荧光定量RT-PCR快速检测N_2亚型流感病毒的研究

Rapid detection of influenza virus N_2 subtype by fluorescence quantitative RT-PCR

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 Developing a rapid method to detect influenza virus N2 subtype by fluorescence real-time quantitative RT-PCR. Methods According to conservative sequences of NA gene of influenza virus N2 subtype, a pair primers and Taq-man probe were designed, respectively. Using one step RT-PCR kit to set up real-time RT-PCR system for detection of influenza virus N2 subtype, after the standard quantitative curve of the assay was established using 10-fold serial dilution of TCID50, the sensitivity was determined. The specificity and test for influenza virus illness（ILI） samples were also oetermined using this RT-PCR system. Results The sensitivity of detection influenza virus N2 subtype was 2.56 × 10^-6 and the regression coefficient of the quantitative curve was 0.997. The amplification efficiency and specificity of this assay was 99.9% and 100% , respectively. The real-time RT-PCR for N2 subtype results of ILI samples was ih accordance with HAI method completely. Conclusion The detection system based on real-time RT-PCR could be utilized to rapidly and sensitively detect influenza virus N2 subtype.