丙型肝炎病毒环介导恒温扩增可视化快检方法的应用研究

Study on reverse transcription-loop-mediated isothermal amplification assay for rapid and visible detection of hepatitis C virus

目的:建立一种环介导等温扩增测定法,以快速,可视化的检测丙型肝炎病毒(HCV)。方法:通过比对分析,选择了高度保守的HCV 5’UTR序列作为设计逆转录环介导等温扩增法(RT-LAMP)引物的合适靶标。确定样品前处理方法,反应体系的最佳条件,进行方法学评价。HCV RT-LAMP分析产物以实时扩增曲线和显色反应呈现。结果:扩增反应在64℃下进行60 min,加入FD染料可以实现反应产物不开盖,肉眼可见。RT-LAMP的结果表明,检测HCV的灵敏度约为50拷贝(200 ag)~500拷贝(2 fg)。HCV与HIV,HBV之间无交叉反应。结论:RT-LAMP测定法具有快速扩增,简单操作和可见检测而无污染的优势。它为评估HCV RT-LAMP在特殊环境和有限资源(如发展中国家的献血检查)中的可行性奠定了基础。

Objective:To develop a reverse loop-mediated isothermal amplification assay for rapid and visible detection of Hepatitis C virus(HCV). Method:With an alignment analysis,a highly conserved sequence of the HCV 5’UTR was chosen as a suitable target to design RT-LAMP primers.The optimal condition of reaction system,methods of sample pretreatment and methodological evaluation were designed.The HCV RT-LAMP assay products appeared in a real-time amplification curve and a color rendering. Result:The amplification reaction was carried out at 64℃ for 60 min.The products could be visible to the naked-eye by adding FD.The results of RT-LAMP indicated about 50 copies(200 ag)-500 copies(2 fg)sensitivity for detecting HCV.There was no cross-reaction for the RT-LAMP method between HCV and HIV,HBV.Conclusion:The RT-LAMP assay may be superior for rapid amplification,simple operation,and visible detection without contamination.It lays the foundation for evaluating the feasibility of HCV RT-LAMP for special environments and limited resources,such as blood donation screening for the developing country.