单核细胞增生李斯特菌重组酶聚合酶恒温扩增快速检测方法的建立

Establishment of the detection method of Listeria monocytogenes by recombinase polymerase amplification

为建立基于重组酶聚合酶等温扩增(RPA)技术的单核细胞增生李斯特菌快速检测方法,本研究针对单增李斯特菌的基因组序列设计特异性引物,通过对反应体系的优化确定最佳反应温度为42℃和最佳反应时间为15 min。特异性试验结果显示:本研究建立的RPA方法与伊氏李斯特菌、无害李斯特菌、斯氏李斯特菌、格氏李斯特菌、肠致病性大肠埃希菌O157、沙门氏菌、蜡样芽孢杆菌、金黄色葡萄球菌均无交叉反应;敏感性试验结果显示:该方法对单增李斯特菌基因组DNA和菌液的最低检出限分别为3×102拷贝/μL和103 cfu/mL。利用本研究建立的RPA快速检测方法分别对50份人工污染的牛奶、牛肉和鱼肉样品进行检测,并与荧光定量PCR检测结果进行对比,结果一致。本研究建立的单增李斯特菌RPA检测方法具有反应快速、灵敏度高、特异性强、操作简便的特点,适用于基层实验室和现场快速检测。

To develop a fast detection method for Listeria monocytogenes based on the technology of recombinase polymerase amplification(RPA),the present study designed specific primers targeting to the genome sequence of L.monocytogenes.After conditions optimization,the optimal reaction temperature was determined to be 42℃and the optimal reaction time was 15 min.The established detection method had no cross-reaction with Listeria ivanovii,Listeria innocua,Listeria seeligeri,Listeria grayi,Escherichia coli O157,Salmonella enteritidis,Bacillus cereus,or Staphylococcus aureus.Results of sensitivity tests showed that the minimum detection limits of L.monocytogenes were 3×102 copies/μL and 103 cfu/mL.The established RPA rapid detection method in this study was used to detect fifty artificially contaminated samples of milk,beef and fish,and the results were consistent with those of real-time fluorescence quantitative PCR(qPCR).The RPA detection method for L.monocytogene established in this study is characterized by rapid reaction,sensitive detection,high specificity and simple operation,and is suitable for primary-level laboratories and on-site detection.