Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

Abstract

Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10 CFU/g). The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence.

Food Biodefense

“…Recent research and development work using immunomagnetic capture have shown the successful extraction and concentration of anthrax spores from five food matrices with high sensitivity [1]. In addition, novel methods involving the use of sequence based assays have been developed for the detection of Bacillus anthracis spores and Yersinia pestis from food matrices [2,3]. These tools will allow the rapid, specific, and sensitive detection and screening of these bio threat agents in food matrices.…”

Dynabeads protein G antibody conjugates combined with modified brain heart infusion broth for the enrichment and separation of Bacillus cereus in artificially contaminated vegetables

“…The immunomagnetic separation (IMS) method uses immunomagnetic beads (IMBs) as the capture reagents to isolate pathogens from food samples (13)(14)(15). IMS could effectively separate target bacteria from competitive microflora in the food samples, thus reducing the sample volume (16,17).…”

Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR

“…Magnetic-bead-based methods for the separation and enrichment of targets include two types: specific and non-specific separations. Currently, the most widely used application is specific separation; this technology has been used in biomedical applications [8][9][10], food safety [11,12], environmental monitoring [13,14], and plant virus detection [15]. Previous studies have shown the development of magnetic-bead-based methods for the detection of CGMMV; this virus was successfully detected in cucumber seeds through specific virus adsorption with immunomagnetic beads (IMB) (coated with CGMMV antibody) followed by RT-PCR [16] and in cucumber leaves using an RT-PCR method combined with non-specific magnetic separation (MNP) (no CGMMV antibody coating) [17].…”