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目的建立多联病毒核酸荧光PCR检测方法,为我国核酸检测技术的应用前景提供技术支持。方法使用逆转录实时荧光PCR定量方法(QRT-PCR)检测HCV、HIV病毒,在成功扩增定量单一病毒的基础上,通过主要调整Mg(2+)离子、dNTPs、Taq的浓度等优化反应体系,建立同时检测两种病毒核酸的逆转录实时荧光PCR定量检测方法(一步法双检),并分析双检体系的扩增灵敏度。结果通过优化筛选反应条件,使用QRT-PCR方法成功扩增双模板HCV/HIV RNA,且检测特异性好,灵敏度较高,能达到HCV RNA 20IU/ml与HIV RNA 80IU/ml的检测灵敏度。结论本试验成功建立能同时检测两种病毒核酸的QRT-PCR方法,为后续研究HBV、HCV、HIV多联荧光病毒核酸检测系统提供了理论依据与技术支持。
Objective To establish a multi-linked virus nucleic acid fluorescence PCR detection method for the application of nucleic acid detection technology in China to provide technical support. Methods The HCV and HIV viruses were detected by reverse transcription real-time PCR (QRT-PCR). Based on the successful amplification of quantitative single virus, the optimal reaction system was optimized by mainly adjusting the concentrations of Mg 2+, dNTPs and Taq. , A reverse transcription-based real-time fluorescent quantitative PCR assay (one-step double-check) was established to detect both virus nucleic acids at the same time, and the amplification sensitivity of the double detection system was analyzed. Results The double-template HCV / HIV RNA was successfully amplified by QRT-PCR method with optimized specificity and sensitivity. The detection sensitivity of 20IU / ml HCV RNA and 80IU / ml HIV RNA was achieved. Conclusion In this study, a QRT-PCR method for simultaneous detection of two viral nucleic acids was successfully established, which provided theoretical basis and technical support for the subsequent study of nucleic acid detection system of HBV, HCV and HIV polyvalent fluorescent virus.