产毒素多杀性巴氏杆菌菌落双重PCR检测方法的建立  

Detection of toxigenic Pasteurella multocida with a colony diplex PCR assay

作　　者：李伟杰[1] 赵耘[1] 杜昕波[1] 康凯[1] 陈敏[1] 

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《中国预防兽医学报》2010年第4期298-300,共3页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2006BAD06A11)

摘　　要：为建立快速特异的PCR方法以及同时检测并区分产毒素与非产毒素多杀性巴氏杆菌,本研究根据GenBank登录的多杀性巴氏杆菌KMT1基因和toxA毒素基因序列,设计合成了2对特异引物。特异性试验表明产毒素多杀性巴氏杆菌C51-6扩增出了460bp和1854bp的2条目的片段,而不产毒素多杀性巴氏杆菌、大肠埃希菌、胸膜肺炎放线杆菌、猪链球菌、支气管败血波氏杆菌、副猪嗜血杆菌和鸡白痢沙门菌的扩增均为阴性;敏感性试验表明该PCR方法能从含450CFU的菌液中扩增出相应的目的片段。同时用豚鼠皮肤坏死试验和小鼠致死试验对该PCR方法进行了验证。A duplex PCR assay was developed using two sets of specific primers derived from the KMT1 gene and toxA gene of Pasteurella multocida in order to differentiate toxigenic and nontoxigenic P. multocida at the same time. The assay could specifically amplify both 460 bp and 1 854 bp DNA products from thee toxigenic P. multocida C51-6,but not from nontoxigenic P. multocida,E. coli,Actinobacillus pleuropneumoniae,Streptococcus suis,Bordetella bronchiseptica,Haemophilus parasuis and Salmonella enterica subsp. It is highly sensitive and could detect as little as 450 CFU bacteria. The assay was verifiable by Guinea pig skin test and mouse lethal test.

关 键 词：产毒素多杀性巴氏杆菌 双重PCR KMT1基因 toxA基因 

分 类 号：S852.61[农业科学—基础兽医学]