Development and evaluation of a multiplex real-time PCR (qPCR) assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs Narjol González-Escalona ⁎, Eric W. Brown, Guodong Zhang Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, 20740, USA abstract article info Article history: Received 8 November 2011 Accepted 10 March 2012 Available online 23 March 2012 Keywords: Salmonella Produce Real-time PCR qPCR Food safety Eggs Contamination of foods, especially produce and eggs, with Salmonella spp. is a major concern for public health. Therefore the development of a rapid method for Salmonella detection in those two important food commodities is urgently needed. The main objective of our study was to develop and evaluate a multiplex TaqMan-based real time PCR assay for detection of Salmonella spp. targeting invA gene and ttrRSBCA locus. The detection limit of this assay, 13 copies of genomic invA gene and ttrRSBCA locus, was determined by 10-fold dilutions of DNA from S. Typhimurium (strain SARA9). Inclusivity and exclusivity of the assay were assessed using 101 Salmonella spp. (including all Salmonella species and subspecies) and 48 non-S. enterica strains, respectively. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomatoes and jalapeno peppers were seeded with four different Salmonella serovars at levels of 1–20 (low level) and 10 5 (high level) CFU/25 g. The inoculated samples were assayed according to the FDA Salmonella culture method in the Bac- teriological Analytical Manual (BAM). Eggs were seeded with two different S. Enteritidis strains at the level used for the produce samples and were assayed according to the USDA Laboratory Guidebook Salmonella cul- ture method. The contaminated samples were analyzed with the multiplex TaqMan-based assay developed in this study. Comparable results were obtained by the qPCR and the two bacteriological methods. Levels as low as 2 CFU/25 g for produce were detected with the qPCR method according to the BAM and 5 CFU/100 g for eggs were detected with the qPCR method according to the USDA Laboratory Guidebook. False negatives (inhibition of PCR reaction) were ruled out through the use of a DNA internal amplification control (IAC). The qPCR multiplex assay developed in this study allows rapid and accurate detection of Salmonella spp. in six high-risk produce commodities and eggs, and has the potential to be used with other food matrices. Published by Elsevier Ltd. 1. Introduction Food contamination with Salmonella enterica is a significant public health concern worldwide. Salmonella infects thousands of people every year in the US (Scallan et al., 2011) and S. Enteritidis (SE) has emerged as a major egg-associated pathogen. Transmission of Salmonella spp. to humans has been linked mainly to the consump- tion of contaminated foods. In the case of SE, transmission to humans has been usually associated with contaminated foods containing undercooked eggs (Howard, O'Bryan, Crandall, & Ricke, 2011; Rabsch et al., 2000). SE is the second most commonly serotype isolat- ed from human illnesses in the US (Howard et al., 2011). Consump- tion of fresh fruit and produce in the US rose by almost 50% between 1970 and 1994 (Bureau of the Census U.S. Department of Commerce, 1996) remaining stable from 2000 to 2009 (Grimm et al., 2010), and the per capita consumption of eggs and egg products in the US has increased to approximately 246 eggs/year (American Egg Board, 2011, http://www.aeb.org/egg-industry/industry-facts/ egg-industry-facts-sheet). Fresh produce is particularly vulnerable to contamination during the “farm to fork” process and fresh eggs can be contaminated easily with SE through cracks in the shell by contact with chicken feces or by transovarian infection (Howard et al., 2011). These events could have contributed to the observed in- creases of foodborne outbreaks (Altekruse, Cohen, & Swerdlow, 1997), including recent Salmonella outbreaks in the US involving jala- peno peppers in 2008 (Mody et al., 2011) and a large multistate out- break of SE that was associated with contaminated eggs (http://www. cdc.gov/salmonella/enteritidis/se_timeline_092010.pdf) in 2010. Traditional microbiological culture methods are the accepted methods for detection of Salmonella in foods. Two such methods are described in the FDA Bacteriological Analytical Manual (Chapter 5, Salmonella; http://www.cfsan.fda.gov/~ebam/bam-5.html) and in Food Research International 48 (2012) 202–208 ⁎ Corresponding author at: FDA, CFSAN, 5100 Paint Branch Parkway HFS-712, Col- lege Park, MD 20740, USA. Tel.: +1 240 402 1937. E-mail address: narjol.gonzalez-escalona@fda.hhs.gov (N. González-Escalona). 0963-9969/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.foodres.2012.03.009 Contents lists available at SciVerse ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres