副猪嗜血杆菌与猪链球菌2型双重LAMP检测方法的建立  

Development of duplex LAMP assay for the simultaneously detection of Haemophilus parasuis and Streptococcus suis type 2

作　　者：刘博婷[1] 李娜 彭凌[1,2] 郑娅莉 柯璐菲 LIU Bo-ting;LI Na;PENG Ling;ZHENG Ya-li;KE Lu-fei(Yingdong College of Life Science,Shaoguan University,Shaoguan 512005,China;Joint Laboratory ofA nimal Infectious Diseases Diagnostic Center Sponsored by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences and Shaoguan University,Shaoguan 512005,China)

机构地区：[1]韶关学院英东生命科学学院,广东韶关512005 [2]中国农业科学院哈尔滨兽医研究所-韶关学院动物疫病诊断中心联合实验室,广东韶关512005

出　　处：《中国兽医科学》2018年第9期1080-1085,共6页Chinese Veterinary Science

基　　金：广东省公益研究与能力建设专项资金资助项目(2014A020208140);粤北生猪生产及疫病防控协同创新发展中心资助项目[粤教科函(2014)26号-16];2016年度广东省大学生创新创业训练计划资助项目(201610576028)

摘　　要：根据副猪嗜血杆菌的infB基因和猪链球菌2型的Cps2j基因各设计1套LAMP引物，在同一体系中进行双重LAMP扩增，采用琼脂糖凝胶电泳和SYBRGreenⅠ荧光检测，并对双重LAMP扩增产物进行熔解曲线的分析，建立了副猪嗜血杆菌与猪链球菌2型双重LAMP检测方法并进行了特异性和灵敏度试验。结果显示，该方法的灵敏度比常规PCR高两个数量级。通过熔解曲线特征峰的分析可以判断感染的确切目标菌株，且与其他的病原菌无交叉反应。对29份临床样品检测的结果显示，普通PCR和双重LAMP的检出率分别为17．0％和31．0％。结果表明，该方法具有良好的灵敏度和特异性，可实现两种病原的同步快速检测，对于临床疫病的初筛有一定的帮助。To develop a duplex LAMP assay for the simultaneously detection of Haemophilus parasuis and Streptococcus suis 2,two sets of loop-mediated isothermal amplification（LAMP） primers were designed according to the inf B gene of H.parasuis and Cps2j gene of S. suis. The amplification products were then analyzed by electrophoresis,fluorescence observation and dissociation curves. The duplex LAMP assay could specifically detect H. parasuis and S. suis with a sensitivity that was about 100 times higher than the conventional PCR and the LAMP assay had no cross-reactions with the related pathogens. The exact pathogens could be also determined by the dissociation curve. A total of 29 clinical samples with respiratory disease were tested by the conventional PCR and the duplex LAMP and the positive rates were 17.0% and31. 0%,respectively. Theseresultsdemonstratedthat the duplex LAMP assay wasauseful technique for rapid and sensitive etiological diagnosis for H. parasuis and S. suis simultaneously and provided an effective method for the preliminary screening of clinical diseases.

关 键 词：副猪嗜血杆菌 猪链球菌2型 双重LAMP 常规PCR 

分 类 号：S852.612[农业科学—基础兽医学]