副猪嗜血杆菌TaqMan实时荧光定量PCR检测方法的建立及应用  

Development and application of TaqMan real time PCR assay for detection of Haemophilusparasuis

作　　者：李富祥[1] 熊和丽[1] 姚俊[1] 李华春[1] 

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《中国兽医科学》2013年第12期1268-1273,共6页Chinese Veterinary Science

基　　金：云南省重大科技专项(2012ZA017);云南省现代农业生猪产业技术体系建设项目(云财农[2009]171号);云南省农业科技创新工程(2008LA019)

摘　　要：根据副猪嗜血杆菌16SrRNA基N的保守序列设计特异性引物和TaqMan探针，通过反应条件优化和特异性、敏感性、重复性试验以及对临床样品的检测，建立了副猪嗜血杆菌TaqMan实时荧光定量PCR检测方法。结果表明，该方法与猪肺炎支原体、巴氏杆菌、链球菌、葡萄球菌、大肠杆菌、沙门氏菌、肠球菌、乳杆菌、解淀粉芽孢杆菌、奇异变形杆菌以及猪瘟病毒、猪繁殖与呼吸综合征病毒无交叉反应；标准品浓度在6．92×10^8-6．92×10^3 copies／μL范围内具有良好的线性关系，最低可检测到6．92×10^1copies／μL的标准品阳性质粒；批内和批间变异系数均小于3％。临床样品检测结果表明，该方法具有敏感性高、特异性好、稳定性强和快速的优点，可用于副猪嗜血杆菌感染的早期诊断和流行病学调查以及副猪嗜血杆菌的定量分析。A pair of primers and one TaqMan probe were designed according to the conserved region of the 16 S rRNA gene of Haemophilus parasuis （HPS） in this study. A real-time fluorescent quantitative PCR assay was developed by optimization of reaction conditions, and by specificity, sensitivity, reproducibility test and clinical sample tests. Under the optimized reaction conditions, the test results showed that this method was specific to detect HPS with a detection limit of 6.92 × 10^2 copies/μL,and no cross-reactions with Mycoplasma hyopneumoniae, Pasteurella multocida, Streptococcus, Staphylococcus, E. coli, Salmonella, Enterococcus, Lactobacillus, Bacillus amylolique f aciens , Proteus mirabilis , classical swine fever virus and porcine reproductive and respiratory syndrome virus. The repeatability test indicated that the inter-and intra-variations were less than 3%. The clinical application of the TaqMan real-time PCR assay indicated the sensitive, specific, repeatable and rapid detection method could be applied to early diagnosis of HPS infections,epidemiological survey and quantitative analysis of HPS.

关 键 词：副猪嗜血杆菌 TaqMan实时荧光定量PCR 16SRRNA基因 早期诊断 流行病学调查 

分 类 号：S852.612[农业科学—基础兽医学]