## 2 VBNC L. _monocytogenes_ Detection Methods
### Molecular Detection Methods
Therefore, sodium deoxycholate (SD), an anionic surfactant that disrupts the cell membranes of damaged or dead cells, was used to enhance \(L\). _monocytogenes_ dead cell permeability. APMA concentration of 20 \(\upmu\)M and 0.01% SD with an exposure time of 5 min were found to optimally protect the viable cells while at the same time inhibiting amplification from non-viable cells. However, an SD concentration higher than 0.02% caused the inhibition of amplification also from viable cells [38]. In some studies, sodium dodecyl sulfate (SDS) was used to decrease amplification from dead cells before treatment with PMA by enhancing the permeability of the membranes, but this practice is not easily applicable to bacteria at low concentrations and complex food matrices [35]. Immunomagnetic separation (IMS) allows researchers to selectively bind and separate foodborne pathogens from a food suspension, thus eliminating the matrix interference effect. However, its efficiency can be reduced by minor changes in pH, monodispersity, and cross-linking time, which influence the activity of the antibody adsorbed on the immunomagnetic beads. Therefore, an IMS method based on the streptavidin-biotin system was developed to enhance capture capability. However, antibodies were required in high amounts to enhance the capture efficiency. A novel biotin exposure-based IMS assay coupled with SDS and multiplex vPCR with PMA was developed for the rapid detection of viable bacterial pathogens including _L. monocytogenes_ in milk. Biotinylated antibodies were used to bind to the cells; then, streptavidin-coated magnetic beads (SA-MB) were used to concentrate the pathogens that were treated with SDS and PMA. Finally, the genomic DNA was extracted and used in multiplex vPCR. The sensitivity of detection ranged between 10 and 10\({}^{3}\) CFU/mL for the target bacteria and the limit of detection (LOD) in artificially contaminated milk was 10 CFU/mL for all pathogens, showing an increased sensitivity compared to the test without IMS [35]. Gold nanoparticles (AuNPs), silver nanoparticles ( AgNPs), silver nanowires, and polyethylenimine-coated Fe\({}_{3}\)O\({}_{4}\) (PEI@Fe\({}_{3}\)O\({}_{4}\)) were tested as qPCR signal enhancers able to increase the normalized fluorescence (\(\Delta\)Rn) value. AuNPs with 20 nm particle size and 100-fold dilution displayed the highest signal enhancement with an increase of 20%. The sensitivity of the AuNPs-SD-PMA-qPCR assay was 5 \(\times\) 10\({}^{2}\) CFU/mL for _L. monocytogenes_ detected simultaneously with _Salmonella enterica_ in enrichment cultures from dairy products. Thermal lysis for 15 min for DNA extraction reduced the detection time and the number of needed reagents by improving portability for on-site detection. The use of AuNPs can be easily implementable because it is non-toxic, highly biocompatible, not expensive, and easily produced and modified [38]. Isothermal amplification techniques coupled with intercalating dyes were introduced in recent years as an alternative to vPCR. One of these is loop-mediated isothermal amplification (LAMP), a cyclic strand displacement DNA synthesis process that allows the synthesis of 10\({}^{6}\)-10\({}^{9}\) copies of target DNA in less than 1 h at a constant temperature of 60-65 \({}^{\circ}\)C. Amplification is visualized in real-time for the formation of a white magnesium pyro-phosphate precipitate that can be measured with a turbidimeter or by adding a fluorescent dye, such as Sybr Green I, to visualize amplification under UV light. The LAMP test is less sensitive to inhibition by the food matrix compared to PCR and can be carried out in water baths or heaters, which makes it a portable method [28; 30; 33; 39]. The specificity is high since up to six specific primers matching the target regions are used. These include forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3), forward loop primer (FLP), and backward loop primer (BLP).