摘要
为建立一种能快速检测奶牛乳房炎主要病原菌(大肠埃希氏菌、无乳链球菌、肺炎克雷伯氏菌、金黄色葡萄球菌和肠炎沙门氏菌)的多重PCR检测方法,根据大肠埃希氏菌phoA基因、无乳链球菌ef-tu基因、肺炎克雷伯氏菌Khe基因、金黄色葡萄球菌Nuc基因和肠炎沙门氏菌invA基因设计特异性引物,优化PCR反应体系与反应程序并进行特异性试验,同时分别构建含病原菌基因的pMD-19T重组质粒进行灵敏性试验。结果显示,多重PCR反应的最适引物工作浓度为0.8μmol/L,最适退火温度为59.6℃,最佳延伸时间为40 s,最合适的循环数为30次;优化后的多重PCR反应体系可扩增出5种目标病原菌的特异性条带;5种病原菌PCR检测灵敏度可达到5 copies/μL。进一步利用该多重PCR反应体系对采集的108份乳房炎乳样进行检测,检测结果显示,108份样本的阳性检出率为77.8%,大肠埃希氏菌、无乳链球菌、金黄色葡萄球菌、肺炎克雷伯氏菌和肠炎沙门氏菌的检出率分别为23.1%、8.3%、34.3%、8.3%、5.6%。结果表明,建立了一种具有良好特异性和灵敏性的多重PCR检测方法,在临床生产实践中可用于快速鉴定奶牛乳房炎的致病菌,为奶牛乳房炎的诊断、防控与流行病学调查提供了方法基础。
To develop a multiplex PCR assay for the rapid detection of the main pathogens of dairy mastitis(E.coli,S.agalactiae,K.pneumoniae,S.aureus and S.enterica),in this study,specific primers were designed according to the phoA gene of E.coli,ef-tu gene of S.agalactiae,Khe gene of K.pneumoniae,Nuc gene of S.aureus and invA gene of S.enterica.PCR reaction system and reaction procedure were optimized and specific tests were performed.At the same time,pMD-19T recombinant plasmids containing pathogenic bacteria genes were constructed for sensitivity tests.The results showed that the optimal working concentration of primers was 0.8μmol/L,the optimal annealing temperature was 59.6℃,the optimal extension time was 40 s,and the optimal number of cycles was 30.The optimized multiplex PCR system can amplify the specific bands of five target pathogens.The PCR sensitivity of the five pathogens was 5 copies/μL.The multiplex PCR system was further used to detect 108 samples of milk from cows with mastitis.The results showed that the positive detection rate of 108 samples was 77.8%.The detection rates of E.coli,S.agalactiae,S.aureus,K.pneumoniae and S.enterica were 23.1%,8.3%,34.3%,8.3%and 5.6%,respectively.This study established a multiplex PCR detection method with good specificity and sensitivity,which can be used to rapidly identify the pathogenic bacteria of dairy cow mastitis in clinical production practice,and provide a methodological basis for the diagnosis,prevention and control of dairy cow mastitis and epidemiological investigation.
作者
王雪容
张宇
张海森
杨王浩
靳亚平
陈华涛
WANG Xue-rong;ZHANG Yu;ZHANG Hai-sen;YANG Wang-hao;JIN Ya-ping;CHEN Hua-tao(Key Laboratory of Animal Biotechnology of Ministry of Agriculture and Rural Affairs/College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2025年第1期48-55,共8页
Progress In Veterinary Medicine
基金
国家自然科学基金面上项目(32373088)。
关键词
奶牛
乳房炎
病原菌
多重PCR
Cow
Mastitis
Pathogenic bacteria
Multiplex PCR
