(541b) Direct Detection of Nucleic Acids without Amplification

Rapidly detecting the source of an infection remains challenging. Techniques using nucleic acid amplification have promise in providing answers quickly, but remain vulnerable to contamination and lack thermal stability because of their reliance on enzymatic proteins. We are developing a sensitive, inexpensive and portable instrument to automate the direct detection and identification of pathogenic nucleic acids, which will enable health professionals to rapidly identify infections in humans and subsequently accelerate the rational treatment of such infections. The current prototype performing the assay produces results from non-invasive samples in one hour and requires only minimally-processed crude samples. Our method promises to be robust for field and physician office applications, allowing distributed healthcare decisions outside of traditional laboratories.

All bacteria, viruses and fungi have within them nucleic acids (DNA, RNA) that determine their properties (benign or pathogenic) – and detecting the presence of specific nucleic acids is the fastest and the most optimal method to detect the source of an infection. Our methodology, called CAPTURE™ (Confirming Active Pathogens Through Unamplified RNA Expression), is unique in that it works in the presence of unknown amounts of unrelated nucleic acids, proteins and cellular debris. The design of the custom DNA ‘capture’ probes used in the assay is arguably the most integral step in our process, and as such, requires the most detailed method. Through a combination of locally compiled databases and proprietary software, ideal candidate sequences from pathogen genomes can be determined which exhibit the appropriate thermodynamic behavior required of the panel. The CAPTURE™ method is currently being used to identify several dozen specific pathogens and also to quickly determine the presence of any bacteria in a patient sample. The ability to determine universal bacterial signatures in a sample is a powerful tool for combating antimicrobial resistance, as it has the potential to limit the prescription of unnecessary antibiotics.

We have developed a CAPTUREâ„¢ panel for the analysis of urinary tract infections (UTI) that correctly identifies and differentiates the gram-negative bacteria Escherichia coli (which cause over 85% of UTI), Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa; the gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus; and fungal Candida species. The use of multiple captors for each pathogen allows for a statistical cluster analysis and introduces systematic redundancy to ensure efficacy. Our CAPTUREâ„¢ technique offers health care professionals a reliable and a quick way to screen for pathogens in clinical samples.