实时定量PCR检测乙型肝炎病毒拉米夫定耐药突变及其与临床指标的相关性

目的采用实时定量PCR检测接受与未接受拉米夫定治疗的乙肝患者血清中乙肝病毒(HBV)突变情况,并将检测结果与临床指标进行相关性分析。方法采用touch-down PCR反应程序,并结合SYBR Green Ⅰ染料进行实时定量PCR,检测115份乙肝患者血清标本(其中50名患者未服用过拉米夫定,65名患者服用过拉米夫定)。随机选择115份标本中的30份用同样的方法提取血清DNA,进行PCR扩增及测序,并与实时定量PCR检测结果进行比较。将乙肝病毒YMDD突变实时定量PCR检测结果与临床各指标之间进行相关性分析。结果115份血清标本中,75份含有野生型HBV(其中49份来自未接受过拉米夫定治疗的患者),40份含有YMDD突变的HBV(其中1份来自未接受过拉米夫定治疗的患者);180位点的突变检测中,98份标本含有野生型HBV,17份标本含有突变型HBV。随机选取的30份标本测序结果与实时定量PCR检测结果完全一致。乙肝病毒拉米夫定耐药突变与患者的性别、血清DNA水平、病毒基因型之间无相关性,而乙肝病毒YMDD变异与患者服用拉米夫定的时间长短有相关性,用药时间越长,发生突变的几率越大。乙肝患者服用拉米夫定后,180和204位点突变具有相关性。结论实时定量PCR可以高效、准确地检测拉米夫定治疗后乙肝病毒的耐药突变。

Determination of Lamivudine-resistant Mutation of Hepatitis B Virus by Real-time Quantitative PCR and Analysis of Relationship between Mutation and Clinical Indexes

Objective To determine the lamivudine-resistant mutation of hepatitis B virus(HBV) by real-time quantitative PCR and analyze the relationship between the mutation and clinical indexes. Methods The serum samples from 115 patients with HB, of whom 65 were treated and 50 were untreated with lamivudine, were determined by touch-down real-time quantitative PCR with SYBR Green Ⅰ. DNAs were extracted from 30 of the 115 samples for PCR amplification and sequencing, and the results were compared with that of real-time quantitative PCR. Analyze the relationship between determination result of YMDD mutation of HBV by real-time quantitative PCR and various clinical indexes. Results Of the 115 serum samples, 75 were positive for wild HBV, of which 49 were from the patients untreated with lamivudine; and the other 40 were positive for YMDD mutants, of which only one was from a patient untreated with lamivudine. However, 98 of the 115 samples showed no mutation at site 180, while the other 17 showed mutation at this site. The lamivudine-resistant mutation of HBV was unrelated to the sex, serum DNA level and virus genotype of patients, but was related to the time for treatment with lamivudine. The frequency of mutation increased with the increasing time for treatment. In the patients treated with lamivudine, the mutation at site 180 was related to that at site 204. Conclusion Real-time quantitative PCR may be used for effective and accurate determination of lamivudine-resistant mutation of HBV in the patients treated with lamivudine.