小反刍兽疫病毒可视化RT-LAMP检测方法的建立

Development of visual reverse transcription-loop-mediated isothermal amplification assay for the detection of peste des petits ruminants virus

为建立小反刍兽疫病毒（PPRV）可视化RT-LAMP检测方法,本研究根据PPRV基质蛋白（M）基因保守序列设计2对种特异性引物,通过反应条件优化和特异性、敏感性和临床样品的检测,建立了PPRV可视化RT-LAMP检测方法。结果显示,该方法最佳反应条件为62℃1 h,与口蹄疫病毒、蓝舌病病毒、鹿流行性出血热病毒、阿卡斑病毒、狂犬病病毒和犬瘟热病毒这5种羊常见病毒无交叉反应,最低可检测到2.4×10^（-9）ng/L的RNA,敏感性是普通RT-PCR检测方法的1 000倍。该方法与普通RT-PCR的总符合率为95.23%。结果表明,该方法具有敏感性高、特异性好、操作简便、快速和可视化的优点,可用于羊PPRV感染的现场可视化快速诊断检测。

To establish a rapid visual detection method for peste des petits ruminants virus（PPRV）,two pairs of species-specific primers were designed according to the conserved region of the matrix protein gene of PPRV,and the reaction conditions were optimized in this study.In result,the RT-LAMP reaction was finished at 60 ℃ for 1 h,and this method was specific for detecting PPRV with a detection limit of 2.4×10^（-9） ng/L RNA,which was 1 000-fold more sensitive than the conventional RT-PCR.It had no cross-amplifications with food and mouth disease virus,blue tongue virus,epizootic hemorrhagic disease virus,akabane virus,rabies virus and canine distemper virus,respectively.The total coincidence rate between RT-LAMP and the conventional RT-PCR was 95.23%.The above-mentioned results indicated that the sensitive,specific,simple,rapid and visual RT-LAMP,devolped in this study,could be applied in on-site diagnosis of PPRV infections in sheep and goat.