三种方法检测乙型肝炎YMDD变异的比较研究及临床结果分析

Compurative studies on detection of YMDD mutation in hepatitis B virus by three methods and their clinical results

目的对检测乙型肝炎病毒(HBV)YMDD变异的3种方法进行灵敏度和特异性比较,并结合患者治疗前HBV DNA载量、丙氨酸氨基转移酶(ALT)水平免疫标志物和YMDD变异后HBV DNA载量等临床信息进行结果分析。方法分别以荧光定量PCR(FQ-PCR)、通用模板信号扩增PCR(UT-PCR)和聚合酶链反应微板核酸杂交-酶联免疫吸附法(PCRmnh-ELISA),对拉米夫定治疗过程中血清出现HBV DNA由阴转阳的52例慢性乙型肝炎患者,进行HBV YMDD变异检测,比较3种方法的敏感性,并对3种方法检测结果不一致的标本进行测序,比较他们的特异性。结果FQ-PCR、UT-PCR和PCRmnh-ELISA对YMDD变异的检出率分别为:53.85%、48.08%、73.07%,FQ-PCR和UT-PCR均与PCRmnh-ELISA差异有统计学意义(P<0.05),FQ-PCR与UT-PCR差异无统计学意义(P>0.1)。检测结果不一致的17例标本进行了测序,FQ-PCR、UT-PCR、PCRmnh-ELISA与检测结果的符合率分别为94.1%、70.6%、29.4%,3者之间差异有统计学意义(P<0.05)。结论3种方法比较,FQ-PCR检测HBV YMDD变异具有较高的灵敏性和特异性,并能同时检测出野生株和变异的混合感染,与测序结果相符,是诊断HBV YMDD变异较好的方法。

Objective To compare the sensitivity and specificity of three methods used to detect YMDD mutation in hepatitis B virus（HBV） , and to analyse the detection results combined with HBV DNA, alanine aminotransferase （ALT） levels,serum markers vefote and after treatment. Methods The sensitivity and specificity of fluorescence quantitative polymerase chain reaction （ FQ-PCR ） and signal universal template polymerase chain reaction （UT-PCR） were com- pared with those of polymerase chain reaction microplate hybridization enzyme-linked immunosorbent assay （ PCRmnh- ELISA） through detection of HBV YMDD mutation in 52 serums from chronic hepatitis B patients rceiving lamivudine monotherapy at the time of viral breakthrough. The inconsistent samples detected by three methods were sequenced. Resuits The rates of HBV YMDD mutation detected by FQ-PCR,UT-PCR and PCRmnh-ELISA were 53.85% ,48.08% and 73. 07% , respectively. There were significant differences between FQ-PCR and PCRmnh-ELISA, UT-PCR and PCRmnh-ELISA （P 〈0.05 ）. However,there was not significant difference between FQ-PCR and UT-PCR （P 〉 0. 1 ）. The rates of consistency with sequencing in 17 samples were 94.1% by FQ-PCR,70.6% by UT-PCR and 29.4% by PCRmnh-ELISA, respectively, and there were significant differeces among them （ P 〈 0.05 ）. Conclusions FQ-PCR is a very good approach for detect ion of HBV YMDD mutations due to the higher sensitivity and specificity,and for the detection of coinfection of wild type and YMDD mutations.