Establishment of a Sensitive and Visual Detection Platform for Viable Salmonella in Wastewater That Combines Propidium Monoazide with Recombinase Polymerase Amplification-CRISPR/Cas12a System.

Urban sewage, aquaculture wastewater, and medical wastewater are significant reservoirs and transmission sources of Salmonella. Rapid detection of Salmonella is crucial for effectively reducing the risk of disease transmission and safeguarding public health. Differentiating viable Salmonella from inactivated cells presents significant challenges, affecting the accurate assessment of pathogen risks. Moreover, current detection methods face several limitations, including lengthy detection periods, high costs, and limited applicability, underscoring the need for rapid, sensitive, and visual detection diagnostic approaches. In this study, we combined propidium monoazide (PMA) with recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeats (CRISPR)/Cas12a systems to develop a rapid detection system for viable Salmonella targeting the fimY gene. DNA of viable Salmonella was amplified and visually detected within 60 min and dead cells were effectively excluded. We assessed the specificity and sensitivity of the PMA-RPA-CRISPR/Cas12a assay. The results showed that the assay had a high level of specificity, with no reactions observed with other pathogens. The application of PMA has no effect on the sensitivity of RPA-CRISPR/Cas12a technology and the visibility of the fluorescence reporting system. We successfully detected viable Salmonella in wastewater with a minimum detection limit of 10¹ CFU/mL. In summary, the PMA-RPA-CRISPR/Cas12a system developed in this study allows for the rapid and visual detection of viable Salmonella in wastewater at concentrations as low as 10¹ CFU/mL. By integrating PMA with the RPA-CRISPR/Cas12a technology, this system offers valuable technical support for the efficient, sensitive, and clear detection of viable Salmonella in wastewater.