错配PCR-RFLP检测乙型肝炎病毒X基因/C基因启动子变异

Detection of double point mutations in the core promoter sequence of hepatitis B virus by mismatched PCR in combination with a restriction fragment length polymorphism assay

设计错配PCR-限制性片段长度多态性分析（PCR－RFLP）检测中国HBV每株C基因启动子（BCP）区第2个AT丰富区的一对点突变；核苷酸（nt）1762的碱基由A→T和1764的碱基由G→A变异。结果发现在32例慢性HBV感染者中BCP变异者有10例，总检出率为31．2％．提示在我国HBV毒株BCP区这一联合点突变较常见．PCR－RFLP技术与序列分析结合，对于研究这一新发现的热点变异与临床的关系具有重要的实用价值．

The core promoter of hepatitis B virus (HBV) controls the transcription of both precore RNA and core RNA. The presumed core promoter mutations resulting in decreased HBeAg synthesis might be one of factors leading to persistence of HBV infection. In this study,a new method for detection of double point mutations in the core promoter sequence by mismatched PCR in combination with a restriction fragment length polymorphism assay was developed. This novel method uses mismatched oligonucleotide primers in a PCR to generate an artificial restriction site,thus an artificially created Bcl Ⅰ cleavage site is introduced if the double mutation is present. By using this rapid detection method,the double mutation of nucleotides (nt) 1762 and 1764 in the core promoter from A to T and G to A,respectively,can be frequently observed in HBV sequences isolated from HBeAg negative patients with chronic hepatitis B. This method for detection of the double mutation in core promoter should be useful for the evaluation of the dynamic changes of core promoter variant population during the course of HBV infection.