Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanotechnology

Background:

• Respiratory viral infections can be misdiagnosed as bacterial infections, leading to unnecessary antibiotic use.
• Antimicrobial resistance is a growing global health threat, particularly in low-income settings.
• Accurate and rapid viral diagnostics are essential to guide appropriate treatment and reduce antibiotic misuse.

Purpose of the Study:

• To develop and validate a rapid, affordable, and multiplexed diagnostic assay for respiratory viruses.
• To combine isothermal amplification with a paper-based microarray for sensitive viral detection.
• To demonstrate the utility of this method for clinical sample analysis.

Main Methods:

• Isothermal amplification of viral DNA at 37°C using recombinase polymerase amplification (RPA).
• Detection of amplicons using a paper-based vertical flow microarray (VFM) with functionalized gold nanoparticles.
• Colorimetric detection of amplified viral DNA sequences.

Main Results:

• Validated oligonucleotide probes for microarray detection down to 50 nM using synthetic templates.
• Demonstrated adenoviral detection of four species associated with respiratory infections using the VFM format.
• Achieved detection at 1 ng of starting material with low intra- and inter-assay coefficients of variation (CV ≤ 9% and ≤ 13%, respectively) in clinical samples.

Conclusions:

• The combination of RPA, paper microarray analysis, and nanoparticle-based colorimetric detection is a promising strategy for diagnostics.
• This method enables rapid and affordable multiplexed viral diagnostics.
• The developed assay shows potential for use in clinical settings, particularly in resource-limited environments.