Simultaneous Detection ofEscherichia coli O157:H7,Salmonella, andShigella in Apple Cider and Produce by a Multiplex PCR†

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157: H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 × 10−1 CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 × 101 CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.