Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa in food

Food and beverage poisoning is detrimental to people's health since it can lead to fever, stomachaches, and even death. To rapidly detect the presence of foodborne pathogens, conventional PCR assays are currently widely employed. Meanwhile, isothermal PCR methods, in which the amplification reactions take place at a low and constant temperature, have lately emerged as effective and alternative means for quickly identifying pathogens in low-resource settings. Staphylococcus aureus and Pseudomonas aeruginosa are two of the most concerning foodborne bacterial infections. In this work, an isothermal PCR assay based on the Recombinase Polymerase Amplification (RPA) method was developed to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit of detection for multiplex RPA was 10 and 30 fg/reaction of S. aureus and P. aeruginosa genomic DNA, respectively. Furthermore, the reaction time was reduced to only 25 minutes, with a low incubation temperature of 39°C. Multiplex RPA reactions, in particular, were successful in directly identifying as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively, without the need for DNA genome extraction. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice, and bottled water samples. In conclusion, the direct multiplex RPA reported in this work offers a quick, easy, sensitive, and effective alternative approach for detecting the presence of S. aureus and P. aeruginosa without the requirement of a pricey instrument or highly-trained personnel.