Establishment and evaluation of a rapid detection method on viable cells of Salmonella enterica: A potential POCT applicable in various food systems

As a common foodborne pathogen, Salmonella enterica can enter into the viable but nonculturable (VBNC) state, thus cause false negative detection by culture-based methods. This study aimed to establish a propidium monoazide (PMA)-polymerase spiral reaction (PSR) for rapid detection on viable cells of S. enterica. Firstly, to ensure the stability and effectiveness of PMA-PSR assay, optimization of PSR assay which is critical for stability was performed. The optimized PSR assay was established with reaction time at 60 min, reaction temperature at 65 degrees C, betaine concentration at 0.6 M and chromogenic reagent with calcein and Mn2+ ratio at 1:4. Calcein was used as an indicator to ensure naked eye result determination and avoid false positive detection. Secondly, the specificity and sensitivity of PSR assay were examined to ensure effective detection of S. enterica. The optimized PSR assay had 100 % specificity and the detection limit was 10 copies of plasmid pINVA and 123 pg/mu L of genomic DNA. Thirdly, considering the complexity of food matrix, 12 S. enterica frequently contaminated food systems covering liquid and solid foods were included to construct artificially contaminated food models. The detection limit of the optimized PSR assay was 103 CFU/mL in liquid food samples and 104 CFU/mL in solid food samples. Fourthly, PMA treatment, which is critical for specific identification of viable cells, was combined with PSR assay to establish PMA-PSR assay. Lastly, the PMA-PSR assay was applied in 12 food types to ensure its applicability to detect viable S. enterica cells in various food systems. PMA-PSR assay was successfully established to detect VBNC cells of S. enterica and applied in various food systems. Considering its rapidity, sensitivity and simplicity, especially naked eye results determination, the PMA-PSR assay is a potential POCT for viable cells detection in various food systems.