## 2 VBNC L. _monocytogenes_ Detection Methods
### Molecular Detection Methods
The amplification products are detected by turbidimetry and electrophoresis or with fluorescent dyes such as calcein and SYBR Green [33]. A PMA-CPA assay with a sensitivity similar to a PCR-based assay was applied for the direct detection of VBNC _L. monocytogenes_ cells in artificially contaminated rice flour. Dead bacterial cells killed by high temperature (121 \({}^{\circ}\)C) and high pressure (100 kpa) were included as a negative control and genomic DNA was included to verify no amplification from extracellular DNA. PMA was used in a final concentration of 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, with incubation at room temperature in the dark for 5 min, and exposure to light from a 500 W halogen lamp for 15 min. AfterDNA extraction, the CPA assay could quantify bacterial cells in rice flour suspension in a concentration range of 10\({}^{4}\)-10\({}^{8}\) CFU/mL [33]. A disadvantage of vPCR and other molecular viability tests based on DNA amplification is that sub-injured cells with a damaged membrane are not detected. Colorimetric sensors can aid in vPCR to detect only viable pathogens by using a streptavidin magnetic bead-dual aptamers (SMB-Apts) sandwich coupled with an AuNPs colorimetric sensor [30]. One limitation of PMA-based vPCR was the poor discrimination between viable and dead cells when applied to high levels of \(L\). _monocytogenes_ heat-treated at 60 \({}^{\circ}\)C for 6 min. False positive results were attributed to the shadowing of non-viable cells in the photo-activation step. In addition, this method might overestimate the number of viable cells since it can misidentify dead cells with an intact membrane as viable cells [41]. To increase vPCR accuracy, the use of a stable internal process control (ISPC), similar to the one developed for _Campylobacter jejuni_, was recommended to normalize and subtract the signal originating from dead cells [42]. An alternative approach to vPCR is the amplification of mRNA by specific reverse transcription followed by qPCR (RT-qPCR) tests [43]. Since mRNAs have only a few minutes of half-life, they are only detectable when cells are viable through reverse transcription to cDNA and PCR amplification [30]. A comparison among commercial RNA extraction kits based on 260/280 and 260/230 optical density (OD) ratios, RNA integrity (RIN) values, and the absence of residual gDNA after DNAse treatment was performed to select the most suitable for the quantitative detection of \(L\). _monocytogenes_. Subsequent multiplex RT-qPCR targeted on _hly_, _act_A, and a non-competitive internal amplification control (NC-IAC) allowed for obtaining LOD\({}_{50}\) and LOD\({}_{95}\) values of 1.2 CFU/25 g and 5.1 CFU/25 g, respectively, from enrichment cultures of artificially spiked smoked salmon [43]. Other studies also used _hly_ and _inLA_-targeted RT-qPCR to analyze the viability of \(L\). _monocytogenes_ in food production conditions [41; 44]. Limitations of mRNA usage for viable bacteria detection are its instability and the risk of contamination with DNA. Moreover, RNA extraction from complex food matrices still requires optimization [30]. Figure 1 reports a simplified scheme highlighting the experimental workflows that were applied for the detection of viable \(L\). _monocytogenes_ cells. Figure 1: Experimental workflows to detect viable cells of \(L\). _monocytogenes_[14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44]. The images shown were modified from illustrations available at the following websites: [[https://www.thermofishercom](https://www.thermofishercom)]([https://www.thermofishercom](https://www.thermofishercom));  
[[https://www.raybiotech.com](https://www.raybiotech.com)]([https://www.raybiotech.com](https://www.raybiotech.com)); [[https://www.neb.com](https://www.neb.com)]([https://www.neb.com](https://www.neb.com)) (accessed on 20 January 2025). The amplification curves, used exclusively for explicative purposes, were obtained from an author's experiment.