Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR

A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 101 CFU/ml for P. aeruginosa, 102 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 103 CFU/ml for B. cereus, respectively. In addition, with a 6–8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.