Method to remove non-target nucleic acids for enhanced pathogen detection in food and clinical samples

Overview

This technology offers a sample preparation methodology designed to dramatically improve the specificity and sensitivity of nucleic acid-based pathogen detection. The method selectively removes non-target DNA—including human host DNA, dead bacterial DNA, and DNA from non-viable cells—while preserving DNA from viable target bacteria. This addresses a critical bottleneck in pathogen diagnostics, particularly in complex matrices where target bacterial DNA may be present at extremely low concentrations (as low as 1 CFU/mL in blood) alongside overwhelming amounts of background and non-viable DNA.

The workflow is PCR-compatible, rapid (completed in under one hour), and inexpensive, relying on readily available reagents and materials. It has already been validated in clinical compartments including blood, urine, and sputum, and the developers anticipate straightforward adaptation to food matrices. Potential applications include clinical diagnostics for bloodstream infections, urinary tract infections, and respiratory infections, as well as food safety testing where low-level pathogen contamination must be detected amid high background DNA.

Technical specifications

Key features:

• Selective depletion of human host DNA, dead bacterial DNA, and DNA from non-viable bacterial cells
• High retention of target viable bacterial DNA for downstream PCR-based detection
• Rapid workflow completed in under one hour
• Low-cost reagents and readily available materials
• Direct compatibility with standard PCR workflows (validated at 35 cycles)
• Adaptable across multiple sample types including blood, urine, sputum, and potentially food matrices

Proof-of-concept results:

In controlled equimolar mixture experiments in PBS, the method successfully differentiated between DNA and intact cells of E. coli and S. aureus. Treated mixtures showed amplification only of the intended target organism, while untreated mixtures showed amplification of both. Subsequent testing in clinical samples (post-culture) confirmed the method's effectiveness in real-world matrices.

Technology readiness level

The technology is currently at a proof-of-concept stage with qualitative validation completed in PBS and clinical compartments. The developers have proposed a one-year validation plan that includes: (1) generating quantitative data on percentage depletion of non-target DNA and percentage retention of target DNA in PBS and blood, (2) executing sample transfer and confidentiality agreements to enable methodology disclosure, (3) testing with partner-provided samples with qualitative and quantitative assessment, and (4) transferring reagents, materials, and protocols for partner-side testing and further optimization. The method is not yet validated in food matrices, though the developers do not anticipate significant hurdles in adapting the approach for food safety applications.