【作者】 刘宝明； 李彤； 董建平； 胥婕； 李晓光； 严平； 王凤水； 闫玲； 李文鹏； 金晖； 杨靖娴； 庄辉；

【Author】 LIU Bao-ming~*,LI Tong,DONG Jian-ping,et al.*Department of Microbiology,Peking University Health Science Center,Beijing 100083,China

【机构】 北京大学医学部病原生物学系； 北京市海淀医院感染疾病科； 北京大学第三医院感染疾病科； 北京市地坛医院；

【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(>5.0×10~2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1.2×10~3、7.0×10~2、6.0×10~2和6.0×10~2拷贝/ml。snPCR检测HBV DNA>5×10~2拷贝/ml的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10~3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HBV A～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 respectively,were used for determining the conditions and the sensitivity of this snPCR.Forty-four sera from chronic hepatitis B patients with positive HBV DNA(>5.0×10~2 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 1.2×10~3,7.0×10~2,6.0×10~2 and 6.0×10~2 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 10 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. 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