鉴别猪胸膜肺炎放线杆菌2/6/8/12型的多重PCR方法的建立  

Establishment of a multiplex PCR for detection of Actinobacillus pleuropneumoniae serotype 2/6/8/12

作　　者：孙学飞[1,2] 王丹丹 周俊明[1] 祝昊丹[1] 何孔旺[1] 李彬[1] 倪艳秀[1] SUN Xue-fei;WANG Dan-dan;ZHOU Jun-ming;ZHU Hao-dan;HE Kong-wang;LI Bin;NI Yan-xiu(Veterinary Research Institute,Jiangsu Academy of Agricultural Sciences,Nanjing 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：《中国兽医科学》2021年第6期689-694,共6页Chinese Veterinary Science

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

摘　　要：为快速准确鉴别猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)血清2、6、8、12型,从已发表的文献中筛选出4对引物,通过扩增体系和扩增程序的优化来建立APP血清2、6、8、12型的多重PCR检测方法,并对其特异性和敏感性进行了研究。结果发现,所建立的多重PCR方法特异性良好,对APP血清1~15型进行PCR检测,除血清2、6、8、12型外,均不能扩增出任何条带,检测猪链球菌、副猪嗜血杆菌、猪多杀性巴氏杆菌和猪大肠杆菌也呈阴性,对APP血清2、6、8、12型的细菌纯培养物、组织样品的检测敏感性分别都为1×10^(3) CFU/mL、1×10^(3) CFU/g。本PCR方法快速有效,可在4.5 h左右直接从肺脏组织中得到检测结果。本方法的建立为APP的分子分型和流行病学调查提供了有力的技术支撑。In order to quickly and accurately identify Actinobacillus pleuropneumoniae(APP)serotype 2,6,8 and 12,4 pairs of specific primers selected from published literature were synthesized.The multiplex PCR detection method of APP serotype 2,6,8 and 12 was established by optimizing the amplification system and amplification program,and its specificity and sensitivity were studied.The results showed that the multiplex PCR method had good specificity,and PCR detection of APP serotypes 1—15 couldn’t amplify any bands except for serotypes 2,6,8 and 12.Detection of genomic DNA from Streptococcus suis,Haemophilus parasuis,Pasteurella multocida and Escherichia coli were also negative.The detection sensitivity of bacterial pure cultures and tissue samples of APP serotypes 2,6,8 and 12 were 1×10^(3) CFU/mL and 1×10^(3) CFU/g,respectively.This PCR method was fast and effective,and the test results can be obtained directly from the lung tissue in about 4.5 h.The establishment of this method provides strong technical support for molecular typing and epidemiological investigation of APP.

关 键 词：猪胸膜肺炎放线杆菌 血清型 多重PCR 

分 类 号：S852.1[农业科学—基础兽医学]