产单核细胞李氏杆菌RPA-LF快速检测方法的建立

Rapid detection of Listeria monocytogenes with isothermal recombinase polymerase amplification and lateral flow analysis

为建立一种基于侧流层析的重组酶聚合酶扩增技术(RPA-LF)的快速检测产单核细胞李氏杆菌的方法,以该菌溶血素基因hly为靶序列设计用于RPA试验的引物及探针。利用筛选到的引物和探针对RPA反应进行优化,结果表明RPA反应可在25~45℃较大温度范围内进行,10~25 min即可完成,其扩增产物在试纸条上仅需5 min即可获得检测结果。因此,在最佳反应温度(37℃)和最佳反应时间(15 min)条件下,RPA-LF总反应时间为20 min。该检测方法不与其他12种常见的食源性病原微生物发生交叉反应,对产单核细胞李氏杆菌基因组的最低检测限可低至400 pg。本研究中建立的产单核细胞李氏杆菌的RPA-LF检测方法具有检测时间短,反应灵敏,特异性好,结果直观,无须昂贵仪器等优点,可为后期产单核细胞李氏杆菌的现场检测应用打下坚实基础。

To establish a novel isothermal recombinase polymerase amplification with lateral flow(RPA-LF) method for detection of Listeria monocytogenes,several pairs of primers were designed according to the hemolysin gene(hly) of L.monocytogenes and a methodology evaluation was performed.The results showed that the RPA assay based on the primers was successfully applied in L.monocytogenes detection.The RPA-LF assay can be performed within 10-25 min at 25-45 ℃.The amplification product was visualized by LF strip within 5 min using the specific probe added to the RPA reaction system.After optimization,the RPA-LF assay could be used to detect L.monocytogenes within 20 min,including DNA amplification with RPA for 15 min at 37 ℃ and visualization of the amplicons by LF strips for 5 min.No cross-reaction was observed with 12 other foodborne pathogenic microorganisms,indicating the good specificity of this method.The limit of detection of RPA-LF assay was as low as 400 pg of genomic DNA of L.monocytogenes.The developed RPA-LF assay established several attractive characteristics,such as high selectivity,high specificity,easy to read the results and no special equipment requirement.The proposed detection system provided a potential method for L.monocytogenes in point-of-care test.