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Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification
Fan-En Chen1, Alexander Y Trick1, Alexander C Hasnain1
1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.
Abstract:
Polymerase chain reaction (PCR)-based diagnostic testing is the gold standard method for pathogen identification (ID) with recent developments enabling automated PCR tests for point-of-care (POC) use. However, multiplexed identification of several pathogens in PCR assays typically requires optics for an equivalent number of fluorescence channels, increasing instrumentation's complexity and cost. In this study, we first developed ratiometric PCR that surpassed one target per color barrier to allow multiplexed identification while minimizing optical components for affordable POC use. We realized it by amplifying pathogenic targets with fluorescently labeled hydrolysis probes with a specific ratio of red-to-green fluorophores for each bacterial species. We then coupled ratiometric PCR and automated magnetic beads-based sample preparation within a thermoplastic cartridge and a portable droplet magnetofluidic platform. We named the integrated workflow POC-ratioPCR. We demonstrated that the POC-ratioPCR could detect one out of six bacterial targets related to urinary tract infections (UTIs) in a single reaction using only two-color channels. We further evaluated POC-ratioPCR using mock bacterial urine samples spiked with good agreement. The POC-ratioPCR presents a simple and effective method for enabling broad-based POC PCR identification of pathogens directly from crude biosamples with low optical instrumentation complexity.
Insights
This study introduces ratiometric Polymerase Chain Reaction (PCR) for affordable point-of-care (POC) pathogen identification. This novel method uses color ratios for multiplexed detection, reducing complexity and cost for diagnostics.
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.
