环介导逆转录等温扩增技术(RT-LAMP)在丙型肝炎病毒基因检测中的应用

Detection of HCV Gene by Reverse Transcription Loop-mediated Isothermal Amplification Method

该文介绍了一种便捷、灵敏而又特异的环介导逆转录等温扩增基因检测技术，该技术分别使用特异对应于靶序列中8个基因区段的3对特殊引物，并在反转录酶和Bst-DNA聚合酶的作用下对靶序列进行等温核酸扩增反应，整个检测反应只需1～2h。利用这种技术成功检测了丙型肝炎病毒基因，对60份经Real-timePCR或RT—-PCR验证阳性的血清样品检测，阳性符合率为98％。同时，对扩增终产物进一步进行酶切分析，并与HIV、HBV和不同亚型流感病毒RNA进行交叉反应和特异性测试，均与预期结果吻合。将Real-timePCR定量后的RNA系列稀释后对检测方法的灵敏度进行了测试。结果显示，该技术的检测灵敏度在理论上可达到10个拷贝的RNA分子。以上结果证明，RT-LAMP扩增技术是一种检测程序简单、灵敏度和特异性较高的基因检测手段，在丙型肝炎病毒的快速检测方面具有一定的开发潜力。

A simple, sensitive RT-LAMP （Reverse Transcription Loop-mediated Isothermal Amplification） method for detection of hepatitis C virus（HCV）RNA is described. The method employs a set of six specially designed primers that recognize eight distinct sequences of the target for sensitive, specific amplification of nucleic acid under isothermal conditions. The whole reaction can be completed within 1 - 2 h. In this study, sixty specimens from HCV patients that had been tested positive by Real-time PCR or RT-PCR were analyzed by RT- LAMP, the results showed a good correlation among Real-time PCR, RT-PCR and RT-LAMP（98%）.The specificity of the RT-LAMP assay was further validated by cross-reaction with different virus genomes and restriction analysis of the amplified products. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from clinical sample, it was found that the RT-LAMP method was able to achieve theoretically a sensitivity of 10 RNA molecules. Thus, we conclude that this established RT-LAMP assay has potential usefulness for rapid detection of HCV.