单核细胞增生李斯特氏菌实时荧光RPA检测方法的建立及应用

Development and Application of the Real-time Recombinase Polymerase Amplification Assay for Detection of Listeria monocytogenes

本研究旨在建立一种快速检测单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)的实时荧光重组酶聚合酶扩增(real-time RPA)方法。基于LMhly A基因保守序列,设计合成特异性RPA引物和exo探针,所建立的LM real-time RPA方法能够在37℃等温条件下20 min内完成扩增。该方法仅对LM基因组DNA为阳性扩增,对其它20种致病菌基因组DNA的扩增均为阴性;以LM纯培养菌液的基因组DNA进行10倍列梯度稀释,并以之作为模板,其灵敏性可达到5×10-1 pg,与实验室已建立的real-time PCR方法一致。人工污染实验中,对于LM接菌量为3 CFU/25 g的羊肉和生菜样品,增菌14 h即可通过建立的real-time RPA方法检出,与传统培养方法和real-time PCR检测结果均一致,但是前者所需时间明显少于后者。本文建立的LM real-time RPA方法特异性强,灵敏性高,操作简单,反应迅速,并能够摆脱对价格昂贵的荧光PCR仪和专业实验室的需求,可应用于食品突发事件中LM的现场快速检测,对保障我国食品安全具有重要意义。

The aim of this study was to establish a method for the rapid detection of Listeria monocytogenes（LM） by real-time recombinase polymerase amplification（real-time RPA）. The primers and exo probe real-time RPA were designed basing on the conserved region of the hly A gene of LM, and the assay was performed successfully at 37 for 20 min. The real℃-time RPA assay could specifically amplify the genomic DNA of LM, but not other 20 bacteria. Using 10-fold serial dilutions of the genomic DNA of LM, the data showed that the limit of detection of the assay was 5×10^-1 pg, which was the same as the real-time PCR. In the artificial contamination assays, the Listeria monocytogenes could be detected after 14 hours enrichment culture by the real-time RPA for the mutton and lettuce samples, which were inoculated initially with LM at the concentration of 3 CFU/25 g. The detection results of real-time RPA were the same as those of the traditional culture method and real-time PCR, while the reaction time was much less than the latter two assays. The developed real-time RPA assay is highly specific, highly sensitive, rapid, easy to perform, and is independent of the expensive fluorescence thermal cycler and good laboratory circumstance, which could be applied in the on-site detection of LM in the food safetyoutbreaksand is of great significance for ensuring the food safety.