焦磷酸测序技术检测HBV基本核心启动子变异的研究

Rapid detection of the basic core promoter mutatios of hepatitis B virus using pyrosequencing technology

目的建立HBV基本核心启动子(BCP)区变异的快速检测方法。方法采用焦磷酸测序(pyrose-quencing)技术,设计一对引物,其中一条经生物素标记,PCR扩增产物含HBVBCP A1762T/G1764A双突变的区域,借用生物素包被葡聚珠纯化单链PCR产物,设计一条测序引物,运用焦磷酸测序技术对A1762T/G1764A两个变异点进行变异分析。通过对已知变异类型的HBV分离株进行测定分析,评价所建立方法的检测敏感性和特异性。结果PCR扩增后,可在2 h内得到A1762T/G1764A两个变异点的突变情况。所建立方法的检测灵敏度达到HBV-DNA血清中的含量为103copies/ml;在所分析的HBV BCP区均可检测出变异株、非变异株及混合株,且所检测的结果与直接测序方法结果符合率为95%。结论Pyrosequencing技术用于检测病毒基因突变有高通量、自动化程度高和结果准确等特点,适用于对HBV BCP区突变进行快速高通量检测。

[Objective] To establish a rapid and simple method to detect the basic core promoter（BCP） mutations of hepatitis B virus（HBV） with pyrosequencing technology. [ Methods] One pyrosequencing sequencing primer and one pair of polymerase chain reaction（PCR） primers were chosen for pyrosequencing analysis. One of the primers was marked with biotin. The area including the BCP of HBV was amplified by PCR. Two points（A1762T/G1764A） mutations were analyzed by pyrosequencing technology. The sensitivity and specificity of pyrosequencing technology were estimated by analyzing the known HBV sequencing. [ Results] Amplified with PCR, the mutations of A1762T/G1764A can be detected in 2 hours. The sensitivity of pyrosequencing technology was determined by assaying PCR products generated from the HBV-DNA （as low as 103copies/ml in blood serum）. The variant strains and non-varlant strains and mixed strains were detected in all the analyzed HBV BCP areas. The consistent ratio was 95% compared pyrosequencing technology with direct sequencing technology. [ Conclusions]The pyrosequencing technology can be used as a highly- automatic, high-through and efficient method for the investigation of virus gene and the BCP mutations of HBV.