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 (MPXV) in clinical samples based on isothermal amplification together with clustered recurrently interspaced short palindromic repeats (CRISPR) CRISPR-associated protein (Cas) technology.

The assay was found to be highly sensitive and specific, and it performed well in comparison with the gold-standard quantitative polymerase chain response (qPCR) assay. An optimized assay form may very well be used to detect MPXV in low-resource and community settings.

Study: Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study. Image Credit: Dotted Yeti/Shutterstock.com

Background

MPXV, a big double-stranded deoxyribonucleic acid (DNA) virus, causes a zoonotic disease named mpox (formerly monkeypox). Mpox cases have been primarily reported in men who’ve sex with men but have also been observed in women and infants.

Between July 2022 and May 2023, the World Health Organization (WHO) declared mpox a public health emergency. Although the present transmission rate of MPXV is low, its sudden emergence, rapid spread, and lack of understanding on vaccine efficacy call for stringent vigilance, rapid diagnosis, and isolation of cases to forestall future outbreaks.

The present gold-standard diagnostic method for MPXV detection is qPCR, which requires specialized equipment and trained personnel. Moreover, many of the current diagnosis occurs in specialized laboratories, and it takes several days after sample collection to acquire results, especially in low-resource settings.

Regardless of the event of other point-of-care tests, the info on their efficacy are limited. In an attempt to handle this gap, in the current 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.

Concerning the study

Genomic DNA (gDNA) was extracted from 185 anonymized clinical samples, including oral, anal, and skin lesion specimens collected from individuals who underwent qPCR testing for mpox. The qPCR assay consisted of pan-orthodox primers and an MPXV-specific probe to discover the hemagglutinin (B2R) gene. 

A database of 523 MPXV genomes was downloaded from the National Center for Biotechnology Information (NCBI) and used to design recombinase polymerase amplification (RPA) primers and CRISPR guide ribonucleic acids (gRNAs) for the MPXV-CRISPR assay, targeting the highly conserved genes of MPXV.

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.

At 40 minutes and across three experimental repeats, the assay endpoint was measured using a fluorescence-based readout followed by a lateral flow detection. Three varieties of control samples were used in the course of the testing: no-guide control, no-template control, and at the very least one non-MPXV viral gDNA control.

The outcomes from the lateral-flow strips were evaluated blinded to the fluorescence results, manually by eye and computationally using ImageJ software.

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 a pair of, molluscum contagiosum virus, enterovirus (coxsackie B2), orf virus, and varicella zoster virus.

Moreover, intra-assay and inter-assay precision was estimated using average fluorescence values.

Results

The mean coefficient of variation was 5.2% (± 3·5) for the intra-assay evaluation and 20.8% (± 11.3) for the inter-assay evaluation. While the optimal run duration was 45 minutes, the optimal time to fluorescence readout was 2–4 minutes based on receiver operating characteristic (ROC) curve evaluation.

Within the blind concordance study on genomic DNA extracted from 185 clinical samples, the assay displayed 100% clinical sensitivity (1 copy per μL) and 99.3% specificity using the fluorescence readout in comparison with the qPCR assay and a big panel of viral and bacterial pathogens.

The lateral-flow readout with the chosen primer-guide set also achieved a 100% sensitivity (1 copy per μL) and 98.6% specificity for MPXV, with no cross-reactivity against a panel of other viruses.

The fluorescence and lateral flow readouts showed 96.8% concordance, barring viral-load-related discrepancies. Nevertheless, the study is restricted by the low variety of MPXV-positive cases (40) of the 185 samples analyzed and the modularized workflow for gDNA extraction, amplification, and detection.

Conclusion

The MPXV-CRISPR assay developed on this study combines RPA-based amplification with CRISPR-Cas12a-mediated DNA goal recognition and single-stranded DNA cleavage. It might probably be read out using fluorescence-based and lateral-flow strip methods.

Overall, this recent assay might be potentially utilized in low-resource settings for point-of-care diagnosis of MPXV, as it may well be performed in roughly 45 minutes and has high clinical sensitivity and specificity compared with the usual qPCR test.

Further research with multiplexed gRNA primers targeting a wider range of circulating MPXV lineages could be useful to help robust and early diagnostic tests against MPXV for improved public health outcomes.

Journal reference:

• Low, Soo Jen, Matthew T O’Neill, William J Kerry, Marcelina Krysiak, Georgina Papadakis, Lachlan W Whitehead, Ivana Savic, et al. (2023) Rapid Detection of Monkeypox Virus Using a CRISPR-Cas12a Mediated Assay: A Laboratory Validation and Evaluation Study., The Lancet Microbe, (2023). doi: https://doi.org/10.1016/S2666-5247(23)00148-9. https://www.thelancet.com/journals/lanmic/article/PIIS2666-5247(23)00148-9/fulltext