National Research Council, Institute of Sciences of Food Production (CNR-ISPA), Bari, Italy.
Int J Food Microbiol. 2011 Jan 5;144(3):528-37. doi: 10.1016/j.ijfoodmicro.2010.11.016. Epub 2010 Nov 13.
A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc(+)S. aureus log-phase broth culture showed a good linearity of response (R²≥0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV≤3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc(+)S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc(+) mixture in the presence of 10⁶ cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ=100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ=10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc(+)S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.
开发了 TaqMan 和 SYBR Green 实时 PCR（rt-PCR），用于可靠地鉴定和定量检测携带肠毒素基因簇（egc）的金黄色葡萄球菌（S. aureus）菌株，无论其变体如何。这两种方法均显示出针对 70 个参考菌株的 100%特异性，包括 29 株临床和食源性金黄色葡萄球菌菌株，这些菌株携带了迄今为止已知的所有 egc 变体，4 株 egc⁻金黄色葡萄球菌菌株和 37 株亲缘关系密切和较远的种的菌株。通过从 egc（+）金黄色葡萄球菌对数期肉汤培养物中稀释 10 倍的基因组 DNA 或细胞制作的标准曲线，对于六个数量级的反应具有良好的线性响应（R²≥0.993），相对准确度约为 100%，且检测内变异性低（CV≤3.02）。这两种 rt-PCR 检测方法（约 100%的 PCR 效率；运行时间 30 分钟）的总体定量限（LOQ）均为 100 cfu 或 10 个基因组当量/反应混合物，尽管以 33.33%的概率检测到 1 cfu 或 1 个基因组当量。在人工污染有单个 egc（+）金黄色葡萄球菌菌株或携带迄今为止已知的所有 egc 变体的金黄色葡萄球菌菌株混合物的生奶中，对这些性能进行了确认。在存在与葡萄球菌食物中毒相关的一些最常见肠毒素基因的 egc⁻金黄色葡萄球菌菌株的 10⁶ cfu/mL 的情况下，基于生奶的金黄色葡萄球菌 egc（+）混合物标准曲线也得到了类似的结果。然而，与基于 SYBR Green 的测定法（LOQ=10 个 cfu 当量/反应混合物）相比，TaqMan 测定法的灵敏度较低（LOQ=100 cfu 当量/反应混合物）。当应用于实际牛奶样品时，与基于传统培养的 PCR 方法相比，这两种 PCR 检测方法均提供了良好的反应，具有 100%的诊断特异性和 96-107%的相对准确性。由于高特异性，宽检测动态范围以及在复杂且潜在高度污染的生奶基质中仍具有高灵敏度，因此 SYBR Green rt-PCR 测定法是一种快速，高通量且可靠的常规筛选 egc（+）金黄色葡萄球菌分离物的诊断工具。此外，在生奶中基于 SYBR Green 的这些病原体的定量检测可以极大地阐明它们在葡萄球菌食物中毒和与牛奶和牛奶产品消费相关的其他临床综合征中的实际作用。
