湖南农业大学学报（自然科学版）2025，Vol.51Issue(3)：71-78,8.DOI:10.13331/j.cnki.jhau.2025.03.010
团头鲂皮特不动杆菌环介导等温扩增技术(LAMP)快速检测方法建立
Development of a rapid loop-mediated isothermal amplification(LAMP)detection method for Acinetobacter pittii in Megalobrama amblycephala
摘要
Abstract
A rapid detection method based on loop-mediated isothermal amplification(LAMP)was developed for Acinetobacter pittii in blunt snout bream(Megalobrama amblycephala).Specific primers targeting the rpoB housekeeping gene were designed,and the reaction system and conditions,including temperature,time,Mg2+concentration,and primer ratios,were optimized.The results showed that the optimal condition settings,Mg2+concentration at 6.0 mmol/L,with an inner,outer and loop primer ratio of 8∶1∶4.The protocol could detect A.pittii within 30 min at 63℃,with optimal results obtained after 60 min.The sensitivity was 100-fold that of conventional PCR,with a detection limit of 1.4 cfu/mL.The method showed high specificity for A.pittii,with no cross-reactivity against seven common fish pathogens,including Aeromonas hydrophila,Staphylococcus aureus,Citrobacter freundii,Klebsiella pneumoniae,Morganella morganii,Pseudomonas putida and Yersinia ruckeri.Results could be directly visualized by color change profile or verified via gel electrophoresis.This LAMP protocol could provide high sensitivity,specificity,and simplicity,making it suitable for on-site detection of bacterial septicemia caused by A.pittii in aquaculture.关键词
团头鲂/皮特不动杆菌/细菌性败血症/环介导等温扩增技术(LAMP)/rpoB/反应体系Key words
Megalobrama amblycephala/Acinetobacter pittii/bacterial septicemia/loop-mediated isothermal amplification(LAMP)/rpoB/reaction system分类
农业科技引用本文复制引用
张瑞轩,曹景龙,王卫民,罗毅,黎洁..团头鲂皮特不动杆菌环介导等温扩增技术(LAMP)快速检测方法建立[J].湖南农业大学学报（自然科学版）,2025,51(3):71-78,8.基金项目
国家重点研发计划项目(2022YFD2400604) （2022YFD2400604）
