Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule...

Area of Science:

• Biotechnology
• Molecular Biology
• Microfluidics

Background:

• Accurate nucleic acid quantification is crucial but challenging with existing methods like qPCR and dPCR.
• qPCR struggles with accuracy at low concentrations and nonspecific amplification.
• dPCR is expensive and limited to high concentrations.

Purpose of the Study:

• To develop a method for accurate nucleic acid quantification across a broad concentration range.
• To leverage microfluidic technology to combine the strengths of qPCR and dPCR.
• To introduce and validate a novel on-site PCR (osPCR) phenomenon for enhanced quantification.

Main Methods:

• Performing PCR in silicon-based microfluidic chips.
• Observing and characterizing the on-site PCR (osPCR) phenomenon at low template concentrations.
• Developing a sectioning algorithm to improve signal amplitude and quantify results.

Main Results:

• Demonstrated high quantification accuracy in a large concentration range using microfluidic PCR.
• Identified osPCR as a quasi-single molecule phenomenon with consistent ct values.
• Enabled simultaneous measurement of ct values and absolute template concentration.
• Showed improved detection of COVID in patient samples through enhanced quantification and removal of nonspecific amplification.

Conclusions:

• Microfluidic PCR with osPCR offers a powerful solution for accurate nucleic acid quantification.
• osPCR facilitates precise measurement and identification of individual template molecules.
• This approach significantly improves quantification accuracy and diagnostic capabilities, as demonstrated in COVID detection.