## 2 VBNC L. _monocytogenes_ Detection Methods
### Molecular Detection Methods
The sensitivity of LAMP was found to be higher than that of qPCR with an LOD of 6 CFU/reaction for _L. monocytogenes_. LAMP coupled with PMA or EMA proved faster and 100-fold more sensitive than vPCR for food samples [30]. Most LAMP methods do not include an internal amplification control (IAC) due to the difficulty of multiplexing the reaction, and LAMP amplification products are differentiated based on their melting temperatures. However, an _L. monocytogenes_-specific test was developed to comprise an IAC constituted by a plasmid containing the original target fragment with the inserted sequence CCATCCGACGGCGGCGGCGCC, which increased the Tm of the IAC fragment, allowing its distinction by melting curve analysis. This LAMP method effectively blocked amplification from up to 10\({}^{6}\) CFU/mL dead cells, and its LOD\({}_{95}\) defined by plate counts was 2.7 CFU/25 mL of milk. The performance of the method was tested on a total of 59 spiked milk samples but not on VBNC cells [39]. Competitive annealing mediated isothermal amplification (CAMP) is another isothermal amplification reaction with incubation at 60-65 \({}^{\circ}\)C for 60 min and with a simpler primer design compared to LAMP. It was tested for the detection of _L. monocytogenes_ in milk and milk powder and was coupled with PMA cross-linking or to PMA and hydroxynaphthol (HNB) to visualize amplification by color change (violet to blue). Visual detection in this method was also achieved by using gold nanoparticle probes (AuNP) whose dispersion and aggregation can be visually detected based on color change. In negative samples, nanoparticles form aggregates conferring a red or blue-grey color to the solution, while in positive samples, the AuNP probe hybridizes with the target and remains dispersed with no color change [30]. A novel technology was developed to detect viable _L. monocytogenes_ based on PMA-LAMP with four primers combined with nanozyme strips that are cost-effective and equipment-free with the possibility of visual detection. This technology includes using BIO- and FITC-modified primers targeting the _lhyA_ gene and a magnetic nanozyme modified with a biotin-conjugated antibody specifically for _L. monocytogenes_. The peroxidase-like activity of the magnetic nanozyme generated a visually readable color, and quantitative detection could be achieved by measuring the optical intensity of the test line (T line) by ImageJ with a detection limit of 5.4 fg/mL [40]. Fe\({}_{3}\)O\({}_{4}\) NPs' S15 nanozyme were tested for viable _L. monocytogenes_ detection after mixing with the amplification products of PMA-LAMP and allowed to react with nitrocellulose (NC) membranes on which anti-mouse immunoglobulin G (IgG) and anti-FITC antibodies were immobilized to form C and T lines, respectively. If _L. monocytogenes_ was present in the sample, the PMA-LAMP products conjugated with the nanozyme probe by the combination of BIO and anti-biotin antibodies. One side of positive products was labeled with Fe\({}_{3}\)O\({}_{4}\) NPs while the other side was labeled with FITC so that the amplification products could be captured by anti-FITC antibody on the T line while the free nanozyme probes stopped in the C line. The color reaction was enhanced by the enzymatic reaction between the nanozyme and 3,3-diaminobenzzidine (DAB)/H\({}_{2}\)O\({}_{2}\) enzymatic substrate. If viable _L. monocytogenes_ was absent, only the C line appeared; if no color appeared, the result was not considered valid. The method allowed the detection of 1.164 \(\times\) 10 CFU/mL and 1.021 \(\times\) 10 CFU/mL _L. monocytogenes_ in biofilms developed on stainless steel and lettuce, respectively [40]. Other isothermal amplification techniques include polymerase spiral reaction (PSR) and cross-priming amplification (CPA). The CPA assay was applied to the _L. monocytogenes_ _hylA_ gene using calcein for product visualization. It involves several primers and probes, with one or more cross primers, and exploits the _Bst_ DNA polymerase for strand displacement of the target DNA.