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Title: Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay

item Rasooly, Reuven
item Stanker, Larry
item Carter, John
item Do, Paula
item Cheng, Luisa
item He, Xiaohua
item Brandon, David

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2008
Publication Date: 5/13/2008
Citation: Rasooly, R., Stanker, L.H., Carter, J.M., Do, P.M., Cheng, L.W., He, X., Brandon, D.L. 2008. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food. International Journal of Food Microbiiology. 126 (2008):135-139

Interpretive Summary: Botulinum neurotoxins (BoNT) produced by some bacteria, are a cause of food poisoning. Because these toxins are extremely poisonous, they might be used for intentional contamination of food in an incident of terrorism. This paper describes detection of BoNT in juice and milk using an improved method. The method is based on a unique material that is converted by the toxin into a highly fluorescent dye. The improved method is sensitive enough to detect very small amounts of BoNT that are dangerous to humans. Because it is relatively fast and inexpensive, this method could be used for large scale screening of BoNT in food. It could also replace the widely-used live mouse test for BoNT.

Technical Abstract: The World Health Organization (WHO) and U.S. Centers for Disease Control and Prevention (CDC) have labeled botulinum toxins as a high priority biological agent that may be used in terrorist attacks against food supplies. Due to this threat there is an increased need to develop fast and effective methods to detect active botulinum neurotoxins (BoNTs). This study reports the successful use of internally quenched fluorogenic peptide as a fast, simple and inexpensive alternative to the mice bioassay. Our results show that in less than 15 minutes we are able to detect 9.4 ng of BoNT-A in liquid food items similar to those that caused a recent outbreak of botulism in Georgia and Florida. The detection level is far below the human lethal oral dose of 70 ug of toxin for 70 kg of body weight. Our results also show that by using immunomagnetic beads containing monoclonal antibodies that target the toxin heavy chain, we are able to concentrate the toxin without neutralizing its enzymatic activity. This fast and effective tool could be used for large scale screenings for the detection of BoNT-A.