Progress 08/01/13 to 03/31/14
Target Audience: Executive Summary The purpose of this Phase 1 SBIR-funded project for the USDA is to produce more sensitive lateral flow (LF) or chromatographic test strips for the major foodborne pathogenic bacteria including the newly acknowledged “Big 6” non-O157 Shiga toxin-producing serotypes of E. coli or “STEC” (i.e., O26, O45, O103, O111, O121, and O145). Operational Technologies Corporation (OpTech) proposed coupling of its extant foodborne pathogen aptamers (U.S. Patent Application No. 13/136,820) and new aptamers reported herein (Appendix 1) which bind the Big 6 non-O157 STEC lipopolysaccharides (LPS) with fluorescent nanoparticles (FNPs; latex or other) or quantum dots (Q dots) in LF test strips to greatly enhance detection sensitivity versus conventional colloidal gold LF test strips. During Phase 1 Operational Technologies Corporation (OpTech) developed numerous new anti-Big 6 non-O157 STEC LPS aptamer sequences which are reported herein (Appendix 1) and for which OpTech will file a patent application or add these sequences to its existing patent application as a continuation in part. These nascent anti-LPS aptamers are longer (~ 200 bases) and therefore potentially more specific than previous shorter aptamers, because they can bind more epitopes on one chain. OpTech screened numerous existing aptamers for general E. coli, Listeria monocytogenes, and Salmonella enterica (formerly S. typhimurium) LF strip assays by conventional aptamer-colloidal gold LF methods and arrived at the best or promising sandwich aptamer combinations in each case. OpTech has also solved several problems which were previously impeding progress on aptamer-LF strip developmentsuch as: 1) the development of robust control lines by the use of digoxigenin-labeled conjugate aptamers on colloidal gold to give a strong red control line when anti-digoxigenin antibody is dried onto the nitrocellulose analytical membrane instead of relying on aptamer and primer hybridization, 2) selection of the best analytical membrane for speed and sensitivity (Millipore High Flow or HF-180), and 3) full mobility of clumped Q dots into nitrocellulose membranes by addition of 5% Tween 20 detergent to the Q dot-aptamer conjugate stock to disperse clumps. The E. coli LF assay was the best studied during Phase 1 and resulted in detection of as few as 3,000 E. coli strain 8739 cells per LF test using colloidal gold. This sensitivity was improved to < 300 E. coli cells by conversion to a Q dot-aptamer-based fluorescent LF assay. Due to diminishing funds near the end of Phase 1, OpTech was only able to explore one of the six STEC (the O111 E. coli) LF system, but found several combinations of new and existing aptamers which would detect the O111 serotype. The best aptamer sandwich combination for O111 also weakly detected O45 and O103 serotypes as well, but not O26, O121, O145 and O157:H7, thus making it semi-specific. There were some failures as well. For example, OpTech was not able to detect Campylobacter jejuni or botulinum toxins in the LF sandwich format despite previous success with these same aptamers in other fluorescence assay formats. However, these initial failures can probably be turned into successful aptamer-LF test strip assays by trial of other extant C. jejuni or botulinum toxin aptamers and further development in Phase 2. Due to the very good progress during Phase 1, OpTech attracted the attention of its LARTA sponsored business aide who invited OpTech to present at a late March 2014 agricultural investors’ forum in St. Louis (www.InvestMidwestforum.com) which could lead to substantial venture capital funding for OpTech in the future. OpTech also plans to add any promising new Big 6 aptamers for sale on its existing and profitable aptamer sales website: www.OTCBiotech.com. In addition, an open access peer-reviewed journal article was published based on the major accomplishments of Phase 1. The article is: Bruno J.G. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold. Pathogens. 3:341-355, 2014. URL: http://www.mdpi.com/2076-0817/3/2/341/ Changes/Problems:
What opportunities for training and professional development has the project provided?
How have the results been disseminated to communities of interest? Results were disseminated via an open access journal article as cited below: Bruno J.G. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold. Pathogens. 3:341-355, 2014. URL: http://www.mdpi.com/2076-0817/3/2/341/ What do you plan to do during the next reporting period to accomplish the goals?
What was accomplished under these goals?
The following are teh accomplishments and conclusions gleaned from the Phase 1 project: Much stronger and more robust control lines or dots can be achieved by the use of dual biotin and digoxigenin end-labeled aptamers on streptavidin-coated colloidal gold or Q dots in conjunction with an air-dried anti-digoxigenin monoclonal antibody in the control line or dot versus the former aptamer and primer capture or hybridization scheme. A 5’- or 3’-primary amine with 6 or 12 carbon length linker and 15 minute UV baking of the capture line or dot at ~ 1.5 µg of capture aptamer on nitrocellulose is necessary for robust capture of bacteria. Aptamers must be UV baked onto the analytical membrane or they will wash away during wicking. Streptavidin or aptamer-coated Q dots probably clump making it difficult for them to migrate all the way consistently into or through even large porosity nitrocellulose analytical membranes. However, this problem was solved by addition of 5% Tween 20 detergent to the stock aptamer-biotin-streptavidin-Q dot suspension as suggested by Berlina et al. (Anal. Biochem. Chem. 405:4997-5000, 2013). Millipore’s High Flow (HF)-180 with a 180 second (3 minute) transit time emerged as the optimal analytical membrane for its combination of speed and full mobility of colloidal gold and Q dots through the 4 cm length of the nitrocellulose membrane. Several promising sandwich aptamer combinations for general E. coli (strain 8739), Listeria monocytogenes (19115) grown at room temperature so as to express flagella, and Salmonella enterica 14028 have been identified. The best E. coli sandwich combination (EcO 3R-reporter conjugate and EcO 4F-capture aptamer) appears to produce a visible LOD of ~ 3,000 cells by colloidal gold and perhaps < 300 cells per LF test with Q dot 655 and UV excitation. LPS from the Big 6 non-O157 STEC were extracted, characterized on silver-stained polyacrylamide electrophoresis gels and used for 10 successful rounds of LPS-magnetic bead SELEX aptamer development with negative selection using a cocktail of the other 5 serotypes. Numerous Big 6 LPS aptamer sequences were obtained and are reported in Appendix 1 along with their secondary structures. The secondary structures show a greater number of loops or potential binding sites for the 200 base aptamers vs. the older 72 base aptamers which could enhance affinity, avidity and specificity. A set of preliminary experiments led to semi-specific LF detection of live E. coli O111 using the O111-9R aptamer for capture and the EcO 3R aptamer for reporting. This combination also detected live E. coli O45 and O103, but not the other Big 6 or O157 serotypes. The specificity can be enhanced further in Phase 2 by more rounds of negative selection and SELEX aptamer development. Overall the project was very successful and encouraging for the ultimate development of Big 6 aptamer-based colloidal gold and fluorescent (Q dot-based) LF test strips.
1.Bruno J.G. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold. Pathogens. 3:341-355, 2014. URL: http://www.mdpi.com/2076-0817/3/2/341/