Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field

Abstract

SummaryThe increase in foodborne outbreaks highlights the need for rapid, sensitive and specific methods for food safety monitoring, enabling specific detection and quantification of viable foodborne pathogens. Real-time PCR (qPCR) combined with the use of viability dyes, recently introduced, fulfils all these requirements. The strategy relies on the use of DNA-binding molecules such as propidium monoazide (PMA) or ethidium monoazide (EMA) as sample pretreatment previous to the qPCR. These molecules permeate only membrane-compromised cells and have successfully been applied for different types of foodborne pathogens, including bacteria and viruses. Moreover, those dyes have been explored to monitor different food manufacturing processes as an alternative to classical cultural methods. In this review, state-of-the-art information regarding viability PCR (v-PCR) is compiled.

Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples

“…This highlights the importance of implementing new methodologies to assess infectivity in food samples. So far, viability PCR has successfully been applied for bacterial detection in several types of food (reviewed by Elizaquível et al, 2014) but not for enteric viruses. This study shows for the first time the potential of PMA to discriminate between thermally inactivated HAV and infectious HAV in food applications.…”

“…A promising new strategy to assess viral infectivity relies on the use of nucleic acid intercalating dyes, or viability dyes, such as ethidium monoazide (EMA) or propidium monoazide (PMA) as a sample pretreatment before applying molecular techniques (reviewed by Elizaquível et al, 2014). Theoretically, these compounds cannot penetrate intact capsids but are able to penetrate damaged or destroyed capsids.…”

Dead or Alive: Molecular Assessment of Microbial Viability

“…Of the two methods discussed in this review, viability PCR is by far the more extensively evaluated and vetted. In one of several reviews (9, 30-33), Elizaquível et al (31) cataloged over 30 published studies that applied the method to food safety models alone. Viability PCR has also been extensively optimized.…”

“…The extent of signal reduction or ⌬C T correlates with the Rapid physical inactivation of a narrow range of targets, resulting in rapid loss of viability followed by slower decay (over hours or days) of cellular components, including the cytoplasmic membrane UV, solar disinfection, low-temp pasteurization 4 Inactivation of a specific and essential target (e.g., DNA, RNA, and protein biosynthetic enzymes), resulting in rapid loss of viability followed by slower decay (over hours or days) of cellular components, including the cytoplasmic membrane Antibiotics such as rifamycins, macrolides, aminoglycosides, and quinolones portion of the target DNA in the sample that is associated with inactivated cells (34). The utility of viability PCR has been expanded by modifications to this basic strategy (31). Dithiothreitol cotreatment was reported to facilitate PMA penetration of inactivated Bacillus subtilis spores, thereby improving the ability to discern the viability of spores (35).…”

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

“…A possible explanation on the statistical significant difference between qPCR and plate count at 28 days is the presence of dead cells that could not be distinguished from viable cells by qPCR, since it amplifies DNA from both dead and viable bacteria as DNA remains stable after the death of bacteria (Li et al 2013). An alternative approach to detect only viable bacteria is viability qPCR using dyes that intercalate DNA of dead cells, such as ethidium monoazide and propidium monoazide (Barbau-Piednoir et al 2014;Elizaquivel et al 2014). Another strategy to detect viable bacteria is the analysis of 16S transcripts by RNAseq (Gosalbes et al 2011), although it is an expensive strategy.…”