Multiplex real-time PCR coupled with sodium dodecyl sulphate and propidium monoazide for the simultaneous detection of viable Listeria monocytogenes, Cronobacter sakazakii, Staphylococcus aureus and Salmonella spp. in milk

Abstract

A sodium dodecyl sulphate (SDS)-propidium monoazide (PMA)-multiplex real-time PCR (mRT-PCR) assay for accurate and rapid detection of viable Listeria monocytogenes, Cronobacter sakazakii, Staphylococcus aureus and Salmonella spp. was established. The SDS and PMA pretreatments were used to eliminate the false-positive results caused by dead cells in mRT-PCR, in which the SDS could improve the membrane permeability of dead cells to PMA without affecting viable cells. Optimisation studies indicated that SDS and PMA at 100 μg mL−1 and 40 μm, respectively, were the most appropriate. Using the optimised parameters, the detection limit of SDS-PMA-mRT-PCR for L. monocytogenes, C. sakazakii, S. aureus and Salmonella spp. in spiked milk was 102 cfu mL−1, and the total assay time took less than 7 h. In conclusion, the SDS-PMA-mRT-PCR method developed in this study can accurately and quickly detect the combined contamination of viable L. monocytogenes, C. sakazakii, S. aureus and Salmonella spp. in milk.