Abstract：
The accurate quantification of viable pathogens in food is crucial for ensuring food safety. This study mainly aimed to investigate the quantification of viable pathogens using PMA-qPCR and RT-qPCR, taking into account bacterial species, food matrices, and inactivation methods. The detection limit of PMA-qPCR for Salmonella serovars in simple matrices, such as culture broth, lake, or tap water, was found to be 102 cells per ml. Regarding the detection of Staphylococcus aureus and Escherichia coli in culture broth, as well as Salmonella in more complex matrices, such as juices and lab-made broth, both methods exhibited a detection limit of 103 cells per ml. Besides that, in adverse situations, there was a risk of overestimating the number of viable pathogens using PMA-qPCR. In addition, a conspicuous discrepancy between the results of PMA-qPCR/RT-qPCR and those of the plate counting assay was observed when Salmonella was exposed to isopropanol, H2O2, NaClO, sonication, or thermosonication. This suggests that it may survive in a viable but non-culturable state and poses a challenge for accurate quantification of viable cells using plate counting assay. Therefore, the results obtained by RT-qPCR were more objective compared to PMA-qPCR due to potential influences from bacteria species, surrounding media, and inactivation methods.
摘要：
准确定量食品中的活病原体对于确保食品安全至关重要。本研究的目的是探讨PMA-qPCR和RT-qPCR对病原菌的定量,考虑到细菌种类，食物矩阵，和失活方法。简单基质中沙门氏菌血清变型的PMA-qPCR检测限，如培养肉汤，湖,或者自来水，被发现是每毫升102个细胞。关于培养肉汤中金黄色葡萄球菌和大肠杆菌的检测，以及更复杂的基质中的沙门氏菌，比如果汁和实验室制作的肉汤，两种方法的检出限均为每毫升103个细胞。除此之外,在不利的情况下，使用PMA-qPCR有高估活病原体数量的风险.此外,当沙门氏菌暴露于异丙醇时，PMA-qPCR/RT-qPCR的结果与平板计数测定的结果之间存在明显差异，H2O2,NaClO,超声处理，或热声处理。这表明它可以在VBNC状态下存活,并且对使用板计数测定准确定量活细胞提出挑战。因此,由于细菌种类的潜在影响，与PMA-qPCR相比，RT-qPCR获得的结果更客观，周边媒体,和失活方法。
