Development of a multiplex real‐time PCR for simultaneous detection of <scp><i>Bacillus cereus</i></scp>, <scp><i>Listeria monocytogenes</i></scp>, and <scp><i>Staphylococcus aureus</i></scp> in food samples

<jats:title>Abstract</jats:title><jats:sec><jats:label/><jats:p> <jats:styled-content style="fixed-case"><jats:italic>Bacillus cereus</jats:italic></jats:styled-content>, <jats:styled-content style="fixed-case"><jats:italic>Listeria monocytogenes</jats:italic></jats:styled-content>, and <jats:styled-content style="fixed-case"><jats:italic>Staphylococcus aureus</jats:italic></jats:styled-content> 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<jats:sup>8</jats:sup> to 10<jats:sup>2</jats:sup> cfu/ml) with high amplification efficiencies ranging from 94.2 to 105.4% with <jats:italic>R</jats:italic>‐squares over 0.999. When multiplex qPCR was applied for artificially contaminated cherry tomato, milk, and spam samples, a detection limit of 10<jats:sup>3</jats:sup> 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 <jats:styled-content style="fixed-case"><jats:italic>B. cereus</jats:italic></jats:styled-content> 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 <jats:styled-content style="fixed-case"><jats:italic>B. cereus</jats:italic></jats:styled-content>, <jats:styled-content style="fixed-case"><jats:italic>L. monocytogenes</jats:italic></jats:styled-content>, and <jats:styled-content style="fixed-case"><jats:italic>S. aureus</jats:italic></jats:styled-content> in food samples.</jats:p></jats:sec><jats:sec><jats:title>Practical applications</jats:title><jats:p> <jats:italic><jats:styled-content style="fixed-case">Bacillus cereus</jats:styled-content>, <jats:styled-content style="fixed-case">Listeria monocytogenes</jats:styled-content>,</jats:italic> and <jats:styled-content style="fixed-case"><jats:italic>Staphylococcus aureus</jats:italic></jats:styled-content> are three major foodborne pathogens and have been found in a wide range of foods. We developed a multiplex qPCR specific targeting <jats:italic>groEL</jats:italic>, <jats:italic>iap</jats:italic>, and <jats:italic>nuc</jats:italic> genes with a high amplification efficiency. Different food samples (cherry tomato, milk, and spam) were tested for evaluating the performance of the multiplex qPCR. The detection results by qPCR were compared with the conventional culture methods and no significant differences were found using Mantel–Haenszel chi‐square test. This study provides the information of the developed multiplex qPCR for detecting three specific foodborne pathogens and applications in food samples, which will be helpful for further studies about simultaneous detection of several targets in food samples using multiplex PCR.</jats:p></jats:sec>