鸭疫里默氏菌病和大肠杆菌病多重PCR诊断方法的建立  

Establishment of multiplex PCR assay for the diagnosis of Riemerella anatipestifer and Escherichia coli infection in ducks

作　　者：王春平[1,2,3] 韦强[1] 鲍国连[1] 崔言顺[2] 刘燕[1] 邵泽香[1,2] 季权安[1] 肖琛闻[1] 李建亮[2] 

机构地区：[1]浙江省农科院畜牧兽医研究所,浙江杭州310021 [2]山东农业大学动物科技学院,山东泰安271018 [3]山东省日照市畜牧兽医局,山东日照276826

出　　处：《中国兽医学报》2010年第3期352-355,共4页Chinese Journal of Veterinary Science

基　　金：浙江省重点科技攻关项目(2006C12026);浙江省农科院创新能力提升工程项目

摘　　要：参考GenBank中鸭疫里默氏菌和大肠杆菌的外膜蛋白A(OmpA)基因序列,应用PrimerPremier5.0软件在二者高度保守区设计了2对引物,建立了适合鸭疫里默氏菌和大肠杆菌的快速检测的多重PCR检测方法。以该方法对已分离并保存的鸭疫里默氏菌和大肠杆菌进行PCR扩增,分别扩增出与试验设计相符的670、408bp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鸭疫里默氏菌和大肠杆菌OmpA基因序列。该方法对鸭疫里默氏菌和大肠杆菌的检测下限分别为4×104CFU/mL和3×104CFU/mL。表明所建立的PCR方法具有特异、快速和敏感的特点,可用于诊断鸭疫里默氏菌、大肠杆菌以及两者的混合感染。According to the outer membrane protein A （OmpA） gene sequences of Riemerella anatipestifer and Escherichia coli recorded in GenBank,two pairs of primer with highly conservative region in the two strains were designed by Primer Premier 5.0 software,rapid multiplex PCR detection method of Riemerella anatipestifer and Escherichia coli was established,the 670 and 408 bp specific DNA fragment could be amplified by the PCR method we established,from their genome of Riemerella anatipestifer and Escherichia coli those were isolated respectively,which consistent with experimental design.The amplified DNA fragments were cloned and sequenced and matched with Riemerella anatipestifer and Escherichia coli gene sequences.The detection limit of Riemerella anatipestifer and Escherichia coli were 4×10^4 CFU/mL,3×10^4 CFU/mL,respectively.The established PCR method was specific,rapid and sensitive,it can be used for the diagnosis of Riemerella anatipestifer,Escherichia coli and mixed infection in ducks.

关 键 词：鸭疫里默氏菌 大肠杆菌 多重PCR 

分 类 号：S852.61[农业科学—基础兽医学]