禽流感病毒4个亚型一步法多重RT-PCR检测方法研究

Multiplex RT-PCR for rapid identification of four subtypes of avian influenza virus

为了建立简便、快速的AIV检测方法,根据禽流感病毒H5、H7、H9亚型HA基因和N1亚型NA基因的保守序列,设计了4对RT-PCR引物,对H5、H7、H9、N1 4个亚型进行了多重RT-PCR扩增,通过多重RT-PCR敏感性和特异性试验,建立了多重RT-PCR检测方法;应用所建立的多重RT-PCR方法和病毒分离的经典方法对395份(4省20多个地区)口岸检样进行了平行检测,并对其检测结果进行了比较。结果表明,H5、H7、H9、N1 4个亚型的扩增产物大小分别为380,641,493和328 bp,对NDVI、BV、ARVI、BDV、MDV、FPV扩增均为阴性,平行检测结果完全吻合,说明所建立的多重RT-PCR检测方法具有较好的敏感性和特异性。

In order to develope a simple, rapide avian influenza virus detection method, four pairs of primers were designed according to the conserved region sequences of the hemagglutinin （HA） and neuraminidase（NA）genes of H5, HT, H9 ,and N1 subtypes of avian influenza virus （AIV）. A multiplex RT-PCR method was developed for rapidly detecting H5, HT, H9, and N1 subtypes of AIV. The sensitivity and specificity of the method were evaluated by using different viral strain of H5, HT, H9, N1 subtypes of AIV and NDV, IBV, ARV, IBDV, MDV and FPV. The results showed that the specific amplicons of H5, HT, H9 and N1 subtypes of AIV were 380,641,493 and 328 bp in size, respectively, the method was also used to detect 395 avian field samples from more than 20 regions of 4 different provinces, and the detection results was consistent with the conventional virus isolation method, which indicated the method was very specific and sensitive.