Annexing isothermal nucleotide amplification enables probe-guided amplification and direct detection at ambient temperature

Read the full open-access article at Nature Communications.

Abstract

Isothermal nucleic acid amplification offers advantages over qPCR for decentralized diagnostics but remains constrained by rigid primer design, detection complexity, and equipment dependence. Recombinase-based systems operate at lower temperatures than other isothermal methods but require long primers and auxiliary enzymatic processing for detection. Here we introduce Annexing Isothermal Nucleotide Amplification (ANINA), a probe-guided recombinase-based framework integrating amplification and detection at ambient temperatures within a single lyophilized reaction. An Annexing Probe recruits short primers through spatial annexation, enabling efficient amplification at 25 °C within 30  minutes and direct lateral flow detection without auxiliary enzymatic processing. We show this framework detects attomolar WSSV and EBV DNA in abundant host gDNA, supports target-specific detection of DNA and RNA viruses and bacteria in contrived matrices, enables quantitative real-time detection comparable to qPCR, and facilitates a fully equipment-free 45-minute sample-to-answer workflow that detects early viral infection in a natural host model with qPCR-level sensitivity and improved performance over a commercial antigen test.