title: Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.
abstract: Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Janzen
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Thomas
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.
authors.forename: Noriko
authors.initials: N
authors.lastname: Goji
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shields
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4.
authors.forename: Kristen R
authors.initials: KR
authors.lastname: Hahn
authors.affiliation: Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4. kingsley.amoako@inspection.gc.ca.
authors.forename: Kingsley K
authors.initials: KK
authors.lastname: Amoako
mesh_terms: D000818:Animals; D001408:Bacillus anthracis / Q000235:genetics / Q000302:isolation & purification*; D001628:Beverages / Q000382:microbiology*; D023482:Bioterrorism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D060766:Drinking Water / Q000382:microbiology; D005516:Food Microbiology / Q000379:methods*; D018189:Immunomagnetic Separation; D008892:Milk / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013171:Spores, Bacterial / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D060766:Drinking Water