## 2 VBNC L. _monocytogenes_ Detection Methods
### Molecular Detection Methods  
Molecular methods have been widely used in detecting VBNC bacteria in food matrixes and water for their high sensitivity, specificity, and rapidity. The most commonly adopted method is quantitative polymerase chain reaction (qPCR) applied to cells treated with DNA intercalating dyes before DNA extraction [29; 30]. This method is designated as vPCR and allows an accurate quantification of only viable bacteria. It consists of applying a specific qPCR test to DNA extracted from cells treated with dyes, which enter into the cells with a damaged membrane and bind to their DNA blocking amplification after exposure to light. In theory, DNA intercalating dyes cannot penetrate intact cell membranes [18; 30; 33; 34; 35]. Unlike standard culture detection methods applied to bacterial pathogen detection in food, vPCR provides results before products with a short shelf life are sold out [30]. If qPCR is carried out with and without pre-treatment with a DNA intercalating dye, total and viable cells can be quantified separately [36]. This method must be validated for each matrix to avoid the interference of dead cells with intact membranes on VBNC cell quantification [29]. Ethidium monoazide (EMA) was the first DNA intercalating dye to be used to prevent the detection of dead cells [30; 34] but presented limitations such as the extent of penetration influenced by the species of microorganisms and cytotoxic effects on viable cells [29; 30]. Moreover, the usefulness of EMA is also limited by the ability of the dye to penetrate viable cells with consequent underestimation of their number [18]. Propidium monoazide (PMA) has a lower toxicity than EMA and a higher selectivity for cells with damaged membranes for its double positive charge, differently from EMA, which has only one positive charge [30]. The azide group of PMA allows its crosslinking with the DNA of dead cells, which is permanently modified while the DNA of viable cells remains intact and can be detected in the subsequent PCR reaction. The PMAxx derivative of PMA exerts better discrimination between viable and dead cells in complex matrices [37], and a combination of EMA and PMAxx improves selectivity for viable cells. Indeed, EMA is effective in distinguishing dormant cells that lack active efflux pumps to expel the dye. The mixture of these two dyes is commercially available under the name PEMAX [29]. In the vPCR, dye-treated samples are exposed to light, for example, from the blue light PMA-Lite LED or a 500-650 W halogen lamp, and dye crosslinking to DNA is usually allowed to occur for 5-15 min. Bacterial cells are concentrated by centrifugation and used for DNA extraction. These can also be stored at \(-20\)\({}^{\circ}\)C until DNA extraction. The theoretical limit of quantification (LOQ) for \(L\). _monocytogenes_ in water and fresh produce by vPCR with PMAxx was 2.4 and 1.0 Log cells/100 mL or gram, respectively, with primers targeting the Listeriolysin O _hly_ gene. The numbers of VBNC cells were determined as the difference between viable bacteria quantified by vPCR and plate counts [29; 30; 20]. Before the vPCR tests, several sample pretreatments must be carried out. For example, solid dairy products are homogenized in phosphate buffer saline (PBS) or trisodium citrate solution, fat and proteins are removed by centrifugation, and the pellet is collected. For liquid samples such as milk and fermented milk, no homogenization is needed. The pellets are then re-suspended in a solution transparent to light mixed with the dye solution, incubated in the dark for a minimum of 5 min at room temperature, and exposed to light while held on ice for 2-5 min to induce dye crosslinking. Finally, pelleted cells are used for DNA extraction and vPCR [29]. It was reported that PMA treatment does not completely abolish amplification from dead cells possibly because cell debris hinders its penetration through the membrane.