Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration.

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Digital PCR (dPCR) is crucial for absolute nucleic acid quantification.
• Existing dPCR methods face challenges with liquid loss and data interpretation (e.g., "rain").
• There is a need for more sensitive and accurate dPCR techniques.

Purpose of the Study:

• To develop a novel dPCR apparatus, centrifugal force real-time dPCR (crdPCR), for improved nucleic acid detection and quantification.
• To enhance the accuracy and efficiency of dPCR by minimizing liquid loss and addressing data interpretation issues.
• To achieve higher sensitivity in absolute micro-analysis.

Main Methods:

• Established a novel crdPCR system utilizing centrifugal force for sample dispensing.
• Implemented real-time analysis of micro-well data.
• Applied artificial intelligence to distinguish true/false positives and mitigate "rain" artifacts.

Main Results:

• The crdPCR system demonstrated significantly reduced liquid loss (2.14%).
• Achieved limits of detection and quantification of 1.38 and 4.19 copies/μL, respectively.
• Showcased two-fold higher sensitivity compared to other dPCR devices.

Conclusions:

• The crdPCR system offers a more efficient and accurate method for nucleic acid quantification.
• The integration of AI for data analysis addresses key limitations of traditional dPCR.
• This technology holds significant potential for next-generation PCR applications in absolute micro-analysis.