EMA-Real-Time PCR as a Reliable Method for Detection of Viable Salmonella in Chicken and Eggs
ABSTRACT Culture-based Salmonella detection takes at least 4 d to complete. The use of TaqMan probes allows the real-time PCR technique to be a rapid and sensitive way to detect foodborne pathogens. However, unlike RNA-based PCR, DNA-based PCR techniques cannot differentiate between DNA from live and dead cells. Ethidium bromide monoazide (EMA) is a dye that can bind to DNA of dead cells and prevent its amplification by PCR. An EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells. The aim of this study was to design an accurate detection method that can detect only viable Salmonella cells from poultry products. The sensitivity of EMA staining coupled with real-time PCR was compared to that of an RNA-based reverse transcription (RT)-real-time PCR. To prevent false negative results, an internal amplification control was added to the same reaction mixture as the target Salmonella sequences. With an optimized EMA staining step, the detection range of a subsequent real-time PCR was determined to be 10(3) to 10(9) CFU/mL for pure cultures and 10(5) to 10(9) CFU/mL for food samples, which was a wider detection range than for RT-real-time PCR. After a 12-h enrichment step, EMA staining combined with real-time PCR could detect as low as 10 CFU/mL Salmonella from chicken rinses and egg broth. The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results and represents a simple, yet accurate detection tool for enhancing the safety of food.
- SourceAvailable from: Elodie Barbau-Piednoir[Show abstract] [Hide abstract]
ABSTRACT: In this study, the complete CoSYPS Path Food workflow including all steps, namely swab sample enrichment, SYBR®Green qPCR detection of Salmonella spp. and Listeria spp., isolation and confirmation of the detected strain, was validated on beef carcass swabs. To perform the validation, the results of the complete workflow were compared, according to the ISO 16140:2003, with the ISO reference methods for detection, isolation and confirmation of Listeria monocytogenes and Salmonella spp. The results showed that the relative level of detection and the limit of detection of the complete workflow and ISO reference methods are in a range from 2 to 16 CFU/swab for both bacteria. The relative specificity, sensitivity and accuracy identified during this validation were all 100% since the results obtained with the complete CoSYPS Path Food workflow and the ISO reference methods were identical (Cohen's kappa index = 1.00). In addition the complete CoSYPS Path Food workflow is able to provide detection results (negative or presumptive positive) in half the time needed as for the ISO reference methods. These results demonstrate that the performance of the complete CoSYPS Path Food workflow is not only comparable to the ISO reference methods but also provides a faster response for the verification of beef carcasses before commercial distribution.International Journal of Food Microbiology 01/2015; 192:103–110. DOI:10.1016/j.ijfoodmicro.2014.09.018 · 3.16 Impact Factor
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ABSTRACT: The microbiological standard for detection of Salmonella relies on several cultural steps and requires more than 5 days for final confirmation, and as consequence there is a need for an alternative rapid methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, ready to eat lettuce salad and raw sheep milk cured cheese. Three main parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the incubation times (6, 10 and 18 hours) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column). The results obtained demonstrate that a combination of an incubation in buffered peptone water for 18 h of a 25 g-sample coupled to a DNA extraction by boiling and a real-time PCR assay detected down to 2-4 Salmonella spp. CFU per sample in less than 21 hours in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method.International Journal of Food Microbiology 08/2014; 184:113–120. DOI:10.1016/j.ijfoodmicro.2014.03.021 · 3.16 Impact Factor
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ABSTRACT: The 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, fulfills 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 in order to monitor different food manufacturing processes as an alternative to classical cultural methods. In this review, state-of- the-art informationregarding viability PCR (v-PCR) is compiled. This article is protected by copyright. All rights reserved.Journal of Applied Microbiology 10/2013; DOI:10.1111/jam.12365 · 2.39 Impact Factor