Simultaneous direct detection of Salmonella spp., Listeria Monocytogenes and Escherichia Coli o157 in milk samples by magnetic extraction and multiplex PCR

The aim of our work was to develop a new molecular method for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 directly in milk samples. Three specific target sequences were chosen for a multiplex polymerase chain reaction (PCR) assay: a 155-bp region of the Salmonella spp. tetrathionate reductase (ttr) locus; a 173-bp region of the L. monocytogenes listeriolysin O gene (hlyA); a 217-bp region of the E. coli O157 lipopolysaccharide gene (rfbE). An internal amplification control was also included to detect PCR inhibition. In addition, a magnetic-based extraction method was also developed to isolate PCR-ready DNA from milk. The assay was able to detect, whether alone or mixed, also with a difference of 2 log units, as few as 102 cells of each pathogen per 10 mL of spiked milk.