猪流感病毒分型基因芯片的建立和初步应用

根据流感病毒血凝素（HA）和神经氨酸酶（NA）亚型基因序列间的差异性,分别设计H1、H3、H9、N1和N2的特异分型引物,并根据M基因设计A型流感病毒的通用引物。RT-PCR扩增猪流感病毒各亚型HA、NA和M基因的特异片段,克隆至pMD18-T构建重组质粒,制备探针。将各探针按一定的阵列点加到硝酸纤维素膜上,制成基因芯片。并对217份不同地区的样品进行检测。结果表明：该芯片可以同时检测待检样品中5种亚型A型流感病毒,并显示了较高的灵敏度和特异性,灵敏度比PCR高1个稀释度,比病毒分离高2个稀释度。该方法的建立为猪流感的检测及猪流感病毒各亚型在猪群中的流行病学调查提供了一种快速、灵敏和高通量的手段。

Establishment and Initial Application of Low Density DNAarray for Diagnosing the Subtypes of SIV

The hemagglutinin(HA) and neuraminidase(NA) gene of five subtypes influenza A virus were amplified by RT-PCR with primers based on the diversity of HA and NA gene.Meanwhile conserved cDNA fragments of M gene was amplified by RT-PCR with universal primer of influenza A virus.Each fragments were cloned and inserted into pMD18-T to construct recombinant plasmids for DNA probe preparation.Recovered DNA probes were spotted on located sites on nitrocellulose filter to prepare DNAarrays.217 different samples of area was detected by this DNAarray.The results showed that this DNAarray could identify five subtypes of Influenza A virus in samples and show high sensitivity and specificity.This DNAarray provids a quick,sensitive and high-flux method for large-scale epidemiology investigation and quarantine of swine influenza virus.