Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food

Abstract

BackgroundAlthough a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples.ResultsWe investigated the relationship between amplicon length and inhibitory effect of PMA treatment to prevent DNA amplification from dead cells while allowing for DNA amplification from live cells, and found that the two factors are well correlated with each other. An amplicon that is 130 bp in length was determined to be optimal for PMA treatment and was selected for further PMA-qPCR assay development. A PMA-qPCR assay was established by utilizing this amplicon and adopting a modified PMA-treatment procedure. The PMA-qPCR assay provided excellent inhibition of DNA amplification from dead cells (a 17-CT-value, or 128,000-fold reduction) while only a slight DNA amplification difference (0.5 CT value) was noted between the PMA-treated and untreated live cells. This assay has been validated through stringent inclusivity and exclusivity studies using a large number of (n = 167) Salmonella, including all strains of SARA and SARB collections, and non-Salmonella strains (n = 36). This PMA-qPCR assay is capable of detecting live Salmonella cells in live/dead cell mixtures, or 30 CFU/g live Salmonella cells from enriched spiked spinach samples as early as 4 h.ConclusionsA 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR. This PMA-qPCR assay provides a sensitive, specific, and efficient method for detecting live Salmonella cells in foods and environmental samples and may have an impact on the accurate microbiological monitoring of Salmonella in foods and environment samples.

Diversity and Antimicrobial Resistance of Salmonella enterica Isolates from Surface Water in Southeastern United States

“…A part of the culture was saved as seeds for the picked colonies, and the other part of the culture was used for DNA extraction, which was performed using PrepMan Ultra sample preparation reagent (Life Technologies) according to the manufacturer's instructions. In experiment A, a qPCR assay was performed to confirm the presumptive Salmonella-positive colonies as described above (30), whereas in experiment B, conventional methods were used as described in the BAM (29) (Fig. 2).…”

“…Initially, 0.5 ml of the preenriched culture was centrifuged at 10,000 ϫ g for 5 min; the supernatant was then removed, and the cell pellet was washed with 1 ml of phosphate-buffered saline (PBS), centrifuged, and suspended in 150 l of PrepMan solution (Life Technologies, Grand Island, NY) for DNA extraction. The extracted DNA was subjected to a qPCR assay (30) to screen for the presence of Salmonella. Briefly, reaction mixtures consisted of 12.5 l of 2ϫ Universal Master Mix (Life Technologies), 200 nM forward and reverse primers targeting the invA gene in Salmonella, and 100 nM probe.…”

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

“…Recently, DNA‐intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) have been proposed as useful molecular methods to quantify intact bacterial cells in environments (Nogva et al ., 2003; Rudi et al ., 2005; Nocker et al ., 2006). PMA or EMA with PCR technique has been widely used to assess cell viability in water, soil, air and food (Rogers et al ., 2008; Bae and Wuertz, 2009; Miotto et al ., 2012; Kaushik and Balasubramanian, 2013; Li and Chen, 2013). …”

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

“…43 Alonso and others (2014) reported a more effective exclusion of dead cysts of Giardia duodenalis in a qPCR assay with longer amplicons. Likewise, Li and Chen (2013) found a good correlation between amplicon length and the signal inhibitory effect of PMA treatment on dead cells of Salmonella spp, concluding that the best qPCR signal reduction was obtained with the larger amplicon, albeit with a slight loss of technique efficiency. As suggested by Soejima and others (2011) 44 and Contreras and other (2011), 45 the beneficial effect of targeting longer DNA sequences is likely due to the increased probability of dye binding in the targeted region, resulting in a stronger inhibition of the amplification.…”

“…E-mail: rfisa@ub.edu principally in the fields of environment and food. [26][27][28][29][30][31] Lately, they have also been applied in bacterial studies on clinical samples, indicating that this method constitutes a potential alternative to diagnosis by microscopy and culture, as well as in monitoring early treatment response 32,33 or in drug experimental assays. 34,35 However, to date, this methodology has had only scant application in parasites, for example, oocysts of Cryptosporidium, cysts of Giardia duodenalis and trophozoites and cysts of Acanthamoeba castellani in clinical and environmental samples.…”