实时荧光PCR监测HBV感染患者拉米夫定治疗病毒YMDD变异  

A real-time PCR method for monitoring YMDD motif mutations associated with lamivudine resistance in patients with HBV infection

作　　者：孙余婕[1] 沈佐君[1] 吴赟[1] 高人焘[2] 李宜[2] 杨志军 

机构地区：[1]安徽医科大学附属省立医院 安徽省临床检验中心,合肥230001 [2]安徽省立医院感染科 [3]上海复星医学科技发展有限公司

出　　处：《中华检验医学杂志》2007年第5期533-537,共5页Chinese Journal of Laboratory Medicine

基　　金：安徽省自然科学基金(050430902);安徽省人才开发资金(20052040)

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HBVDNA阳性血清157例，HBVDNA含量为2．0×10^3-8．0×10^8拷贝／ml，HBVDNA阴性血清30例，然后采用TaqMan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（cyclethreshold，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HBVDNA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HBVDNA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。Objective We established a real-time PCB 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 ll were mixed mutants （YIDD and YVDD）. The results of real-time PCB and sequencing were concordant in 68 of 69 samples, while only 1 of 69 samples were discordant. Conclusion The real-time PCB method with TaqMan probe and selective primers is a rapid sensitive method for detection of lamivudine-resistant mutants.

关 键 词：肝炎病毒 乙型 聚合酶链反应 突变 拉米夫定 

分 类 号：R512.6[医药卫生—内科学]