Detection of Listeria monocytogenes in food by fluorescent recombinase-aided amplification

Abstract:

[Background] Listeria monocytogenes is a common contaminated foodborne pathogen in meat and dairy products. The traditional culture method can not meet the rapid detection requirements of large quantities of food at the port. It is very important to establish a simple, sensitive, fast and field operable technology. [Objective] To establish a rapid and simple method for the detection of Listeria monocytogenes by recombinase-aided amplification (RAA) to meet the actual needs of port rapid customs clearance and supervision. [Methods] According to the conserved region of hlyA gene of Listeria monocytogenes, the specific primers and probes were designed. The best primer combination with the best amplification efficiency and sensitivity was selected by the combination of primers and probes. The optimal reaction conditions were determined by optimizing the reaction temperature and the concentration of primers and probes. The established fluorescence RAA method was applied to the detection of food matrix and actual samples, and compared with the national standard GB 4789.30-2016. [Results] The optimum reaction temperature of fluorescence RAA of Listeria monocytogenes was 42 ℃, and the final concentration of primer and probe was 400 nmol/L. The established fluorescence RAA method showed high specificity and the sensitivity, and the detection limit of the method was 3×10² CFU/mL in pure culture. The LOD for Listeria monocytogenes was 0.3 CFU/mL, 3 CFU/mL and 30 CFU/mL original concentrations under 4 h LB2 enrichment in artificially contaminated beef, Atlantic salmon and processed cheese respectively. The RAA assay produced a positive signal in 5 min, and the whole assay could be completed in approximately 20-30 min. The speed and sensitivity were significantly higher than those of the national standard method. [Conclusion] The fluorescence RAA method can be used for rapid detection and monitoring of Listeria monocytogenes at ports or other places.