禽流感病毒N1和N2亚型神经氨酸酶RT-PCR鉴别方法的建立

Differentiation of neuraminidase in N1 subtype from that in N2 subtype of avian influenza A virus

针对家禽中流行较为广泛、危害相对大的禽流感病毒(AIV)N1、N2亚型神经氨酸酶基因设计了2对引物,建立了一种RT PCR鉴别方法,其目的片段大小分别为358bp、377bp。经对A/goose/Guangdong/1/96(H5N1)、A/Turkey/England/N28/73(H5N2)、A/Africanstarling/983/79(H7N1)和A/Turkey/Wiscosin/1/66(H9N2)病毒株的鸡胚尿囊液样品进行扩增,扩增片段的大小与预期大小完全相符。经对国内不同地区分离的16株AIVN1亚型和21株AIVN2亚型用RT PCR方法检测,阳性检出率分别为87.5%(14/16)和95.2%(20/21);对50份样品进行盲检,准确率为98%(49/50)。

Avian influenza viruses (AIV) are divided into different subtypes based on antigenicities of the surface protein hemagglutinin (HA) and neuraminidase (NA), and 15 HA subtypes and 9 NA subtypes have been isolated from birds. At present, AIVs with N1 (H5N1) or N2 (H5N2 and H9N2) NA are widely isolated in poultry. In this study, we developed anRT-PCR method to differentiate N1 subtype AIVs from N2 subtype AIVs. The target PCR product was 358 bp and 377 bp respectively. The reference virus strains serve as positive control, which include A/goose/Guangdong/1/96(H5N1), A/Turkey/England/N28/73(H5N2), A/African starling/983/79(H7N1) and A/Turkey/Wiscosin/1/66(H9N2). We use this method inspect 37 influenza virus isolates which include 16 N1 subtype viruses and 21 N2 subtype (viruses). The PCR positive ratios were 87.5%(14/16) and 95.2%(20/21) respectively. Resultsshowed that the PCR primers have much advantages. In addition, we tested 50 positive samples stochastic, the (ratio) of veracities was 98.0%(49/50).