兔源强毒力金黄色葡萄球菌环介导等温扩增快速检测方法的建立  

Development of a loop-mediated isothermal amplification assay for rapid detection of hyper-virulent Staphylococcus aureus strains in rabbits

作　　者：王锦祥[1] 孙世坤[1] 陈岩峰[1] 陈冬金[1] 桑雷[1] 谢喜平[1] WANG Jin-xiang;SUN Shi-kun;CHEN Yan-feng;CHEN Dong-jin;SANG Lei;XIE Xi-ping(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：《中国预防兽医学报》2021年第6期621-625,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：福建省自然科学基金(2020J01346);福建省科技计划公益类专项(2019R1026-9);国家兔产业技术体系专项(CARS-43-G-5)。

摘　　要：为建立检测兔源强毒力金黄色葡萄球菌的快速检测方法,本实验根据GenBank中登录的金黄色葡萄球菌pvl基因保守序列设计了2对引物,经反应条件优化,建立了检测兔源强毒力金黄色葡萄球菌的环介导等温扩增(LAMP)快速检测方法。结果显示,该LAMP快速检测方法最优反应条件为:外引物和内引物的浓度比为1.7,反应温度为65℃,反应时间为60 min。利用细菌基因组DNA提取试剂盒提取兔源强毒力金黄色葡萄球菌、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、肺炎克雷伯菌和大肠杆菌DNA,利用该方法检测,结果显示仅兔源强毒力金黄色葡萄球菌检测为阳性结果,其他均为阴性,无交叉反应,该方法特异性较强;将兔源强毒力金黄色葡萄球菌基因组DNA的浓度设置为1×10^(8)拷贝/μL~1×10^(0)拷贝/μL,利用本研究建立的方法检测,结果显示对兔源强毒力金黄色葡萄球菌基因组DNA的最低检出限为1×10^(2)拷贝/μL,其敏感性为双重PCR方法的100倍,敏感性高;利用该方法进行批内和批间重复性试验,结果均一致,重复性较好。利用该方法检测88份临床样品,结果显示与已报道的双重PCR方法检测结果的符合率为100%。本研究在国内首次建立了兔源强毒力金黄色葡萄球菌LAMP方法,为该病原的快速检测提供了有效的技术手段。In order to develop a method for rapid detection of hyper-virulent Staphylococcus aureus(S.aureus)in rabbits,a loop-mediated isothermal amplification(LAMP)assay was established with two sets of primers targeting the conserved sequence of pvl gene.The optimized ratio of outer and inner primers,incubation temperature and incubation duration were 1.7,65℃and 60 min,respectively.The LAMP assay with optimized reaction conditions had no cross-reaction activities with low-virulent S.aureus,Pasteurella multocida,Bordetella bronchiseptica,Klebsiella pneumonia or Escherichia coli,which were isolated from rabbits.The LAMP assay was highly sensitive,and could detect up to 1×10^(2)copies/μL of genomic DNA of hyper-virulent S.aureus,which was 100-fold higher than that of the conventional duplex PCR assay.The LAMP assay was repeatable and the coefficients of both intra-and inter-assay variations were identical.Moreover,the results of the LAMP assay based on the 88 clinical samples showed 100%consistency with that of the reported duplex PCR assay.In conclusion,the LAMP assay provides an effective method for the rapid 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 LAMP检测方法 

分 类 号：S852.61[农业科学—基础兽医学]