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.

1 Follower
31 Reads
  • Source
    • "(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. "
    [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 · 3.08 Impact Factor
  • Source
    • "detect the presence of Salmonella in foods (Hein et al., 2006; Jakociune et al., 2014; Josefsen et al., 2007; Krämer et al., 2011; Liming and Bhagwat, 2004; Löfström et al., 2004, 2009, 2012; Rodriguez-Lazaro et al., 2003; Seo et al., 2004; Wang and Mustapha, 2010). The main advantages of RTi-PCR are high sensitivity and specificity, excellent efficiency , and no post-PCR steps that reduce risks of cross-contamination (Rodríguez-Lázaro and Hernandez, 2013; Rodríguez-Lázaro et al., 2007, 2013). "
    [Show abstract] [Hide abstract]
    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 · 3.08 Impact Factor
  • Source
    • "SE is considered as the only bacterium that routinely causes human infection through intact chicken eggs [9,14]. Although an egg has its own protection mechanisms including both physical and chemical barriers, with bactericidal factors such as lysozyme, ovotransferrin, nuclease and β-defensin-11existed in egg albumen that can kill most bacteria. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Salmonella enterica serovar Enteritidis (SE), as a major cause of foodborn illness, infects humans mainly through the egg. However, the symptom of laying hens usually is not typical and hard to diagnosis. In the present study, it is studied that the influences of SE infection on layers' performance, egg quality and blood biochemical indicators. It will help us to improve the strategy to control SE infection in commercial layers. One hundred layers at 20 wks of age were divided into 2 groups, 60 hens for experiment and others for control. The experiment group was fed with the dosage of 108 CFU SE per hen. The specific PCR was used to detect the deposition of SE. On the 8d after SE infection, 10 hens from the control group and 30 hens from the experimental group were slaughtered to detect the SE colonization. The production performance, egg quality and blood biochemical indices were also analyzed. The results showed that the colonization rate of SE was highest in caecum contents (55.17%) and lowest in vagina (17.24%). For the eggs the detection rate of SE was highest on the eggshell (80.00%) and lowest in yolk (18.81%). SE infection had no significant influence on production performance and egg qualities (P > 0.05). The difference of laying rate between the experimental and control groups was less than 0.30%, and both were approximately equal to 82.00%. The blood analysis showed that the aspartic aminotransferase (AST) and alanine aminotransferase (ALT) of experimental group was significantly higher than those of control group (P < 0.05). For experimental and control groups AST values were 236.22 U/l and 211.84 U/l respectively, and ALT values were 32.19 U/l and 24.55 U/l. All of coefficients were less than 20%. The colonization of SE in organs increases the enzyme activities of AST and ALT in blood. SE in feed could invade the oviduct and infect the forming eggs. It significantly increased the concentration of ALT and AST in blood. However,SE infection was hard to be observed from the appearances of layer and egg. It might be a dangerous risk to human health.
    Journal of Animal Science and Biotechnology 01/2014; 5(1):4. DOI:10.1186/2049-1891-5-4 · 1.68 Impact Factor
Show more