H9N2亚型禽流感病毒三重RT-PCR诊断技术的建立与应用

Develop a Multiplex RT-PCR Method to Amplify Three Full-length Genes from H9N2 Avian Influenza Virus

为建立H9N2亚型禽流感病毒三重RT-PCR诊断技术,参考Gen Bank登录的H9N2亚型禽流感病毒(AIV)的HA、NA、M基因序列,利用Primer Premier5.0软件设计3对可扩增HA、NA、M基因的特异性引物以及反转录引物。3对引物扩增的c DNA片段大小分别为1 742、1 410、1 027 bp。结果:通过对多重RT-PCR扩增条件的优化,建立了H9N2亚型禽流感病毒三重RT-PCR诊断技术,与目的片段大小相符的片段,经测序均正确,且与其他常见禽病病原不存在交叉反应。该方法对HA基因的最低检出量为10-2μg/μL c DNA,对NA基因的最低检出量为10-2μg/μL c DNA,对M基因的最低检出量为10-3μg/μL c DNA。通过对30份H9N2阳性病毒液进行三重PCR,均可同时扩出HA、NA、M基因。结果表明,本试验建立了H9N2亚型禽流感病毒三重RT-PCR诊断技术,为提高H9N2亚型禽流感病毒HA、NA、M基因序列分析的效率奠定基础。

In order to develop a triplex RT-PCR method for H9N2 avian influenza virus, the sequences of H9N2 subtype avian influenza vinis HA, NA and M were searched in GeneBank.The homology among these genes was analyzed by Gene Star software. Primer Prenier 5.0 software was used to design the specific oligo nucleotide primers for PCR and primers for reverse transcription. Three specific DNA bands, 1 742 bp, 1 410 bp, 1 027 bp were detected simuhaneously by this multiplex RT-PCR method. And there was no eross-reaetion with other avian pathogens.The minimal amount of template was 10-2 μg/μL eDNA for HA, 10-2 l,μg/μL eDNA for NA and 10-3μg/μL cDNA for M gene, respectively.The efficiency of this method was proved by thirty H9N2 avian influenza virus samples.