H5和H7亚型禽流感病毒多重反转录聚合酶链反应快速检测及鉴别方法的建立

Development of multiplex RT-PCR for identification of H5 and H7 subtypes of avian influenza virus

参考禽流感病毒(AIV)M基因和HA基因序列设计了3对引物,其中1对为针对不同HA亚型AIV的通用引物,另外2对为分别针对AIVH5和H7亚型的特异性引物。这些引物所扩增的cDNA片段大小分别为244、860和634bp。利用这3对引物,通过对多重RTPCR扩增条件的优化,成功建立了快速检测鉴别AIVH5、H7亚型的多重RTPCR技术。特异性和敏感性试验结果表明,该技术对AIVH5亚型同时扩增出2条大小分别为244bp和860bp的cDNA片段;对AIVH7亚型同时扩增出2条大小分别为244bp和634bp的cDNA片段;对AIVH5和H7亚型混合样品能同时扩增出3条大小分别为244、860和634bp的cDNA片段;对其他AIVHA亚型只扩增出1条244bp的cDNA片段;对其他常见禽病病原扩增均为阴性;该多重RTPCR对AIVRNA、AIVH5和AIVH7亚型RNA的最低检出量分别为10、100和100pg。

A multiplex RT-PCR was developed to detect H5 subtypes of avian influenza virus (AIV). Three sets of specific oligonucleotide primers were designed for the detection of AIV H5 and H7 subtypes. The primers XZ145-2 and XZ146 based on the highly conversed region of the matrix gene could be used to amplify cDNA bands of 244 bp for all subtypes AIV, the primers XZ H5-1 and XZ H5-5 based on the relatively conserved region of the hemagglutinin gene of H5 subtype could ~only be used to amplify cDNA bands of 860 bp for H5 subtypes AIV, and the primers XZ H7-1 and XZ H7-2 based on the relatively conserved region of the hemagglutinin gene of H7 subtype could only be used to amplify cDNA bands of 634bp for H7 subtypes AIV. Two specific cDNA bands of 244 bp and 860bp were observed simultaneously only for RNA extracted from H5 subtypes AIV, two specific cDNA bands of 244bp and 634bp were observed ~simultaneously only for RNA extracted from H7 subtypes AIV, three specific cDNA bands of 244bp, 860bp and 634bp were observed simultaneously only for the samples containing RNA extracted from both H5 and H7 subtypes AIV, one specific cDNA band of 244 bp was observed for RNA extracted from other subtypes of AIV and no specific cDNA bands were observed for RNA/DNA extracted from other avian pathogens or tissues of SPF embryos after amplification, and as little as 10 pg RNA of AIV, 100pg RNA of H5 and H7 subtypesAIV could be identified by this multiplex RT-PCR.