Integrating isothermal amplification with magnetic flocculation and DNA-guided PfAgo for SARS-CoV-2 detection

The recurrent threat of respiratory infectious disease outbreaks creates an urgent need for diagnostic tests that are rapid, portable, and user-friendly, applicable across diverse settings-from central laboratories and hospitals to field deployments, resource-limited regions, and home use.

In response, we developed three novel nucleic acid detection platforms-RT-RAA-MPs, RT-RAA-F-PfAgo, and RT-RAA-L-PfAgo-that integrate reverse transcription-recombinase-aided amplification (RT-RAA) with distinct instrument-free readout mechanisms. These systems utilize custom-designed primers and guide DNA (gDNA) targeting the SARS-CoV-2 N and ORF genes.

Performance evaluation demonstrated high analytical sensitivity, with detection limits of 10² copies/μL for RT-RAA-MPs and 5 copies/μL for both RT-RAA-F-PfAgo and RT-RAA-L-PfAgo. All assays exhibited excellent specificity, showing no cross-reactivity with nine common respiratory pathogens. Clinical validation using 23 patient samples revealed perfect agreement with RT-qPCR (100% concordance), supported by a Kappa value of 1.00, indicating outstanding diagnostic consistency and statistical robustness.

With lyophilized reagent compatibility and minimal equipment requirements, these platforms are readily adaptable for a wide range of applications, including clinical diagnosis at all hospital levels, point-of-care screening, border control inspection, and environmental monitoring. This work establishes a versatile, reliable, and deployable toolkit for effective outbreak response and decentralized molecular diagnostics.