Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Medical Devices

Background:

• Polymerase Chain Reaction (PCR) is the gold standard for pathogen identification.
• Multiplexed PCR assays often require complex and costly optical systems for multiple fluorescence channels.
• Point-of-care (POC) diagnostics need simplified, cost-effective solutions for pathogen detection.

Purpose of the Study:

• To develop a ratiometric PCR method for multiplexed pathogen identification with reduced optical complexity.
• To integrate ratiometric PCR with automated sample preparation for a POC diagnostic platform.
• To enable affordable and simplified POC PCR identification of multiple pathogens.

Main Methods:

• Developed ratiometric PCR using hydrolysis probes with specific red-to-green fluorophore ratios for each bacterial species.
• Integrated ratiometric PCR with automated magnetic bead-based sample preparation in a thermoplastic cartridge.
• Utilized a portable droplet magnetofluidic platform for the integrated workflow, named POC-ratioPCR.

Main Results:

• Demonstrated POC-ratioPCR's ability to detect one of six bacterial targets for urinary tract infections (UTIs) in a single reaction using only two-color channels.
• Achieved good agreement when evaluating POC-ratioPCR with spiked mock bacterial urine samples.
• Validated the simplified approach for pathogen identification directly from crude biosamples.

Conclusions:

• POC-ratioPCR offers a simple and effective method for broad-based POC PCR identification of pathogens.
• The approach minimizes optical instrumentation complexity, making it suitable for affordable POC use.
• This technology has the potential to significantly improve infectious disease diagnostics at the point of care.