Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Digital PCR (dPCR) offers absolute quantification but is limited by the number of detectable targets per assay.
• Current dPCR systems typically detect one target per optical channel, restricting multiplexing capabilities.
• Increased multiplexing in dPCR can enhance utility, especially for limited samples, and reduce costs.

Purpose of the Study:

• To develop a novel probe design for robust multiplexing in digital PCR.
• To create a multiplex digital PCR (mdPCR) assay capable of distinguishing multiple nucleic acid targets simultaneously.
• To evaluate the performance of the novel mdPCR assay in terms of accuracy, sensitivity, and reproducibility.

Main Methods:

• Development of a novel melt-based hairpin probe design for multiplexing.
• Implementation of a prototype mdPCR assay on a 16-well microfluidic digital PCR platform.
• Validation using samples with 10,000 human genome equivalents to determine limit of blank and analytical limit of detection.

Main Results:

• The mdPCR assay accurately distinguished and quantified 12 nucleic acid targets per well.
• The probe-specific ranges for limit of blank were 0.00%-0.13%.
• The probe-specific ranges for analytical limit of detection were 0.00%-0.20%, with excellent inter-laboratory reproducibility (r² = 0.997).

Conclusions:

• The novel melt-based hairpin probe design provides a robust solution for multiplexing in digital PCR.
• This mdPCR technology significantly expands the number of targets detectable per well, overcoming previous limitations.
• The developed mdPCR assay demonstrates high performance, offering potential to revolutionize dPCR applications in research and diagnostics.