# US 2014/0154812 A1 · Real-time assay for the detection of botulinum toxin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154812 A1 - Document ID: US20140154812A1 - Status: Published application - Kind: A1 - Application number: 13/839,827 - Filed: 2013-03-15 - Published: 2014-06-05 - Earliest claimed: 2012-12-03 - Assignees: SRC, Inc. - Applicants: Src, Inc. - Inventors: Kenton Arthur Doctor; Stacey Ann Massulik; Frances Louise Stites; Timothy Francis Moshier; Jeffrey Harold Mills; Lisa Helen Chamberlin; Deborah L. Plochocki; Olivia Jennifer Barrett; Huda Sirageldin Suliman - Web page: https://patentgrep.com/patent/US20140154812A1 ## CPC - C12Q 1/37: involving peptidase or proteinase - C07K 7/00: Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof - G01N 33/56911: {Bacteria} - G01N 2333/33: from Clostridium (G) - G01N 2333/952: derived from bacteria - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A real-time portable and rapid detection assay to identify the presence of biologically active toxins such as botulinum toxins. The proteolytic activity of BoNT/A is measured using a peptide cleavage assay, where a fluorescent substrate is cleaved by BoNT/A, resulting in increased fluorescence. This fluorescence can be monitored in real-time using a fluorescence detection instrument, such as a real-time PCR system that has been modified to implement a detection algorithm specific to the identification of the target toxin. ## Claims ### Claim 1 A device for detecting the presence of biologically active botulinum toxin, comprising: a chamber for receiving a test sample, said chamber including a peptide substrate labeled with a fluorophore and a quencher and having a cleavage site responsive to biologically active botulinum toxin positioned between said fluorophore and said quencher; a detector for measuring the amount of fluorescence emitted from said sample and outputting a corresponding fluorescence reading positioned proximately to said chamber; a microcontroller interconnected to said detector that is programmed to acquire said fluorescence reading and to determine whether any biologically active botulinum toxin is present in said sample based on said fluorescence reading. ### Claim 2 (depends on 1) The device of claim 1, wherein said peptide substrate comprises SEQ. ID NO. 1. ### Claim 3 (depends on 1) The device of claim 1, wherein said chamber comprises a real-time PCR detector and said microcontroller comprises a microcontroller associated with said PCR detector that has been additionally programmed to acquire said fluorescence reading after a plurality of cycles and to determine whether any biologically active botulinum toxin is present in said sample based on the change in said fluorescence reading over a plurality of said cycles. ### Claim 4 (depends on 1) The device of claim 1, further comprising a negative control sample positioned in said chamber that has fluorescence that is separately detectable by said detector. ### Claim 5 (depends on 4) The device of claim 4, wherein said negative control sample is a peptide selected from the group consisting of SEQ. ID NO. 2, SEQ. ID NO. 3, SEQ. ID NO. 4, and SEQ. ID NO. 5. ### Claim 6 (depends on 4) The device of claim 4, wherein said microcontroller is programmed to determine whether biologically active botulinum toxin in present in said sample based on the change in magnitude of said fluorescence reading of said test sample over a plurality of said cycles relative to the change in magnitude of said fluorescence reading of said negative control sample over said plurality of said cycles. ### Claim 7 (depends on 6) The device of claim 6, wherein said microcontroller is further programmed to determine whether biologically active botulinum toxin in present in said sample based an average change in slope of said fluorescence of said negative control sample over said plurality of said cycles and an average change in slope of said fluorescence of said test sample over said plurality of said cycles. ### Claim 8 A method for detecting the presence of biologically active botulinum toxin, comprising the steps of: providing a peptide substrate labeled with a fluorophore and a quencher and having a cleavage site responsive to biologically active botulinum toxin positioned between said fluorophone and said quencher; adding a test sample to said peptide substrate so that any biologically active botulinum toxin contained in said test sample will cleave said peptide substrate, thereby allowing said peptide substrate to fluoresce; incubating said peptide substrate; detecting the amount of fluorescence emitted from said sample and outputting a corresponding fluorescence reading; determining whether any biologically active botulinum toxin is present in said sample based on said fluorescence reading. ### Claim 9 (depends on 8) The method of claim 8, wherein said peptide substrate comprises SEQ. ID NO. 1 ### Claim 10 (depends on 9) The method of claim 9, wherein the step of determining whether biologically active botulinum toxin is present in said sample comprising calculating the change in magnitude of said fluorescence reading over time. ### Claim 11 (depends on 10) The method of claim 10, further comprising the step of providing a negative control sample in said chamber that has fluorescence that is separately detectable by said detector prior to. ### Claim 12 (depends on 11) The method of claim 11, wherein said negative control sample is a peptide selected from the group consisting of SEQ. ID NO. 2, SEQ. ID NO. 3, SEQ. ID NO. 4, and SEQ. ID NO. 5. ### Claim 13 (depends on 10) The method of claim 10, wherein the step of determining whether biologically active botulinum toxin is present in said sample comprising calculating the change in magnitude of said fluorescence reading of said test sample relative to the change in magnitude of said fluorescence reading of said negative control sample. ### Claim 14 (depends on 13) The method of claim 13, wherein the step of determining whether botulinum toxin is present in said sample comprising calculating an average change in slope of said fluorescence of said negative control sample and an average change in slope of said fluorescence of said test sample. ### Claim 15 (depends on 14) The method of claim 14, wherein said peptide substrate is provided in a real-time PCR detector having a controller that is programmed to incubate said test sample at a predetermined temperature, to detect the amount of fluorescence emitted from said sample, to output a corresponding fluorescence reading, and to determine whether any biologically active botulinum toxin is present in said sample based on said fluorescence reading. ### Claim 16 An isolated peptide comprising a sequence selected from the group consisting of SEQ. ID NO. 1, SEQ. ID NO. 2, SEQ. ID NO. 3, SEQ. ID NO. 4, and SEQ. ID NO. 5.