山羊痘病毒属病毒荧光LAMP检测方法的建立与应用

Establishment and application of fluorescent LAMP for detection of Capripoxvirus

旨在建立一种荧光LAMP快速检测方法用于检测羊痘病毒属病毒(CaPV)。根据羊痘病毒属病毒ORF068基因高度保守区域,设计特异性引物,优化反应温度、反应时间,进行特异性及灵敏度验证,建立了一种基于SYTO9荧光染料的CaPV荧光LAMP快速检测方法。结果表明,在60℃下扩增40 min为最适反应条件,最低检测限为3 copies/μL,与小反刍兽疫病毒、羊口疮病毒、牛病毒性腹泻病毒、口蹄疫病毒均无交叉反应,变异系数低于5%。选取36份模拟样品,采用该方法和实时荧光PCR方法一起对其进行检测,均检出阳性26份,其结果符合率为100%。本研究所建立的CaPV荧光LAMP检测方法灵敏度高、特异性好、操作简便,40 min内即判定结果,可用于临床CaPV感染的快速诊断,为开展相关疾病流行病学研究提供了技术支持,具有较好的应用前景。

This study was to establish a fluorescent loop-mediated isothermal nucleic acid amplification(LAMP)method for rapid detection of Capripoxvirus(CaPV).According to the highly conserved region of the ORF068 gene of capripoxvirus CaPV,specific primers were designed,reaction temperature and time were optimized,specificity and sensitivity were verified,and a fluorescent lamp rapid method for detection of poxvirus based on SYTO9 fluorescent dye was established.The results showed that the optimum reaction condition of the method was amplification at 60℃for 40 minutes.The method possessed high sensitivity and its minimum detection limit was 3 copies/μL.Its specificity was good,and there was no cross-reaction with PPRV,ORFV,BVDV and FMDV.Its repeatability was good,and the coefficient of variation was less than 5%.36 simulated samples were selected and detected by this method together with real-time fluorescent PCR.26 samples were positive,and the coincidence rate of the results was 100%.The fluorescent lamp method of CaPV detection established in this study had the advantages of high sensitivity,good specificity,simple operation and determination of results within 40 minutes.Therefore,this method was suitable for quick diagnosis of clinical samples,and it provided technical support for the detection,diagnosis and epidemiological investigation of CaPVs prevailing in China.