在DNA芯片平台上探测AIV不同亚型 cDNA.pdfVIP

中国农业科学 2005,38(2):394-398 Scientia Agricultura Sinica 在DNA芯片平台上探测AIV不同亚型 cDNA 王秀荣，邓国华，于康震，石 锐，刘丽玲，乔传玲，陈化兰，孔宪刚 ( 中国农业科学院哈尔滨兽医研究所/农业部动物流感重点开放实验室/兽医生物技术国家重点实验室，哈尔滨 150001) 摘要：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。 关键词：禽流感病毒；基因芯片；亚型鉴定 Detection of cDNA of Avian Influenza Virus by DNA Microarray WANG Xiu-rong, DENG Guo-hua, YU Kang-zhen, SHI Rui, LIU Li-ling, QIAO Chuan-ling, CHEN Hua-lan, KONG Xian-gang (Animal Influenza Laboratory of Ministry of Agriculture / National Key Laboratory of Veterinary Biotechnology / Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001) Abstract: 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