T4 DNA ligase-mediated RAA coupled with RNA aptamer-driven cascade signal amplification for ultra-sensitive monkeypox...

The global dissemination of monkeypox virus (MPXV) poses a formidable challenge to public health systems worldwide, disrupting surveillance and containment strategies. This crisis underscores the urgent demand for rapid diagnostic technologies that balance high sensitivity for detecting low viral loads and strict specificity to prevent cross-reactivity. To address this unmet need, the present study developed a cascade signal amplification system-named "Monkeypox Fluorescent T4-Ligase Assay (MFTA)"-by integrating recombinase-aided amplification (RAA) and fluorescent RNA aptamer sensing technology, with T4 DNA ligase as the core mediator.

MFTA generates single-stranded DNA (ssDNA) templates through 5'-phosphorylated primers combined with Lambda nuclease treatment and employs two specifically designed bifunctional probes: Probe-L, which incorporates a T7 promoter, and Probe-R, which contains a DNA Mango aptamer sequence. In the presence of MPXV, T4 DNA ligase specifically catalyzes probe ligation to form a complete transcription template, further initiating T7 RNA polymerase-mediated in vitro transcription. This process produces abundant RNA Mango aptamers, which specifically bind the TO1 fluorophore to generate a detectable fluorescent signal. The "Identification-Ligation-Transcription-Fluorescence" cascade enables dual verification: RAA ensures target-specific amplification of MPXV sequences, while T4 DNA ligase guarantees precise probe ligation and reduces non-specific background noise. Experiments confirmed MFTA has a detection limit of 1 copy/μL for MPXV, with results consistent with those of qPCR, and effectively discriminates MPXV from other orthopoxviruses.

MFTA innovatively integrates the precise specific recognition of target sequences by T4 DNA ligase with the efficient signal amplification capability of RNA aptamers, successfully establishing a novel technical platform for pathogen detection. Its high sensitivity, strong specificity, and favorable scalability make it a valuable tool for MPXV diagnostics, supporting targeted public health responses to outbreaks and aiding in curbing virus spread.