ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Current digital nucleic acid amplification tests (digital NAATs) typically use a "one-color-one-target" approach, limiting multiplexing due to spectral overlap issues.
• Scaling multiplexability in digital NAATs is challenging, hindering the simultaneous detection of multiple nucleic acid targets.

Purpose of the Study:

• To develop a multiplexed digital NAAT platform capable of detecting multiple nucleic acid targets simultaneously.
• To overcome the limitations of single-plex detection in current digital NAAT platforms.

Main Methods:

• Developed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), a padlock probe-based nucleic acid detection assay.
• Encoded each nucleic acid target with a unique two-color fluorescence signature using two fluorophores.
• Performed digital amplification in microfluidic droplets to detect the encoded fluorescence signatures.

Main Results:

• Demonstrated broad-based, two-plex, four-plex, and six-plex detection of sexually transmitted infection (STI) targets with single-molecule resolution.
• Successfully synthesized six distinct padlock probes, each yielding a unique two-color fluorescence signature.
• Validated the ddRFC platform's ability to achieve high multiplexing without redesigning amplification primers or fluorescent molecular beacons.

Conclusions:

• The ddRFC platform offers a cost-effective strategy to scale multiplexability in digital NAATs by adjusting molecular beacon binding sites.
• This innovative approach has the potential for highly multiplexed nucleic acid detection with potentially unrestricted multiplexability.
• The ddRFC platform could serve as a valuable diagnostic tool for a wider range of diseases in the future.