短片段PCR-ELISA液相杂交法的建立及检测HBV YMDD变异的研究

Establishment of short segment PCR-ELISA liquid phase hybridization and research of detecting variation of HBV YMDD

目的 建立短片段PCR ELISA技术 ,检测HBVP基因C区变异位点 ,以了解拉米呋啶治疗过程中与HBV耐药有关突变株的产生。方法 通过定点诱变获得的重组质粒作为标准对照 ,采用短片段PCR ELISA方法对拉米呋啶治疗的 6 0例乙肝患者血清标本进行HBV聚合酶P基因的变异检测 ,同时观察其HBVDNA定量、肝功能指标的变化。结果 所测 6 0份血样中有 8份检出突变株 ,经测序证实YMDD变异存在 ,其中 4例由ATG变为GTG ,1例变为ATT ,3例变为ATC ,并出现HBVDNA定量反跳及肝功能的变化。结论 短片段PCR ELISA法监测乙肝病毒YMDD变异 ,其方法与一般PCR相比 ,时间仅为其五分之一 。

Objective To establish a technique of short segment PCR ELISA for detecting the variation locus at C section of P gene of HBV to analyze the relationship between lamivudine and HBV resistance to lamivudine in the course of therapy. Methods Variation of P gene of HBV polymerase was determined by short segment PCR ELISA according to the recombined plasmid which was acquired by the site directed mutagensis. Results Sixty blood samples of patients of hepatitis B were determined and their wild type is 'ATG'. Eight of them had mutated, and it is proved by sequencing that there was variation of YMDD. Among the eight mutants, four turned to 'GTG', one turned to 'ATT', the other three turned to 'ATC'. It was also proved that there was bounce of quantity of HBV DNA and variation of liver function. Conclusion The technique of short segment PCR ELISA for detecting the variation of HBV YMDD is more sensitive and spend only one fifth time in comparision with common PCR.