奶牛乳房炎大肠埃希氏菌和肺炎克雷伯菌及奇异变形杆菌三重TaqMan荧光定量PCR检测方法的建立  

Establishment of A Triple Taq Man Fluorescence Quantitative PCR Method for Detection of Escherichia coli,Klebsiella pneumoniae and Proteus mirabilis in Dairy Mastitis

作　　者：朱安基 李世宏[2] 徐浩 杨亚军[2] 白莉霞 李剑勇[1,2] 许笑 ZHU An-ji;LI Shi-hong;XU Hao;YANG Ya-jun;BAI Li-xia;LI Jian-yong;XU Xiao(College of Veterinary Medicine,Gansu Agricμltural University,Lanzhou,Gansu,730070,China;Lanzhou Institute of Animal Husbandry and Pharmaceutical Science,Chinese Academy of Agricultural Sciences/Key Laboratory of New Animal Drug Discovery,Ministry of Agriculture and Rural Affairs/Key Laboratory of New Animal Drug Project of Gansu Province,Lanzhou,Gansu,730050,China;Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs District,Shenzhen,Guangdong,518045,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州畜牧与兽药研究所/农业农村部兽用药物创制重点实验室/甘肃省新兽药工程重点实验室,甘肃兰州730050 [3]深圳海关动植物检验检疫技术中心,广东深圳518045

出　　处：《动物医学进展》2024年第12期19-25,共7页Progress In Veterinary Medicine

基　　金：中国农业科学院基本科研业务费专项所级统筹项目(1610322023016);中国农业科学院科技创新工程项目(25-LZIHPS-02)。

摘　　要：为建立检测奶牛乳房炎主要革兰氏阴性致病菌的快速诊断方法,选择大肠埃希氏菌phoa基因,肺炎克雷伯菌rcsa基因以及奇异变形杆菌urer基因设计合成特异性引物与探针,建立了奶牛乳房炎大肠埃希氏菌和肺炎克雷伯菌及奇异变形杆菌三重Taq Man荧光定量PCR检测方法。该方法可在120 min内通过一管反应体系特异性鉴定出3种常见的奶牛乳房炎革兰氏阴性致病菌,最低检测限可达1×10^(1)copies/μL且特异性和重复性良好,以此方法对80份奶牛乳房炎生乳样进行检测,大肠埃希氏菌检出率为35%(28/80),肺炎克雷伯菌检出率为21.2%(17/80),奇异变形杆菌检出率为11.2%(9/80)。结果表明,所建立的奶牛乳房炎大肠埃希氏菌和肺炎克雷伯菌及奇异变形杆菌三重Taq Man荧光定量PCR检测方法具有操作简单、特异性强、灵敏度高的特点,适用于奶牛乳房炎的临床诊断,对革兰氏阴性致病菌的快速检测及合理用药具有指导意义。To establish a rapid diagnostic method for detecting the primary Gram-negative pathogenic bacteria that cause mastitis in dairy cows,this study designed and synthesized specific primers and probes for the Escherichia coli phoa gene,the Klebsiella pneumoniae rcsa gene,and the Proteus mirabilis urer gene,and developed a tripled Taq Man fluorescence quantitative PCR detection method.Using a single reaction system,this approach can precisely identify three prevalent Gram-negative pathogenic bacteria associated with cow mastitis in 120 minutes.It has good repeatability and specificity,with a minimum detection limit of 1×10^(1)copies/μL.The detection rates of Escherichia coli,Klebsiella pneumoniae and Proteus mirabilis were 35%(28/80),21.2%(17/80)and 11.2%(9/80)respectively.According to this study,The triplex Taq Man fluorescence quantitative PCR method for detection of Escherichia coli,Klebsiella pneumoniae and Proteus mirabilis established in this study has the characteristics of simple operation,strong specificity and high sensitivity,which is suitable for clinical diagnosis of dairy mastitis,and has guiding significance for rapid detection and rational drug use of Gram-negative pathogenic bacteria.

关 键 词：奶牛乳房炎 实时荧光定量PCR 大肠埃希氏菌 肺炎克雷伯菌 奇异变形杆菌 

分 类 号：S852.61[农业科学—基础兽医学] S857.26[农业科学—兽医学]