Final Report: Hand-Held Fluorometer Using SELEX DNA Aptamer Strip Assays To Detect Cryptosporidium and Encephalitozoon

EPA Contract Number: 68D01044
Title: Hand-Held Fluorometer Using SELEX DNA Aptamer Strip Assays To Detect Cryptosporidium and Encephalitozoon
Investigators: Bruno, John G.
Small Business: OmniSite BioDiagnostics Inc.
EPA Contact: Manager, SBIR Program
Phase: I
Project Period: April 1, 2001 through September 1, 2001
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2001) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

OmniSite BioDiagnostics, Inc. (OmniSite), a wholly owned subsidiary of Systems & Processes Engineering Corporation (SPEC), proposed to develop receptors composed of DNA oligomers (called "aptamers") from a combinatorial library for binding and detecting Cryptosporidium parvum oocysts and species of Encephalitozoon microspores. Aptamers were generated by the Systematic Evolution of Ligands by EXponential enrichment (SELEX) combinatorial chemistry technique. SELEX aptamers offer several major advantages over antibody technology; most notably eliminating the use of animal hosts, resulting in reduced cost as well as higher affinity and specificity to target pathogens of interest. In Phase I, OmniSite proposed to generate a set of specific DNA aptamers to bind and detect oocysts and spores. Aptamer assay development was designed for use on a magnetic bead-based cartridge for enhanced sample separation into various aptamer capture regions with subsequent detection using SPEC's proprietary hand-held fluorometer. In Phase II, OmniSite will clone and sequence the aptamers and optimize assays for field and home use. In addition, OmniSite will add the ability to transfer data from the fluorescence reader at remote locations to a Web site either by hardwire or via the wireless Internet.

Summary/Accomplishments (Outputs/Outcomes):

OmniSite has made significant strides toward the use of DNA aptamers coupled to fluorescence polarization (FP) for the detection of C. parvum and related waterborne parasites during this Phase I SBIR project. Specifically, OmniSite has:
  • Developed a set of DNA aptamers to C. parvum and E. cuniculi.
  • Demonstrated binding of aptamers to parasite targets by fluorescence microscopy, spectrofluorometry, and FP.
  • Developed a tabletop and a portable FP reader instrument.
  • Demonstrated highly reproducible FP data with a Cy5-labeled protein slowed in increasing concentrations of glycerol.
  • Established a commercial tie with Quantum Design Corporation to aid in the Phase II development of a magnetic bead-based aptamer cartridge for the detection of C. parvum and E. cuniculi.

Conclusions:

OmniSite has developed a very technically and commercially viable technology that combines SELEX DNA aptamers (that have significant advantages over antibodies in terms of ease of manufacture and cost); FP for homogenous, one-step binding detection; and the purification ability of a magnetic microbead-based cartridge. Together, these technologies offer a powerful new tool for detection of a variety of eukaryotic, bacterial, and even viral waterborne pathogens. This conclusion is supported by Foresight Science and Technology's assessment of OmniSite's FP aptamer magnetic bead technology as well.

Supplemental Keywords:

aptamers, assays, Cryptosporidium, DNA, Encephalitozoon, fluorescence polarization, magnetic bead, SELEX, water., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Chemistry, Monitoring/Modeling, Environmental Monitoring, Drinking Water, Engineering, Engineering, Chemistry, & Physics, Environmental Engineering, Pathology, cryptosporidium parvum oocysts, pathogens, encephalitozoon, aptamer, detect, cryptosporidium , microbial risk management