State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China.
J Dairy Sci. 2017 Nov;100(11):8804-8813. doi: 10.3168/jds.2017-13362. Epub 2017 Aug 31.
Escherichia coli O157:H7 and Salmonella spp. in milk are 2 common pathogens that cause foodborne diseases. An accurate, rapid, specific method has been developed for the simultaneous detection of viable E. coli O157:H7 and Salmonella spp. in milk. Two specific genes, namely, fliC from E. coli O157:H7 and invA from Salmonella spp., were selected to design primers and probes. A combined treatment containing sodium deoxycholate (SDO) and propidium monoazide (PMA) was applied to detect viable E. coli O157:H7 and Salmonella spp. only. Traditional culture methods and SDO-PMA-multiplex real-time (mRT) PCR assay were applied to determine the number of viable E. coli O157:H7 and Salmonella spp. in cell suspensions with different proportions of dead cells. These methods revealed consistent findings regarding the detected viable cells. The detection limit of the SDO-PMA-mRT-PCR assay reached 10 cfu/mL for Salmonella spp. and 10 cfu/mL for E. coli O157:H7 in milk. The detection limit of SDO-PMA-mRT-PCR for E. coli O157:H7 and Salmonella spp. in milk was significantly similar even in the presence of 10 cfu/mL of 2 nontarget bacteria. The proposed SDO-PMA-mRT-PCR assay is a potential approach for the accurate and sensitive detection of viable E. coli O157:H7 and Salmonella spp. in milk.
大肠杆菌 O157:H7 和沙门氏菌是引起食源性疾病的 2 种常见病原体，存在于牛奶中。本研究建立了一种快速、特异、准确检测牛奶中活的大肠杆菌 O157:H7 和沙门氏菌的方法。选择大肠杆菌 O157:H7 的 fliC 基因和沙门氏菌的 invA 基因作为靶基因，设计引物和探针。采用含脱氧胆酸钠（SDO）和丙啶单核酸（PMA）的联合处理方法，特异性检测活的大肠杆菌 O157:H7 和沙门氏菌。分别采用传统培养方法和 SDO-PMA-多重实时 PCR（mRT-PCR）法检测不同比例死细胞悬液中活的大肠杆菌 O157:H7 和沙门氏菌的数量。这 2 种方法检测活细胞的结果一致。SDO-PMA-mRT-PCR 法检测牛奶中沙门氏菌和大肠杆菌 O157:H7 的检测限均为 10 cfu/mL。即使在存在 10 cfu/mL 非靶标 2 种菌的情况下，该方法检测牛奶中大肠杆菌 O157:H7 和沙门氏菌的检测限也无显著差异。本研究建立的 SDO-PMA-mRT-PCR 方法可用于准确、灵敏地检测牛奶中的活的大肠杆菌 O157:H7 和沙门氏菌。
