A portable CRISPR-based assay developed during the 2025 Mpox outbreak in Sierra Leone demonstrated complete concordance with quantitative polymerase chain reaction (qPCR) in a field evaluation, according to a study published in Nature Communications.
The assay, called Mpox SHINE, was designed to address diagnostic challenges in decentralized settings where access to molecular testing may be limited. The platform combines recombinase polymerase amplification and CRISPR-Cas13 detection. It uses lyophilized reagents, ambient-temperature sample preparation, and automated fluorescence readout on a portable device, ensuring ease of use at the point of need.
Researchers designed the assay using genome sequences from the outbreak strain, Mpox clade IIb. Analytical testing showed a limit of detection between approximately one and 10 viral copies per microliter, with consistent detection at 10 copies/µL.
Clinical performance was evaluated at Kenema Government Hospital using 56 lesion swab specimens. Reference qPCR testing identified 45 positive and 11 negative samples. When performed on extracted DNA, Mpox SHINE achieved 100 percent sensitivity and 100 percent specificity relative to qPCR, correctly classifying all samples. Positive specimens were detected in an average of 11.4 minutes, and all positives were identified within 37 minutes.
The investigators also assessed a simplified workflow using chemically lysed, unextracted lesion swabs. In a pilot analysis of 16 specimens – eight positive and eight negative by qPCR – the assay again demonstrated 100 percent sensitivity and 100 percent specificity. Mean time to detection was 27.9 minutes, with all positive samples detected within 44 minutes.
Unlike conventional qPCR, which typically requires centralized laboratory infrastructure, the Mpox SHINE workflow was designed for use in lower-resource settings. The portable DxHub device provides real-time fluorescence monitoring and automated result interpretation, while the assay can be performed using minimally processed samples.
The study demonstrates the potential of CRISPR-based diagnostics to provide molecular testing with performance comparable to laboratory-based methods while reducing infrastructure requirements. The assay maintained accuracy across clinical samples with qPCR cycle threshold values ranging from 15 to 36.
The authors noted several limitations. The unextracted-sample evaluation included only 16 specimens, resulting in wide confidence intervals, and the current system is not a fully automated sample-to-answer platform. Additional studies will be needed to assess performance in larger cohorts and to evaluate long-term reagent stability under field conditions.
The researchers concluded that the study demonstrates the feasibility of developing, validating, and deploying a CRISPR-based molecular assay during an active outbreak, with results available in less than 30 minutes from minimally processed samples.
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