焦磷酸测序技术检测乙型肝炎病毒多个基因型耐药突变方法的建立及初步评价

The development and evaluation of a pyrosequencing system for the detection of drug resistance mutations of hepatitis B virus

目的建立焦磷酸测序(Pyrosequencing,PyroS)法检测HBV反转录酶基因区(rt区)与核苷(酸)类似物(NAs)耐药相关基因突变的检测方法。方法针对HBVDNArt区10个已知常见耐药突变位点的12种突变形式,分别构建野生型和突变型质粒作为标准品;设计通用PCR扩增引物和针对不同突变位点的焦磷酸测序引物,建立基于PyroS技术的HBV耐药突变检测方法。分别采用传统的双脱氧测序法和我们构建的PyroS法对接受/未接受NAs治疗的慢性乙型肝炎患者95份血清标本进行检测比较,并采用克隆测序法进行结果验证。结果 1.构建了HBVrt区常见耐药突变的野生株和变异株标准质粒,并建立了能对12种常见NAs耐药突变同时进行检测的PyroS方法;证实当标准质粒浓度≥1000拷贝/mL时,PyroS法对耐药突变位点的检出率、特异性和重复率均可达100%;2.对95份临床血清标本共检测了950个耐药相关氨基酸置换位点,PyroS法检测结果与直接测序法相符位点939个(符合率98.84%),PyroS法较直接测序法多检出8份标本中共11个变异位点,克隆测序法证实了焦磷酸测序法结果。结论成功建立了能同时定量检测10个已知NAs相关耐药基因突变的PyroS技术,可用于临床抗病毒疗效的监测、及早发现基因型耐药突变,并指导治疗方案的及时调整。

Objective To develop a reagent for detection of nucleos（t） ide analogues （NAs） resistance-associated mutations within the reverse transcriptase （RT） gene of hepatitis B virus（HBV） with pyrosequencing assay（PyroS）. Methods Four kinds of plasmids, including wild strain, rtA181V mutant, rtM204I mutant and multi-position mutant covering 10 NAs-resistance hotspots within HBV RT-gene, were constructed as plasmid standards. Universal PCR amplifying primers and pyrosequencing primers were designed for the establishment of PyroS. Ninety five serum samples of NAs-treated/non- treated chronic hepatitis B patients were detected and compared with PyroS and dideoxy sequencing method （direct sequencing）, rseparately, and T-A cloning sequencing was employed for further confirmation. Results When the concentration of standard plasmids was ≥1 000 copies/mL, all NAs-resistance mutations could be detected by PyroS we established. Of the overall 950 sites of drug-resistance amino-acid substitutions of the 95 clinical serum samples, 939（98.84%） were detected concordantly between PyroS and direct sequencing, and 11 more sites of substitutions in 8 serum samples were detected by PyroS, which was confirmed by cloning sequencing. Conclusion We established a Pyrosequencing system for quantitative detection of multiple NAs resistance mutations of HBV, which is more sensitive than direct PCR sequencing.