实时荧光聚合酶链反应检测HBV基因型方法的建立和临床研究

目的 建立实时荧光聚合酶链反应（PCR）检测乙型肝炎病毒（HBV）基因型方法并对检测的慢性乙型肝炎（CHB）患者基因型进行临床分析。方法 根据GenBank中已发表的明确分型的143株HBV全序列，设计特异的组合引物探针，建立实时荧光PCR方法，检测128例慢性乙型肝炎患者基因型；同时检测HBVDNA、HBV-M。结果 （1）128例标本中B型检出率为20．3％（26／128），C型占71．9％（92／128），D型占7．8％（10／128）；18株HBV克隆标本S基因测序结果与本分型法完全一致。（2）男性与女性的HBV基因型构成比无明显差异（P=0．561）。B型与C型比较，C基因型HBVDNA含量（P〈0．05）和HBeAg阳性率（P〈0．01）明显高于B基因型。基因型B具有更高的HBeAb水平（P〈0．05）。结论 （1）实时荧光聚合酶链反应HBV基因分型法能够简便、灵敏、快速、准确地鉴定HBV基因型。（2）HBV基因型主要以C型为主，其次为B型、D型。在CHB患者中，性别不是基因型构成差别的因素，C基因型易引起较重的肝脏病变，B型易发生免疫逃逸，致病情较轻。

Distribution of hepatitis B virus genotypes in lanzhou region assayed by the real time fluorimetry polymerase chain reaction and its clinical investigation

Objective To explore a new method to determine genotypes B-D of hepatitis B virus (HBV) by use of real time fluorimetry PCR and study the clinical significance of hepatitis B virus genotype.Methods The PCR primers and probes were designed according to the analysis of 143 complete HBV genomes from GeneBank.128 samples were detected for HBV genotypes by real time fluorimetry PCR.At the same time, their HBV DNA and HBV-M were detected.Results Based on the real-time PCR method, 26 (20.3%) were genotype B, 92 (71.9%) were genotype C, 10 (7.8%) were genotype D in the 128 samples.At one time, Each S gene of 18 PCR-produced clones was sequenced.The results of sequencing were completely in accordance to those of fluorimetry PCR.Genotype distribution was similar between male and female groups (P=0.561).However, levels of HBV DNA and positivity rate for HBeAg were higher significantly in patients infected with genotype C them those with genotype B (P<0.05). Conclusion The PCR method described here was proved to be convenient, sensitive, rapid and accurate.Genotype C was the main genotype, followed by genotype C and D in the research.The difference of HBV genotype may not be caused by sex.In the patient of CHB, genotype C can easily cause serious disease of liver and genotype B can easily occur immune escape which resulted in slight state of an illness.