## 7 Discussion and Future Perspectives  
From the recent scientific studies, it emerged that most investigations were aimed at identifying the stress factors that induce _L. monocytogenes_ VBNC formation in conditions mimicking those existing in food and food production plants, especially analyzing the effects of the most common sanitizers [20; 29; 37; 47]. The evidence that four of these, i.e., chlorine, PAA, BC, and CIO2, led to the formation of VBNC populations appears particularly worrisome for the consequences that can arise from the presence of viable _L. monocytogenes_ in RTE products that go undetected by the standard culturing procedures. Those findings indicated that the VBNC issue should not be ignored and that the highly sensitive molecular methods of VBNC cell detection, such as FC coupled with viability staining, vPCR, isothermal amplification methods coupled with DNA intercalating dyes, and mRNA detection, should be optimized and validated to develop shared procedures for routine application together with the standard methods for the control of this pathogen. In particular, the methods based on mRNA detection, which are those requiring a minimum number of phases compared to the DNA-based methods, were applied only in a few instances and have not yet been compared to the DNA-based methods for the sensitivity and specificity parameters [41; 43; 44]. In addition, a highly sensitive detection method not prone to the presence of amplification inhibitors--namely, Droplet digital PCR (ddPCR) --though being widely used to detect foodborne pathogens with low concentration in samples, has not yet been applied for the detection of VBNC _L. monocytogenes_. In this technique, the PCR mixture is divided into nanodroplets, and the target DNA copy number in the sample can be absolutely quantified according to the number of positive droplets without the need for a standard curve [28]. The application of ddPCR coupled with PMA treatment has already been tested with _Salmonella_ Typhimurium with promising results in terms of sensitivity [54]. Beyond mRNA, rRNA precursors (Pre-rRNA), which account for more than 25% of total rRNA in the cells, are used as targets in a technique named molecular viability test (MVT). In this technique, Pre-rRNAs are detected when pathogens are growing actively. The quantification of viable cells is achieved on the basis of quantification cycle values produced by RT-qPCR before and after nutritional stimulation. MVT successfully detected viable _L. monocytogenes_ and other pathogens in milk [30]. Also, for RNA detection, an isothermal amplification method named nucleic acid sequence-based amplification (NASBA) is available. This method requires three enzymes--i.e., T7 RNA polymerase, RNase H, and the Avian myeloblastosis virus (AMV) reverse transcriptase--and two primers--i.e., one including the T7 promoter sequence and one complementary to the target sequence--and a constant temperature of 41 \({}^{\circ}\)C. The entire reaction can be completed in 2 h through cycles of reverse transcription, RNA degradation, double-stranded DNA synthesis, and target transcription [30]. Sequencing-based techniques are increasingly being applied to detect foodborne pathogens in microbial communities, also allowing source-tracking in the food industry. Direct metatranscriptome RNA-seq and multiplex RT-PCR were compared in detecting viable _E. coli_ O157:H7, _Salmonella_ Enteritidis, and _L. monocytogenes_ in Romaine lettuce and juice extract with nanopore MinION sequencing. Metatranscriptome analysis, which includes the steps of RNA extraction, library preparation, and sequencing, allowed the defining of the physiological state of the cells by mapping the sequences to metabolic databases (e.g., Kyoto Encyclopedia for Genes and Genomes (KEGG) database), although the sensitivity was lower than for multiplex RT-PCR.