Abstract A sodium dodecyl sulphate (SDS)-propidium monoazide (PMA)-multiplex real-time PCR (mRT-PCR) assay for accurate and rapid detection of viable Listeria monocytogenes, Cronobacter sakazakii, Staphylococcus aureus and Salmonella spp. was established. The SDS and PMA pretreatments were used to eliminate the false-positive results caused by dead cells in mRT-PCR, in which the SDS could improve the membrane permeability of dead cells to PMA without affecting viable cells. Optimisation studies indicated that SDS and PMA at 100 μg mL−1 and 40 μ m , respectively, were the most appropriate. Using the optimised parameters, the detection limit of SDS-PMA-mRT-PCR for L. monocytogenes, C. sakazakii, S. aureus and Salmonella spp. in spiked milk was 102 cfu mL−1, and the total assay time took less than 7 h. In conclusion, the SDS-PMA-mRT-PCR method developed in this study can accurately and quickly detect the combined contamination of viable L. monocytogenes, C. sakazakii, S. aureus and Salmonella spp. in milk.

中文翻译：

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多重实时 PCR 结合十二烷基硫酸钠和单叠氮化丙啶，可同时检测单核细胞增生李斯特菌、坂崎克罗诺杆菌、金黄色葡萄球菌和沙门氏菌。在牛奶中

摘要 一种十二烷基硫酸钠 (SDS)-单叠氮化丙啶 (PMA)-多重实时 PCR (mRT-PCR) 检测，用于准确和快速检测可行的单核细胞增生李斯特菌、坂崎克罗诺杆菌、金黄色葡萄球菌和沙门氏菌。建立了。SDS和PMA预处理用于消除mRT-PCR中由死细胞引起的假阳性结果，其中SDS可以提高死细胞对PMA的膜通透性而不影响活细胞。优化研究表明，分别为 100 μg mL-1 和 40 μm 的 SDS 和 PMA 是最合适的。使用优化的参数，SDS-PMA-mRT-PCR 对单核细胞增生李斯特菌、阪崎肠杆菌、金黄色葡萄球菌和沙门氏菌的检测限。加标牛奶中的浓度为 102 cfu mL-1，总检测时间不到 7 小时。综上所述，本研究开发的 SDS-PMA-mRT-PCR 方法可以准确快速地检测活的单核细胞增生李斯特氏菌、阪崎肠杆菌、金黄色葡萄球菌和沙门氏菌的联合污染。在牛奶中。