Forghani Fereidoun, Wei Shuai, Oh Deog-Hwan
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.
Department of Bioconvergence Science and Technology, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea.
J Food Prot. 2016 May;79(5):810-5. doi: 10.4315/0362-028X.JFP-15-428.
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 .
三种重要的食源性病原体，即蜡样芽孢杆菌、单核细胞增生李斯特菌和金黄色葡萄球菌，是食品安全领域极为关注的对象。它们也可能在食品基质中共存，而且就蜡样芽孢杆菌和金黄色葡萄球菌而言，由于症状相似，由此引发的疾病可能彼此相似。因此，同时检测它们在成本节约和速度方面可能具有优势。在此背景下，开发了一种用于同时检测食品中这三种病原体的快速多重实时聚合酶链反应高分辨率熔解曲线分析方法。该分析方法在单一反应中成功检测到了蜡样芽孢杆菌（gyrB）、单核细胞增生李斯特菌（hly）和金黄色葡萄球菌（nuc），其平均熔解温度分别为76.23、80.19和74.01°C。SYTO9染料的应用以及缓慢的熔解曲线分析升温速率（0.1°C/秒）使得能够产生尖锐的、易于彼此区分的高分辨率熔解曲线峰。在食品（牛奶、大米和生菜）中的检测限在不进行富集步骤时为3.7×10³CFU/克，在10小时富集后为3.7×10¹CFU/克。因此，这里开发的分析方法具有特异性和灵敏性，为在食品中同时检测蜡样芽孢杆菌、单核细胞增生李斯特菌和金黄色葡萄球菌提供了一种有效的工具。
