猪传染性胸膜肺炎放线杆菌LAMP方法的建立  

Establishment of rapid loop-mediated isothermal amplification for detection of actinobacillus pleuropneumoniae

作　　者：刘亚娟[1,2] 聂福平[2,3] 杨俊[2,3] 王昱[2,3] 石宝石 保雨 叶自霞 王国民[2] 李贤良[2] 李应国[2] 肖进文[2] 刘力[1] 

机构地区：[1]西南大学动物科技学院,重庆北碚400715 [2]重庆出入境检验检疫局/重庆市进出口食品安全工程中心,重庆江北400020 [3]重庆大学生物工程学院,重庆沙坪坝400030

出　　处：《中国兽医学报》2016年第2期200-205,共6页Chinese Journal of Veterinary Science

基　　金：质检公益性资助项目(201310093);国家质检总局科技资助项目(2014IK247;2013IK049)

摘　　要：为建立快速检测猪传染性胸膜肺炎放线杆菌（APP）的鉴别方法，衣研究根据APP ApxIVA基因的保守区设计6条环介导等温扩增（LAMP）引物，利用Loopamp Realtime Turbidimeter LA-320c实时浊度仪对反应体系和条件进行优化，建立了APP的LAMP方法。结果显示：该方法在64℃下反应15rainDNA即可出现扩增，扩增后2～3min内即可判定结果，最低检测限为0．307ng／L，具有良好的重复性及稳定性，与其他病原菌无交叉反应，同时，试验结果可实现肉眼可视化观察。采用建立的LAMP方法与SN／T14472011标准公布的方法同时对36份临床疑似APP样品进行检测分析，结果显示：APP阳性10份，阴性26份，两种方法的符合率为100％。本研究建立的方法为防止猪传染性胸膜肺炎的传播提供了有效的检测手段。In the present study, six primers of loop-mediated isothermal amplification （LAMP） were designed based on the conservative sequence of ApxlVA gene of Actinobacillus pleuropneumoniae （APP）. After the optimized reaction system and condition by Loopamp Realtime Turbidimeter LA-320c,the I.AMP for rapid detection of APP nucleic acid was established. The results showed that APP nuceic acid could be amplified in about 15 minutes under 64℃ and be determined whether is positive after 2 to 3 minutes. The developed LAMP had a good stability and repetition with a detection limit of 0. 307 ng/L DNA,and no cross-reactions with other related bacteria. The amplification can be observed with naked eyes. Thirty six clinical samples were detected by using the LAMP assay developed in this study. The 10 of 36 were APP positive and 26 were negative by LAMP assay, which was 100% identity with the standard method （the standard SN/T 1447- 2011）. The successful development of LAMP assay provided an efficient detection tool for preventing the spread of porcine contagious pleuropneumonia.

关 键 词：猪传染性胸膜肺炎放线杆菌 环介导等温扩增技术(LAMP) ApxIVA基因 

分 类 号：S852.65[农业科学—基础兽医学]