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