Development of a multiplex real-time PCR for simultaneous detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in food samples

摘要

Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus caused problems in public health and food safety. A multiplex real-time PCR (qPCR) for simultaneous detection of these three pathogens in different kinds of food was developed. A high specificity (100%) was obtained using 21 target strains and 19 nontarget strains. Standard curves for pure cultures covered seven orders of magnitude (from 10(8) to 10(2) cfu/ml) with high amplification efficiencies ranging from 94.2 to 105.4% with R-squares over 0.999. When multiplex qPCR was applied for artificially contaminated cherry tomato, milk, and spam samples, a detection limit of 103 cfu/g or ml was obtained for these three bacteria. When low levels (0.4-5.5 cfu/25 g or ml) of bacteria were inoculated in three kinds of food samples and cultured in tryptic soy broth for 24 hr, results obtained from multiplex real-time and conventional culture methods were not significantly different for all three food matrices based on Mantel-Haenszel chi-square test. Only for B. cereus in milk, positive portions detected by qPCR were significantly higher than those detected by culture method. Hence, the multiplex qPCR developed in this study is highly specific and effective for simultaneous detection B. cereus, L. monocytogenes, and S. aureus in food samples.