猪胸膜肺炎放线杆菌及血清1、3、5、7型多重PCR检测方法的建立及应用  

Establishment and Application of a Multiplex PCR for Detection of Actinobacillus pleuropneumoniae and Serotype 1,3,5 and 7

作　　者：孙学飞[1,2] 谭业平 王丹丹[1] 周俊明[1] 祝昊丹[1] 吕立新[1] 俞正玉[1] 何孔旺[1] 李彬[1] 温立斌[1] 张雪寒[1] 倪艳秀[1] SUN Xue-fei;TAN Ye-pin;WANG Dan-dan;ZHOU Jun-ming;ZHU Hao-dan;LV Li-xing;YU Zheng-yu;HE Kong-wang;LI Bin;WEN Li-bin;ZHANG Xue-han;NI Yan-xiu(Veterinary Research Institute,Jiangsu Academy of Agricultural Sciences,Nanjing,Jiangsu,210014,China;Shanghai Balantek Biotechnology Limited Corporation, Shanghai,201402,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]上海百立生物科技有限公司,上海201402

出　　处：《动物医学进展》2020年第12期17-22,共6页Progress In Veterinary Medicine

基　　金：国家重点研发计划(2018YFD0500101);江苏现代农业生猪产业技术体系[JATS(2018)259];公益性行业(农业)科研专项(201303034-8)。

摘　　要：针对猪胸膜肺炎放线杆菌种特异性基因ApxⅣ和血清1、3、5、7型的特异Cps基因设计5对引物,通过扩增条件的优化,建立了猪胸膜肺炎放线杆菌及血清1、3、5、7型的五重PCR检测方法。特异性试验显示,对血清1~15型参考菌株均扩增出相应的预期目的条带,对6种引起猪发病的主要病原菌均未扩增出任何条带。敏感性试验表明,对各血清型细菌纯培物的敏感性皆为103 CFU/mL;对1、5型感染样品的敏感性为104 CFU/g,对3、7型感染样品的敏感性为103 CFU/g。可在4.5 h左右直接从病猪肺脏中得到检测结果。方法的建立为相关临床病例快速诊断及流行病学调查提供了有效的技术支持。5 pairs of primers were designed according to the species-specific gene ApxⅣof Actinobacillus pleuropneumoniae(APP)and the capsular polysaccharide(CPS)biosynthesis genes of serotype 1,3,5 and 7.A five-fold PCR assay for detecting APP and serotypes 1,3,5 and 7 was established by optimization of amplification conditions.The specificity test results showed that the corresponding expected bands were amplified from APP serotype 1-15 reference strains,while no specific bands were detected from the six major pathogenic bacteria causing swine disease.The sensitivity test results showed that the minimum concentration of detecting the pure cultures of APP serotype 1,3,5 and 7 was 103 CFU/mL and that of detecting the infection samples of APP serotype 1,3,5 and 7 were 104 CFU/g,103 CFU/g,104 CFU/g and 103 CFU/g,respectively.The test results can be directly obtained from the lungs of sick pigs in about 4.5 hours by this method.It provides effective technical support for rapid diagnosis and epidemiological investigation of relevant clinical cases.

关 键 词：猪胸膜肺炎放线杆菌 血清1 3 5和7型 多重PCR 

分 类 号：S852.619[农业科学—基础兽医学] S828.9[农业科学—兽医学]