单核细胞增生李斯特氏菌荧光RPA快速检测方法的建立  

Development of Fluorescence Recombinase Polymerase Amplification for the Rapid Detection of Listeria monocytogenes

作　　者：郭正洋 陈晶[1] 刘小青[1] 任佳影 陈佳平 王远洋 陈血建[1] 陈国培[1] Guo Zhengyang;Chen Jing;Liu Xiaoqing;Ren Jiaying;Chen Jiaping;Wang Yuanyang;Chen Xuejian;Chen Guopei(Shenzhen Academy of Metrology&Quality Inspection,Shenzhen,518131;School of Food Science and Technology,Henan University of Technology,Zhengzhou,450001)

机构地区：[1]深圳市计量质量检测研究院,深圳518131 [2]河南工业大学粮油食品学院,郑州450001

出　　处：《基因组学与应用生物学》2020年第12期5599-5605,共7页Genomics and Applied Biology

基　　金：广东省质量技术监督局科技项目(2018CZ20)资助。

摘　　要：本研究旨在建立一种单核细胞增生李斯特氏菌荧光RPA检测方法。根据NCBI数据库中单核细胞增生李斯特氏菌actA基因序列的比对分析,选择同源性好的片段以此设计引物及探针,建立检测单核细胞增生李斯特氏菌的RPA方法。结果表明,本实验所建立的方法特异性较强,只对单核细胞增生李斯特氏菌为检出阳性,对其他菌种无扩增,该方法的检出限为2.3×10^(2)cfu/mL。在对实验室分离菌株的检测分析中,其检测结果与传统培养方法一致。在人工污染样品的检测中,经过4 h的培养后,该方法可检出DNA浓度为2.3×10^(2)cfu/mL的单核细胞增生李斯特氏菌。本研究所建立的RPA检测方法能够快速准确地检测单核细胞增生李斯特氏菌,为基层检测实验室和疫情突发现场单核细胞增生李斯特氏菌的快速检测提供了有效的技术手段。The aim of this study was to establish a fluorescence recombinase polymerase amplification assay for detection of Listeria monocytogenes.Based on the result of analysis on the published L.monocytogenes actA sequences on NCBI database,primers and probes were designed to establish the RPA method for the detection of L.monocytogenes.The results showed the method developed in this study had strong specificity,only L.monocytogenes be amplified.The limit of detection by RPA was 2.3×10^(2) cfu/mL.In the analysis of the isolated strains,the results were consistent with the traditional culture methods.In the simulated detection of artificially contaminated samples,the limit of detection of RPA to L.monocytogenes achieved 2.3×10^(3) cfu/mL,after 4 hours of incubation.The RPA method established in this study can rapidly and accurately detect L.monocytogenes,providing an effective technical means for the rapid detection of L.monocytogenes in the laboratory and the outbreak site with poor experimental equipment and in the outbreak site.

关 键 词：单增李斯特菌(Listeria monocytogenes) RPA 检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学]