两种实验方法检测乙型肝炎病毒前C区A1896变异的比较研究

目的建立错配PCR-限制性片段长度多态性(mPCR-RFLP)检测乙型肝炎病毒(HBV)前C区A1896变异的方法,与直接测序法比较,评估其应用价值。方法利用错配PCR的原理扩增HBV前C区长194bp的基因片段,扩增产物经限制性内切酶Bsu36I酶切,琼脂糖凝胶电泳,根据酶切图谱多态性,建立检测HBV前CA1896变异的方法,对134份乙肝患者血清进行分析,酶切同时测序。2份标本用克隆测序以验证酶切鉴定变异株、野株混合感染的准确性。结果134份血清中117(87.31%)份能用酶切、109(81.34%)份能用测序成功分析HBV前CA1896状况。酶切与测序均成功的101份血清中,54份为前C区A1896变异株,47份为前C区G1896野株,酶切与测序的结果完全一致。1份酶切鉴定为单纯前C区A1896变异株的5个克隆子均为前C区A1896变异株;1份酶切鉴定为混合感染标本的5个克隆子中,1个为前C区A1896变异株,另外4个为前C区G1896野株。酶切分析结果与克隆测序结果完全相符。结论与测序法相比,本方法简便、特异性强,能鉴定混合感染,适合大样本分析,可用于临床及流行病学调查。

Comparative study on detection of hepatitis B virus mutants in precore region with two methods

Objective To establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detction of hepatitis B virus (HBV) mutation in precore A1896,and compare with direct sequencing for evaluating its applicability. Methods According to the principle of mPCR,194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis.A method for detecting procore A1896 mutation was established by restricted fragment length plymorphism. Tolally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with preocre wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing. Result From 134 sera,117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method,109 could be analyzed by sequencing.In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains.Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains.The results of mPCR-RFLP were verified by cloning.Conclusion Compared with sequencing, the mPCR-RFLP method is simple,accurate and can be used in large-scale surveys and clinical research.