Multiplex qPCR for simultaneous quantification of the main psychrotrophic bacteria and their enzymes in raw milk

Milk spoilage is caused by proteolytic enzymes produced by psychrotrophic bacteria during storage at low temperatures. However, the current enumeration method is cultured-based and time-consuming. In this study, we developed a dual-quadruplex qPCR assay targeting both housekeeping and enzyme-coding genes of key psychrotrophic bacteria, including Pseudomonas spp., Acinetobacter spp., Stenotrophomonas maltophilia , and Bacillus cereus . The results showed that all primers and probes exhibited high specificity. A linear relationship between Ct values and colony counts in both singleplex and multiplex qPCR was confirmed, with R 2 values > 0.98 and amplification efficiencies ranging between 95% and 115%. The multiplex qPCR assay had detection limits of approximately 10 1 –10 2 CFU/mL in pure culture and spiked milk samples. The detection of psychrotrophic bacterial loads in 30 raw milk samples further validated the assay’s efficacy. This study provides a time-efficient approach for monitoring four main psychrotrophic bacteria and their enzymes in raw milk, enabling the prediction of spoilage potential. • Revised Highlights • We developed multiplex qPCR for detecting psychrotrophic bacteria and their enzymes in raw milk. • The assay is an efficient tool for predicting spoilage potential in milk and dairy products. • It is more time-efficient compared to traditional methods. • Testing on spiked and raw milk samples confirmed feasibility. • High sensitivity at 10 1 CFU/mL and specificity were demonstrated.