Detection of Listeria monocytogenes and Staphylococcus aureus via duplex recombinase polymerase amplification

Foodborne illnesses associated with foodborne pathogens Listeria monocytogenes and Staphylococcus aureus occur at high frequencies. In this study, species-specific primers and probes sets for the detection of L. monocytogenes and S. aureus were designed based on the nucleotide sequences of the actin-assembly inducing protein precursor (actA) and clumping factor A (clfA) genes, respectively. We established a duplex RPA assay that is capable of simultaneously detecting L. monocytogenes and S. aureus within 15 min. With the plasmid standard, the sensitivity of the double RPA to L. monocytogenes and S. aureus can reach 10(2) copies/mu L. The method also detected DNA extracted from samples inoculated with L. monocytogenes and S. aureus. After 4 hr of incubation, the sensitivity of the food model to L. monocytogenes and S. aureus reached 230 and 270 cfu/reaction, respectively. The assay was highly specific and had no cross-reaction between closely related bacteria. Further assessment of this method on strains isolated within the lab collection showed that the duplex RPA provided the same testing results as microbiological methods. Therefore, the assay is effective for the rapid detection and preliminary screening of L. monocytogenes and S. aureus. Practical applications Listeria monocytogenes and Staphylococcus aureus are two major foodborne pathogens and have been found in a wide range of foods. We developed a duplex RPA specific targeting actA and clfA genes with high-amplification efficiency. We evaluated the performance of duplex RPA by building a food model. The detection results by the duplex RPA were compared with PCR and no significant differences were found. This study provides the information of the developed duplex RPA for detecting two specific foodborne pathogens, which will be helpful for further studies about simultaneous detection of several targets using duplex RPA.