禽流感病毒H1、H3、H5、N2亚型多重RT-PCR检测方法的初步研究

Simultaneous detection of H1,H3,H5 and N2 subtypes of avian influenza virus by multipex RT-PCR

目的根据禽流感病毒H1、H3、H5亚型的HA基因和N2型NA基因的保守序列,设计出四对RT-PCR引物,建立一步法多重RT-PCR对禽流感H1、H3、H5、N2四亚型进行快速检测。方法利用所设计的四对引物,通过对该方法扩增条件的优化,成功建立快速检测禽流感病毒H1、H3、H5、N2四亚型的一步法多重RT-PCR。利用禽流感H1、H3、H5、N2四亚型毒株和其它相关标准毒株进行敏感性和特异性试验。结果与结论所建立的一步法多重RT-PCR具有较高的特异性和敏感性,与禽流感其它亚型和NDV、IBV、ARV?IBDV的核酸均无交叉反应。用该方法检测现场样品395份(4省20多个地区),结果与经典检测方法一致。

Based on the conserved sequences of hemagglutinin（HA） and neuraminidase（NA）genes of H1, H3, H5 and N2 subtype avian influenza virus（AIV） ,4 sets of specific primers were designed and the one step-multiplex RT-PCR assay was established and optimized for rapid and simultaneous detection of these genes. The sensitivity and specificity of this method was evaluated with the H1, H3, H5 and N2 subtypes of AIV and standard strains of other related viruses. It was demonstrated that the sensitivity and good specificity,there was no cross-reaction with other subtypes of AIV as well as NDV, IBV, ARV and IB- DV. The method was used to detect the 395 clinical samples taken from over 20 areas of 4 provinces and the results were completely consistent with those of the conventional virus isolation method.