猪胸膜肺炎放线杆菌环介导等温扩增检测方法的建立与应用  

Detection of Actinobacillus pleuropneumoniae by Newly Developed Loop-mediated Isothermal Amplification Method

作　　者：李佳禾[1] 李一婧[2] 付萍[1] 张跃伟[1] 黄书林[1] 郭盼盼[1] 蒋菲[1] 吴文学[1] 

机构地区：[1]中国农业大学动物医学院,北京100193 [2]沈阳药科大学,沈阳110016

出　　处：《农业生物技术学报》2009年第6期948-953,共6页Journal of Agricultural Biotechnology

基　　金：农业部重点项目(No.2008-G57);国家大学生创新性实验计划;中国农业大学URP计划资助

摘　　要：以1型猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)ApxIV基因片段为靶序列设计了一组引物,建立了敏感、特异的猪胸膜肺炎放线杆菌环介导等温扩增检测方法。在63℃等温条件下反应45min,最低可检测到102个拷贝的目的基因,敏感性是PCR方法的10倍。通过对1～10型猪胸膜肺炎放线杆菌、多杀巴氏杆菌(Pasteurella multocida)、链球菌(Strepto-coccussuis)、支原体(Mycoplasma hyopneumoniae)等18种病原微生物的检测,证明该法具有很强的种特异性。对8头猪胸膜肺炎放线杆菌实验感染猪和127份临床发病猪的鼻拭子进行检测,发现该方法对鼻拭子中猪胸膜肺炎放线杆菌的检出率为100%,优于PCR方法。The loop-mediated isothermal amplification （LAMP） assay with high sensitivity and specificity for the detection of Actinobacillus pleuropneumoniae was established. Based on the ApxⅣ gene of Actinobacillus pleuropneumoniae serotype 1, a set of six primers were designed, The detection limit of the LAMP assay completed within 45 min at 63 ℃ was 10^2 copies of the target sequence, which was 10 times more sensitive than that of the PCR assay. High species-specificity of the LAMP method were confirmed by the assay of 18 pathogens such as A. pleuropneumoniae serotypes 1-10, Pasteurella multocida, Streptococcus suis and Mycoplasma hyopneumoniae. In addition, all the nasal swab samples from 8 experimental pigs infected by Actinobacillus pleuropncumoniae and 127 clinical cases were detected by LAMP assay, which deteleion rate was 100%, and the sensitivity is preferable to the PCR assay.

关 键 词：环介导等温扩增(LAMP) 猪胸膜肺炎放线杆菌 PCR 检测 

分 类 号：S188[农业科学—农业基础科学]