Bohaychuk V M, Gensler G E, McFall M E, King R K, Renter D G
Agri-Food Laboratories Branch, Food Safety Division, Alberta Agriculture and Food, 6909-116 Street, Edmonton, Alberta, Canada.
J Food Prot. 2007 May;70(5):1080-7. doi: 10.4315/0362-028x-70.5.1080.
Conventional culture methods have traditionally been considered the "gold standards" for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.
传统上，常规培养方法一直被视为食源性病原体分离和鉴定的“金标准”。然而，培养方法 labor-intensive 且耗时。我们开发了一种实时 PCR 检测方法，用于检测各种食品和食用动物基质中的沙门氏菌。该实时 PCR 检测方法基于沙门氏菌 invA 基因序列设计了引物和杂交探针，并使用荧光共振能量转移技术以确保获得高度灵敏和特异的结果。该方法正确分类了 51 株沙门氏菌实验室分离株和 28 株非沙门氏菌菌株。该方法还通过大量现场样本进行了验证，这些样本包括猪粪便和盲肠内容物、猪胴体、牛粪便和牛胴体、家禽盲肠内容物和胴体、马粪便、动物饲料以及各种食品。将 3388 份样本在缓冲蛋白胨水中进行预增菌，然后在四硫磺酸盐和 Rappaport-Vassiliadis 肉汤中进行选择性增菌。将选择性增菌肉汤的等分试样合并用于 DNA 提取，并通过实时 PCR 检测进行分析。与培养方法相比，该 PCR 检测方法对各种基质的诊断敏感性范围为 97.1%至 100.0%，诊断特异性范围为 91.3%至 100.0%。Kappa 值范围为 0.87 至 1.00，表明实时 PCR 检测方法与培养方法具有极好的一致性。时间和人力的减少使得这种高度灵敏和特异的实时 PCR 检测方法成为常规培养方法的极佳替代方法，可用于监测和研究以提高食品安全。
