A highly sensitive detection platform for scrub typhus using multi-fluorescence nucleic acid probes and STORM imaging

Nucleic acid testing (NAT) is essential for pathogen identification, disease diagnosis, and biomedical research. Traditional amplification-based methods, while widely used, often face challenges such as long processing times and susceptibility to false positives. Additionally, single-molecule detection approaches are limited by weak signal intensities and rapid photobleaching. To address these limitations, we present an amplification-free biosensing platform for the highly sensitive and specific single-molecule detection of Orientia tsutsugamushi DNA.

This platform employs multi-fluorophore nucleic acid probes synthesized via click chemistry to significantly enhance signal intensity and photostability. Specifically, 5-Ethynyl-dUTP (5-EdUTP) was incorporated into PCR amplicons to enable conjugation with Cy5 fluorophores. Stochastic optical reconstruction microscopy (STORM) was employed as the readout method, enabling the localization and quantification of tens of thousands of single-molecule events and thereby achieving substantial signal amplification. The assay achieved a detection limit of 2.74 aM (equivalent to 27.4 ymol in a 10 μL system) with excellent specificity. Clinical validation with 11 samples demonstrated 100% sensitivity and specificity.

This approach offers a simple, rapid, and robust solution for single-molecule detection, providing an invaluable tool for detecting low-abundance biomarkers and holding great promise for early disease diagnosis.