猪肺炎支原体、多杀性巴氏杆菌和猪胸膜肺炎放线杆菌三重PCR检测方法的建立  

Development of multiplex PCR for detection of swine Mycoplasmal pneumonia, Pasteurella multocida and Actinobacillus

作　　者：李鹏[1] 马艳娇[1,2] 夏伟[1] 郜冬雪[2] 倪宏波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]讷河市畜牧水产技术推广中心,黑龙江讷河161300

出　　处：《中国预防兽医学报》2010年第8期607-610,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：大庆市科学技术局项目(SGG2007-048);黑龙江省农垦总局攻关项目(HNKXIV-08-03-01)

摘　　要：为建立简便、快速及灵敏度高的同时对猪肺炎支原体(Mh)、多杀性巴氏杆菌(PM)和猪传染性胸膜肺炎放线杆菌(App)三重PCR的检测方法,本研究针对这3种病原的基因分别设计3对特异性引物,通过PCR方法分别扩增出116bp(Mh)、253bp(PM)和473bp(App)的目的片段,其最低检出量为4个拷贝;而对肺炎双球菌、支气管败血波氏杆菌、副猪嗜血杆菌和链球菌的PCR扩增则结果均呈阴性。表明该方法具有很好的敏感性和特异性。利用该检测方法对某屠宰场472头猪肺组织病料进行检测,其结果显示:Mh、PM和App的阳性检出率分别为19.27%、5.5%和2.75%;而ELISA检测的阳性检出率分别为18%、5.08%和1.9%。这两种方法对阳性的样品检测的平均符合率为90%;此外,多重PCR与病原分离培养方法的符合率为100%。A simple, rapid and sensitive multiplex PCR method for detection of swine Mycoplasmal pneumonia （Mh）, Pasteurella multocida （PM） and Actinobacillus （App） was developed using primers specific to the respective pathogen. The assay could specifically amplify PCR fragments of 116 bp, 253 bp and 473 bp from Mh, PM and App respectively, while no PCR products were detected for Streptococcus pneumoniae, Bordetella bronchi, Haemophilus parasuis and Streptococcus. A total of 472 samples of swine lung tissue were detected by the PCR assay and compared with ELISA method. The positive detection rate of Mh, PM and App by the PCR method were 19.27 %, 5.5 % and 2.75 % respectively, which were comparable to those of ELISA method （18 %, 5.08 % and 1.9 %）. Furthermore, the results of the multiplex PCR and the bacteria isolation by culture method had 100 % consistency.

关 键 词：猪支原体肺炎 多杀巴氏杆菌病 猪传染性胸膜肺炎 三重PCR诊断 

分 类 号：S855[农业科学—临床兽医学]