弗氏柠檬酸杆菌可视化LAMP检测方法的建立  

Establishment of Visual Loop-mediated Isothermal Amplification(LAMP)Assay for Detection of Citrobacter freundii

作　　者：公翠萍 娄剑锋 孙威 郭梦雅 胡大雁 刘晓丹 GONG Cuiping;LOU Jianfeng;SUN Wei;GUO Mengya;HU Dayan;LIU Xiaodan(Huzhou Academy of Agricultural Sciences,Huzhou 313000,China;Huzhou Nanxun District Agricultural Technology Extension Service Center,Huzhou 313000,China;College of Animal Science and Technology,Yangzhou University,Yangzhou 225000,China;International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions,Yangzhou University,Yangzhou 225000,China)

机构地区：[1]湖州市农业科学研究院,浙江湖州313000 [2]湖州市南浔区农业技术推广服务中心,浙江湖州313000 [3]扬州大学动物科学与技术学院,江苏扬州225000 [4]扬州大学江苏高校动物重要疫病和重要人兽共患病防控技术国际合作联合实验室,江苏扬州225000

出　　处：《水产学杂志》2025年第1期76-81,共6页Chinese Journal of Fisheries

基　　金：国家自然科学基金青年项目(32002420);江苏高校动物重要疫病和重要人兽共患病防控技术国际合作联合实验室资助[5];中国博士后科学基金(2021M702761);扬州大学“青蓝工程”(2019);2022年市级农科院联盟区域示范性项目(2022SJLM08);湖州市公益性应用研究项目(2021GZ31).

摘　　要：为了建立一种快速鉴别诊断弗氏柠檬酸杆菌(Citrobacter freundii)的环介导恒温扩增(loop-mediated isothermal amplification,LAMP)检测方法,根据OmpA基因保守序列设计1套LAMP特异性检测引物,以弗氏柠檬酸杆菌DNA为模板、羟基萘酚蓝为显色剂,建立弗氏柠檬酸杆菌可视化LAMP检测方法,并优化反应体系和反应条件,检测该方法的特异性及灵敏度。结果显示:所建立的弗氏柠檬酸杆菌可视化LAMP检测方法与大肠杆菌(Escherichia coli)、鲁氏不动杆菌(Acinetobacter lwoffi)、鲁氏耶尔森氏菌(Yersinia ruckeri)、蜂房哈夫尼亚菌(Hafnia alvei)、迟缓爱德华氏菌(Edwardsiella tarda)、温和气单胞菌(Aeromonas sobria)、杀鲑气单胞菌(Aeromonas salmonicida)、维氏气单胞菌(Aeromonas veronii)和嗜水气单胞菌(Aeromonas hydrophila)均无交叉反应,最低检测量为1.43×10^(2) copies。为了进一步缩短检测该菌的过程,将HNB染料添加到LAMP反应体系中建立可视化弗氏柠檬酸杆菌LAMP检测方法,55 min内在可见光下通过肉眼直接观察颜色变化来判断结果。本研究建立的弗氏柠檬酸杆菌可视化LAMP检测方法操作简便、快速、特异性及灵敏性高,对弗氏柠檬酸杆菌引起的水产动物疾病的快速诊断具有重要的实践意义。In order to develop and evaluate a loop-mediated isothermal amplification(LAMP)assay for rapid visual detection of Citrobacter freundii,a set of LAMP primers were designed utilizing the conserved region of OmpA gene sequence and was applied for detection of C.freundii.The LAMP assay was shown to be specific,without cross-reactivity with other bacteria including Escherichia coli,Acinetobacter lwoffi,Yersinia ruckeri,Hafnia alvei,Edwardia tarda,Aeromonas sobria,Aeromonas salmonicida,Aeromonas veronii,and Aeromonas hydrophila.Amplification results were visualized by a color change after addition of HNB and sensitivity test results showed that the minimum detection volume of this method was 1.43×10^(2) copies,with the reading of positive reaction was simply with the naked eye through color changes under visible light within 55 min.The visual LAMP assay is featured by further short time of C.freundii detection process,visual reading of positive responses through color changes,and suitable for quarantine and field detection. The finding provides effective technical support for rapid diagnosis of viral pathogens of C. freundii in culture site and laboratory.

关 键 词：弗氏柠檬酸杆菌 环介导恒温扩增技术 快速检测 

分 类 号：S917[农业科学—水产科学] S941.4