Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

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

“…) and even prior to a multiplex PCR method that successfully detects three different Salmonella serotypes (Yang et al . ).…”

“…; Yang et al . ) and EMA (Rudi et al . ,b; Wang and Mustapha ), and in all these studies, the use of viability dyes allowed the detection of viable pathogens without any interference from the natural microbiota of these complex food products.…”

“…; Yang et al . , ). The application of viability dyes on vegetables for the selective detection of live foodborne bacterial pathogens by qPCR was first described for E. coli O157:H7, L. monocytogenes and Salmonella using PMA in spinach and mixed salad (Elizaquível et al .…”

Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process

“…However, common qPCR does not have the ability to differentiate viable from dead cells. Therefore, DNA from dead cells that have been killed by heat or freeze processing also served as qPCR template during the amplification phase; these have caused inaccurate quantification of actual bacterial level of contamination (Yang et al, 2012;Zhang et al, 2013). To overcome this drawback, researchers used propidium monoazide (PMA; that selectively penetrates compromised cells and binds to DNA) treatment to remove the interference from dead cells in a PMAqPCR assay (Nocker, Cheung, & Camper, 2006).…”

“…To overcome this drawback, researchers used propidium monoazide (PMA; that selectively penetrates compromised cells and binds to DNA) treatment to remove the interference from dead cells in a PMAqPCR assay (Nocker, Cheung, & Camper, 2006). This PMA-qPCR assay had been reported to detect various microorganisms including E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables (Elizaquível, Sánchez, & Aznar, 2011), Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium in Freshwater (Bae & Wuertz, 2012), Vibrio parahaemolyticus in raw seafood (Zhu, Li, Jia, & Song, 2012), S. enterica serovars (Yang et al, 2012), E. coli, Staphylococcus aureus and L. monocytogenes from artificially contaminated food processing surfaces (Martinon, Cronin, Quealy, Stapleton, & Wilkinson, 2012). However, when bacteria was subjected to heat or refrigerated, it was found that numbers of viable cells determined from colony counts did not correlate with the results of PMA-qPCR (Løvdal, Hovda, Björkblom, & Møller, 2011;Yang, Badoni, & Gill, 2011) which indicated that not all DNA from dead cells were inhibited during the PCR amplification stage.…”