Application of Pyrosequencing® in Food Biodefense
The perpetration of a bioterrorism attack poses a significant risk for public health with potential socioeconomic consequences. It is imperative that we possess reliable assays for the rapid and accurate identification of biothreat agents to make rapid risk-informed decisions on emergency response. The development of advanced methodologies for the detection of biothreat agents has been evolving rapidly since the release of the anthrax spores in the mail in 2001, and recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence-based approaches such as Pyrosequencing®, which has the capability to determine short DNA stretches in real time using biotinylated PCR amplicons, have potential biodefense applications. Using markers from the virulence plasmids and chromosomal regions, my laboratory has demonstrated the power of this technology in the rapid, specific, and sensitive detection of B. anthracis spores and Yersinia pestis in food. These are the first applications for the detection of the two organisms in food. Furthermore, my lab has developed a rapid assay to characterize the antimicrobial resistance (AMR) gene profiles for Y. pestis using Pyrosequencing. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence, thus enabling rapid risk-informed decisions to be made. A typical run yields 40–84 bp reads with 94–100 % identity to the expected sequence. It also provides a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The method described is proposed as a novel detection system for potential application in food biodefense.
Key wordsBacillus anthracis Yersinia pestis Antimicrobial resistance Pyrosequencing®
Thank you to all the personnel in the Amoako lab who provided technical assistance in the work on the application of Pyrosequencing for the detection of Bacillus anthracis and Yersinia pestis in food. The assistance of Matthew Thomas in compiling this work is acknowledged. This work was funded by the Defence Research Development Canada Centre for Security Science, Chemical, Biological, Radiological, Nuclear and Explosive Research Technology Initiative (CRTI) funding from CRTI 08-0203RD.
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