荧光定量RT-PCR快速检测A型流感病毒

目的利用荧光定量RT-PCR技术建立一种快速检测A型流感病毒的方法。方法根据A型流感M2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,建立、优化反应体系后,利用十倍稀释法检验方法的灵敏度并建立相对定量标准曲线并与普通RT-PCR法进行比较;特异性检验后进行临床标本的检验。结果A型流感病毒检测反应的灵敏度为2·56×10-6TCID50,标准曲线相关系数为0·999,扩增效率为108·5%,5种非A型流感病毒病原体检测均为阴性,说明此方法具有很好的稳定性、重现性和特异性。结论本研究建立荧光定量RT-PCR技术可以准确检测A型流感病毒,不仅灵敏度高、稳定性好,而且可以对病毒滴度进行定量检测。

Rapid detection of influenza virus A by fluorescence quantitative RT-PCR

OBJECTIVE: To develop a method to rapidly detect influenza virus type A by fluorescence real-time quantitative RT-PCR. METHODS: According to conservative sequence of influenza virus type A M2 gene, a pair primers and Taqman probe were designed, respectively. After the quantitative curve of the assay were established using tenfold serial dilution of TCID50, the sensitivity and the specificity of clinic samples were determined. RESULTS: The detection sensitivity of the assay was 2.56 x 10(-5) TCID50 and the regression coefficient of the quantitative curve was 0.999. The specificity was determined by testing five other specimens, all of which yielded negative results. CONCLUSION: The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.