禽流感病毒H1、H3亚型和M基因三重PCR检测方法的建立

Simultaneous detection of H1, H3 subtypes and M gene of avian influenza virus by multiplex PCR

【目的】建立用于检测禽流感病毒(Avianinfluenzavirus,AIV)且能同时区分H1和H3亚型AIV的方法,为有效防控禽流感(Avianinfluenza,AI)奠定基础。【方法】根据H1亚型、H3亚型AIV-HA基因和M基因的保守核苷酸序列,分别设计3对特异性引物,以含有H1亚型AIV和H3亚型AIV的cDNA为模板,对三重PCR扩增条件进行优化,验证其特异性和敏感性,并对临床样品进行AIV检测。【结果】H1和H3亚型AIV混合模板经三重PCR扩增可获得3条特异性条带,其中302bp为H1亚型HA基因、453bp为M基因、626bp为H3亚型HA基因;含有H1或H3亚型AIV的模板均出现两条特异性条带,大小分别为302和453bp、453和626bp;含有其他亚型AIV的模板仅出现一条特异性条带(453bp);而其他禽呼吸道疾病病毒均未扩增出任何条带。优化后的三重PCR最低能同时检测出7.00pg的H1亚型AIV、3.62pg的M基因和20.60pg的H3亚型AIV;对100个临床样品进行AIV检测,AIV总阳性率为36%,其中H1亚型阳性率4%、H3亚型阳性率9%、其他亚型阳性率23%。【结论】禽流感病毒H1、H3亚型和M基因三重PCR检测方法具有快速、敏感、特异等特点,适用于AIV的检测及H1和H3亚型AIV的分型检测。

[Objective]A method of detecting avian influenza virus（AIV） and separating H1 and H3 subtype was deter- mined to provide references for effectively preventing and controlling AI. [Method]Three sets of specific primers were de- signed according to the conserved sequences of AIV matix （M） gene, and the hemagglutinin （HA） genes of H1 and H3 subtype AIV. A multiplex PCR assay system was developed and optimized for rapid and simultaneous detection of these genes. The specificity and sensitivity of this method was evaluated. This method was used to test the AIV infection from clinical samples. [ Result ] It was shown that all samples containing HI and H3 subtype AIV could be amplified into three specific bands by this multiplex PCR. The lengths of these bands were 302 bp H1 AIV HA,453 bp（M） and 626 bp（H3 AIV）, respectively. Samples containing H1 or H3 subtype AIV could be amplified into two specific bands, which were 302 bp and 453 bp as well as 453 bp and 626 bp, respectively. Samples containing other subtypes AIV could be amplified into a 453 bp specific band. However, no specific band was amplified from other avian pathogenic virus. As little as 7.00 pg of H1 subtype A1V, 3.62 pg of M subtype and 20.60 pg of H3 subtype AIV could be detected by this multiplex PCR. Thirty-six clinical samples （36/100, 36%） were found to be infected with AIV, of which 4 were H1 subtype AIV, 9 were H3 subtype AIV and 23 were other type AIV. [Conclusion]Detecting H1, H3 and M subtypes of avian influenza by multiplex PCR was appropriate, rapid, sensitive and specific.