Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain

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Abstract

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and < 54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of < 62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.

Introduction

The microbiological safety of food is a significant concern of consumers and industries today. The rapid and accurate identification of bacterial pathogens in foods is important, both for quality assurance and to trace bacterial pathogens within the food supply (Bhagwat, 2003). Grain is considered to be a product with a low risk of contamination with pathogenic bacteria due to its low water activity (Berghofer et al., 2003). Although grain storage practices are not conducive to growth of bacteria, several studies have indicated the presence of low levels of Escherichia coli, Salmonella spp., Bacillus cereus and various food spoilage microorganisms in wheat and flour due to both pre- and post-harvest contamination (Eyles et al., 1989, Richter et al., 1993, Berghofer et al., 2003). The microbiological quality of the grain is considered to have an impact on the quality of the end product (Berghofer et al., 2003), and many processors monitor the microbial load of the raw grain. The sampling protocols employed and the extent of the information sought vary between companies and are not typically in the public domain. Buyers of grain can place the types and numbers of microorganisms into a contract specification, which then requires that the shipment in question be tested for those organisms. Interest in the microbial load of a product may take the form of an inquiry into the historic record, from which one can prepare a statement of assurance that does not require testing of a particular shipment. Customer standards for acceptable levels of contamination are variable, and may be needless or ill-advised (International Commission on Microbiological Specifications for Foods, 1986). Inquiries from buyers and processors are not always based on a knowledge of science, and can encompass organisms known to be absent from grain to specifications, such as free from bacteria and moulds, that are impossible to meet. There are recommended tolerances for some pathogens in grain, although the emphasis is typically on the finished product (International Commission on Microbiological Specifications for Foods, 1986). Grain sellers who know which organisms are present in their product, and their frequency, are better able to respond quickly and effectively to the inquiries and concerns of the grain trade.

Salmonella strains can cause general infection, food poisoning and Salmonellosis, a zoonotic disease of considerable importance (Davies and Hinton, 2000). Although E. coli is the predominant facultative anaerobe of the human colonic flora, some strains are responsible for enteric disease (Abd-El-Haleem et al., 2003, Bischoff et al., 2005). Major disease outbreaks and numerous sporadic cases of listeriosis occurring world-wide have implicated Listeria monocytogenes as another major food borne pathogen. Although L. monocytogenes has been isolated from a variety of foods (Norrung et al., 1999, Inoue et al., 2000, Rocourt et al., 2000, Maijala et al., 2001), it has not been found on grain. However, its importance in human disease has resulted in requests from grain buyers that grain be tested for this organism. In a recent survey of bacteria on milling wheat from Canada Salmonella spp. and E. coli were not detected, but coliform bacteria have been found in about 25% of samples of western milling wheat (Blaine Timlick, Canadian Grain Commission, personal communication).

Most studies of pathogenic bacteria in grain have used conventional, culture-based methods (Eyles et al., 1989, Richter et al., 1993, Berghofer et al., 2003). Those methods are time-consuming and have low accuracy. Polymerase chain reaction (PCR) technology has proven to be valuable for the detection of bacteria in foods. With its high levels of sensitivity and specificity, PCR can be used for the rapid detection of pathogenic bacteria contaminating various foods. Multiplex PCR assays employ multiple sets of primers to amplify more than one target sequence simultaneously in a single reaction. Multiplex PCR assays have been used to detect and/or identify one organism by amplification of more than one gene, or multiple organisms can be detected simultaneously by targeting unique sequences from each organism (Fratamico, 2001).

The aim of this study was to establish a rapid and simple method for simultaneous detection of E. coli, L. monocytogenes and S. typhimurium in artificially inoculated wheat grain using PCR.

Section snippets

Bacterial strains and preparation of inoculum

Salmonella typhimurium (# 03-5608), S. agona (# 03-0890), and S. hadar (# 03-4494) were originally from the National Microbiology Laboratory (Canadian Science Centre for Human and Animal Health, Winnipeg, MB, Canada). A non-pathogenic strain of E. coli was from the Food Product Development Center, Portage la Prairie, MB, Canada, and L. monocytogenes (# 19112) was from the American Type Culture Collection, Manassas, Virginia, USA. All isolates were provided by Dr. Greg Blank of the Department of

Results and discussion

All strains produced typical growth when inoculated onto their respective selective media. PCR with the salinvA primer set produced a PCR product of the expected size from S. typhimurium, S. agona, and S. hagar (data not shown). As shown in Fig. 1 (lanes 2, 3 and 4), a mixture of the three primer pairs in a PCR reaction containing a DNA template of a single bacterial pathogen amplified the expected PCR amplicons.

When multiple target organisms were included in the reaction containing the mixture

Acknowledgements

We would like to thank Dr. Greg Blank for providing us with the bacterial strains used in this study; Dr. Sung-Jong Lee for helpful suggestions for PCR set-up, and Drs. Bill Scowcroft and Daniel Perry for reviewing the manuscript and providing valuable comments.

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