Analytical methods : advancing methods and applicationsApr 7, 2026

Fast and sensitive one-step test using CRISPR for detecting monkeypox virus

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Apr 8, 2026

Abstract

The novel RAA-CRISPR/Cas12a assay detects monkeypox virus at a sensitivity of 0.5 copies per µL within 35 minutes.

The assay shows excellent specificity, accurately distinguishing monkeypox virus from other viruses like vaccinia virus without cross-reactivity.
A lateral flow assay format enables visual detection of as low as 5 copies per µL within 40 minutes.
In validation with simulated clinical samples, the assay achieved 100% accuracy in differentiating positive and negative results.
This approach eliminates the need for costly thermal cyclers, simplifying the detection process and reducing contamination risks.
The low template volume requirement of just 1 µL enhances detection efficiency and allows for repeated testing without compromising results.

Simplified

Monkeypox (mpox) is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks historically concentrated in West and Central Africa. The current global outbreak of MPXV highlights the urgent need for an efficient detection system. This study presents a novel single-tube recombinase-aided amplification (RAA)-CRISPR/Cas12a assay for the rapid, sensitive, and specific detection of MPXV. Optimized to achieve a fluorescence-based sensitivity as low as 0.5 copies per µL within 35 min at 37 °C, the system demonstrates excellent specificity, accurately distinguishing MPXV from other viruses, including vaccinia virus, with no cross-reactivity observed. Additionally, a lateral flow assay (LFA) format was developed, enabling visual detection of concentrations as low as 5 copies per µL within 40 min. Validated with simulated clinical samples, the assay achieved 100% accuracy in distinguishing positives from negatives. This integrated single-tube approach eliminates the need for costly thermal cyclers, simplifying the detection process, reducing contamination risks, and delivering reliable results in a short time, making it ideal for point-of-care testing (POCT) in resource-limited settings. Furthermore, the RAA-CRISPR/Cas12a platform offers significant cost savings by requiring fewer reagents for virus detection. The extremely low template volume requirement of just 1 µL maximizes detection efficiency. This configuration allows for repeated testing without compromising result integrity, further enhancing the utility of this approach for surveillance and outbreak control, particularly in low-resource environments.

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Fast and sensitive one-step test using CRISPR for detecting monkeypox virus

Abstract

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