Dinh Thanh Mai, Agustí Gemma, Mader Anneluise, Codony Francesc
Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany.
Technical Department, Reactivos para Diagnóstico, S.L., Polígono Industrial Mas d'En Cisa, Sentmenat, Barcelona, Spain.
PLoS One. 2025 May 23;20(5):e0324819. doi: 10.1371/journal.pone.0324819. eCollection 2025.
Staphylococcus (S.) aureus is a prominent foodborne pathogen that can cause food poisoning due to its staphylococcal toxins. Controlling the viable levels of S. aureus is crucial for ensuring food safety. The detection of S. aureus during routine quality control is still primarily conducted using traditional culture-based methods, which are time-consuming and unable to detect viable but non-culturable cells. Viability PCR (vPCR) - a combination of traditional (or quantitative) PCR with photo-reactive DNA-intercalating dye(s) - has been introduced as an alternative to detect viable cells by excluding those with compromised membranes using molecular methods. Despite the success of the vPCR methodology, avoiding false-positive results remains a significant challenge. To enhance the accuracy of vPCR results for S. aureus, several approaches have been proposed by various researchers in the past decade; however, complete PCR signal suppression of dead cells has not been achieved. In this study, we developed a strategy to detect only viable S. aureus cells by combining double PMA treatment with a low PMA concentration and performing a tube change between the last dark incubation and light exposure to improve the vPCR protocol. For pure cultures, the optimized protocol was able to completely suppress DNA signals from 5.0 × 107 dead cells in a final reaction volume of 200 µl. For artificially contaminated food samples with such a high dead cell count, complete PCR signal reduction was observed in ground pepper, - oregano, and infant milk powder, while ground paprika, - allspice, and - pork exhibited PCR signals close to the detection limit. To simulate conditions in real samples, we artificially contaminated ground paprika, - pork, and milk powder with a low number of viable cells (1.9 cfu/ml) and a high number of heat-inactivated S. aureus (4.8 × 10⁶ cells/ml). The results showed that the optimized protocol is effective in detecting only the desired live cells, even in the presence of a high dead cell count. Our findings highlight that vPCR can be an accurate and reliable method with strong potential for high-throughput detection of live S. aureus cells in certain food matrices.
金黄色葡萄球菌是一种重要的食源性病原体，因其产生的葡萄球菌毒素可导致食物中毒。控制金黄色葡萄球菌的存活水平对于确保食品安全至关重要。在常规质量控制过程中，金黄色葡萄球菌的检测仍主要采用传统的基于培养的方法，这些方法耗时且无法检测到活的但不可培养的细胞。活力PCR（vPCR）——传统（或定量）PCR与光反应性DNA嵌入染料的结合——已被引入作为一种替代方法，通过分子方法排除细胞膜受损的细胞来检测活细胞。尽管vPCR方法取得了成功，但避免假阳性结果仍然是一个重大挑战。为了提高金黄色葡萄球菌vPCR结果的准确性，在过去十年中，不同的研究人员提出了几种方法；然而，尚未实现对死细胞的完全PCR信号抑制。在本研究中，我们开发了一种策略，通过结合低浓度PMA的双重PMA处理，并在最后一次黑暗孵育和光照之间进行换管操作来改进vPCR方案，从而仅检测活的金黄色葡萄球菌细胞。对于纯培养物，优化后的方案能够在最终反应体积为200微升的情况下完全抑制5.0×10⁷个死细胞的DNA信号。对于死细胞数量如此之高的人工污染食品样品，在磨碎的胡椒、牛至和婴儿奶粉中观察到了完全的PCR信号降低，而磨碎的辣椒粉、多香果和猪肉的PCR信号接近检测限。为了模拟实际样品中的情况，我们用少量活细胞（约1.9 cfu/ml）和大量热灭活的金黄色葡萄球菌（约4.8×10⁶个细胞/ml）人工污染磨碎的辣椒粉、猪肉和奶粉。结果表明，即使在死细胞数量很高的情况下，优化后的方案也能有效地仅检测到所需的活细胞。我们的研究结果突出表明，vPCR可以成为一种准确可靠的方法，在某些食品基质中对活的金黄色葡萄球菌细胞进行高通量检测具有很大潜力。
