Georgetown University Medical Center

Sequence-Specific Detection of Nucleotide Sequences for Rapid Detection of Pathogens

Section: For Industry

Categories: "Diagnostics" "Viruses, Chronic & Infectious Diseases" "Research Tools"

Reference #: 2004-002

OTC Contact: Ruchika Nijhara Ph.D. (Directory Information | Send a Message)

Description

The invention describes a sensitive method of identifying single- or double-stranded DNA/RNA sequences. Such a method can be used to detect the presence of any microorganism 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 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.

Applications

  • Medical diagnostics- can detect the presence of any microorganism from any biological sample.
  • Bio-defense tool- can be used for a rapid, specific, and sensitive detection of any biological threat agent such as anthrax or plaque.
  • Food safety
  • SNP analysis
  • Genotyping
  • Biological research tool

Advantages

  • High specificity- based on the complete complementarity between the probe and the target DNA and specificity of nick endonucleases.
  • Excellent sensitivity- can detect a single DNA/RNA target at very low levels (less than 30 cfu).
  • Reaction is rapid; full cleavage of probe occurs within one minute.
  • Method is amenable to automation.
  • The method can be multiplexed allowing the detection of multiple sequences (multiple genes in one organism or individual genes in multiple organisms).
  • The assay could also be used to identify both DNA and RNA sequences.
  • Real time assays can be performed that are less time consuming than end-point assays and offer the possibility of quantification.
  • Assays can be performed in parallel and are inexpensive

Stage of Development

Studies 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 completed to develop a Dengue virus assay which can detect Dengue serotypes in a highly sensitive single-tube multiplex assay.

Relevant Publications

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA); Danielsen M et al; Nucleic Acids Res. 2007;35(18):e117.

Three manuscripts under review.

INVENTORS: Mark Danielsen, Eugene Davidson, and Kenneth L. Dretchen

Patent Status

US Patent application # 11/884,366, entitled, “Sequence-Specific Detection Of Nucleotide Sequences” filed on August 15, 2007