【摘要】 目的建立适用于扩增我国常见的乙型肝炎病毒(HBV)A～D基因型全长逆转录酶(RT)区(包含全长HBsAg编码区)的聚合酶链反应(PCR)法,并确定其在分析临床标本中的应用价值。方法建立我国HBV基因序列库,设计适用于扩增A～D基因型HBV全长RT区引物,以A～D基因型HBV重组质粒为模板建立半巢式PCR(snPCR)法,并确定该法灵敏度。用所建立的snPCR对44份HBV DNA定量阳性(>5.0×102拷贝/ml)慢性乙型肝炎患者血清标本进行扩增及PCR产物直接测序。结果琼脂糖凝胶电泳及测序证实,snPCR可扩增A～D基因型HBV全长RT区,对A、B、C和D基因型HBV质粒扩增的灵敏度分别为1.2×103、7.0×102、6.0×102和6.0×102拷贝/ml。snPCR检测HBV DNA>5×102拷贝/ml血清标本的阳性率为88.64%(39/44),扩增阴性血清标本HBV DNA滴度均处于103拷贝/ml水平。扩增目的产物经直接测序确证无误。生物信息学分析显示,样品中B和C基因型分别占35.90%(14/39)和64.10%(25/39);其中15.38%(6/39)发生RT区变异,包括4例为已知耐药变异;7例(17.95%)存在HBsAg编码区变异,其中2例为已知免疫逃逸变异;6例(15.38%)发生RT区和HBsAg编码区"镜像改变"。结论建立了一种新的扩增全长RT区(涵盖全长HBsAg编码区)的snPCR。该法结合直接测序法,可同时分析我国HBVA～D基因型已知和潜在的耐药变异位点。更多还原

【Abstract】 Objective To establish a polymerase chain reaction (PCR) assay for amplifying the full-length reverse transcriptase (RT) region (full-length HBsAg coding region included) of genotypes A~D of hepatitis B virus (HBV), and to estimate its application for analyzing clinical samples. Methods Three primers for semi-nested PCR (snPCR) were designed for amplifying RT region of HBV genotypes A~D based on HBV sequence database. Four HBV recombinant plasmids containing A~D genotypes full-length HBV genomes were used respectively for determining the conditions and the sensitivity of this snPCR. Forty-four sera from chronic hepatitis B patients with positive HBV DNA (>5.0×102 copies/mL) were analyzed by this snPCR amplification and then by PCR product direct sequencing. Results The successful amplification of the full-length RT regions of HBV genotypes A~D was confirmed by agarose gel electrophoresis and sequencing. The sensitivity of the assay for genotypes A, B, C and D plasmids were 1.2×103, 7.0×102, 6.0×102 and 6.0×102 copies/mL, respectively. The amplification positive rate of the serum samples was 88.64% (39/44). The unsuccessful amplification resulted from the low HBV DNA titer in the sera (at the level of 103 copies/mL). The amplified target fragments were confirmed by direct sequencing. Bioinformatics analyses revealed that the genotypes B and C accounted for 35.90% (14/39) and 64.10% (25/39) respectively in all of the HBV sera samples. Mutations in RT region were detected in 6 samples (6/39, 15.38%), in which 4 samples had known drug resistant mutations. Mutations in HBsAg coding region were detected in 7 samples (7/39, 17.95%), in which 2 had known immune escape mutations. In addition, 6 (6/39, 15.38%) "RT-HBsAg mirror changes" were found. Conclusion A novel snPCR assay for amplifying RT region of HBV genotypes A~D was established. Combined with direct sequencing, the assay may be used for simultaneously analyzing the potential and all known drug resistant mutations of HBV genotypes A~D.更多还原