中国兽医科学2017，Vol.47Issue(10)：1207-1213,7.DOI:10.16656/j.issn.1673-4696.2017.10.001
犬冠状病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立
Establishment of SYBR Green Ⅰ real-time RT-PCR for the detection of canine coronavirus
摘要
Abstract
According to the conserved sequence of S gene of the canine coronavirus(CCoV)in GenBank,a pair of specific primers were designed for amplifying the specific fragment from CCoV.ASYBR Green Ⅰ real-time reverse transcriptase polymerase chain reaction assay(real-timeRT-PCR)was established for detecting CCoV,which was rapid,sensitive and specific.The results showed that the method was highly sensitive to detect as little as 4×101-5×101 copies/μL,and it was 10 times more sensitive than the conventional RT-PCR.There was no amplification from canine distemper virus,canine adenovirus type 2,canine parainfluenza virus and canine parvovirus.The intra-and inter-coefficients of variation were less than 1.20％.Seventy-six clinical samples were detected by the real-time RT-PCR and the conventional RT-PCR,and the positive rate was 78.9％and 69.7％for CCoV.Two out of 60 positive faeces samples were inoculated into CRFK cells after neutralizing other exogenous viruses with positive serum and caused typical cytopathic effects.The partical M gene of the isolated viruses were cloned and se quenced.The result showed a high similarity with other CCoVs.In conclusion,the method can provide an effective way for the rapid diagnosis and epidemiological investigation of CCoV.关键词
犬冠状病毒/荧光定量RT-PCR/S基因Key words
canine coronavirus/real-time RT-PCR/S gene分类
农业科技引用本文复制引用
王静,梁琳,逄春华,罗亚坤,袁维峰,崔尚金..犬冠状病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立[J].中国兽医科学,2017,47(10):1207-1213,7.基金项目
国家重点研发计划项目(2016YFD0501003 （）
2017YFD0500905) （）
中国农业科学院创新工程项目(ASTIP-IAS-15) （ASTIP-IAS-15）
中国农业科学院基本科研业务费(2016ywf-yb-12) （2016ywf-yb-12）
