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目的利用荧光定量RT-PCR技术建立快速检测甲型流感病毒N2亚型的方法。方法根据N2亚型流感病毒HA基因的相对保守序列,分别设计一对引物及其相应的Taqman探针,利用一步法RT-PCR试剂盒建立、优化反应体系后,采用十倍稀释法检验建立体系的灵敏度并建立相对定量标准曲线;采用N1亚型毒株核酸进行方法特异性检验并对临床疑似流感样病例样本进行检测。结果N2亚型流感病毒的检测灵敏度为2.56×10-6,扩增效率为99.9%,标准曲线r=0.997,特异性为100%。临床ILI样本检测结果与HAI鉴定结果相符。结论本研究建立的荧光定量RT-PCR技术可以快速、准确的检测N2亚型流感病毒。
Objective To establish a rapid detection method of influenza virus subtype N2 by real-time fluorescence quantitative RT-PCR. Methods According to the conserved sequence of HA gene of N2 subtype influenza virus, a pair of primers and their corresponding Taqman probes were designed respectively. After one-step RT-PCR kit was established and the reaction system was optimized, the tenfold dilution method was used to establish the system Sensitivity and to establish a relative quantitative standard curve; N1 subtype strain nucleic acid was used for method-specific tests and clinical samples of suspected influenza-like cases were detected. Results The detection sensitivity of N2 subtype influenza virus was 2.56 × 10-6, the amplification efficiency was 99.9%, the standard curve was r = 0.997 and the specificity was 100%. Clinical ILI sample test results and HAI identification results. Conclusion The fluorescence quantitative RT-PCR assay established in this study can detect N2 subtype influenza virus rapidly and accurately.