TaqMan探针荧光RT-PCR检测狂犬病病毒

摘要

根据GenBank已公布的狂犬病病毒(rabies virus,RV)核蛋白(N)基因序列设计并合成一对特异性的引物和探针,建立基于TaqMan探针的荧光RT-PCR检测狂犬病病毒方法.对狂犬病疫苗提取核酸后进行RT-PCR扩增,将目的条带切胶回收,克隆测序,重组质粒作为标准阳性对照.对建立的TaqMan探针荧光RT-PCR方法做灵敏度、特异性、重复性及稳定性试验.结果显示,该方法可以达到10拷贝/μL的灵敏度,可以将狂犬病病毒与犬瘟热病毒、犬细小病毒、犬腺病毒、犬冠状病毒和犬副流感病毒分开,方法重复性好,稳定可靠.%To establish the method of TaqMan probe real-time RT-PCR for detection of rabies virus, a pair of primers and probe were designed based on the nucleoprotein sequences of rabies virus that published in GenBank. RNA was extracted from rabies virus vaccine, and then amplified by RT-PCR, cloned the target fragment and took the recombination plasmid as standard positive control. The results showed that the method can distinguished rabies virus from canine distemper virus, canine parvovirus, canine adenoviruses, canine coronavirus and canine para influenza virus, the sensitivity could attained 10 copies/μL and the stability test was good.