锁核酸捕获TaqMan探针实时PCR和PCR-RFLP检测乙型肝炎病毒基因变异的研究  

Gene mutations of hepatitis B virus were detected by locked nucleic acid-mediated TaqMan probes real-time PCR and PCR-RFLP

作　　者：余文辉[1] 周小梅[1] 周大桥 李顺民[1] 贺劲松 李爱娣[1] 

机构地区：[1]深圳市中医院,518033 [2]国家级肝病重点专科

出　　处：《中华微生物学和免疫学杂志》2007年第11期977-982,共6页Chinese Journal of Microbiology and Immunology

基　　金：深圳市科技计划项目（200603200）

摘　　要：目的评价锁核酸捕获TaqMan探针实时聚合酶链反应（PCR）和聚合酶链反应-限制性片段长度多态性（PCR-RFLP）检测乙型肝炎病毒（HBV）基因变异的特点。方法分别用直接测序法、锁核酸（LNA）捕获TaqMan探针实时PCR和PCR-RFLP检测77例接受拉米夫定（LAU）治疗6个月以上的慢性乙型肝炎患者的血清样本。结果锁核酸捕获TaqMan探针实时PCR和PCR-RFLP都能检测HBV野生型YMDD及其变异型YIDD和YVDD。此外，两种方法尚能鉴定HBV野生型和变异型混合种群。更为重要的是，在检测HBV野生型和变异型共生变异方面，锁核酸捕获TaqMan探针实时PCR比PCR-RFLP更敏感、更省时。结论锁核酸捕获TaqMan探针实时PCR是一种检测HBV YMDD变异的灵敏度和特异性都高的快速法，在监测HBV患者LAM抗病毒治疗的疗效及发现新耐药病毒基因型等方面具有广泛的应用前景。Objective To evaluated the role of real-time polymerase chain reaction（PCR） using locked nucleic acid（LNA）-mediated TaqMan probes and polymerase chain reaction-restriction fragment length polymor- phism（PCR-RFLP） in detecting the mutation of hepatitis B virus（HBV） DNA. Methods Seventy-seven serum samples investigated in this study were obtained from 77 patients with chronic hepatitis B virus infection, who had admitted department of liver diseases, Shenzhen Traditional Chinese Medicine Hospital, and had received lamivu-dine（3 TC） therapy for at least 6 months. All specimens were detected by LNA-mediated TaqMan probes real-time PCR, PCR-RFLP and direct sequencing. Results Both PCR-RFLP and LNA-mediated TaqMan probes real-time PCR could detect wild type tyrosine-methionine-aspartate-aspartate（YMDD）, and its mutants, tyrosine-isoleucine-aspartate-aspartate （YIDD） and tyrosine-valine-aspartate-aspartate（YVDD）. In addition, the mixtures of the wild-type virus and its mutants in the serum sample were detected. Real-time PCR is less time-consuming, and more sensitive for the detection of mixed populations than PCR-RFLP. Conclusion The real-time PCR with LNA-mediated TaqMan probes is a more sensitive, more specific and faster detection method for mutations at the YMDD motif, which is essential for monitoring lamivudine effective and a novel viral mutants in patients undergoing lamivudine antiviral therapy.

关 键 词：聚合酶链反应 YMDD基因 乙型肝炎病毒 变异 限制性片段长度多态性 锁核酸 

分 类 号：R686[医药卫生—骨科学]