Detection and Quantification of Viable but Non-culturable .

can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the gene to detect and quantify in the VBNC state. First, we optimized the concentration of PMA (20 μM) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC in poultry products. This technique can give insight into the prevalence of VBNC in the environment and agri-food production system.