4种家兔呼吸道致病菌多重PCR的建立及初步应用  

Establishment and application of multiplex PCR method for detection of four respiratory pathogenic bacteria in rabbits

作　　者：仇汝龙 范志宇[1] 胡波[1] 宋艳华[1] 魏后军[1] 陈萌萌[1] 朱伟峰 薛家宾[1] 王芳[1] QIU Rulong;FAN Zhiyu;HU Bo;SONG Yanhua;WEI Houjun;CHEN Mengmeng;ZHU Weifeng;XUE Jiabin;WANG Fang(Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程重点实验室,江苏南京210014

出　　处：《中国兽医学报》2021年第10期1958-1962,1969,共6页Chinese Journal of Veterinary Science

基　　金：现代农业产业技术体系建设专项资金资助项目(CARS-43-C-1)。

摘　　要：为建立检测家兔铜绿假单胞菌(Pseudomonas aeruginosa)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、多杀性巴氏杆菌(Pasteurella multocida)和支气管败血波氏杆菌(Brodetella bronchiseptica)的多重PCR检测方法,参考GenBank数据库中登录的铜绿假单胞菌gyrA基因、肺炎克雷伯氏菌rcsA基因、多杀性巴氏杆菌KMT1基因和支气管败血波氏杆菌flaA基因,设计4对特异性引物,建立能同时扩增4种家兔呼吸道致病菌核酸的多重PCR检测方法,并进一步对其特异性和敏感性进行检测。特异性检测结果显示,该方法能同时扩增出4种家兔呼吸道致病菌的核酸,而对兔出血症病毒、金黄色葡萄球菌、大肠杆菌、产气荚膜梭菌、奇异变形杆菌与肠炎沙门菌无特异性扩增条带,说明该方法特异性良好。敏感性检测结果显示,该方法对铜绿假单胞菌、肺炎克雷伯氏菌、多杀性巴氏杆菌和支气管败血波氏杆菌检测的灵敏度分别为2.5×10^(-1),2.5×10^(0),2.5×10^(-2),2.5×10^(-2)μg/L。用建立的多重PCR方法对临床样本进行检测,结果表明,该方法可以快速并且特异地鉴别4种细菌病原的单独或混合感染,与单一PCR检测方法相比无显著性差异(P>0.05)。该方法的建立为家兔呼吸道疾病临床鉴别诊断和4种细菌病原的分离鉴定提供重要的技术手段。To establish a multiplex PCR method of Pseudomonas aeruginosa,Klebsiella pneumoniae,Pasteurella multocida and Brodetella bronchiseptica,four pairs of specific primer were designed referring to gyrA,rcsA,KMT1 and flaA gene sequences from GenBank.We next optimized the multiplex PCR method by adjusting the annealing temperature and primer concentrations.The results of the specificity assay showed that the method could simultaneously amplify nucleic acids of four respiratory pathogenic bacteria in rabbits,while there was no specific amplification of RHDV,S.aureus,E.coli,C.welchii,P.mirabilis and S.enteritidis,indicating that the method has good specificity.The sensitivity of the multiplex PCR method to P.aeruginosa,K.pneumoniae,P.multocida,B.bronchiseptica were 2.5×10^(-1),2.5×10^(0),2.5×10^(-2),2.5×10^(-2)μg/L,respectively.Furthermore,the results of the detection of clinical samples using the multiplex PCR method indicated that the four respiratory pathogenic bacteria could be quickly identified in single or mixed infection samples with no significant difference compared to the single PCR method(P>0.05).Collectively,the establishment of new multiplex PCR method provides an important technical tool for the clinical differential diagnosis of respiratory diseases in rabbits and the isolation and identification of four bacterial pathogens.

关 键 词：兔呼吸道疾病 多重PCR 铜绿假单胞菌 肺炎克雷伯氏菌 多杀性巴氏杆菌 支气管败血波氏杆菌 

分 类 号：S852.61[农业科学—基础兽医学]