Wang Rong-Sheng, Zhang Hua, Zhu Yu-Fen, Han Bei, Yang Zhi-Jun
Jiangsu Oil Field Hospital, Shaobo, Jiangdu, China.
World J Gastroenterol. 2006 Feb 28;12(8):1308-11. doi: 10.3748/wjg.v12.i8.1308.
To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection.
We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions (reaction V and reaction I) and total hepatitis B viruses were detected in another reaction for control (reaction C). Results were determined by deltaCt < 3.5 (deltaCt = Ct of reaction V or I - Ct of reaction C). Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing.
As many as 1000 copies per milliliter of wide-type plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples.
This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.
建立一种快速、准确检测乙型肝炎病毒拉米夫定耐药突变的方法，并在慢性乙型肝炎病毒感染患者接受拉米夫定治疗期间监测拉米夫定耐药情况。
我们建立了一种实时聚合酶链反应方法，使用通用模板和TaqMan探针检测YMDD突变体。通过单独反应（反应V和反应I）检测YVDD和YIDD变体，并在另一个对照反应（反应C）中检测总乙型肝炎病毒。通过deltaCt < 3.5（deltaCt = 反应V或I的Ct - 反应C的Ct）确定结果。对含有不同YMDD突变的HBV聚合酶基因克隆进行了检测。使用该方法对163例接受拉米夫定治疗的慢性乙型肝炎病毒感染患者的血清样本进行检测，并通过DNA测序确认结果。
每毫升检测到多达1000拷贝的野生型质粒，排除了非特异性引物。在163例接受拉米夫定治疗患者的样本中，51例检测到拉米夫定耐药突变。
这种通用的实时聚合酶链反应是一种快速、准确的方法，可用于定量拉米夫定治疗患者中HBV病毒的YMDD突变体，并可用于在拉米夫定治疗前和治疗期间监测拉米夫定耐药突变。
