SYBR Green Ⅰ荧光RT-PCR法检测H5亚型禽流感病毒

Real-time RT-PCR detect H5 subtype Avain influenza virus

用2株不同致病力的H5亚型禽流感病毒(AIV)感染Madin-Darby canine kidney(MDCK)细胞,收集感染后6,12,24,48,72 h的病毒,提取总RNA,利用Rotor Gene RG3 000实时定量PCR仪对H5亚型AIV的HA基因进行检测,试图建立快速检测H5亚型AIV的real-time RT-PCR方法,并用所建立的方法对来自全国各地的疑似H5亚型AIV的病料和咽喉拭子、泄殖腔拭子346份进行检测。结果表明：农业部动物流感重点开放实验室建立的HA基因的teal-time RT-PCR方法可以检测到细胞培养物中和临床样品中是否含有H5亚型AIV,与常规RT-PCR比较,该方法更加特异、准确、快速。

Two strains different pathogenicity H5 subtype AIV infected MDCK cells,total RNA were extracted from fluids after infected 6 b,12 b,24 b,48 b, 72 h and throat, tracheal swabs samples from chicken experimentally infected by H5 subtypes AIV,real -time RT - PCR of HA gene was done with Rotor Gene RG3000 PCR amplifier. We attempted to establish a real - time RT - PCR method to detect H5 subtype AIV. The results showed that RT - PCR method we established could detect H5 subtype AIV of cell culture supematant and swabs samples from chicken experimentally infected . As such, the described method is reliable and provides faster disease diagnostics than the conventional RT - PCR procedure to detect AIV.