Evaluation of Fluorescence Detection Algorithms for Efficient ROI Setting in Low-Cost Real-Time PCR Systems

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Optical Engineering

Background:

• Conventional real-time PCR systems are costly and complex, limiting their use in point-of-care (POC) settings.
• A need exists for low-cost, compact fluorescence detection systems for accessible on-site diagnostics.

Purpose of the Study:

• To develop and validate a region of interest (ROI) setting method for accurate fluorescence detection in a compact real-time multiplex fluorescence PCR system.
• To address challenges in fluorescence signal accuracy caused by well-to-well variations in simple, low-cost PCR systems.

Main Methods:

• Developed a low-cost, compact real-time PCR system utilizing an open-platform CMOS camera and a Fresnel lens.
• Proposed two ROI image processing algorithms to extract fluorescence signals and assess well variations.
• Validated the ROI method through comparative analysis of real-time DNA amplification and fluorescence dye images.

Main Results:

• The proposed ROI algorithms reliably extracted fluorescence signals, minimizing ROI distortion.
• Evaluations confirmed stable fluorescence detection and accurate quantitative analysis, comparable to manual detection.
• The method effectively identified the need for physical correction by comparing ROI deviations.

Conclusions:

• The developed ROI setting method enhances the accuracy and efficiency of fluorescence detection in compact PCR systems.
• This approach contributes to improving the performance and accessibility of point-of-care diagnostic devices.
• The study demonstrates the potential for simple algorithms to achieve stable quantitative fluorescence analysis in resource-limited settings.