芯片电泳快速检测乙型肝炎病毒YMDD变异  

Rapid detection of hepatitis B virus YMDD mutation by using microchip electrophoresis

作　　者：王秋平[1] 陈斌[1] 雷秀霞[1] 刘大渔[1] 魏绍静[2] 周小棉[1] 

机构地区：[1]广州医学院附属广州市第一人民医院检验科,广东广州510180 [2]广州市第八人民医院检验科,广东广州510060

出　　处：《中华医院感染学杂志》2011年第4期636-639,共4页Chinese Journal of Nosocomiology

基　　金：国家"十一五"传染病防治重大专项(2008ZX10004-004);国家自然基金(30870753);广东省科技计划项目(73090);广东省自然基金(9151008901000190);广东省医学科研基金(A2007499;A2009518);广州市医药卫生基金(2008-YB-003;重点项目:2009-Zdi-10;2009-Zdi-21);广州市中医药;中西医结合科研资助项目(2009-A-07)

摘　　要：目的利用芯片电泳高分辨力和分析快速的优点,建立一种基于芯片电泳的乙型肝炎病毒YMDD耐药性突变的检测方法。方法利用序列特异性PCR联合芯片电泳检测乙型肝炎病毒YMDD变异;将YVDD、YIDD标准质粒分别按梯度稀释108～100进行检测,将105拷贝/μl YVDD、YMDD质粒及YIDD、YMDD质粒分别按99∶1、50∶50、20∶80、10∶90、5∶95、1∶99混合检测,评价该方法的灵敏度;用芯片电泳法检测86份经拉米夫定治疗慢性乙型肝炎患者血清样品中YMDD突变情况,并将其结果与荧光定量PCR比较;两种方法结果不一致的标本进行DNA测序鉴定。结果芯片电泳法能够在3 min内实现HBV YMDD变异检测,标准YVDD、YIDD质粒的检测限均为101拷贝/μl;不同比例(V∶M或I∶M)的混合质粒,最小比例突变检测率YVDD为5.0%,YIDD为10.0%;在86份标本中,芯片电泳检出总变异65份,突变率为75.6%,其中YVDD变异32份、YIDD变异23份、YVDD+YIDD混合变异为10份,荧光定量PCR检出总变异62份,突变率为72.1%,其中YVDD变异30份、YIDD变异29份、YVDD+YIDD混合变异为3份,两种方法检测结果差异无统计学意义(Kappa=0.609);两种方法检测结果不一致的标本20份,经测序结果显示,YMDD野生株6份、YVDD变异7份、YVDD+YIDD混合变异7份。结论芯片电泳法检测YMDD变异具有简单、快速、灵敏度和准确度高的优点,适合于临床实验室开展乙型肝炎病毒YMDD变异的检测。OBJECTIVE To establish a rapid, high performance method for detecting hepatitis B virus YMDD resistance mutation by high-resolution and fast analysis advantages of microchip electrophoresis. METHODS Combined with sequence-specific PCR and microchip electrophoresis to detect hepatitis B virus YMDD variation, the standard plasmids with YVDD, YIDD were diluted to 108 --10o , with l0s copies/ml YVDD, YMDD plasmids and YIDD. The sensitivity of this method was evaluated with YMDD mixed plasmids of 99 ： 1, 50 ： 50, 20 ： 80, 10 ： 90, 5 ： 95, 1 ： 99. Eighty-six cases from the chronic hepatitis B patients treated with lamivudine in the YMDD mutation were detected and their results were compared with the fluorescent quantitative PCR. The specificity of the two methods was compared. The results inconsistent was performed the direct DNA sequencing. RESULTS Microchip electrophoresis could detect hepatitis B virus YMDD mutations within 3 minutes, and its detection sensitivity was 101 copies/ml standard template of the product. The minimum mutation rate of YVDD

关 键 词：乙型肝炎病毒 拉米夫定耐药变异 芯片电泳 

分 类 号：R512.62[医药卫生—内科学]