甲型流感病毒荧光RT-PCR检测方法的设计和检定

Design and rapid evaluation of a TaqMan assay for the detection of influenza A viruses

目的 设计甲型流感病毒TaqMan荧光RT-PCR检测方法并对其进行检定。方法 用DNAStar和PrimerPremier5. 0软件设计甲型流感病毒荧光PCR检测所用引物和探针 ;在GenBank中进行Blast以及电子对比证明其具有高度的特异性和保守性 ;和标准RT-PCR进行比较 ,检测此方法的灵敏度。结果 所设计的引物和探针高度特异和保守 ,灵敏度比标准RT-PCR方法高 3～ 27倍 ,并且反转录和PCR可合并为一步。结论 设计了甲型流感病毒TaqMan检测方法 ;该方法具有特异。

Objective To design and rapidly evaluate a TaqMan assay for detecting influenza A viruses. Methods The probe and the primers of the assay were designed with the software packages of DNA Star and Primer Premier 5.0. Their specificity and conservation were verified through Blast in GenBank and electronic hybridization. The assay′s sensitivity was compared with the standard RT-PCR. Results The designed primers and probe were confirmed to be very specific and conserved. The assay was 3-27 folds more sensitive than the standard RT-PCR. The RT and PCR steps could be simplified into one step. Conclusion The TaqMan Real-time PCR assay is specific, sensitive and easy to perform.