CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Global spread of monkeypox virus (MPXV) necessitates rapid detection.
• Existing MPXV detection technologies are limited in speed, specificity, and sensitivity.
• Urgent need for accessible diagnostic tools for MPXV surveillance.

Purpose of the Study:

• To develop a rapid, specific, and ultrasensitive assay for MPXV detection.
• To enable on-site screening of MPXV carriers.
• To create a user-friendly diagnostic tool for emerging viral outbreaks.

Main Methods:

• Combined recombinase-aided amplification (RAA) with CRISPR/Cas12a technology.
• Targeted the highly conserved MPXV F3L gene.
• Optimized probes and reaction conditions for high specificity and sensitivity.
• Integrated assay with lateral flow strips for naked-eye detection.

Main Results:

• Achieved a low limit of detection (LOD) of 10¹ copies/μL for MPXV DNA.
• Demonstrated high selectivity, differentiating MPXV from other orthopoxviruses.
• Developed a kit enabling naked-eye MPXV detection with a LOD of 10⁴ copies/μL.
• Assay requires no sophisticated equipment or complex procedures.

Conclusions:

• The RAA-Cas12a-MPXV assay provides a sensitive and specific tool for MPXV detection.
• The developed kit facilitates rapid on-site screening and surveillance.
• This assay can be deployed for effective MPXV monitoring during outbreaks.