H1N1亚型猪流感病毒HA和NA基因双重RT-PCR定型检测方法的建立与应用

摘要

To develop a reliable diagnostic method for H1N1 subtype Swine influenza virus, double RT-PCR methods were developed using primers designed according to the sequences of HA and NA genes of H1N1 Subtype swine influenza virus in GenBank. The amplified gene fragments were 428 bp for HA and 678 bp for NA. The reaction conditions were optimized and minimal detectable RNA concentration was 2.9×10-5μg/μL. Meanwhile, the method had no reaction with H3N2 subtype Swine influenza virus, classical Swine fever virus and Porcine reproductive and respiratory syndrome virus. This method developed here is suitable for double gene typing detection and also for early diagnosis and epidemiological investigation of H1N1 subtype Swine influenza virus.%为了建立适用于临床诊断的H1N1亚型猪流感病毒快速检测方法，本研究根据GenBank已登录的H1N1亚型猪流感病毒HA和NA基因序列设计RT-PCR扩增引物，以H1N1亚型猪流感病毒、H3N2亚型猪流感病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒为试验对照，通过优化RT-PCR反应条件和反应体系，建立了H1N1亚型猪流感病毒HA和NA基因双重RT-PCR定型检测方法。同时，运用H1N1亚型猪流感病毒血凝和血凝抑制试验方法和本研究建立的方法对165份猪病料样品进行了对比验证。结果表明，本研究建立的H1N1亚型猪流感病毒双重RT-PCR具有良好的特异性、敏感性、重复性，所扩增的目的基因片段大小分别为428 bp和678 bp左右，可检出最小基因组RNA浓度为2.9×10-5μg/μL。本研究建立的方法和H1N1亚型猪流感病毒血凝和血凝抑制试验方法均从同一份猪肺脏样品中检测出H1N1亚型猪流感病毒，其余样品中均未检出H1N1亚型猪流感病毒，两种方法符合率为100%。本研究建立的方法适用于H1N1亚型猪流感病毒双基因定型检测，可在H1N1亚型猪流感病毒流行病学调查和临床诊断中应用。