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

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.