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目的利用荧光定量RT-PCR技术建立快速检测流感病毒N1、N2亚型的方法。方法根据N1、N2亚型流感病毒NA基因的相对保守序列,设计两对引物及其相应的Taqman探针,利用一步法RT-PCR试剂盒建立、优化反应体系后,采用十倍稀释体外转录RNA检验建立体系的灵敏度和重复性并建立相对定量标准曲线;利用多种流感病毒和具有相似临床症状的呼吸道病毒检验建立体系的特异性。结果 N1、N2亚型流感病毒的检测灵敏度为10拷贝/μl,扩增效率分别为102.21%和101.78%,标准曲线相关系数大于99%,重复性良好,特异度实验未发现有非特异性扩增。结论双重荧光定量RT-PCR技术可以快速、准确的检测N1、N2亚型流感病毒。
Objective To establish a rapid method for the detection of N1 and N2 subtypes of influenza virus by real-time fluorescent quantitative RT-PCR. Methods Two pairs of primers and their corresponding Taqman probes were designed according to the relative conserved sequences of NA genes of N1 and N2 subtypes of influenza viruses. After one-step RT-PCR kit was established and the reaction system was optimized, ten-fold dilution of in vitro transcription RNA The sensitivity and reproducibility of the established system were tested and a relative quantitative standard curve was established; the specificity of the established system was tested using various influenza viruses and respiratory viruses with similar clinical symptoms. Results The detection sensitivity of N1 and N2 subtypes was 10 copies / μl, the amplification efficiencies were 102.21% and 101.78% respectively. The correlation coefficient of the standard curve was more than 99%. The repeatability was good and no specific amplification was found . Conclusion Double fluorescent quantitative RT-PCR technique can detect N1 and N2 subtypes rapidly and accurately.