禽多杀性巴氏杆菌、副鸡嗜血杆菌和大肠杆菌多重PCR检测方法的建立及应用  

Establishment and Application of Multiplex PCR Assay for Detecting Pasteurella multocida,Haemophilus paragallinarumand Escherichia coli

作　　者：张曼[1] 韩飞[1] 沈文正[1] 何亚鹏 

机构地区：[1]杨凌职业技术学院,陕西杨凌712100

出　　处：《西北农业学报》2018年第2期163-168,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省咸阳市重大科技创新项目(2016K01-43);杨凌农业高新技术示范区重大科技项目(2016-XY-16)~~

摘　　要：为了建立鉴别禽多杀性巴氏杆菌(Pasteurella multocida)、副鸡嗜血杆菌(Haemophilus paragallinarum)和大肠杆菌(Escherichia coli)的多重PCR检测方法,参照GenBank中登录的3种细菌基因组序列,合成3对特异性引物,通过优化多重PCR反应条件,建立快速检测3种细菌的多重PCR方法。特异性试验结果表明,可分别扩增出3种细菌对应的目的片段,对Ⅰ群禽腺病毒4型(FAV-4)、金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonellaspp)、鸡胚成纤维细胞(DF-1)的DNA和灭菌双蒸水均无扩增;敏感性试验结果表明,最低检测量分别为24.3pg/μL禽多杀性巴氏杆菌、21.4pg/μL副鸡嗜血杆菌和28.6pg/μL鸡大肠杆菌的基因组DNA;应用该方法对83份临床病料进行检测,结果与其他已建立的单重PCR检测一致,说明所建立的方法可用于临床上3种细菌的鉴别诊断。A multiplex polymerase chain reaction assay was developed for the detection of Pasteurella multocida,Haemophilus paragallinarum,and Escherichia coli.According to the genome sequence of P.multocida,H.paragallinarum,and Escherichia coli in GenBank,three pairs of primers were synthetized.The multiplex PCR reaction condition was optimized and the multiplex PCR for simultaneously detecting P.multocida,H.paragallinarum,and Escherichia coli was established.The specificity test showed that target fragments were obtained from the genomic DNA of three bacteria respectively.Three fragments were amplified from the mixed DNA sample of P.multocida,H.paragallinarum,and Escherichia coli.simultaneously,and no amplification was accepted from FAV-4,Staphylococcus aureus,Salmonella,DF-1,and ddH2 O.The sensitivity test showed that the multiplex PCR could detect 24.3 pg/μL of P.multocida DNA,21.4 pg/μL of H.paragallinarum DNA and 28.6 pg/μL of Escherichia coli DNA.Tested on 83 clinical samples,the results of multiplex PCR were consistent with the established PCR detection method.These results indicated that multiplex PCR can be used for detection of P.multocida,H.paragallinarum,and E.coli.in clinic.

关 键 词：多重PCR 多杀性巴氏杆菌 副鸡嗜血杆菌 大肠杆菌 检测 

分 类 号：S855.1[农业科学—临床兽医学]