A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.
Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .
Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea; Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Fereidoun
F
Forghani
Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea.
Shuai
S
Wei
Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea. deoghwa@kangwon.ac.kr.
Deog-Hwan
DH
Oh
D000818:Animals; D001409:Bacillus cereus; D005516:Food Microbiology; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics*; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus