乙型肝炎病毒基因的巢式聚合酶链反应-限制性片段长度多态性分型及初步应用

HBV genotyping based on nested-PCR-RFLP and its primary clinical application

目的 用改进的乙型肝炎病毒 (HBV)巢式聚合酶链反应 限制性片段长度多态性 (PCR RFLP)的基因分型方法初探广州地区HBV基因型分布状态。方法 设计巢式PCR扩增前S基因 ,经限制性内切酶AvaⅡ和DpnⅡ酶切鉴别HBVA～G基因型 ;并对 14 0份乙型肝炎患者血清进行基因分型 ,其中随机挑选 3份进行克隆测序 ,并对 2 0份患者血清进行巢式PCR RFLP重复分型试验 ,以验证此分型技术的稳定性和特异性。同时采用PCR和巢式PCR对 92份乙型肝炎病毒e抗原 (HBeAg)阴性血清进行HBVDNA阳性率检测比较。结果 基因分型与测序结果一致 ;基因分型重复试验符合率10 0 % ;92份HBeAg阴性血清经PCR和巢式PCR扩增HBVDNA的阳性率分别为 2 0 7%和 6 8 5 %。14 0份乙型肝炎患者血清中HBV基因型以C型 (94份 )和B型 (4 2份 )为主 ,偶见A型 (4份 )。结论 巢式PCR RFLPHBV基因分型方法具有良好的敏感性、特异性、稳定性 ,且操作简便 ,适用于临床和流行病学调查研究。

Objective A method was improved and established for hepatitis B virus (HBV) genotyping, based on the nested polymerase chain reaction-restriction fragment length polymorphism (nested PCR-RFLP). We use this method to investigate HBV genotype in Guangzhou area. Methods Design nested PCR to amplify the pre-S gene, HBV genotype can be identified from the restriction patterns which produced by the restrictive enzymes, AvaⅡand DpnⅡ. 140 serums were genotyping by this nested-PCR-RFLP method. Verify the results by choosing 3 serums randomly for gene sequencing. And 20 serums were chosen from 140 serums to repeat the nested-PCR-RFLP genotyping test. Serums come form 92 patients with HBeAg negative were used to amplify HBV DNA by PCR and nested-PCR respectively. Results The results of nested-PCR-RFLP genotyping methods were coincide with their gene sequencing. The repeat experiment showed that the genotyping result of the same HBV isolate was identical. 20.7 and 68.5 HBV DNA of 92 HBeAg negative serums were amplified positive by PCR and nested-PCR respectively. Among 140 serums 94 are genotype C and 42 are genotype B, 4 are genotype A.Conclusion The nested-PCR-RFLP method for HBV genotyping was proved to be sensitive, specific and stable. And it is also simple and economic. It can be use well in clinical practice and epidemic study.