摘要:
为建立一种能同时快速检测H5、H9亚型禽流感病毒(avian influenza virus,AIV)和鸭坦布苏病毒(duck Tembusu virus,DTMUV)的多重PCR检测方法,根据GenBank发表的H5、H9亚型禽流感病毒(AIV)HA基因和DTMUV的NS5基因序列,分别设计了3对特异性引物,通过优化扩增条件,建立了同时检测H5亚型AIV、H9亚型AIV和DTMUV的多重PCR检测方法,对其特异性及敏感性进行检验,并在临床中进行初步应用。结果表明,该多重PCR方法具有良好的特异性和敏感性,可同时扩增出大小分别为732bp(H9亚型AIV)、380bp(H5亚型AIV)、250bp(DTMUV)的特异片段,利用本试验建立的多重PCR对其他鸭病病原体进行扩增结果均为阴性,利用这3对引物对H5、H9亚型AIV和DTMUV进行敏感性检测,结果显示最低检测极限分别为533pg·μL-1、56pg·μL-1和6.6ng·μL-1。对临床200份样品的检测结果表明该多重PCR检测方法具有快速、敏感、特异性强等优点,适用于临床检测应用。
Abstract:
The aim of this study is to establish a rapid multiplex PCR assay for detection of H5,H9 subtype avian influenza virus(AIV)and duck Tembusu virus(DTMUV).According to the HA gene sequences of H5 and H9AIV and NS5 gene of DTMUV in GenBank,three primer sets specific to these three kinds of viruses were designed,respectively.The multiplex PCR for simultaneous detection of AIV(H5,H9)and DTMUV was established,and was applied to clinical testing for three kinds of viruses.The specificity and sensitivity of the multiplex PCR were tested.The results showed that the assay could specifically amplify the gene fragments of H5AIV(380bp),H9 AIV(732bp),and DTMUV(250bp),and were all negative for detection of other duck viruses.This method showed a sensitive of 533pg·μL-1 for H5 AIV,56pg·μL-1 for H9,and 6.6ng·μL-1 for DTMUV.Additionally,the detection results of 200 clinical samples indicated that this multiplex PCR method proved a a rapid,sensitive and specific tool for clinical samples detection.
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