Towards on-site detection of nucleic acids for pathogen monitoring.

摘要

As a leading cause of death in developing countries and a persistent problem elsewhere, pathogenic organisms are as ubiquitous as they are dangerous. Significant worldwide resources are directed toward their detection and eradication, leading to a broad coalition of government agencies, healthcare providers, academic researchers, and food manufacturers dedicated to providing the best-available prevention strategies for mitigating exposure risk. At the forefront of this effort is the field of pathogen detection. Our work directly addresses the current lack of simple, rapid, sensitive, and selective pathogen detection methods needed for frontline intervention in the most at-risk populations. To begin, we focused on the common food- and water-borne contaminant, E. coli. Using a resonance energy transfer system incorporating a bioluminescent protein and quantum dots, we demonstrated that adjacent hybridization of sequence-specific, labeled probes could detect E. coli 16s rRNA at concentrations as low as 2.1 nM in only 5 minutes. Continuing, we developed a paper-based platform for Epstein-Barr virus (EBV) detection using a target-bridged capture scheme in which EBER-1 RNA from EBV linked a tethered probe to a fluorescent reporter probe for a low nanomolar detection limit. Finally, we developed a novel tuberculosis (TB) biosensor in both microtiter plate and paper-based microfluidic platforms that utilized zinc finger proteins as selective capture reagents for the detection of two different TB DNA biomarkers. The dual-platform design afforded either quantitative (microtiter plate, 1.0-20.0 nM) or qualitative (paper microfluidic) detection. While divergent in design and target, these assays achieve the aims of current pathogen detection research while providing cost-effective options for deployment in resource-poor environments.