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目的:建立检测耐阿德福韦突变(rtN236T)的HBV感染的聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法。方法:分析阿德福韦抑制HBV复制的作用位点,寻找相应限制性内切酶酶切位点;对野毒株(L49)进行定点突变,构建耐阿德福韦乙肝病毒变异毒株;建立相应的PCR-RFLP检测方法,分别对野毒株和突变毒株进行PCR-RFLP的分析。结果:在阿德福韦作用位点存在限制性内切酶酶切位点,成功对野毒株进行定点诱变,PCR-RFLP方法可以检测HBV野毒株和突变毒株,PCR-RFLP的方法的准确性经测序的方法确认,可以检出5~10%的弱势毒株。结论:用PCR-RFLP的方法可以快速、准确、简便的检测耐药突变毒株。
Objective: To establish a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the detection of HBV-resistant adefovir-resistant mutations (rtN236T). Methods: The site of adefovir inhibition of HBV replication was analyzed to search for the corresponding restriction enzyme sites. Site-directed mutagenesis of wild-type strain L49 was carried out to construct a mutant strain of adefovir-resistant hepatitis B virus. The corresponding PCR-RFLP detection methods were established, and the PCR-RFLP analysis of wild-type and mutant strains respectively. Results: Adefovir sites were identified by restriction enzyme digestion, and site-directed mutagenesis of wild-type strains was successfully performed. PCR-RFLP was used to detect the presence of wild-type and mutant strains of HBV. PCR-RFLP The accuracy of the method was confirmed by sequencing method, we can detect 5 to 10% of the vulnerable strains. Conclusion: The PCR-RFLP method can rapidly, accurately and easily detect drug-resistant mutant strains.