二重逆转录-聚合酶链反应及微孔板反向杂交法检测丙型与庚型肝炎病毒  

Detection of hepatitis C virus and hepatitis G virus RNA by multiplex reverse transcription-polymerase chain reaction and microtiter plate reverse hybridization

作　　者：王惠民[1] 张冬雷[1] 徐芳芹[1] 汤伟[1] 

机构地区：[1]江苏省南通医学院附属医院

出　　处：《中华实验和临床病毒学杂志》1998年第3期261-264,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的为适应临床上需同时检测庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）感染情况，建立了二重逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）及微孔板反向杂交法。方法根据ＨＣＶ与ＨＧＶ基因高度保守区自行设计引物，建立了用二重ＲＴ－ＰＣＲ同时检测ＨＣＶ与ＨＧＶＲＮＡ方法，并分别用ＨＣＶ与ＨＧＶ特异探针微孔板反向杂交检测ＰＣＲ产物。结果ＰＣＲ产物经测序，ＨＣＶ与Ｔａｋａｍｉｚａｗａ等及Ｃｈｏｏ等报道的核苷酸同源性分别为９３１％～９４１％与９２５％～９３７％，ＨＧＶ与Ｓｉｍｏｎｓ等、Ｌｉｎｎｅｎ等及常锦红等的核苷酸同源性分别为９０７％～９２５％、９２０％～９２１％与９４３％～９４５％。测定敏感度约是单式扩增电泳法的１００倍，２０次重复测定ＨＣＶ与ＨＧＶ的ＣＶ值分别为８９％与９８％。微孔板杂交ＮａＯＨ最佳终浓度为０１～０１５ｍｏｌ／Ｌ，最佳杂交时间为３０～５０分钟。１３７例血清样本检测表明，在肝炎患者与血透患者ＨＣＶ单独阳性为１３９％，ＨＧＶ为５１％，ＨＣＶ与ＨＧＶ同时阳性为５８％。结论本文用微孔板杂交酶呈色技术检测ＨＣＶ与ＨＧＶ二重ＲＴ－ＰＣＲ产物敏感、特异、快速，重复性良好，无溴乙锭污染，经?Multiplex reverse transcription polymerase chain reaction for the simultaneous detection of HCV and HGV RNA has been established,in which primers were deduced from high conservative region of HCV and HGV and PCR amplicons were detected by microtiter plate reverse hybridization with HCV or HGV specific probe.Sequence analyses showed that amplicons of HCV were 93.1%～94.1% and 92.5%～93.7% of nucleotide homology compared with Takamizawa and Choo,and amplicons of HGV were 90.7%～92.5%、92.0%～92.1% and 94.3%～94.5% of nucleotide homology with Simons,Linnen and Chang.The detective sensitivity was 100 times over that of electrophoretic assay with single amplification.CVs of HCV and HGV were 8.9% and 9.8%,respectively,and the optimal concentration of NaOH for hybridization was 0.1～0.15mol/L.The optimal time for hybridization was 30～50minutes.The results of detection with multiplex PCR showed the presence of infection with HCV or HGV alone and coinfection with both % them.

关 键 词：肝炎 病毒学 HGV HCV 聚合酶链反应 

分 类 号：R373.21[医药卫生—病原生物学] R446.5[医药卫生—基础医学]