嗜肺军团菌LF-RPA检测方法研究  

Research on LF-RPA detection method of Legionella pneumophila

作　　者：高璟瑜 李秋阳 姚瑶 谢远红[2] GAO Jingyu;LI Qiuyang;YAO Yao;XIE Yuanhong(Science and Technology Research Center of China Customs,Beijing,100026,China;Beijing University of Agriculture)

机构地区：[1]中国海关科学技术研究中心,北京100026 [2]北京农学院

出　　处：《质量安全与检验检测》2025年第1期79-84,90,共7页QUALITY SAFETY INSPECTION AND TESTING

摘　　要：基于重组酶聚合酶扩增(recombinase polymerase amplification,RPA)与侧流层析(lateral flow,LF)相结合的技术,建立一种灵敏、准确、快速的嗜肺军团菌的检测方法。根据嗜肺军团菌mip基因设计合成特异性重组酶聚合酶扩增(RPA)引物,通过筛选引物,优化RPA反应时间、反应温度和引物浓度,建立LF-RPA反应体系。分别选取标准质粒浓度为30 copies/反应、60 copies/反应、90 copies/反应、120 copies/反应和150 copies/反应作为模板,对已建立的方法进行灵敏度评价。选取3种军团菌和6种常见的食源性致病菌的DNA为模板,对已建立的方法进行特异性评价。结果显示,该方法对嗜肺军团菌的检测限为90 copies/反应,与其他军团菌及常见的致病菌无交叉反应,特异性良好。本研究所建立的嗜肺军团菌LF-RPA检测方法可用于嗜肺军团菌的快速检测。To develop a sensitive,accurate and rapid detection method for Legionella pneumophila by combining recombinase polymerase amplification(RPA)with lateral flow(LF)technology.Specific recombinase polymerase amplification(RPA)primers were designed and synthesized based on the mip gene of Legionella pneumophila.Primers were screened,and RPA reaction time,reaction temperature and primer concentration were optimized to establish the optimal LF-RPA reaction system.Furthermore,the standard plasmids were subjected to five different final concentrations of 30,60,90,120 and 150 copies/reaction,respectively,and the established optimal LF-RPA reaction system was used for sensitivity evaluation.Using DNA from 3 Legionella and 6 common foodborne pathogens as templates,the established optimal LF-RPA reaction system was used for specificity evaluation.It showed no cross reactivity with other Legionella and common pathogenic bacteria,and had good specificity.As a high sensitivity method,the detection limit was 90 copies/reaction for Legionella pneumophila.The method established in this study can be used for rapid detection of Legionella pneumophila.

关 键 词：嗜肺军团菌 重组酶聚合酶扩增法 测流免疫层析 

分 类 号：R123.1[医药卫生—环境卫生学]