中国兽医科学2017，Vol.47Issue(4)：435-441,7.DOI:10.16656/j.issn.1673-4696.2017.04.005
鉴别犬瘟热病毒野毒株与疫苗株的SYBR Green Ⅰ real-time PCR方法的建立
Establishment of real-time PCR for differentiation of wild-type from vaccine isolates of canine distemper virus
曾杨茹 1王娅 1涂蕊 1巫晓峰 1左之才 1邓俊良 1胡延春 1任志华1
作者信息
- 1. 四川农业大学动物医学院,四川温江611130
- 折叠
摘要
Abstract
To differentiate CDV natural infection from vaccination,a SYBR Green Ⅰ real-time PCR assay was established basing on a pair of specific primers targeting the H gene of CDV.Under the optimized reaction conditions,the assay could efficiently distinguish wild-type CDV isolateing from the vaccine strains,and have no cross-reactions with canine adenovirus,canine parvovirus,caine coronavirus and canine parainfluenza virus.The correlation coefficient of the standard curve was 0.987.The sensitivity of qPCR could reach 6.22×101 plasmid copies/μL of initial templates,which was 100 times more sensitive than the conventional PCR.Assay performance was evaluated on 42 CDV strains suspected of infected dogs,41 of which were wild-type strains identified by the SYBR Green Ⅰ real-time PCR assay.Randomly chosen 15 positive samples were cloned and Hemagglutinin protein gene was sequenced.The 15 field strains were Asia-Ⅰ genotype by phylogenetic analysis.Compared with vaccine strains,the homology of nucleotide sequences was only from 90.3％to 91.3％,and the homology of amino acid sequences was from 96.3％ to 97.2％.The developed SYBR Green Ⅰ real-time PCR assay could be used to detect and differentiate wild-type CDV from vaccine strains.关键词
犬瘟热病毒/鉴别检测方法/荧光定量PCRKey words
canine distemper virus/differential assay/real-time PCR分类
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曾杨茹,王娅,涂蕊,巫晓峰,左之才,邓俊良,胡延春,任志华..鉴别犬瘟热病毒野毒株与疫苗株的SYBR Green Ⅰ real-time PCR方法的建立[J].中国兽医科学,2017,47(4):435-441,7.
