Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology

• Primers from the chromosome and plasmids of anthrax were designed for pyrosequencing.
• Specificity of primers was demonstrated using a panel of bacteria.
• High sequence identities were observed when tested on several strains of B. anthracis.
• High sequence identities were also observed when tested on different food matrices.
• Detection limits were about 6 CFU/ml or 6 CFU/g of anthrax spores without enrichment.

The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6 CFU/mL and 6 CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5 h. A typical run on food samples yielded 67–80 bp reads with 94–100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores.