摘要
针对犬瘟热病毒(canine distemper virus,CDV)基因保守序列,设计特异性引物和荧光探针,并进行了敏感性和特异性试验,建立了CDV实时重组聚合酶等温扩增(real-time recombinase polymerase amplification,real-timeRPA)检测方法。结果表明,该实时荧光RPA方法在39℃恒温反应25min能特异性扩增目的基因,与其他犬病毒以及CDV同属的麻疹病毒均未出现交叉反应;该检测方法具有与RT-PCR一致的敏感性;通过对CDV阳性犬的组织样品、体表样品和不同时期分离鉴定的临床样本的检测,证实建立的实时荧光RPA方法具有良好的稳定性和可靠性。与现有的核酸检测方法相比,该实时荧光RPA检测方法在核酸上样25min内即可判读结果,可满足CDV快速检疫的需要。
Using pairs of special primers and probes for conserved sequence of canine distemper virus (CDV),the real-time recombinase polymerase amplification assay (RPA)was developed after tests of sensitivity and specificity.The results showed that the new method could detect target gene specifically at 39℃ within 25 min and there was no cross-reaction with other canine virus and measles virus.The new method had high sensitivity,which was in accordance with RT-PCR.The excellent stability of the new method was confirmed by detection of CDV infected positive and negative samples and clinical samples seperated at different time.Compared with other method,the real-time RPAmethod could finish detection within 25 min after nucleic acid isolated and meet the requirement of rapid detection of CDV.
作者
熊炜
王艳
王楷宬
蒋静
黄保续
田桢干
林颖峥
李健
XIONG Wei;WANG Yan;WANG Kai-cheng;JIANG Jing;HUANG Bao-xu;TIAN Zhen-gan;LIN Ying-zheng;LI Jian(Shanghai Inspection and Quarantine Technology Institute ,Shanghai 200135,China;China Animal Health and Epidemiology Center,Qingdao ,Qingdao 266032,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2019年第1期29-35,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2017YFC1200500)
关键词
犬
犬瘟热病毒
实时荧光RPA方法
canine
canine distemper virus
real-time recombinase polymerase amplification assay