兔巴氏杆菌和波氏杆菌多重PCR检测方法的建立  

Establishment of duplex PCR assay for the diagnosis of Pasteurella multocida and Bordetella bornchispetica infection in rabbits

作　　者：刘燕[1] 韦强[1] 肖琛闻[1] 鲍国连[1] 季权安[1] 庞安娜[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《中国兽医学报》2012年第2期248-251,共4页Chinese Journal of Veterinary Science

基　　金：公益性行业科研专项"肉兔高效饲养研究与示范"基金资助项目(nyhyzx07-040);国家现代农业兔产业体系建设疾病预防与控制岗位基金资助项目(nycytx-44-3-2)

摘　　要：参考GenBank中兔巴氏杆菌16SrRNA和波氏杆菌的fim2的基因序列,应用Premier 5.0软件在二者高度保守区设计了2对引物,建立了适合巴氏杆菌和波氏杆菌快速检测的多重PCR检测方法。以该方法对巴氏杆菌和波氏杆菌参考菌株进行PCR扩增,分别能从各自的基因组中扩增出与试验设计相符的644bp和425bp的特异性DNA片段。将扩增所得的DNA片段进行克隆测序,测序结果表明分别为巴氏杆菌16SrRNA和波氏杆菌fim2基因序列。该方法对波氏杆菌的检测下限为4×102 CFU,对巴氏杆菌的检测下限为6×101 CFU。对兔源性沙门菌、葡萄球菌、大肠杆菌、魏氏梭菌扩增结果为阴性。表明所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鉴别诊断兔巴氏杆菌和波氏杆菌以及2者混合感染。According to the 16S rRNA of Pasteurella multocida gene and fim2 gene of Bordetella bornchispetica recorded in GenBank,two pairs of primer with highly conservative region in the two strains were designed by Premier 5.0 software,rapid multiplex PCR detection method of Pasteurella multocida and Bordetella bornchispetica was established,the 644 and 425 bp specific DNA fragment could be amplified by the PCR method from genome of Pasteurella multocida and Bordetella bornchispetica,respectively.The amplified DNA fragments were cloned and sequenced and matched with Pasteurella multocida and Bordetella bornchispetica gene sequences.The detection limit of Pasteurella multocida and Bordetella bornchispetica were 6×101,4×102 CFU/mL,respectively.There was no cross reaction with Salmonella,E.coli,Staphy1ococcus,Clostridium welchii.The established PCR method was specific,rapid and sensitive,it can be used for the diagnosis of Pasteurella multocida,Bordetella bornchispetica and mixed infection of the two strains of the two strains in rabbits.

关 键 词：巴氏杆菌 波氏杆菌 多重PCR 

分 类 号：S852.61[农业科学—基础兽医学]