兔源强毒力金黄色葡萄球菌荧光定量PCR检测方法的建立  

Development of a real-time PCR assay for detection of hyper-virulent Staphylococcus aureus in rabbits

作　　者：王锦祥[1] 林松华 陈冬金[1] 孙世坤[1] 陈岩锋[1] 桑雷[1] 谢喜平[1] WANG Jinxiang;LIN Songhua;CHEN Dongjin;SUN Shikun;CHEN Yanfeng;SANG Lei;XIE Xiping(Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《中国兽医学报》2023年第2期297-302,共6页Chinese Journal of Veterinary Science

基　　金：福建省自然科学基金资助项目(2020J01346);福建省农业科学院“5511”协同创新工程资助项目(XTCX2021008);福建省农业科学院科技创新团队建设资助项目(CXTD2021007-2);国家现代农业产业技术体系资助项目(CARS-43-G-5)。

摘　　要：为了建立一种快速、特异性强且敏感性高的兔源强毒力金黄色葡萄球菌检测方法,本试验根据金黄色葡萄球菌pvl基因的保守序列设计了特异性的引物和探针,通过反应条件的优化,建立了检测兔源强毒力金黄色葡萄球菌的荧光定量PCR检测方法。结果显示,该方法耗时短,仅需约50 min就能完成检测,较已报道的双重PCR方法和环介导等温扩增方法均省时;该方法特异性强,对兔源低毒力金黄色葡萄球菌、多杀性巴氏杆菌、支气管败血波氏杆菌、肺炎克雷伯菌、魏氏梭菌、大肠杆菌和阴性对照(灭菌ddH2O)均无交叉反应;该方法敏感性高,最低检测限为10拷贝/μL,分别是已报道的双重PCR方法和环介导等温扩增方法的1000倍和10倍。此外,该方法重复性好,批内和批间重复性试验的变异系数均小于2%。应用该方法检测126份已知结果的临床样品,检测结果与已知结果、已报道的双重PCR方法和环介导等温扩增方法的检测结果完全一致。该方法的建立不仅丰富了兔源强毒力金黄色葡萄球菌的检测方法,也为该菌的快速检测提供了更有力的技术支持。To develop a rapid,specific and sensitive method for detection of hyper-virulent Staphylococcus aureus(S.aureus)in rabbits,a real-time PCR assay was developed based on a set of primers and probe targeting the conserved sequences of pvl gene of S.aureus.The results showed that the assay was rapid and could fulfill the test within 50 min,which was time-saving than those of reported conventional duplex PCR assay and loop-mediated isothermal amplification assay.The assay was specific for hyper-virulent S.aureus,and had no cross-reactions with low-virulent S.aureus,Pasteurella multocida,Bordetella bronchiseptica,Klebsiella pneumonia,Clostridieum welchii,Escherichia coli.The detection limit of the assay was 1×10~1 copies/μL of genomic DNA of hyper-virulent S.aureus,which was 1000-fold and 10-fold higher than those of reported conventional duplex PCR assay and loop-mediated isothermal amplification assay,respectively.The assay was repeatable,and the coefficients of both intra-and inter-assay variations were less than 2%.Moreover,the assay was accurate,the results based on 126 known clinical samples showed 100%consistency with the known results and the results of those of reported conventional duplex PCR assay and loop-mediated isothermal amplification assay.Taken together,the assay is efficient and an important supplement to the existing assays for the detection of hyper-virulent S.aureus in rabbits.

关 键 词：兔 强毒力金黄色葡萄球菌 PVL基因 荧光定量PCR检测方法 

分 类 号：S857.26[农业科学—临床兽医学]