Based on the sequences of HA genes of 4 swine influenza A virus subtypes (H1N1 ,H3N2,HSN1 and H9N2) , 4 special pain of primers were designed. According to the sequence of M gene of influenza A virus, one pair of universal primers was designed. And the cD-NA fragments obtained by using RT-PCR were ligated with pMD 19-T Simple Vector to construct recombinant plasmid for DNA probe preparation. The recovered DNA probes were spotted on located sites on nitrocellulose filter to prepare low density DNA array. During the process of multiplex PCR, samples were labeled by Biotin-11-dUTP, then the labeled PCR products were hybridized with DNA arrays, and the hybridization signals were scanned and analyzed by scanning device and software. The results showed that this DNA array could distinguish the 4 viruse subtypes, and was more sensitive than PCR method. This method could be used for lab diagnosis, large-scale epidemiology investigation and quarantine of swine influenza.%根据猪流感病毒(swine influenza virus,SIV) H1 N1、H3N2、H5N1和H9N2亚型的血凝素(HA)基因序列,分别设计各亚型的特异引物,并根据A型流感病毒的M基因序列设计引物,作为4种病毒亚型的通用引物.应用RT-PCR分别扩增各病毒亚型HA基因的特异片段和M基因片段,并克隆到pMD19-T Simple Vector构建重组质粒,经测序后,用于各病毒亚型探针的制备.将各探针按一定的阵列点加到硝酸纤维素膜上,制备成低密度基因芯片.在多重PCR过程中,用生物素标记的dUTP( Bio-11 -dUTP)对待检样品进行标记,扩增产物与制备的基因芯片进行杂交,最后用扫描仪进行读片和分析.结果表明,该方法可以同时检测4种SIV病毒亚型,与PCR方法相比,显示了较高的灵敏度.该方法的建立,为SIV分型和猪流感疫情的监测提供了一种快速、灵敏和高通量的手段.
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