|Dunning, F. mark|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/20/2012
Publication Date: 8/24/2012
Citation: Dunning, F., Rige, D.R., Piazza, T.M., Stanker, L.H., Zeytin, F.N., Ward, T.C. 2012. Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity. Applied and Environmental Microbiology. 78(21):7687-7697. DOI.1128/AEM.01664-12. Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. Of the seven different types of BoNT, four cause human disease, serotypes A, B, E, and F. The standard test for BoNT is the mouse bioassay (MBA). While being very sensitive, the MBA takes between 4-7 days to complete, uses death as an end point, is expensive and only available in a few laboratories. A rapid test for food and clinical samples that measures toxin activity in a few hours is needed. A set of rapid assays that measures activity of toxin serotypes A, B, E, and F is described and its application to food and serum analysis demonstrated. These tests will further our ability to monitor for toxin and improve the safety of the food supply.
Technical Abstract: Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and accuracy. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F activity in complex matrices that offer mouse or near-mouse bioassay sensitivity with total assay times of less than 24 h. Tested matrices include human serum, whole milk, carrot juice, and baby food as well as buffers containing common pharmaceutical excipients. Limits of detection for BoNT/A and BoNT/F were below 1 pM and below 10 pM for BoNT/B in most tested matrices using 200 µl samples, and as low as 10 fM for BoNT/A with increased sample volume. Together, these data describe rapid, robust, and high–throughput assays for BoNT detection that are compatible with a wide range of matrices.