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

Area of Science:

• Molecular biology
• Biotechnology
• Infectious disease diagnostics

Background:

• Global monkeypox virus (MPXV) spread necessitates rapid, sensitive, and specific diagnostic tools.
• Current surveillance and containment strategies are challenged by MPXV dissemination.
• Existing diagnostics may lack the required balance of sensitivity and specificity.

Purpose of the Study:

• To develop a novel cascade signal amplification system for MPXV detection.
• To integrate recombinase-aided amplification (RAA) with fluorescent RNA aptamer sensing.
• To create a rapid diagnostic assay utilizing T4 DNA ligase.

Main Methods:

• Developed the Monkeypox Fluorescent T4-Ligase Assay (MFTA) using RAA and T4 DNA ligase.
• Employed bifunctional probes with a T7 promoter and a DNA Mango aptamer sequence.
• Utilized T4 DNA ligase for specific probe ligation and T7 RNA polymerase for transcription.

Main Results:

• MFTA achieved a detection limit of 1 copy/μL for MPXV.
• The assay demonstrated high specificity, distinguishing MPXV from other orthopoxviruses.
• Results showed consistency with quantitative PCR (qPCR) findings.

Conclusions:

• MFTA offers a novel platform for pathogen detection by integrating T4 DNA ligase specificity with RNA aptamer amplification.
• The assay exhibits high sensitivity and specificity, making it valuable for MPXV diagnostics.
• MFTA supports public health responses to MPXV outbreaks and aids in controlling virus spread.