Development of optimized fluorogenic DNA aptamers for a portable one-pot CRISPR-Cas12a platform for rapid and sensitive detection of monkeypox virus and chikungunya virus.

INTRODUCTION

The recent global outbreaks of monkeypox virus (MPXV) and chikungunya virus (CHIKV) underscore the urgent need for rapid, accessible, and cost-effective diagnostic methods. Conventional CRISPR/Cas fluorescence assays rely on trans-cleavage of ssDNA/RNA reporters labeled with expensive fluorophores and quenchers, which limits widespread application.

OBJECTIVES

This study aims to develop and optimize a label-free, fluorogenic DNA aptamer-based reporter for a portable, one-pot Cas12a detection system capable of highly sensitive detection of MPXV and CHIKV directly from clinical specimens.

METHODS

We evaluated commonly used ssDNA aptamers for their fluorescence emission upon Thioflavin T (ThT) binding and their cleavage efficiency by Cas12a. Through systematic mutagenesis targeting G-rich regions, we enhanced fluorescence emission. Additionally, poly-A linkers were introduced between G-rich motifs to promote Cas12a cleavage efficiency. Circular dichroism (CD) spectroscopy confirmed G-quadruplex (G4) formation in the aptamers. The assay's sensitivity and specificity were assessed using simulated clinical samples, followed by validation with actual clinical specimens. The performance of direct detection from simulated clinical samples was compared to qRT-PCR. A battery-powered heating-pad, a mini-centrifuge, and a flashlight were used to validate its POCT applicability.

RESULTS

We designed and optimized a cost-effective, stable fluorogenic ssDNA aptamer that specifically binds to ThT. The aptamer ThT-3-5.1 exhibited the highest fluorescence enhancement and cleavage efficiency by Cas12a. Leveraging this aptamer, we developed a rapid, portable, one-pot detection platform (ROD-ThT) capable of detecting as few as 1 copy/reaction of MPXV and CHIKV nucleic acids within 35 min. Validation with clinical samples confirmed the assay's reliability without the need for nucleic acid purification.

CONCLUSION

Our simple, efficient, portable, and affordable ROD-ThT platform holds great promise for disease diagnostics and management, particularly in resource-limited settings.