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Biosensor for the specific detection of a single viable B. anthracis spore

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Abstract

A simple membrane strip-based biosensor for the detection of viable B. anthracis spores was developed and combined with a spore germination procedure as well as a nucleic acid amplification reaction to identify as little as one viable B. anthracis spore in less than 12 h. The biosensor is based on identification of a unique mRNA sequence from the anthrax toxin activator (atxA) gene encoded on the toxin plasmid, pXO1. Preliminary work relied on plasmid vectors in both E. coli and B. thuringiensis expressing the atxA gene. Once the principle was firmly established, the vaccine strain of B. anthracis was used. After inducing germination and outgrowth of spores of B. anthracis (Sterne strain), RNA was extracted from lysed cells, amplified using nucleic acid sequence-based amplification (NASBA), and rapidly identified by the biosensor. While the biosensor assay requires only 15-min assay time, the overall process takes12 h for the detection of as little as one viable B. anthracis spore, and is shortened significantly, if larger amounts of spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized oligonucleotide probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second oligonucleotide probe that has been coupled to dye-encapsulating liposomes. The dye in the liposomes then provides a signal that can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1.5 fmol of target mRNA. Specificity analysis revealed no crossreactivity with closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis etc.

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Acknowledgement

The authors would like to thank Dr Randy Worobo, who provided essential expertise and guidance in the field of Microbiology; and Wlodek Borejsza-Wysocki for helping with NASBA. Dr Patrick McDonough of the Cornell Veterinary Diagnostic Laboratory provided technical advice and access to the B. anthracis Sterne strain, without which this research would not have been possible. The authors also would like to thank Dr Steven Leppla of the National Institute of Dental and Craniofacial Research (NIDCR) in Bethesda, Maryland, for kindly providing several bacterial strains. Dr Theresa Koehler of the University of Texas Medical Center in Houston, Texas provided valuable help with B. anthracis growth media and atxA. Finally, we thank Innovative Biotechnology International, Inc. for funding parts of the research and the United States Army for providing Harriet Hartley the time and the funding to attend graduate school and carry out the presented research.

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  1. Dept. of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853–5701, USA

    Harriet A. Hartley & Antje J. Baeumner

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  1. Harriet A. Hartley
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  2. Antje J. Baeumner
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Correspondence to Antje J. Baeumner.

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Hartley, H.A., Baeumner, A.J. Biosensor for the specific detection of a single viable B. anthracis spore. Anal Bioanal Chem 376, 319–327 (2003). https://doi.org/10.1007/s00216-003-1939-5

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  • DOI: https://doi.org/10.1007/s00216-003-1939-5

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