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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月14日

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  • Ariana Human-McKinnon,
  • Madisen Pyper,
  • MiKenzee Frazier,
  • Emily Plant,
  • Harrison Piper,
  • Tyler Archibald,
  • Anindita Roy

摘要

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.