Table 2.

Summary of advantages and limitations of culture-independent nucleic acid– and phage-based approaches for detecting or demonstrating the presence of viable pathogens in food

Type of viability test	Method name	Underlying principle	Advantages	Limitations	Example references
Nucleic acid–based	Viability PCR/qPCR	Pre-incubation of test sample with PMA or EMA dyes, which penetrate into bacteria with compromised cell membranes and bind genomic DNA, making it non-amplifiable.	Gives PCR the capability to differentiate viable and dead cells more quickly than culture. qPCR provides quantitative results.	Dead/inactivated bacterial cells do not always have compromised cell membranes, so false positives may result.	Nocker and Camper (2009) Trevors (2012) Emerson et al. (2017)
	Reverse-transcriptase qPCR (RT-qPCR)	Bacterial transcripts are sensitive to degradation by intra- and extra-cellular RNases, so mRNA levels should rapidly decline after cell death. Thus, detectable mRNA would be limited to the viable and active cells within a sample.	Quick compared to culture, but additional cDNA generation step makes it longer test than viability PCR/qPCR.	Not all studies have demonstrated that mRNA is short-lived, so false positive results may occur. RT-qPCR viability assessment validated for longer (> 200 bp) transcription products, but not necessarily short qPCR products.	Techathuvanan et al. (2010) Baskaran et al. (2016) Omori et al. (2017)
Phage-based	Phage amplification (Plaque) assay	Phages only replicate within viable cells and ultimately lyse these cells to release progeny phages within an agar lawn to form plaques (zones of clearing).	A 24-h test, producing countable plaques giving a quantitative result.	Not suited as a high-throughput test. Laborious, multi-step test, which requires cooled molten agar. Virucidal step is key step, otherwise false positive results may be obtained.	Favrin et al. (2003) Botsaris et al. (2010, 2013, 2016) Foddai and Grant (2017) Gerrard et al. (2018)
	Phage amplification + qPCR	As above, but cell lysis occurs in liquid suspension, releasing progeny phages and host DNA, which can both be detected and quantified by qPCR.	Rapid, one-day test, with option to detect released phages or the host DNA by qPCR to demonstrate that lysis has occurred. Only viable cells lyse. Potentially a quantitative assay.	Important that DNA is released into as small a volume as possible to maximize detection sensitivity, otherwise DNA precipitation and column extraction may be necessary.	Sergueev et al. (2010) Anany et al. (2018)
	Phage amplification + immunoassay	Phage amplification proceeds until cell lysis in liquid suspension, releasing progeny phages which can be detected by ELISA or immunochromatographic test	Rapid, one-day test similar to when qPCR is used. Only viable cells lyse. Potentially a quantitative assay.	Analytical sensitivity more limited compared to qPCR detection after phage amplification.	Stewart et al. (2013) Stambach et al. (2015)
	Phage amplification + enzyme assay	Phage amplification proceeds until viable cells burst to release intracellular components such as ATP or ß-galactosidase, which are measured by enzyme assay.	Rapid, one-day test similar to when qPCR or immunoassay are used. Only viable cells lyse. Potentially a quantitative assay.	May require genetically engineered phages. Not many food testing applications to date.	Alcaine et al. (2015a, b) Franche et al. (2017)