多重PCR检测副猪嗜血杆菌毒力相关三聚自转运蛋白  

Detection of virulence-related trimeric autotransporters from Haemophilus parasuis by using a multiple PCR

作　　者：陈汉锶 李春玲[2,3] 郭海翔[1] 邹创智 宋帅[2,3] 李淼[2,3] 臧莹安[1] 

机构地区：[1]仲恺农业工程学院动物科技学院,广东广州510225 [2]广东省农业科学院动物卫生研究所,广东广州510640 [3]广东省兽医公共卫生公共实验室,广东广州510640

出　　处：《仲恺农业工程学院学报》2015年第3期12-16,共5页Journal of Zhongkai University of Agriculture and Engineering

基　　金：广东省科技计划(2012A020200014);广州市科技计划(201300000066);广东省大学生创新计划(201411347060)的资助项目

摘　　要：为建立快速检测副猪嗜血杆菌(Haemophilus parasuis,HPS)毒力相关三聚自转运蛋白(Trimeric autotransporters,Vta A)的多重PCR方法,根据HPS Vta A Vta A1和Vta A3基因为模板,设计合成了2对特异性引物,进行多重PCR扩增及反应条件的优化,并对多重PCR扩增的敏感性和特异性进行了分析.结果表明:所建立的多重PCR检测方法对HPS Vta A1和Vta A3基因分别能扩增出406和291 bp的目的条带,最小检测限为3.4×107cfu/m L;此多重PCR方法只能检出HPS Vta A1和Vta A3基因400和300 bp大小的目的条带,而不能检出胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)、猪霍乱沙门氏菌(Salmonella suipestifer)、大肠埃希氏杆菌(Escherichia coli)、支气管败血波氏杆菌(Brodetella bronchiseptica)和猪链球菌(Streptococcus suis).由此证明,所建立的多重PCR方法操作简单、快速、特异性强和敏感性高,能够实现对HPS的快速诊断与检测.In order to develop a rapid multiple PCR method to detect virulence-related trimeric autotransporters （VtaA） from Haemophilus parasuis （ HPS）, 2 pairs of specific primers were designed and synthesized using HPS VtaA VtaA1 and VtaA3 genes as templates, the multiple PCR amplification and reaction conditions were optimized, and the sensitivity and specificity of the multiple PCR were analyzed. The resuits showed that the developed multiple PCR method with 3.4 × 10^7cfu/mL of the minimum detection limit to HPS could amplify 406 and 291 bp fragments corresponding to the HPS VtaA1 and VtaA3 loci, respectively, but could only detect 300 bp fragment on HPS VtaA1 and 400 bp on HPS VtaA3, it did not work on detection of Actinobacillus pleuropneumoniae, Salmonella suipestifer, Escheriehia eoli, Brodetella bronchiseptica and Streptococcus suis. It could be concluded that this multiple PCR method had a strong specificity and a high sensitivity and could be used in rapid diagnosis and detection of HPS.

关 键 词：副猪嗜血杆菌(Haemophilus parasuis) 多重PCR VTA A基因 

分 类 号：S852.61[农业科学—基础兽医学]