禽流感病毒 H5、H7和 H9亚型一步法多重RT-PCR检测方法的建立

摘要

To develop a rapid multiplex RT-PCR assay for detection of H5,H7,H9 subtype AIV,specific primers were designed with Primer 5.0 software according to the conserved region of the H5,H7,H9 sub-type AIV hemagglutinin gene.The results of this one step-multiplex RT-PCR demonstrated that the specif-ic bands of 380 bp,501 bp and 732 bp were observed simultaneously for RNA isolated from H5,H7 and H9 subtype AIV.And the reaction conditions were optimized to develop one step-multiplex RT-PCR assay for detection of H5,H7,H9 subtype AIV.The method has high specificity and sensitivity.The minimal amount of detection was 10-4 copies/μL RNA of H5,10-4 copies/μL RNA of H7 and 10-2 copies/μL RNA of H9 subtype AIV,respectively.There was no cross-reaction with other avian pathogens after amplifica-tion.The molecular diagnose method was preliminarily established to detect H5,H7 and H9 subtypes of a-vian influenza viruses rapidly,sensitively and specifically.%为了建立能同时检测 H5、H7和 H9亚型禽流感病毒（AIV）的方法，参考 GenBank中 H5、H7和 H9亚型AIV的 HA基因序列的保守区域，利用Primer 5．0软件设计3对分别针对 AIV H5、H7和 H9亚型的特异性引物，预期特异扩增 H5 AIV片段大小为380 bp、H7 AIV为501 bp、H9 AIV为732 bp。经过反应条件的优化，建立了同时检测 H 5、H 7和 H 9亚型AIV的一步法多重 RT-PCR方法。该方法特异性强，对其他亚型AIV和禽呼吸道病原体检测结果为阴性；敏感性高，H5、H7和 H9亚型 AIV RNA的最低检出量分别为10－4、10－4、10－2拷贝／μL。初步建立了快速、敏感、特异地鉴别检测 H5、H7和 H9亚型禽流感病毒的分子生物学诊断方法。