【摘要】 目的建立一种快速准确的实时荧光 PCR 方法检测 HBV 感染患者经拉米夫定治疗后 YMDD 变异情况。方法首先用 PCR 法筛选 HBV DNA 阳性血清157例,HBV DNA 含量为2.0×10~3～8.0×10~8拷贝/ml,HBV DNA 阴性血清30例,然后采用 TaqMa n探针技术和选择性引物的实时荧光 PCR 对其进行 YMDD 变异检测。通过变异株和对照管的循环阈值(cycle threshold,Ct 值)差来计算血清中总 HBV 中的 YMDD 变异株含量,并用 PCR 产物直接测序方法随机对69例 HBV DNA 阳性标本 RT 区进行基因序列分析,验证所建立方法的准确性。结果 187例接受拉米夫定治疗患者血清标本中,发生 YMDD 变异88例,其中 YIDD 变异39例,YVDD 变异38例,YIDD 与 YVDD 混合变异11例。突变株的含量为10%～100%。30例 HBV DNA 阴性血清的 C 管循环40次,69例测序结果显示68例两种方法检测结果完全相同,符合率为98.55%。结论采用选择性引物和 TaqMan 探针技术建立实时荧光 PCR 方法能快速准确地检测 YMDD 变异情况,并能检测患者变异株在 HBV 总量中的比例,为临床使用拉米夫定治疗乙型肝炎病毒感染监测耐药性提供一种有效方法。更多还原

【Abstract】 Objective We established a real-time PCR method to detect the lamivudine resistant mutants in serum sample from patients with HBV infection.Methods Using the principle of primer selection,We established a real-time PCR method with TaqMan probe real-time PCR able to detect HBV YMDD mutants in 187 serum sample from chronic HBV patients treated with lamivudine.The YMDD mutant in HBV was calculated by the difference between the cycle threshold(Ct)number of mutant and control reactions.To confirm the accuracy of this method,sequence analysis was conducted in 69 sample among 157 patients,which were DNA positive by PCR.Results 88 of 157 HBV DNA positive patients had YMDD mutants.Among them,39 were YIDD,38 were YVDD and 11 were mixed mutants(YIDD and YVDD). The results of real-time PCR and sequencing were concordant in 68 of 69 samples,while only 1 of 69 samples were discordant.Conclusion The real-time PCR method with TaqMan probe and selective primers is a rapid sensitive method for detection of lamivudine-resistant mutants.更多还原