EMA-Real-Time PCR as a Reliable Method for Detection of Viable Salmonella in Chicken and Eggs

Food Science Program, Div. of Food Systems and Bioengineering, 256 WCS Wing, Eckles Hall, Univ. of Missouri, Columbia, MO 65211, USA.
Journal of Food Science (Impact Factor: 1.7). 04/2010; 75(3):M134-9. DOI: 10.1111/j.1750-3841.2010.01525.x
Source: PubMed


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.

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    • "PCR-based detections methods have clearly demonstrated their sensitivity, though DNA contamination remains a challenge . Coupling PCR with a sample pre-treatment of ethidium bromide monoazide or propidium monoazide, dyes that irreversibly bind DNA, has been shown to enable PCR-based differentiation of viable and non-viable cells (Kobayashi et al. 2009a; Rudi et al. 2005; Wang and Mustapha 2010). Studies, however, suggest more research is needed to optimize the successful use of these dyes. "
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    ABSTRACT: Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.
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    • "The real-time PCR assay had a linear quantitative detection and a detection limit of 10 3 CFU/mL, regardless of whether the sample was treated with PMA. The EMA real-time PCR assay established by Wang and Mustapha [24] could Fig. 1 e Real-time polymerase chain reaction C t value change of DNA from dead Salmonella Typhimurium in dairy during storage at ¡18 C. Data are means of three separate determinations, and error bars represent ± standard deviation. "
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    ABSTRACT: Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 103 CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 100 CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.
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    • "(e.g. Hein et al., 2006; Josefsen et al., 2007; Liming and Bhagwat, 2004; Malorny et al., 2004, 2007; Pasquali et al., 2013; Perelle et al., 2004; Seo et al., 2004; Wang and Mustapha, 2010) and L. monocytogenes (e.g. Berrada et al., 2006; Hough et al., 2002; Nogva et al., 2000; O'Grady et al., 2008, 2009; Oravcova et al., 2007; Rossmanith et al., 2006; Rudi et al., 2005) in food products. "
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    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.
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