应用TaqMan荧光定量PCR检测H3N8亚型马流感病毒

Development of real-time fluorescent quantitative PCR assay for rapid detection of H3N8 subtype Equine influenza virus

为建立马流感病毒(EIV)的检测方法,本试验针对H3N8亚型EIV血凝素(HA)基因高度保守序列设计并合成了2对引物和1条TaqMan荧光探针,建立了TaqMan荧光定量PCR方法。经用TaqMan荧光定量PCR、RT-PCR和病毒分离方法分别检测新疆等省135份疑似EIV马鼻拭子样品,其结果表明:3种方法的马流感检出率分别为54.07%、37.78%、0.89%;TaqMan荧光定量PCR可检出马流感病毒基因组RNA的灵敏度可达10拷贝/反应,而且与其他马呼吸道病毒均无交叉反应,具有良好的特异性、敏感性和重复性。该方法为H3N8亚型马流感的早期诊断及分子流行病学调查等提供了一种新的快速、准确的定量检测技术。

A real-time fluorescent quantitative PCR assay was developed based on primers and TaqMan probes derived from highly conserved regions of the hemagglutinin gene of equine influenza virus subtype （H3N8 EIV）. A total number of 135 nasopharyngeal swabs collected from suspected equine influenza in Xinjiang and other provinces were tested by real-time PCR, RT-PCR and viruses isolation. The results showed that the positive detection rates of these methods were 54.07 %, 37.78 %, 0.89 %, respectively. The detection limit of the real-time PCR assay was 10 copies per reaction. This assay was sensitive, specific and reproducible and had no cross-reaction with other equine respiratory diseases. This real-time PCR assay reported here is suitable for rapid and quantitative detection of H3N8 EIV under clinical conditions and molecular epidemiological investigation.