GB2512691A - Real-time assay for the detection of botulinum toxin - Google Patents

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.

Description

REAL-TIME ASSAY FOR THE DETECTION OF BOTULINUM TOXIN

FIELD OF THE INVENTION

[00011 The present invention relates to the detection of biologically active botulinum toxins and, more specifically, to a real-time assay for detecting biologically active botulinum

toxins that can be implemented in the field.

DESCRIPTION OF THE RELATED ART

100021 Botulinum neurotoxins (BoNTs) are proteins produced by the bacteria (iostridiurn houlinurn. BoNTs are powerful toxins that cause the life threatening illness, botulism, in humans.

with BoNT serotype A (BoNT/A) being one of the most potent. BoNTs produce their toxic effects by entering neurons and then cleaving N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. In particular. BoNT/A specifically cleaves SNAP-25 which prevents the formation of a synaptic fusion complex and thereby inhibits the release of acetylcholine.

resulting in muscle fiber paralysis. BoNT exposure is fatal without immediate diagnosis and proper treatment, Due to their ease of production, BoNTs pose a major biological warfare threat.

[00031 Early detection of BoNTs is crucial for bio-security and food safety. Real-time quantitative polymerase chain reaction (qPCR) is a very common detection method used in the biodefense field, qPCR is a very sensitive and quick method for detecting biological organisms by amplifying specific regions of deoxyribonucleic acid (DNA), and can be used to detect the genes coding for BoNTs, However, BoNTs are proteins that do not require the intact organism to cause disease, and can be purified from the organism. The purified toxin, which consists of 100-kDa heavy chain (HC; required for cell entry) joined by a disulfide bond to a 5O-kDa light chain (LC: required for SNAP-25 cleavage), may be completely devoid of DNA and therefore not detectable using qPCR. qPCR has the ability to detect the gene coding for a protein toxin, but it does not directly detectthe presence, or i'nore importantly the activity of protein toxins.

BRThF SUMMARY OF THE INVENTION

100041 It is therefore a principal object and advantage of the present invention to provide a real-time assay for the detection of biologically active botulinum toxins.

[00051 In accordance with the foregoing objects and advantages, the present invention provides a real-time portable and rapid detection assay to identify the presence of biologically active toxin such as botulinum toxins. The detection assay includes a BoNT/A sensing fluorescent substrate, a negative control/interferent sensing fluorescent substrate, a qPCR detection protocol modified for toxin identification, and a toxin detection algorithm, The proteolytic activity of BoNT/A can be measured using a peptide cleavage assay, where a synthesized dual labeled fluoresccnt peptide slLbstratc is cleaved by BoNT/A, resulting in increased fluorescence based on Forester (fluorescence) resonance energy transfer (FRET) principles.

[00061 While assays according to the present invention may usc a commercially available fluorescent peptide substrate that mimics the BoNT/A deavage site of SNAP-25 (such as SNAPtide® peptide avai'able from List Biological Laboratories), the detection assay may be made more stable and more sensitive by using a fluorescent peptide substrate (SEQ. ID NO. I) that mimics both the BoNT/A binding and cleavage sites of SNAP-25. Additionally, a negative control/interf'erent sensing fluorescent peptide substrate (SEQ. ID NO. 2) was designed based on SEQ. ID NO. 1 so that it would be cleaved or inhibited by die same protcases or inhibitors that would affect SEQ. ID NO. I, but at the same time be insensitive to BoNT/A proteolytic activity due to a mutated BoNT/A cleavage site.

[00071 The increase in fluorescence in SEQ. ID NO. 1 caused by BoNT/A activity can be monitored in rcal-fime using any temperature controlled fluorimcter (e.g. the FiltcrMaxiD F5 Multimode Microplate Reader available from Molecular Devices), any lab-based qPCR fluorescence detection instrument (e.g. the Rotor-Gene® Q available from Qiagen) running a qPCR detection protocol modified for toxin identification, or any field-based qPCR fluorescence detection instrument (e.g. the RAZOR® EX available from BioFire Diagnostics or the Genedrive® available from Epistcm) running a qPCR detection protocol modified for toxin identification. The nLggedized RAZOR® EX and the small foim factor Genedrive® arc portable qPCR based p'atforms designed for use outside of a laboratory environment that have the ability to detect fluorescence changes in less than 1 hour for biodefense (RAZOR® EX) and point of care diagnostics (Genedrive®). Fluorescence data generated in the fluorescence detection instruments is then applied to a toxin detection algorithm, which utilizes data from a sample exposed to both SEQ. ID NO. 1 and SEQ. ID NO. 2 to determine if biologically active BoNT/A toxin is present or absent in the test sample, qPCR platfoims are preferred over basic temperature controlled fluorimeters because they allow the operator to use a single instrument to screen one sample using conventional qPCR for genetic detection of biological threat agents (such as the Bacillus anthracis and Franc/sd/a lularensis qPCR assays) and non-conventional activity screening for biological activity detection oftoxins (such as the BoNT/A activity assay describe here).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

100081 The present invention will be more thIly understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which: [00091 Figure 1 is a schematic of an assay for detecting botulinum toxin according to the present invention using the proof-of-principle fluorescent peptide SNAPtide® or the designed fluorescence peptide SEQ. ID NO, 1.

[00101 Figure 2 is a graphical representation of the anticipated fluorescence response and algorithmic data processing of an assay for detecting biologically active botulinum toxin according to the present invention.

[00111 Figures 3A-3B are a series of graphs displaying results acquired on the Rotor-Gene® Q qPCR instrument from the buffer optimization using 10 tM SNAP6de® and 1.9ng.

3.Sng, 7Sng. and l5ng BoNT/A-LC.

[00121 Figures 4A-4C are a series of graphs displaying results acquired on the Rotor-Gene® Q qPCR instrument using 10 jiM SNAPtide® showing the activity of 1.9 ng and 7.5 ng BoNT/A-LC in the presence of ZnC12 with varying concentrations of Dfl and in the presencc of the common PCR inhibitors humic acid or diesel exhaust residue.

100131 Figure 5 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 1iM SNAPtide® showing the activity of a range of BoNT/A-LC amounts (0.11 ng-30 ng) incubated for 55 minutes at 37°C.

[00141 Figure 6 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 tM SNAPtide® showing the activity of a range of BoNT/A-LC (0,06 ng-7.5 ng) using 34,5mM Hepes buffer pH 7.4 -I-0.03%v/v) Tween 20 + 0.31% PBS + 0.02% (v/v) Triton X-100.

[00151 Figure 7 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 jiM SNAPtide® showing the optimization of ZnC12 and DTT concentrations in the BoNT/A-LC detection assay.

100161 Figure 8 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 jiM SNAPtide® showing the detection of 1,9 ng-7.5 ng BoNT/A-LC in the presence of the common qPCR inhibitor diesd exhaust residue.

[00171 Figure 9 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 jiM SNAPtide® showing the detection of 7.5 ng BoNT/A-LC in the presence of the common qPCR inhibitor Arizona road dust.

[00181 Figure 10 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using 10 pM SNAPtide® showing the detection 7.5 ng BoNT/A-LC in the presence of the common qPCR inhibitor humic acid.

[00191 Figure II shows three-dimension& graphical representations of the C-terminal region of SNAP-25 binding to BoNT/A-LC which aided in the design of a new BoNT/A peptide substrate.

[00201 Figures 12A-12C show the protease cleavage maps of newly designed BoNT/A peptide substrates.

[00211 Figure 13 is a graph displaying results acquired on the FilterMax® F5 fluorimeter using the newly designed SEQ. ID NO. 1 pcptidc substrate compared to SNAPtidc® showing the improvement in detection of various amounts of BoNT/A-LC.

D

100221 Figure 14 is a graph displaying results acquired on the FiltcrMaxi) F5 fluorimeter showing the requirement of the SNARE domain in SEQ. ID NO. I (compared to SEQ. ID NO. 6) in the detection of BoNT/A-LC, [00231 Figures 15A-15B show graphs displaying results acquired on the FilterMax® F5 fluorimeter showing that on'y bi&ogically active BoNT/A is detected by the SEQ. ID NO. I BoNT/A peptide substrate.

[00241 Figures 16A-16B show graphs displaying results acquired on the FilterMax® F5 showing that the SEQ. ID NO. 2 peptide substrate can act as a negative controLinterferents sensor for SEQ. ID NO. 1 as it is unresponsive to biologically active BoNT/A-LC, but can detect non-specific protease activity (such as that found in Arizona Road Dust).

[00251 Figure 17 is a graph displaying results acquired on the FilterMax® F5 using SEQ.

ID NO. 1 showing the detection of 5,0 ng BoNT/A-LC in the presence of the common PCR inhibitor diesel exhaust extract.

[00261 Figure 18 is a graph displaying results acquired on the FilterMax® F5 using SEQ.

ID NO. 1 showing the detection of 2,5 ng BoNT/A-LC in the presence of the common PCR inhibitor humic acid.

[00271 Figure 19 is a graph displaying results acquired on the FilterMax® F5 using SEQ.

ID NO. 1 showing the detection of 5.0 ng BoNT/A-LC in the presence of the reducing reagents TCEP and DTT.

[00281 Figure 20 is a graph displaying results acquired on the FilterMax® F5 using SEQ.

ID NO. 1 showing the detection of 5.0 ng BoNT A Holotoxin Complex in the presence of varying concentrations of TCEP.

[00291 Figure 21 is a graph displaying results acquired on the RAZOR® EX portable qPCR instrument using SEQ. ID NO. 1 and SEQ. ID NO. 2 showing the detection of 7.5 ng of BoNT/A-LC.

[00301 Figure 22 is a graph displaying results acquired on the Genedrive® portable qPCR instaLment using SEQ. ID NO. 1 showing the detection various amounts of BoNT/A-LC.

100311 Figure 23 is a graph displaying results acquired on the FilterMax® F5 using SEQ.

ID NO. I showing the limits of detection of BoNT/A-LC.

[00321 Figure 24 is a list of the formulas used in the detechon algorithm of the present invenUon.

[00331 Figure 25 is a graph of the raw data of an example according to the detection algorithm of the present invention.

[00341 Figure 26 is a graph of the adjusted raw data of an example according to the detection algorithm of the present invention.

[00351 Figure 27 is a graph of a discrete slope comparison according to the detection algorithm of the present invention.

[00361 Figure 2 is a graph of a discrete slope delta comparison according to the detection algorithm of the present invention.

[00371 Figure 29 is a graph of a cumu'ative slope comparison according to the detection algorithm of the present invention.

[00381 Figure 30 is a graph of a cumulative slope delta comparison according to the detection algorithm of the present invention.

[00391 Figure 31 is a graph of a s'ope comparison according to the detection &gorithm of the present invention.

[00401 Figure 32 is a graph of a slope deha comparison according to the detection algorithm of the present invention.

[00411 FiglLre 33 is a graph of a cumulative and discrete adjusted slope according to the detection algorithm of the present invention.

[00421 Figure 34 is a graph of a cumulative and discrete mean adjusted slope according to the detection algorithm of the present invention.

[00431 Figure 35 is a graph of the standard deviations according to die detection algorithm of the present invention.

100441 Figure 36 is a graph of the discrete slope standard deviations according to the detection algorithm of the present invention.

[00451 Figure 37 is a graph of the cumulative slope standard deviations according to the detection algorithm of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[00461 Referring now to the drawings, wherein like reference numerals refer to like parts throughout, there is seen in Figure 1 a schematic of a functional protein assay with BoNT/A according to the present invention, where a fluorescently labeled peptide substrate, comprising a peptide substrate labeled with a fluorophore and a quencher, such as the FITC/DABCYL labeled proof-of principle substrate SNAPtide® or SEQ. ID NO. 1 (Table 1), is contacted by a sample containing botulinuni nenrotoxin (BoNT/A). For examp'e, SNAPtide® and SEQ. ID NO. I have an N-tcrminal FITC or internal FAM (fluorophore) tag and a C-terminal DABCYL (quencher) tag.

Upon cleavage by BoNT/A-LC, the fluorophore and quencher become spatially separated, resulting in increased fluorescence. The results may be monitored by an appropriate temperature controlled fluorescence reader, such as a Rotor-Gene® Q (available from Qiagen, Valencia, CA), a FilterMax® F5, a Genedrive®. or a RAZOR® EX system. As seen in Figure 2, the increase of fluorescence over time may be measured, graphically displayed, and/or compared to a negative control sample using an algorithm to indicate whether biologically active botulinum toxin is present in a given sample.

EXAMPLE I

Materials [00471 The assay according to the present invention was initially developed using recombinant Botulinum Neurotoxin Type A Light Chain (BoNT/A-LC) from List Biological Laboratories. Inc. (Campbell. CA) and later confirmed using Botulinum Nenrotoxin Type A complex from BEI Resources (Manassas, VA). Botulinnm Type A Complex Toxoid (BoNT/A Complex inactivated by fonnalin) from Metabiologics. Inc (Madison, WI) along with heat-inactivated BoNT/A-LC (boiled for 30 mm) were used to confirm the specificity of the assay to BoNT/A activity in the studies. Studies were performed using SNAPtide®, a peptide substrate abeIed with the FTTC/DABCYL FRET pair, purchased from List BioIogiea Laboratories, Inc. (Campbell, CA). Later studies were performed using designed and synthesized fluorescent peptide substrates (SEQ. ID NOS. 1 through 6) with the FAM/DABCYL FRET pair (Table 1). Hepes buffer s&ution arid tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (Saint Louis, MO). Dithiothreitol (DTT). Triton X-100, zinc chloride (ZnC12), Tween® 20, Phosphate Buffered Saline (PBS) solution, and Boviue serum albumin (BSA) were purchased from Fisher Scientific (Waltham, MA), Table 1 -Synthesized fluorescent peptide substrates SEQ. ID NO. 1 MDENLEQVSGJIGNLRHMALDMQNE[DTQNRQIDRIMEKADSN(KDabeyl) TRIDEANQRATKML(TCSFarn) SEQ. ID NO. 2 MDENLEQVSGIIGNLRFIMALDMGNIEIDTQNRQIDRIMEKAD SN(KDabcyl) TREDIOATN RAKML(K5Fain) SEQ. TD NO.3 MDENLEQVSGTIGNLRHMALDMGNE[DTQNRQIDRIMEKADSNQCDabcvI) TRFDJQNARTAflvTh(KSFani) SEQ. ID NO, 4 MDENLEQVSQJIGNLRHMALDMQNE[DTQNRQIDRIMEKADSN(KDabevl) TRE DJQN A TA RKML (K5 Fain) SEQ. TD NO, 5 MDENLEQVSQT1GNLRHMALDMQNE[DTQNRQIDRIMEKAD5NKDabcv1) TREDJQNATRAKML(K5Fam) SEQ. ID NO, 6 (KDabcyl)TRIDEANQRATKML(K5Fam) where KDabcvl) = Lys labeling by Dabcyl (or other queueher). (K5Fam) = Lys labeling by 5-Carboxyfluorescein (or other Iluorophore). and QR = BoNT/A clcavagc site 100481 Arizona road dust was purchased from Powder Technology, Inc. (Burnsville, MN).

Arizona road dust was collected on a SASS® 3100 filter eartridge from Research International (Monroe. WA), and extracted into lx PBS containing 0.05% (v/v) Triton X-l00 using the SASS® 3100 Dry Air Samp'er and SASS® 3010 Particle Extractor systems from Research International.

The extracted Arizona road dust was centrifuge for 5 minutes at 5000 rpm in an Eppcndorf table top centrifuge, and the supematant was used for inhibition studies. Arizona road dust was also utilized for protease activity studies, where a lOOmg/mL Arizona road dust mixture was made in H20, vortexed for 1 minute, then allowed to settle for 30 minutes. The resulting supenmtant was collected for use in the fluorescent peptide substrate based protease activity experiments.

100491 Diesel exhaust residue from a tractor was collected onto a SASS® 3100 filter cartridge. The filter contaimng the exhaust residue was extracted with lx PBS containing 0.05% (v/v) Triton X-100 using the SASS® 30 10 Partide Extractor system. The resuhing solution was used direcfly in inhibition studies.

[00501 Humic acid was purchased from Fisher Scientific (Waltham, MA). and was diss&ved in lx PBS containing 0.05% (v/v) Triton X-100 for use in subsequent inhibition studies, BoIVJ2A Assay Development and Optimization in the Rotor-Gene® Q using SN4/Jtide® [00511 Initial experiments tested a series of three SNAPtide® concentrations (5 10, and jiM) and 6 amounts of BoNT/A-LC (0.28. 1,4, 2,8. 7,14, 14,3, and 28.6 ng) in the following assay buffer: 50 mM Hepes buffer pH 7.4 + 0.05% (v/v) Tween® 20. Reactions were incubated in the Rotor-Gene® Q real-time PCR cyder using the following cycling profile: 60 x I minute cycles at 37°C. The Rotor-Gene® Q acquired a fluorescence signal at the end of each cyck arid was ab'e to detect BoNT/A-LC activity in real-time. A 10.tM SNAPtide® was chosen as the optimal peptide substrate concentration for the assay. Initial experiments in the Rotor-Gene® Q were perfonned using 60 x 1 minute cycles, but later were adjusted to 55 x I minute cycles to match the RAZOR® EX settings.

[00521 Next, different assay buffers were tested using the 10 pM SNAPtide® concentration and 3 amounts of BoNT/A-LC. The following buffers were compared: 50 mM Hepes buffer pH 7.4 + 0.05% (vlv) Tween® 20, 50mM Hepes buffer pH 7.4 -I-1 mg/mL BSA. 34.5 mM Hepes buffer pH 7.4 + 0.03% Twecn 20 + 0.31% PBS + 0.02% (vlv) Triton X-100. and 34.5 mM Hepes buffer pH 7.4 -1-0,69 mg/mL BSA + 0.31% PBS + 0.02% (v/v) Triton X-100 (Figure 1).

PBS + Triton X100 is commonly used to extract agents, such as BoNT/A, from collected environmental samples. The assay was optimized for working with 0.31% PBS and 0.02% (vlv) Triton X-100. Tween® 20 and BSA are commonly added to enhance protein stability during analysis. However, BSA caused negative controls containing 10 1.tM SNAPtide® and no BoNT/A-LC to fluoresce, so the use of Tween® 20 was preferred. As seen in Figure 3. assays were incubated at 37°C for 55 cycles (I cyde/minute) with fluorescence acquisition at the end of each cycle, BSA caused the negative controls to fluoresce, so the optimal assay buffer chosen was 34.5 mM Hepes buffer pH 7.4 + 0.03% Tween® 20 + 0.31% PBS + 0.02% Triton X-100. TlnLs. the Exarnpk moved forward using 34,5 mM Hepes buffer pH 7.4 + 0.03% (v/v) Tween® 20 + 0.31% PBS + 0.02% (v/v) Triton X-100 as the assay buffer.

[00531 Finally, the assay was optimized in the Rotor-Gene® Q to work with the full length BoNT/A protein by testing different concentrations of ZnCI2 and DTF. The use of a reducing agent, such as Dfl, is required to detect BoNT/A as the BoNT/A-LC and HC disulfide bonds needs to be broken to allow for BoNT!A-LC mediate peptide cleavage. The addition of ZnCI2 to the assay mix with DYF is necessary as DTT can chelate zinc, which is required for BoNT/A-LC protcasc activity. As seen in Figure 4. the activity of 1.9 ng and 7.5 ng BoNT/A-LC using varying concentrations of ZnCI2 and DTT was tested. The addition of Dli alone reduced BoNT/A-LC activity and 0,3 mM ZnCh was found to be the optimal concentration of ZnC2 required to counteract this reduction in activity (data not shown). Addition of the common PCR inhibitors humic acid (0.16 tg/rnL) mid diesel exhaust residue (1:10 dilution) did not affect the activity of BoNT/A-LC in the presence of various DTT concentrations and 0.3 mM ZnC2. In these experiments, 10 pM SNAPtide® and the following assay buffer were used: 34.5 mM 1-lepes buffer pH 7.4 + 0.03% (vlv) Tween® 20 + 0.31% PBS + 0.02%(v/v) Triton X-I00.

RD/VT/A Assv Development and Optimization in the RAZOR® TX using LSNA hide® 100541 The experiments performed in the Rotor-Gene® Q were translated to the RAZOR® EX. An initial experiment was performed in the RAZOR® EX using a configuration profile created specifically for this assay. This configuration profile used the following cycling parameters: 55 x 1 minute cycles at 37°C while acquiring fluorescence at the end of each cycle.

Referring to Figure 5. a range of BoNT/A-LC amounts (0.11 ng-30 ng) was tested using 10 RM SNAPtide® and 50 mM Hcpcs pH 7.4 + 0.05% (v/v) Twccn® 20 assay buffer. The RAZOR® EX was able to detect BoNT/A-LC activity in real-time. The ability of the RAZOR EX® was then tested to detect BoNT/A-LC activity using the 34.5 mM Hopes buffer pH 7.4 + 0.03% (vlv) Tween?) 20 + 0.31% PBS -F 0.02% (v/v) Triton X-I00 buffer. As seen in Figure 6, a range of BoNT/A-LC (0,06 ng-7.5 ng) + 10 iM SNAPtide® was assayed using 34,5 mM Hopes buffer pH 7.4 + 0.03% (vlv) Tween 20 -F 0.31% PBS + 0.02%(v/v) Triton X-100 buffer. The RAZOR® EX was able to detect BoNT/A-LC activity iii real-time, 100551 In order to optimize the ZnC12 and Dfl concentrations for use in the RAZOR® EX, the assay buffer was altered to 32,2 mM Hopes buffer pH 7.4 + 0,03% Tween 20 + 0,30% PBS + 0,02% Triton XI00, and used 9,64 tiM SNAPtide®, Referring to Figure 7, the combination of 2.5 mM Dfl and 0,3 mM ZnC12 was compatible, as the addition of Dfl is required to detect the full length BoNT/A protein.

100561 The 2.5 mM DTT and 0.3 mM ZnCI2 conditions were chosen as optimal concentrations and therefore utilized to test the performance of the assay in the RAZOR® EX in the presence of common PCR inhibitors, as seen in Figures 8-10, As seen in Figure 8. 1.9 ng-7.5 ng BoNT/A-LC in the presence of diesel exhaust residue established that diesd exhaust residne did not inhibit BoNT/A-LC activity. As seen in Figure 9, 0.04 mg/mL-0.08 mg/mL of Arizona road dust did not inhibit 7.5 ng BoNT/A-LC. Finally, as seen in Figure 10. 7.5 ng BoNT/A-LC estabhshed that the BoNT/A-LC assay is compatible with humic acid concentrations up to 0,23 .ig/mL.

Design of SEQ. Ti) NO. I and SEQ. ID NO. 2 BuNT/A Fluorescent Peptide Substrates 100571 Based on the studies utilizing SNAPtide® in the Rotor-Gene® Q and the RAZOR® EX, the detection of biologically active BoNT/A-LC protein toxin was shown to be possible on a qPCR platform given the correct buffer conditions and PCR parameters. While SNAPtide® is a good candidate for BoNT/A-LC activity, it did have issues with stability.

solubility. and detection signal. The instability of the SNAPtide® peptide was shown in a number of experiments where the negative control samples showed large and unexpected increases in fluorescence, followed by drops in fluorescence signal overtime. Additionally. solubility issues were identified in some of our solutions where precipitation of SNAPtide® was observed in both stock samples and experimental samples. Finally. the detection signal of SNAPtide® was not ideal as a large amount of BoNT-A LC is required in order to generate a high enough signal over noise to allow detection of BoNT-A LC activity based on an algorithmic detennination. Therefore, a new BoNT-A peptide substrate design was utilized.

100581 The present invention includes six fluorescent peptide substrates for BoNT/A detection (Table 1). The SNAPtide® peptide substrate is known to only include a short SEQ. ID NO. (about 15 amino acids in length) resembling the BoNT/A cleavage site on SNAP-25, While BoNT/A can cleave the SNAPtide® peptide substrate, BoNT/A binding to SNAPtide® is not ideal due to the exclusion of BoNT/A binding domains found on SNAP-25, Therefore, to increase the binding efficiency of BoNT/A, a 59 amino acid fluorescent peptide (SEQ. ID NO. 1) based on the SNAP-25/BoNT/A interaction was designed. Shown in the three-dimensional Swiss PDB viewer representations in Figure 11, BoNT/A-LC (in red) interacts with the C-terminal portion (Green/Blue) of the SNAP-25 protein. The BoNT/A cleavage area (dark blue) and cleavage site (light Mue) on SNAP-25 fall within a pocket within BoNT/A-LC protease (Figure I IA-B), Due to the acceptable distance (32.8 Angstroms) between the lysines located on the peptide SEQ. ID NO.

(Figure 1 IB). these residues were chosen as the location of fluorophore and quencher for the FRET pair. While the internal region of the peptide was not shown to have any obvious interactions with the BoNTIA-LC (Figure 1 1C). an alpha-helical structure located in a SNARE region of the SNAP-did interact with BoNT/A-LC (Figure lID). Based on these observations, the SEQ. ID NO. I fluorescent peptide was designed (TaMe I).

[00591 To design a negative control fluorescent peptide, it had to meet two criteria: 1) Cannot be cleaved to BoNTIA-LC and 2) Must be sensitive to any interferents that affect the SEQ.

ID NO. 1 peptide. Based on these criteria, four potential negative control fluorescent peptides were designed (SEQ. ID NOS. 2-5; Table 1). In each of the peptides, the fluor/quencher region was mutated in such a way to destroy the BoNTIA recognition/cleavage site. These SEQ. ID NO. s were then run through the ExPASy cleavage predictor software to determine if the fluor/queneher regions still possessed similar cleavage maps based on the known proteases (Figure 12). While SEQ. ID NO. 1 can be cleaved by 16 different proteases in 36 different potential cleavages, SEQ.

ID NO. 2 was shown to be cleaved by the same number of proteases in the same number of cleavages (Figure 12A). Additionally, the SEQ. ID NO. 2 fluor/quencher contained an S amino acid difference in the peptides BoNT/A cleavage recognition site compared to SEQ. ID NO. 1 (Table I). Therefore, the SEQ. ID NO. 2 negative control/interferents sensing peptide was chosen over the other negative controls due to various reasons as listed in the Figures 128-C.

BoNTA Assay Development and Optimization In the PilierMax® PS iislng SEQ. ID NO. I 100601 Initial experiments with SEQ. ID NO. I were performed to determine its capability in detecting BoNT/A-LC activity compared to the proof-of-principle peptide SNAPtide®, The FilterMax® F5 fluorimeter was used in this testing because it possesses a heat controlled sample detection area (can be regulated from 25 to 45°C) and can precisely detect changes in fluorescence of a broad detection range over a time course. Similar concentrations of SEQ. ID NO. 1 and SNAPtide® (10 pM) were used to detect 3 different amounts of BoNT/A-LC (7.5, IS, and 30 ng) in a 96 well plate (100 jiL per well). Simllar buffer conditions (30 mM Hepes pH 7.4, 0.2% Tween 20), that were optimized from the proof-of-principle studies, were used for all the samples in the assay. The reactions were incubated at 37°C for 60 minutes with fluorescence readings taken at 1 minute mtervals, As seen in Figure 13, a greater increase in fluorescence overtime was observed in all SEQ. ID NO. 1 samples compare to SNAPtide® samples at all BoNT/A-LC conditions tested.

These results show that the SEQ. ID NO. I fluorescence substrate produces a greater signal than that of SNAPtide®. Additionally, based on observational studies SEQ. ID NO. I is more staMe (does not precipitate) and produces very little fluorescence background noise compared to SNAPtide®.

Importance of the BoIVI2I4 Binding Region in SEQ. ID NO. I compared to SEQ. ID NO. 6 100611 As SEQ. ID NO. 1 was designed to possess an alpha-helical binding region to enhance its interaction with BoNT/A (Figure liD), the next test was designed to assess the importance of this region in detection BoNT/A activity, SEQ. ID NO. 6 was designed to lack the BoNT/A binding region that is found in SEQ. ID NO. 1 (Table 1). Similar concentrations of SEQ.

ID NO. 1 and SEQ. ID NO. 6 (10 j.tM) were used to detect 2 different amounts of BoNT/A-LC (15 and 30 ng) in a 96 well plate (100 JIL per wefl; 30 mM Hepes pH 7.4. 0.2% Tween 20) to be analyzed on the FilterMax® Fi fluorimcter. The reactions were incubated at 37°C for 60 minutes.

with fluorescence readings taken at 1 minute intervals. As seen in Figure 14, while SEQ. ID NO. 1 was able to detect BoNT/A-LC, the lack of the BoNTIA binding region on SEQ. ID NO. 6 rendered it incapable of detecting BoNT/A-LC activity, SEQ. 11) NO. I L)elects Biologically Active BoNI A, hut Not Heat or h'ormalin Inactivated BoNI?A 100621 The present invention was designed to produce an assay capaMe of detecting only the biologically active form of BoNT/A. Therefore, versions of BoNT/A that were known to be inactivated either through heating (heat inactivated BoNT/A-LC) or through chemical treatment with formalin (BoNT/A Toxoid) were assessed in our studies with SEQ, ID NO, 1, Due to the high signal produced using SEQ, ID NO, 1, we were able to optimize our conditions to only use 1 JLM instead of 10 SEQ. ID NO, I (data not shown). SEQ, ID NO, I (1 JIM) was used in a detection assay with BoNT/A-LC (I and 5 ng), heat inactivated BoNT/A-LC (5 and 80 ng), and BoNT/A Toxoid (20 and 200 ng) in a 96 well plate (100 p,tL per well; 30 mM Hepes pH 7,4, 0,2% Tween 20) to be analyzed on the FilterMaxCiv F5 fluorirneter. The reactions were incubated at 37°C for 60 minutes, with fluorescence readings taken at I minute intervals. As seen in Figures ISA, SEQ. ID NO. 1 was able to detection biologically active BoNT/A-LC (5 ng), but not heat inactivated BoNT/A-LC. even when present at high concentrations, Similarly, as seen in Figure 15B, SEQ. ID NO, I was able to detect biological'y active BoNT/A-LC (I ng), but not BoNT/A Toxoid, even when present at high concentrations. Therefore. assays utilizing SEQ. ID NO. 1 can differentiate between biologically active and biologically inactive BoNT/A.

Development of SEQ. II) NO, 2 as a Negative Control1nterferent Sensor tbr the BoNTA Assay [00631 The present invention was designed to produce a BoNT/A detection assay capable of producing a signal that can be inputted into an algorithm to determine if a sample possesses or lacks biologically active BoNT/A, In order to make this determination, the algorithm not only needs a signal from an input that can detect the presence or absence of BoNT/A, but it also requires a signal that is a negative control, which provides information regarding die backgrolLnd fluorescence in a system. This negative contro' must be insensitive to BoNT/A mediated cleavage, yet it must be able to registcr the background noise (interference) present in the system which may affect die detecting substrate (SEQ. ID NO. 1). Therefore, SEQ. ID NO. 2 was designed as fins negative control/interference sensor, SEQ. ID NO. I and SEQ. ID NO. 2 (I LM) were used in a detection assay with BoNT/A-LC (5 and 50 ng) in a 96 well plate (100 RL per well; 30 mlvi Hepes pH 7,4, 0.2% Tween 20) to be analyzed on the FilterMax® F5 fluorimcter, The reactions were incubated at 37°C for 60 minutes. with fluorescence readings taken at 1 minute intervals. As seen in Figures 16A. while SEQ. ID NO. 1 was able to detect biologically active BoNT/A-LC (5 and 50 ng), SEQ. ID NO. 2 showed no increase in fluorcsccnce over time in the presence of BoNT/A-LC.

even at high concentrations, Therefore, SEQ. ID NO. 2 is capable of acting as a negative control in die BoNT/A detection assay. in a separate test, SEQ. ID NO. 1 and SEQ. ID NO. 2 (1 jiM) \.vere used in a detection assay with 100 mg/niL Arizona road dust (ARD), a common PCR inhibitor, with observed protease activity, This assay was perfonned in a 96 well plate (100 jiL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20) and analyzed on the FilterMax® F5 fluorimeter at 37°C for 60 minutes, with fluorescence readings taken at 1 niinute intervals. As seen in Figure 16B, ARD non-specifically caused an increase in fluorescence (due to the presence of proteases within the ARD) in SEQ. ID NO. 1 over the time course of the assay. Similar to SEQ. ID NO. 1, ARD also caused an increase in fluorescence overtime in SEQ. ID NO. 2, Therefore, SEQ. ID NO. 2 can be utilized as a negative controi/interferents sensor for the BoNT/A detection assay.

SEQ. JD NO. 1 Detects BoNT'A Achy/tv in the Presence of Common PCR Inhibitors [00641 To determine if common PCR inhibitors affect the ability of SEQ. ID NO. I to detect the activity of BoNT/A-LC in the presence of common PCR inhibitors, diesel exhaust (Figure 17) and humic acid (Figure 18) were tcsted. SEQ. ID NO. 1 (1 pM) was incnbatcd with BoNT/A-LC (2.5 and 5 ng) in the presence of non-diluted diesel exhaust (30% assay volume) or humic acid (250 ng/mL) in a 96 well plate (100 4 per well; 30 mlvi Hepes pH 7.4, 0.2% Twccn 20) and analyzed on die FiltcrMax® ES fluorimeter at 37°C for 60 minutes, with fluorescence readings taken at 1 minute intervals. As seen in Figures 17 and 18, while common PCR inhibitors slightly affect the overall fluorescence in the BoNT/A detection assay, srnafl amounts of BoNT/A-LC were still capable of generating a large increase in fluorescence signal overtime, Therefore, SEQ. ID NO. 1 can detect BoNTIA-LC activity in the presence of common PCR inhibitors.

BoIVII2A Assay I?educingAgent Optimization using TC]±J' and Dli' 100651 Next, the assay was optimized to work with the fill length BoNT/A (containing both heavy and light chains). Initially, the assay was optimized to work with 0.3 mM ZnC12 and the 2.5 mM DYT reducing agent. However, TCEP, a reducing agent more staMe thwi DYT and that does not chelate zinc, was tested in the BoNT/A assay. SEQ. ID NO. 1 (1 jiM) was used in a detection assay with BoNTIA-LC (5 ng) either with DTT (2.5 mM. with 0.3 mM ZnCL). TCEP (2.5 mM) or no reducing agent, in a 96 well plate (100 jiL per wefl; 30 mM Hepes pH 7.4, 0.2% Tween 20). The reactions were incubated at 37°C for 60 minutes in the FilterMax® F5 fluorimeter.

with fluorescence readings taken at 1 minute intervals, As seen in Figure 19, samples with 2,5 mM TCEP perf'omed better than samples with DTT (and zinc), and even better than samp'es containing no reducing agent. Therefore, TCEP was chosen as the reducing agent used in the BoNT/A activity detection assay.

Detection of Full BoN7i/A Holotoxin Complex in the B0N7YA Assay with TCFP 100661 To determine die optimal TCEP concentration required for BoNT/A Holotoxin detection, SEQ. ID NO. I (I RN'l) was used in a detection assay with BoNT/A Holotoxin (5 ng) either with or without TCEP (1,0 and 2,0 mM), in a 96 wefl plate (100 jiL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20). The reactions were incubated at 37°C for 60 minutes in the FilterMaxilv F5 fluorimeter, with fluorescence readings taken at 1 minute intervals. As seen in Figure 20, samples with 1.0 mM TCEP performed better than samples with 2.0 mM TCEP, noted by the greater increase in fluorescence over time. As expected, samples that did not contain TCEP were unable to detect BoNT/A Holotoxin. Therefore, 1.0 mM TCEP was chosen as the reducing agent concentration used in the BoNT/A activity detection assay.

BoNTA Assay Development and Optimization in (he RAZOR® EXusing SEQ. ID NO. 1 and 2 100671 One embodiment of the present invention is an assay to detect bi&ogically active BoNT/A on a qPCR platform. The previous buffer optimizations and designed fluorescence peptide substrates which were optimized on the FilterMaxR) F5 fluorirneter can be translated to perfomi on a qPCR platforni. To test this, the RAZOR® EX qPCR protoc& was optimized (based on information gathered from assays performed on the RAZOR® EX with SNAPtide®). The optimizations consistcd of making an isothennal cycling protocol to be run at 370 C. adapting the read times to be performed at 1 minute intervals, and collecting PCR run data as raw data for subsequent graphing and algorithm processing. As seen in Figure 21, SEQ. ID NO. 1 and SEQ. ID NO. 2 (both at 1 piM) were used in a detection assay with BoNT/A-LC (7.5 ng) in a 6 x 2 polLch (200 1it per well; 30 mM Hepes pH 7,4, 0.2% Tween 20, 1.0 mM TCEP). The reactions were incubated at 37°C for 45 minutcs in the RAZOR® EX. with fluorescence read at 1 minute intervals. Whlie this assay was for a total of 45 minutes. such a strong positive signal was generated against a low background signal that a positive BoNT/A detemiination based on algorithmic determination and weighting (based on overall fluorescence increase and increase in fluorescence slope) could be made within approximately 10 minutes (10 cycles).

BoNTA Assay Development and Optimization in the Genedrive® using SEQ. ID NO. 1 at 42°C [00681 To flLrther test the BoNT/A assay, we utilized the Genedrive® portable qPCR system, To test this assay, we first optimized the Genedrive® qPCR protocol making an isothemal cycling protoc& to be run at 42°C (standardized on this machine), adapting the read times to be performed at 1 second intervals, and collecting PCR run data as raw data for slLbsequent graphing and algorithm processing. As seen in Figure 22, SEQ. ID NO. 1 (1 tM was used in a detection assay with BoNT/A-LC (1.0 and 5,0 ng) in a 3 x 1 Genedrive® cassette (20 RL per well; 30 mM Hepes pH 7.4, 0.2% Tween 20, 1.0 mM TCEP). The reactions were incubated at 42°C for 60 minutes iii the Genedrive®, with fluorescence readings taken at 1 second intervals. While this assay was run for a total of 60 minutes, such a strong positive signal was generated against a background signal that a positive BoNT/A determination based on algorithmic determination and weighting (based on overall fluorescence increase and increase in fluorescence slope) could be made within approximately 15 minutes. Additionally. these results show that the BoNT/A assay can be run at 42°C, as well at 37°C.

BoNTA Assay Limits ofDeteeiion using SEQ. ID NO. 1 100691 To determine the limit of detection of biologically active BoNT/A LC using SEQ.

ID NO. 1, SEQ. ID NO. 1 (1 jiM) was incubated with varying amounts of BoNT/A-LC (0,008, 0.04, and 0,2 ng) in a 96 well plate (100.L per well; 30mM Hepcs pH 7.4. 0.2% Twcen 20, 1,0 mM TCEP) and analyzed on the FilterMax® F5 fluorimeter at 37°C for 60 minutes, with fluorescence readings taken at 1 minute intervals. As seen in Figure 23, the limit of the detection for the SEQ. ID NO. 1 BoNT/A activity assay was at least 0.04 ng BoNTIA-LC over a 60 minute time period. Longer incubations may increase this limit of detection, Development and Optimization of a Toxin Algorithm tbr the Detection of BoN'7A [00701 The algorithm according to the present invention was developed to allow the presence or absence determination of biologicaflv active toxin on a qPCR platform. The detection algorithm that is typically included in qPCR fluorescence detection instruments (e.g. Rotor-Gene® Q, RAZOR® EX, Genedrive®) is optimized to interpret fluorescence data from individual genetic samples that generate exponential increases in fluorescence when a critical threshold (CT) point is achieved. Genetic samples arc callcd positive" by a qPCR instruments algorithm based on this exponential fluorescence increase (graphically displayed as a sigmoidal curve; cycle vs. fluorescence), which can be distinguished from negative samples that do not generate exponential fluorescence increases (graphically displayed as a flat straight line). Conn'ary to genetic samples.

toxin samples generate non-exponential increases in fluorescence (graphically displayed as a straight line with a positive slope; time vs. fluorescence). Additionally, toxin samples require the comparison of thc unknown samples (could be positivc or negative) to a control sample (a negative sample) to determine a signal to noise ratio. Therefore. a qPCR instrument's genetic algorithm.

which inherently lacks the capability to factor in a signal to noise ratio and is wcightcd to distinguish exponential data from non-exponential data, is not usefiLl for data generated from toxin assays.

[00711 The first approach that was applied to the data from the initial SNAPtide experiments was to compare the fluorescence at the end of each cycle to a threshold value. The concept was that biologically active toxin was present in the sample if a specified threshold was exceeded; otherwise, toxin was absent from the sample. Observations of the results from this initial approach (data not shown) showed that this method was unreliable as background noise could exceed the thresh&d under certain conditions (e.g., interferents). These observations helped to illustrate the importance of using a negative control for determining the level of background fluorescence.

[00721 Development of the algorithm began by importing the raw data outputs into Microsoft Excel for 152 sample sets run on the FilterMax® F5 fluorimeter and 15 sample sets run on the RAZOR® EX. The purpose of this was to chart the data for comparison and perforniing investigative calculations to identift trends and conditions of significaiice. The following progression was applied to each of these 167 sample sets. First, the raw fluorescence values were graphed. as seen in Figure 25, for both the assay and the negative control. This graph illustrated that the two lines diverged, but also experienced marked variabihty between consecutive cycles.

Next, the average fluorescence of the negative control was graphed, as seen in Figure 26, to smooth out the variability of the unprocessed data. The backgroundlnegative control data values were then subtracted from the assay data values and graphed, as seen in Figure 26, for comparison.

This illustrated that the backgrolLnd fluorescence could vary somewhat depending on which well or lane the sample was tested in becaiLse some of the subtracfions resulted in a fluorescence value less than zero for the assay, which is not an actual possible condition.

[00731 The divergence of assay fluorescence values from the negative control fluorescence was identified as an important contributor to determining the presence of a biologically active toxin, but it was also identified that the rate at which these values diverged (or the slope of the curve) was also important. To study this. graphs of the slopes at each cycle, as seen in Figure 27, and the change in tile slope over time, as seen in Figure 28, were created. These graphs showed that it could be difficult to compare the slope of the background fluorescence curve with the slope of the assay curve at each cycle with a good degree of accuracy. The cumulative slope, as seen in Figure 29. and the deltas between cycles of the cumulative slope, as seen Figure 30, were then graphed. which demonstrate that there is a higher variability in the first 10 cycles of a run, This finding lead to the conclusion that we needed an algorithm that could even out the high variability over time, Comparisons of the discrete and cumulative slopes for unprocessed values, as see in Figures 3! and 32, and background adjusted values, as seen in Figures 33 and 34, Were graphed to reinforce the idea that the algorithm should be using an averaged slope value instead of the slope at a specified point in time.

100741 Analysis of the standard deviations of fluorescence values for the assay, negative control, and the averages of these showed that there is a more noticeable increase over time for the assay than for the negative control, as seen in Figure 35. This finding started on the path to using standard deviation as the preferred unit of measure for deterniining if biologically active toxin is present in a sample. Next, the standard deviations of the discrete sloped, as seen in Figure 36, and cumulative sloped, as seen in Figure 37. were compared. This confirmed findings from previous comparisons that cumulative slope is a better indicator of the presence of biologically active toxin and that standard deviation is an acceptable unit of measure to use for this determination. All of these findings were used as inputs to developing the toxin detection algorithm.

100751 Tn order to implement the botulinum assay of the present invention in a legacy or conventional real-time PCR detector that is adapted strictly for real-time PCR routines and assays, the present invention also encompasses a detection algorithm to identify the presence of biologically active toxin that is instrument independent. The algorithm of the present invention is intended to be applied periodically at the end of each time cycle during an assay, and the output from the algorithm becomes more reliable as the number of completed cycles increases. The contributing factors to the algorithm are the fluorescence values as measured by the instrument of choice and the change in fluorescence over time. The algorithm requires a negative control assay sample to be run simultaneously on the same instrument, which provides a measure of the background fluorescence in the sample not resulting from peptide deavage activity by a toxin (e.g., BoNT/A).

[00761 More specifically, the algorithm determines if the fluorescence and the change over time in fluorescence for the assay differ significanflv enough from the negative control background fluorescence and the change in background fluorescence to indicate peptide cleavage activity, The magnitude of the delta from the background values is positi,vely conelated to the likelihood of the presence of bi&ogically active toxin. The larger the differences for each of the factors, the higher the likelihood that BoNT/A is present in the sample.

[00771 The algorithm relies on fluorescence values that have been obtained for all completed time cycles. First, the standard deviation and average fluorescence are calc&ated for the negative control fluorescence values. The average fluorescence is also calculated from the sample control values. Using these c&cuated values, the algorithm determines the number of positive standard deviations (up to 10) between the negative contr& average fluorescence and the sample average fluorescence, (A sample average fluorescence lower than the negative control average fluorescence is assigned a resulting value of 0 standard deviations.) The resulting number is then weighted by the fluorescence weight factor to obtain the fluorescence sub-score for that cycle. A fluorescence weight factor is used to assign the level of importance that fluorescence contributes to the determination of the presence or absence of a biologicafly active toxin, The fluorescence sub-scores are added together to achieve the overall fluorescence sub-score.

[00781 Next, the change in fluorescence (slope) between consecutive cycles is evaluated.

The standard deviation and average slope are calculated for the negative control fluorescence values. The average slope is also calculated from the sample control values. Using these calculated values, the algorithm determines the number of positive standard deviations (up to 10) between the negative control average slope and the sample average slope. (A sample average slope lower than the negative control average slope is assigned a resulting value of 0 standard deviations.) The resulting number is then weighted by the slope weight factor to obtain the slope sub-score for that cycle. A slope weight factor is used to assiw the level of importance that slope contributes to the deterniination of the presence or absence of a biologicaflv active toxin. The slope sub-scores are added together to achieve the overall slope sub-score.

The overall fluorescence sub-score and the overall slope sub-score are then added together and then divided by the maximum possibk points to arrive at the likelihood score. This score can then be compared to one or more thresholds to provide a ranking of the presence of biologically active toxin. For instance, comparing the score against a single threshold will indicate positive vs. negative. Similady, comparing the score against three thresholds or ratiges will provide low, medium, high indications of a positive presence of the biologically active toxin,

EXAMPLE 2

100801 The mathematical form&as and variaMes for determining each of the steps of the algorithm may be seen in Fig, 24, Figures 25-37 are graphs of the results of the steps of the algorithm of the present invention as applied to the exemplary raw test data of Table 2 below: Table 2-Raw Test Data 0:00:00 I 17219782 16639028 16929405 16941250 16127615 16534432,5 0:01:03 2 16954224 16570812 16762518 16898200 16291588 16594894 0:02:06 3 17348886 16617691 16983288.5 17064096 16192753 16628424,5 0:03:09 4 17037452 16571507 16804479.5 16868470 16139195 16503832.5 0:04:12 5 17063478 16493083 16778280.5 16974956 16113786 16544371 0:05:15 6 16995928 16555440 16775684 16897506 16102262 16499884 0:06:18 7 16997406 16522870 16760138 16928310 16102754 16515532 0:07:21 8 17049742 16419397 16734569.5 16958966 16182215 16570590.5 0:08:24 9 17187260 16568604 16877932 16991158 16241493 16616325.5 0:09:27 10 17127746 16612530 16870138 17091148 16158359 16624753,5 0:10:29 11 17489694 16721705 17105699.5 17135356 16393144 16764250 0:11:32 12 17073140 16455406 16764273 17003150 16292940 16648045 0:12:35 13 17162860 16651130 16906995 17032674 16263690 16648182 0:13:38 14 17180334 16635167 16907750.5 17168432 16399479 16783955,5 0:14:41 15 17290586 16598597 16944591.5 17113964 16396502 16755233 0:15:44 16 17302706 16724791 17013748.5 17235722 16379507 16807614,5 0:16:47 17 17419846 16856302 17138074 17346484 16612662 16979573 0:17:50 18 17548768 16974448 17261608 17374476 16559293 16966884,5 0:18:53 19 17431 ISO 16769061 17100105.5 17402868 16559305 16981086,5 0:19:56 20 17525446 16787790 17156618 17441596 16634751 17038173,5 0:20:59 21 17443736 16832370 17138053 17433534 16665144 17049339 0:22:02 22 17502950 16827952 1716545! 17464344 16838100 17151222 0:23:05 23 17767910 17002642 17385276 17661374 16788834 17225104 0:24:08 24 17409266 16875390 17142328 17625426 16799602 17212514 0:25:11 25 17439062 16791298 17115180 17603460 16859024 17231242 0:26:14 26 17844372 16958290 17401331 17690426 16814306 17252366 0:27:17 27 17522334 16931420 17226877 17606234 16768304 17187269 0:28:20 28 17472412 16915442 17193927 17705496 16960658 17333077 0:29:23 29 17535798 17028778 17282288 17857644 17060324 17458984 0:30:26 30 17630158 17067400 17348779 17948812 17170650 17559731 0:31:29 3! 17606646 16984718 17295682 17762786 17053586 17408186 0:32:32 32 17582174 16880812 17231493 18037586 17240350 17638968 0:33:34 33 17613928 17060662 17337295 17886246 17218370 17552308 0:34:37 34 17564772 17119882 17342327 17999864 17198184 17599024 0:35:40 35 17985124 17108548 17546836 18146340 17307620 17726980 0:36:43 36 17647100 17003370 17325235 18020932 17235566 17628249 0:37:46 37 17621018 17046734 17333876 18041022 17375910 17708466 0:38:49 38 17636718 17009510 17323114 17949748 17263460 17606604 0:39:52 39 17625832 16817450 17221641 17960704 17244136 17602420 0:40:55 40 17970444 17141602 17556023 18258588 17547610 17903099 0:41:58 4! 17670898 17016084 1734349! 18172040 17581468 17876754 0:43:01 42 17707308 17035982 17371645 18183054 17457358 17820206 0:44.04 43 17683410 17089802 17386606 18330410 17610868 17970639 0:45:07 44 17754202 17058480 1740634! 18275842 17577324 17926583 0:46:10 45 17781308 17112792 17447050 18463180 17673186 18068183 0:47:13 16 17724302 17110662 17417482 18385432 17806612 18096022 0:48:16 47 17716832 17185570 1745120! 18513974 17789102 18151538 0:49:19 48 17733516 17043026 17388271 18561598 17705960 18133779 0:50:22 49 17684590 17028736 17356663 18436202 17776382 18106292 0:51:24 50 17610388 17012552 17311470 18454758 17701072 18077915 0:52:27 51 17635742 17042302 17339022 18607506 17856482 18231994 0:53:30 52 17937962 17134502 17536232 18486846 17739870 18113358 0:54:33 53 17711400 17081744 17396572 18525500 17660466 18092983 0:55:36 54 17746340 17135416 17440878 18654216 17912552 18283384 0:56:39 55 18025748 17261254 17643501 18837796 18026790 18432293 0:57:42 56 17837154 17217404 17527279 18908546 18053532 18481039 0:58:45 57 17759734 17116800 17438267 18795458 18018540 18406999 0:59:48 58 17751840 17179348 17465594 18852418 18077902 18465160 1:00:51 59 17834238 17192950 17513594 18839772 18060456 18450114 1:01:54 60 17781560 17167442 1747450! 18879812 18142536 18511174 1:02:57 61 17897182 17219990 17558586 18978560 18162694 18570627 100811 It should be recognized by those of skill in the art that the algorithm of the present invention may be programmed into or as part of the operating system of a device, such as a real-time PCR detection system, to enable the detection algorithm to be applied to a target sample. For example. the a'gorithm may physically incorporated into a PCR instrument will depend on the PCR instrument and vendor cooperation, and could be done by implementing the algorithm in code modules added to the instrument. Alternatively, the algorithm may be implemented on a laptop connected to the PCR instrument to analyze the data if it is unable to be incorporated directly into the PCR instrument.

Claims (6)

1. CLAIMS: 1. A device for detecting the presence of bi&ogically active botulinum toxin, comprising: a chamber for receiving a test sample said chamber including a peptide substrate labeled with a fluorophore arid 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 sampk based on said fluorescence reading.
2. 2. The device according to claim 1, wherein said peptide substrate comprises SEQ.
3. IDNO, I, 3, The device according to claim I or daim 2, 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 purahtv of cydes and to deterniine whether any' biologically active botulinum toxin is present iii said sample based on the change in said fluorescence reading over a plurality of said cycles.
4. 4, The device according to any preceding claim, thrther comprising a negative control sample positioned in said chamber that has fluorescence that is separately detectable by said detector.
5. 5. The device according to 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.
6. 6. The device according to claim 4 or claim 5, 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 filLorescence reading of said negative control sample over said plurality of said cycles.

   7, The device according to 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, 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 bi&ogically active botulinum toxin positioned between said fluorophone and said qucncher adding a test sanipe to said peptide substrate so that any biologically active botulinuni 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, 9. The method according to claim 8, wherein said peptide substrate comprises SEQ.IDNO. 1.10. The method according to claim 8 or claim 9. wherein the step of determining whether biologically active botulinum toxin is present in said sample comprises calculating the change in magnitude of said fluorescence reading over time.11. The method according to any of claims 8 to 10 fiLrther comprising the step of providing a negative control sample in said chamber that has fluorescence that is separately detectable by said detector prior to adding said test sample.12. 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. 5.13. The method according to claim 11 or claim 12, wherein the step of determining whether biologically active botulinum toxin is present in said sample comprises calculating the chatige in magnitude of said fluorescence reading of said test sample rdative to the change in magnitude of said fluorescence reading of said negative control sample, 14. The method according to any of claims 11 to 13, wherein the step of determining whether botuhnum toxin is present in said sample comprises cakdating 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.15. The method according to any of claims 8 to 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 biologieall active botulinum toxin is present in said sample based on said fluorescence reading.16. An is&ated 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.