Improved sample treatment protocol for accurate detection of live Salmonella spp. in food samples by viability PCR

Abstract

Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals.

Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method

“…The v-qPCR has been efficiently used for the monitoring of microorganisms with biotechnological potential [ 31 ], and for the detection and quantification of human pathogens in food [ 47 ] or in the environment [ 48 ]. In particular, in the case of Xf, different PCR assays are commonly used for the detection and quantification, and v-qPCR methods in combination with EMA or PMAxx reagents were also reported to discriminate between viable and membrane-damaged cells [ 35 , 36 ].…”

A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

“…The number of studies using PMAxx is, however, still limited and has mainly been used in studies with viruses. Also of great interest is the recently developed dye PEMAX, marketed by GenIUL, which takes advantage of two photoreactive dyes and has shown to effectively suppress the signal from dead Salmonella enterica in 33 food samples using a 262 bp amplicon (Thanh et al, 2017). The PEMAX double dye method is based on the finding that use of a combination of EMA and PMA suppresses the qPCR signal from dead cells with intact membranes but no ATP turnover (Codony et al, 2015).…”

“…Some studies interpret a qPCR signal below LOD as a complete suppression of the 'dead signal' and thus depict bars in figures with the measured cell concentration as zero for these observations (Banihashemi et al, 2012;Ditommaso et al, 2015). Others show the obtained Cq value or the increase in Cq without explicit information about the LOD (Martin et al, 2013;Thanh et al, 2017). This Running Header: Long-amplicon PMA-qPCR assay for enumeration of viable L. monocytogenes 28 makes direct comparisons between studies regarding the complete and incomplete suppression of the 'dead signal' difficult.…”

Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method

“…To our knowledge, the application of the PEMAX reagent in the v-qPCR approach has been recently used in viability assessment studies for monitoring bacterial pathogens (e.g., Legionella and Salmonella ) ( 39 , 40 ) but not for the specific quantification of beneficial bacteria in plant environments.…”