Multicolor Bioluminescent Toolbox for Multiplex Glow-in-the-Dark Nucleic Acid Diagnostics

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Point-of-Care Testing

Background:

• Nucleic acid diagnostics are crucial for disease detection.
• Existing methods can be slow, complex, and require specialized equipment.
• The LUNAS (luminescent nucleic acid sensor) platform integrates isothermal amplification with bioluminescent detection.

Purpose of the Study:

• To develop a multiplex LUNAS platform for simultaneous detection of multiple DNA targets.
• To create green- and red-light emitting LUNAS variants for orthogonal detection.
• To validate the multiplex platform for diagnosing sexually transmitted infections.

Main Methods:

• Development of green- and red-light emitting LUNAS variants.
• Integration with duplex recombinase polymerase amplification (RPA) for dual-target amplification.
• Orthogonal detection of two DNA targets in a single reaction.
• Utilized clustered regularly interspaced short palindromic repeats (CRISPR) dCas9-mediated bioluminescent readout.

Main Results:

• Achieved attomolar sensitivity for detecting gonorrhea and chlamydia DNA.
• Demonstrated robust ratiometric readout for accurate quantification.
• Enabled detection of bacterial DNA from clinical samples down to ~100 copies/μL.
• Completed detection within approximately 30 minutes.

Conclusions:

• The multicolor LUNAS platform enables fast, sensitive, and multiplexed nucleic acid detection.
• This technology is suitable for point-of-care applications, especially in low-resource settings.
• The developed duplex assay shows diagnostic potential for common sexually transmitted infections.