Development of multiplex loop-mediated isothermal amplification for three foodborne pathogens

Abstract Staphylococcus aureus, Salmonella, and Shigella are three major foodborne pathogenic microorganisms that cause global public health problems. We developed a multiplex loop-mediated isothermal amplification (mLAMP) assay for simultaneous detection of S. aureus nuc, Salmonella fimY, and Shigella ipaH in fresh fruit juice using three sets of primers. In addition, three different restriction enzyme cleavage sites were designed in each forward inner primer (FIP), namely, XhoI in nuc FIP, KpnI in fimY FIP, and BamHI in ipaH FIP. DNA was amplified using the LAMP assay at 64 °C for 50 min followed by endonuclease restriction digestion to separate the LAMP products of three pathogens. The minimum amount of genomic DNA of S. aureus, Salmonella, and Shigella that could be detected by mLAMP was 100 fg/25 μL, whereas for mPCR, it was 1 pg/25 μL. The artificially contaminated juice can also be detected by mLAMP after enrichment, which had the limit of detection (LOD) of 2 CFU/10 mL. In conclusion, the mLAMP developed in this study could be potentially used in the detection of S. aureus, Salmonella, and Shigella in food, particularly as a primary screening method in developing areas.