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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    • "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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    • "(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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