Researchers developed a point-of-care assay for the rapid detection of mpox virus.
By Susha Cheriyedath, M.Sc.Sep 20 2023Reviewed by Lily Ramsey, LLM In a proof-of-concept study published in The Lancet Microbe, researchers from Australia developed a point-of-care assay for the rapid detection of mpox virus in clinical samples based on isothermal amplification along with clustered regularly interspaced short palindromic repeats CRISPR-associated protein technology.
Background MPXV, a large double-stranded deoxyribonucleic acid virus, causes a zoonotic disease named mpox . Mpox cases have been primarily reported in men who have sex with men but have also been observed in women and infants. In spite of the development of other point-of-care tests, the data on their efficacy are limited. In an attempt to address this gap, in the present study, researchers designed, evaluated, and validated a point-of-care, fluorescence-based, CRISPR-Cas12a assay named “MPXV-CRISPR” which could potentially be deployed in decentralized settings for rapid MPXV detection.
Twenty two sets of RPA primers were obtained, which flanked seven gRNAs. The targets were amplified via RPA in a thermal cycler and detected using CRISPR-Cas12a-based trans cleavage of labeled single-stranded DNA. While the analytical sensitivity of the assay was assessed using serial dilutions of gDNA, the analytical specificity was assessed on several common viral skin pathogens, including vaccinia virus, herpes simplex virus types 1 and 2, molluscum contagiosum virus, enterovirus , orf virus, and varicella zoster virus.Results The mean coefficient of variation was 5.2% for the intra-assay analysis and 20.8% for the inter-assay analysis.
The fluorescence and lateral flow readouts showed 96.8% concordance, barring viral-load-related discrepancies. However, the study is limited by the low number of MPXV-positive cases of the 185 samples analyzed and the modularized workflow for gDNA extraction, amplification, and detection.
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