Reference #: 2004-002 & 2009-010
Researchers at Georgetown University have developed a sensitive method for detecting the presence of any microorganism (such as bacteria or virus) in an infection, contamination or otherwise that affect human and animal health. The method involves the hybridization of a probe and target DNA to create a recognition site for a strand-specific enzyme (endonuclease) that cleaves the probe into two pieces leaving the target DNA intact. Laser-induced fluorescence coupled with capillary electrophoresis is then used to measure the probe cleavage products. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats.
- Diagnosis of Infectious diseases (human, animal or plant diseases)
- Diagnosis of Hospital infections, such as MRSA
- Diagnosis of ESKAPE organisms
- Detection of bioware agents- can be used for a rapid, specific, and sensitive detection of any biological threat agent such as anthrax or plaque.
- Food and water surveillance
- Environmental monitoring
- SNP analysis and Genotyping
- As a biological research tool
- Broad spectrum: Detects bacteria and both DNA and RNA viruses
- Sensitive: Detection levels approaching a single organism
- Specific: No cross-reactivity with closely related organisms
- Fast : Results within hours (currently the assay can be completed in under three hours)
- Robust: Works with unpurified samples
- High throughput: Can be multiplexed and automated
- Stability: Processed samples can be archived for future testing
- Isothermal: Does not use thermocycling
- Multiple applications: Works with environmental, biological and diagnostic samples
- Extremely low false positive and false negatives rates
- Extreme sensitivity towards RNA based microorganisms
- No highly specialized equipment is required
- No unusual training is required.
Stage of Development
Studies are done to demonstrate that the assay can be used to specifically detect the presence of Bacillus anthracis pX01 and pX02 plasmids, E-Coli genomic DNA and Bacillus subtilis genomic DNA. Studies also completed to develop a Dengue virus assay which can detect Dengue serotypes in a highly sensitive single-tube multiplex assay.
The initial work was funded by Department of Defense (DoD) and focused on the development of a stand-alone instrument capable of detecting bioterror threat organism. As a part of this work, Georgetown scientists developed probes against Bacillus anthracis that have been validated by DoD. Also, most recently, the researchers developed and delivered to DoD a semi-automated prototype of a stand-alone detector that uses these probes.
- Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA); Danielsen M et al; Nucleic Acids Res. 2007;35(18):e117.
- 3 manuscripts under review.
Mark Danielsen, Eugene Davidson, and Kenneth L. Dretchen
US Patent application # 11/884,366, entitled, “Sequence-Specific Detection Of Nucleotide Sequences” filed on August 15, 2007; • PCT application, WO 2010/096202, entitled, “Sequence –Specific Detection of Nucleotide Sequences” filed on February 23, 2010