中国兽医科学2017，Vol.38Issue(8)：1027-1031,5.DOI:10.16656/j.issn.1673-4696.2017.08.016
猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR检测方法的建立
Development of multiplex TaqMan real-time PCR for detection of the Erysipelothrix rhusiopathiae and Mannheimia haemolytica
摘要
Abstract
To establish a rapid and accurate method for Erysipelothrix rhusiopathiae and Mannheimia haemolytica,a multiplex TaqMan real-time PCR assay was developed for the bacteria detection with two pairs of species-specific primers and two TaqMan probes designed according to the conserved region of the 16S rRNA gene of the two bacterial species in this study.Under the optimized reaction conditions,the results showed that this method was specific for detecting Erysipelothrix rhusiopathiae and Mannheimia haemolytica and had no cross-amplifications for other pig bacteria.In K rhusiopathiaedetection,the detection limit was 2.07×101 copies/μL pMD-Er-16S recombinant plasmids,and the inter-and intra-variation was less than 3％.In M.haemolytica detection,the detection limit was 1.07×102 copies/μL pMD-MH-16S recombinant plasmids,and the inter-and intra-variation was less than 3％.A total of 47 clinical samples were tested by the multiplex real-time PCR assay comparing with conventional real-time PCR,and the coincidence rate were l00％.The results indicated that the detection method could be applied for rapid and high through-put diagnosis of E.rhusiopathiae and M.haemolytica infections in pig,and fast identification and quantitative analysis of E.rhusiopathiae and M.haemolytica.关键词
猪丹毒杆菌/溶血性曼氏杆菌/TaqMan荧光定量PCR/诊断Key words
Erysipelothrix rhusiopathiae/Mannheimia haemolytica/TaqMan real-time PCR/diagnosis分类
农业科技引用本文复制引用
李富祥,朱凤琼,吴晶,宋建领,杨仕标,赵文华,邵庆勇,李华春..猪丹毒杆菌和溶血性曼氏杆菌二重TaqMan荧光定量PCR检测方法的建立[J].中国兽医科学,2017,38(8):1027-1031,5.基金项目
云南省现代农业生猪产业技术体系建设项目(云财教[2013]160号) （云财教[2013]160号）
云南省现代农业奶牛产业技术体系建设(云财农[2009]171号) （云财农[2009]171号）
