在DNA芯片平台上探测AIV不同亚型cDNA

Detection of cDNA of Avian Influenza Virus by DNA Microarray

对以基因芯片技术为基础的检测H5、H7、H9亚型禽流感病毒的快速诊断技术进行了研究。试验中所使用的病毒为A/Goose/Guangdong/1/96(H5N1)、A/African starling/983/79(H7N1)和 A/Turkey/Wisconsin/1/66(H9N2)。通过RT-PCR获得大约500 bp的禽流感病毒基因cDNAs片段,克隆,从重组质粒扩增DNA片段,并点到玻璃载体上,制成芯片。在病毒RNA反转录过程中,用Cy5标记样品 cDNAs。样品 cDNAs是一个包括禽流感病毒HA和M基因的混合物。依据M基因鉴别型,依据HA基因鉴别亚型。扫描芯片上探针结合位点,杂交信号与预期设想基本一致。结果显示,DNA芯片技术可以提供一种有效的AIV诊断方法。

A rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus (AIV) was developed. The strains used in the experiment were A/Turkey/England/N28/73(H5N2),A/African starling/983/79(H7N1)and A/Turkey/Wiscosin/1/66(H9N2). The cDNAs clone which encode approximately 500bp avian influenza virus gene fragments were obtained by reverse transcription PCR technique, the DNA fragments, which were reamplified from recombinant plasmids, were spotted on a glass-bound microarray. Cy5-labeled fluorescent probes were hybridized to these samples cDNAs, which generated from vRNA during reverse transcription. These samples cDNAs contained multiple fragments of AIV including the hemagglutinin gene and matrix gene. The type of AIV were detected with the matrix gene,and the subtypes of AIV were differentiated with hemagglutinin gene. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results showed that DNA microarray technology could provide a useful diagnostic method for AIV.