1％（1／14），前者明显高于后者（P〈0．05）。结论型特异性引物PCR和INNO-LiPA均可鉴定HBV基因型，但前者较为简便和可靠，且费用较低，可用于临床标本的检测和流行病学调查。

Establishment and application of a type-specific polymerase chain reaction(PCR) for HBV genotyping

Objective To determine the accuracy of a type-specific primer polymerase chain reaction (PCR) for identifying HBV genotypes. Methods A type-specific primer PCR and INNO-LiPA assay were used to identify HBV genotype of 152 serum samples of patients with chronic hepatitis B from Shenzhen, Changchun and Beijing. Fourteen of 20 serum samples with discrepant genotype were analyzed by the sequencing of HBV S gene to determine their accuracy. Results The total coincidence rate between the type-specific primer PCR and INNO-LiPA was 86.8%(132/152), with the discrepancy of 13.2%(20/152). Of 152 samples from patients with HBV infection determined by the type-specific PCR, the proportion of genotype B, C, and B+C were 53.3%(81/152), 38.2%(58/152) and 8.5%(13/152), respectively. The figures for INNO-LiPA were 48.7%(74/152), 40.1% (61/152) and 3.3% (5/152), respectively. In addition, 3(2.0%) genotype A + B, 1(0.7%) B + E, 1 (0.7%) C+D, 1(0.7%) D, 3(2.0%) B+C + D, and 3(2.0%) were unidentified by INNO-LiPA. Of 20 sera with discrepant genotype results by the type-specific primer PCR and INNO-LiPA, 6 had no serum left, and the remaining 14 samples were further analyzed by the sequencing of HBV S gene for genotyping. The total coincidence rate between the type-specific primer PCR and the sequencing of HBV S gene was 71.4% (10/14), which was significantly higher than that of INNO-LiPA(7.1 % , 1/14), P < 0.05. Conclusion Type-specific primer PCR and INNO-LiPA can be used for HBV genotyping. However, the type-specific PCR is more simple and accurate, with low cost. It may be used for test of clinical specimens and for epidemiologiesl investigations.