荧光定量RT-PCR快速检测B型流感病毒的研究

目的:利用荧光定量RT-PCR技术建立一种快速检测B型流感病毒的方法。方法:根据B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,建立、优化反应体系后,利用10倍稀释法检验方法的灵敏度并建立相对定量标准曲线;特异性检验后利用临床标本与普通RT-PCR法进行比较。结果:B型流感病毒检测反应的灵敏度为5.0×10-6TCID50,标准曲线相关系数为0.998,扩增效率为107.8%,5种非B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B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 B BY REAL-TIME QUANTITATIVE RT-PCR

Objective:To develop a method which can rapid detection of influenza virus B by real-time quantitative RT-PCR.Methods:By the HA gene of influenza virus B, a set of primers and Taqman probe were designed, respectively. After the quantitative curve of the assay were established using tenfold serial dilution of TCID_(50), the sensitivity and the specificity were determined.Result:For the influenza virus B, the sensitivity of the assay was 5.010~(-5) TCID_(50) and the regression coefficient of the quantitative curve was 0.998. 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 also could detect the viral load.