基于gcp基因的溶血性曼氏杆菌LAMP检测方法的建立  

Establishment of LAMP detection method for M.haemolyticus based on gcp gene

作　　者：钟舒红[1] 韩林梅[2] 吴翠兰 彭昊[1] 冯世文[1] 贺会利 马春霞[1] 禤雄标[1] 李军[1] 曾芸[2] ZHONG Shu-hong;HAN Lin-mei;WU Cui-lan;PENG Hao;FENG Shi-wen;HE Hui-li;MA Chun-xia;XUAN Xiong-biao;LI Jun;ZENG Yun(Guangxi Veterinary Research Institute/Guangxi Key Laboratory of Veterinary Biotechnology,Nanning 530001,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西兽医研究所广西兽医生物技术重点实验室,广西南宁530001 [2]广西大学动物科学技术学院,广西南宁530004

出　　处：《中国兽医科学》2022年第10期1245-1251,共7页Chinese Veterinary Science

基　　金：广西重点研发计划项目(桂科AB21238003,AB16380106);桂科专项项目(21-6);南宁市重点研发计划项目(20212023);良庆区重点研发计划项目(202109)

摘　　要：为建立一种快速、准确并可定量检测溶血性曼氏杆菌的环介导等温扩增(LAMP)方法,本研究针对溶血性曼氏杆菌gcp基因设计5条特异性引物,在链置换DNA聚合酶的作用下,优化反应条件,并进行特异性和敏感性试验。特异性检测结果表明,建立的LAMP扩增方法可以在65 min内完成,并具有良好的特异性,检测化脓隐秘杆菌、肺炎克雷伯氏菌、大肠杆菌、牛支原体、鼠伤寒沙门菌、溶血性巴氏杆菌等其他6种常见牛呼吸道综合征致病菌和空白对照(水)时结果均为阴性;灵敏性检测结果表明,建立的LAMP方法检测溶血性曼氏杆菌质粒标准品的最低检出浓度为0.855×10^(-4)ng/μL;在对临床样品的检测中,本研究建立的LAMP方法对溶血性曼氏杆菌的检出率为52.63%(10/19)大于PCR方法的检出率47.36%(9/19)。以上结果表明,本研究建立的基于gcp基因的溶血性曼氏杆菌LAMP检测方法为溶血性曼氏杆菌的快速检测提供了一种新的技术手段,该方法特异性强、灵敏度高、快速简便,可将该方法推广至基层和流行病调查一线,应用于该病原的快速检测。In order to establish a rapid,accurate and quantitative loop-mediated isothermal amplification(LAMP)method for the detection of M.haemolyticus,five specific primers were designed for the gcp gene of M.haemolyticus.The reaction conditions were optimized,and the specificity and sensitivity tests were carried out with the action of the chain replacement DNA polymerase.The specificity test results showed that the established LAMP amplification reaction could be completed in 65 min and had good specificity.The detection results were negative for other pathogenic bacteria such as Cryptobacillus pyogenes,Klebsiella pneumoniae,Escherichia coli,Mycoplasma bovis,Salmonella typhimurium,Pasteurella hemolytica and negative control(water).The sensitivity test results showed that the minimum detection concentration of the established LAMP method was 0.855×10^(-4)ng/μL.In the detection of clinical samples,the positive detection rate of LAMP method established in this study was 52.63%(10/19),which was higher than that of PCR method 47.36%(9/19).In conclusion,the LAMP method based on gcp gene established in this study provides a new technical method for rapid detection of M.hemolyticus.This method has strong specificity,high sensitivity,rapid and simple characteristics,and can be extended to the grassroots and the front line of epidemic investigation for rapid detection of the pathogen.

关 键 词：溶血性曼氏杆菌 GCP 环介导等温核酸扩增 PCR 

分 类 号：S852.61[农业科学—基础兽医学]