The urgency behind the work is difficult to overstate. Since 2022, mpox outbreaks have emerged across multiple continents, and in 2024 a newly identified sublineage of clade I, designated Ib, triggered fresh waves of infection in several countries. The World Health Organization declared the ongoing outbreak a Public Health Emergency of International Concern on August 14, 2024, and its surveillance data recorded 52,845 confirmed cases and 215 deaths among confirmed cases globally between January and December 2025 alone. What makes clade I particularly alarming is its epidemiological profile. Transmission is no longer confined to sexual contact; confirmed household spread through close contact threatens pediatric populations, and in the current Democratic Republic of the Congo outbreak, children under 15 account for roughly 70 percent of confirmed cases and a staggering 88 percent of documented fatalities.

Clade I is also clinically distinct from clade II. Comparative studies have associated it with increased virulence, higher mortality, and more severe disease outcomes, demanding closer clinical oversight and earlier therapeutic escalation. This combination of enhanced transmissibility and heightened severity means that rapid clade discrimination is not merely a technical convenience. It is a fundamental requirement for real-time genomic surveillance, precise containment protocols, and rigorous epidemiological inquiry. PCR remains the WHO-endorsed reference assay for mpox detection, but its dependence on expensive thermal cycling equipment and its comparatively long turnaround time leave a critical gap at the point of care, particularly in resource-limited settings where clade I circulates most actively.

The new assay builds on the CRISPR/Cas12a system, an adaptive immune mechanism first identified in bacteria and archaea and now widely repurposed for viral pathogen detection. Cas12a possesses a distinctive property called trans-cleavage: once its guide RNA directs it to a matching DNA target, the enzyme promiscuously shreds nearby single-stranded DNA molecules. By flooding the reaction with single-stranded DNA reporters bearing a fluorescent tag and a quencher, researchers can convert the presence of a single target molecule into an explosive burst of fluorescence. Because Cas12a alone cannot amplify scarce templates, the system is typically paired with a pre-amplification step, and RPA has emerged as a favorite partner. Operating at just 37 to 42 degrees Celsius, RPA achieves exponential amplification within minutes using recombinase enzymes rather than heat-driven denaturation, making it an ideal match for field-deployable diagnostics.

The central engineering challenge in such combined systems is incompatibility. Premixing amplification reagents with CRISPR components in a single homogeneous reaction can actually degrade sensitivity, because premature activation of Cas12a during amplification may consume newly generated amplicons or the target templates themselves, throttling the amplification reaction before it reaches its full potential. The research team circumvented this problem with an elegantly simple physical solution: a compartmentalized tube sleeve consisting of an ordinary 1.5 milliliter microcentrifuge tube with the RPA mixture at its base, and an inverted, capless PCR tube nested inside it, held in place by surface tension and containing the preconfigured CRISPR/Cas12a detection mixture. During the 20-minute amplification at 39 degrees Celsius, the two reactions remain fully separated. A brief centrifugation then releases the CRISPR mixture into the amplified RPA reaction without ever opening the tube, minimizing both cross-contamination and aerosol exposure while allowing a further 20-minute incubation at 37 degrees Celsius to generate the fluorescent signal.

Optimization of the assay was methodical and thorough. The team produced its own LbCas12a protein in bacterial expression cells, achieving a purification purity of 96.98 percent as confirmed by thin-layer scanning, and validated its trans-cleavage activity with established positive and negative controls. Two candidate crRNAs, the guide RNAs that steer Cas12a to its target, were designed against clade I-specific conserved regions identified by comparative analysis of global MPXV genomes, and one, crRNA-I-2, produced markedly stronger fluorescence and was selected for further work. Titration experiments then pinned down the reaction cocktail: 187.5 nanomolar Cas12a protein, 25 nanomolar crRNA, and 1 micromolar single-stranded reporter, concentrations chosen to balance peak signal intensity against reagent cost. Among three RPA primer pairs, RPA-3-F/R showed the highest amplification efficiency, and a temperature gradient across 37, 39, and 42 degrees Celsius established 39 degrees as optimal.

The analytical performance figures are impressive for such a low-tech platform. Under optimized conditions, preliminary screening detected plasmid targets down to 43.7 copies per microliter, and a rigorous probit regression analysis across 20 replicates at each of five concentrations near the detection limit placed the 95 percent limit of detection at 27.16 copies per microliter, with a 95 percent confidence interval of 23.06 to 35.24 copies per microliter. All 20 replicates were positive at 43.70 copies per microliter, while detection fell to 15 of 20 at 21.85 copies and 6 of 20 at 10.93 copies, and vanished entirely below that. Specificity testing against a panel that included clade II plasmids, monkey B virus, cowpox virus, vaccinia virus, and varicella-zoster virus yielded robust fluorescence exclusively for clade I, with no cross-reactivity whatsoever. Repeatability was similarly strong, with intra-assay coefficients of variation ranging from 1.032 to 8.509 percent and inter-assay coefficients spanning 2.279 to 8.483 percent across three independent runs on different days, and negative controls remained negative throughout.

Notably, the assay retained meaningful sensitivity even without any incubator at all. At room temperature, 25 degrees Celsius, the platform reliably detected targets at 437 copies per microliter, though signal strength attenuated at lower concentrations. While this ambient-temperature performance does not match the optimized thermostated conditions, the researchers emphasize that it confirms the assay remains viable in field settings where heating devices are simply not an option. The visual readout itself is deliberately accessible: results are documented with a handheld blue-light transilluminator or even a smartphone camera, and green-channel pixel intensities extracted with image analysis software provide an objective, quantifiable measure that removes subjectivity from result interpretation.

Head-to-head evaluation against qPCR in simulated clinical samples provided perhaps the most compelling evidence of the assay’s practical value. The team spiked recombinant plasmid into total DNA extracted from Vero cells and human embryonic kidney 293T cells, generating a panel of 32 simulated samples including negative controls. Using a pixel-value threshold of 40 to distinguish signal from background, the RPA-CRISPR/Cas12a assay detected 10 of 14 spiked samples in both the Vero cell and 293T cell DNA backgrounds. Standard qPCR, run in parallel with a Ct cutoff of 35 and a requirement for a typical sigmoidal amplification curve, managed only 7 of 14 in the Vero background and 8 of 14 in the 293T background. The researchers attribute the undetected samples in both methods to target concentrations genuinely below each assay’s analytical detection limit rather than random false negatives or instability, but the outcome demonstrates that the low-equipment platform actually outperformed the laboratory-bound reference method on low-copy samples within the simulated matrices.

The authors are candid about the limitations that remain before this assay can reach clinics and field stations. crRNA design is constrained by restrictive protospacer adjacent motif requirements and by the remarkably high sequence homology, 96.8 percent, between clades I and II, though alternative Cas systems such as Cas13 and Cas14 could relax these constraints in future iterations. More significantly, the analytical evaluation relied primarily on recombinant plasmids, and while simulated samples with cellular DNA backgrounds offered a preliminary reality check, they cannot fully reproduce viral particle lysis, extraction efficiency, or the complexities of genuine clinical matrices. Validation with authentic clinical specimens or pseudovirus-based samples was not possible owing to biosafety requirements and the unavailability of clade I clinical material, and the researchers acknowledge that such evaluation will be essential. Even so, the assay fills a specific niche within an integrated diagnostic strategy: a universal mpox test would first confirm infection, after which this clade I-specific assay could stratify risk, guide clinical escalation, and sharpen surveillance of the lineage now driving the world’s most dangerous mpox outbreaks. With a 40-minute workflow, a visual readout, and no need for sophisticated equipment, it represents a practical step toward decentralized detection precisely where it is needed most.

Subject of Research: Development of a rapid one-pot RPA-CRISPR/Cas12a assay with visual readout for clade I-specific detection of monkeypox virus

Article Title: One‐Pot RPA‐CRISPR/Cas12a Assay With Visual Readout for the Ultra‐Specific Detection of Monkeypox Virus Clade I

Article References: Li, B., Liu, L., Jin, K., Huang, Z., Zhang, T., Gao, R., Chen, H., Niu, L., Fan, C., Zhang, H., Huang, P., & Wang, H. (2026). One‐Pot RPA ‐ CRISPR /Cas12a Assay With Visual Readout for the Ultra‐Specific Detection of Monkeypox Virus Clade I. Microbial Biotechnology, 19(9), Article e70437. https://doi.org/10.1111/1751-7915.70437

Image Credits: AI Generated

DOI: 10.1111/1751-7915.70437

Keywords: mpox, monkeypox virus, CRISPR/Cas12a, recombinase polymerase amplification, clade I, point-of-care diagnostics, isothermal amplification, crRNA, outbreak surveillance, limit of detection, visual readout, qPCR

The reference method for mpox diagnosis remains real-time quantitative polymerase chain reaction, or qPCR, which offers high sensitivity and specificity. However, qPCR depends on expensive thermocycling instruments, trained personnel, and biosafety-compliant laboratory facilities, all of which limit its accessibility in decentralized laboratories, outbreak settings, and resource-limited regions. Alternative molecular approaches such as restriction fragment length polymorphism analysis and next-generation sequencing are either labor-intensive, costly, or unsuitable for rapid frontline diagnostics. The World Health Organization has repeatedly emphasized the expansion of laboratory diagnostic capacity and the deployment of rapid, reliable, and accessible assays as a core component of its global mpox preparedness and response strategy, creating an urgent need for tools that can operate outside centralized reference laboratories.

The new assay, described in a study published in New Microbes and New Infections, is built on loop-mediated isothermal amplification, commonly known as LAMP. This nucleic acid amplification technique operates under constant-temperature conditions and enables rapid DNA amplification using a set of primers that recognize multiple regions of the target genome. LAMP-based assays require substantially less equipment than qPCR, offer shorter turnaround times, and allow visual interpretation of results. In the colorimetric format used by the Indian team, amplification is read out through a pH-sensitive dye: the reaction mixture starts pink under alkaline conditions and turns yellow when amplification succeeds, a transition that can be judged by eye without any specialized instrumentation.

To design the assay, the researchers retrieved complete and partial genomic sequences of monkeypox virus and other representative orthopoxviruses, including Vaccinia, Variola, Buffalopox, and Camelpox viruses, from the NCBI Virus database. Multiple sequence alignments were analyzed to identify conserved genomic regions suitable for isothermal amplification. Candidate genes evaluated included H2R, encoding a putative viral transmembrane protein; B6R, encoding an envelope protein; F3L, encoding a double-stranded RNA-binding protein; and F8L, encoding DNA polymerase. After preliminary analytical sensitivity testing and temperature optimization, primer sets targeting the orthopoxvirus genus-specific B6R gene and the monkeypox virus species-specific F3L gene were selected for full assay development. Primers were designed with PrimerExplorer V5, optimized for GC contents of approximately 50 to 60 percent, compatible melting temperatures, and minimal secondary structure or primer-dimer formation.

A defining feature of the assay is its dual-target architecture, which incorporates an internal control targeting the human beta-actin gene to verify sample integrity and amplification suitability. Under the predefined interpretation criteria, a specimen is classified as mpox-positive only if both the genus-specific B6R and species-specific F3L reactions yield positive colorimetric results. Specimens that are B6R-positive but F3L-negative are interpreted as non-mpox orthopoxvirus-positive, while specimens negative for both targets are classified as mpox-negative. This design provides two important advantages: reliable identification of monkeypox virus infections and the ability to differentiate mpox from other circulating orthopoxviruses, a capability that is particularly valuable in regions where multiple orthopoxviruses may co-circulate.

Clinical evaluation was performed using a well-characterized panel of 130 specimens obtained retrospectively from suspected mpox cases received for routine diagnostic testing at the Maximum Containment Facility of ICMR-NIV as part of national surveillance activities. The panel comprised 50 mpox-positive and 80 mpox-negative specimens as determined by qPCR, spanning five specimen categories: nasopharyngeal and throat swabs, vesicle swabs, lesion swabs, crust specimens, and lesion fluid specimens. All specimens had previously been tested by real-time qPCR targeting the B6R and F3L genes, which served as the reference standard. Sample processing and nucleic acid extraction were carried out in biosafety level-3 and level-4 containment laboratories in accordance with national biosafety guidelines, and the study received approval from the Institutional Ethics Committee of ICMR-NIV.

The results were striking. The LAMP assay correctly identified all 50 qPCR-positive specimens as positive and all 80 qPCR-negative specimens as negative, achieving complete concordance with the reference method. Sensitivity, specificity, positive predictive value, negative predictive value, and overall percent agreement were each 100 percent, with a Cohen’s kappa of 1.00 indicating perfect agreement beyond chance. Likelihood ratio analysis yielded a positive likelihood ratio of 160.4 and a negative likelihood ratio of 0.0099, corresponding to a diagnostic odds ratio of approximately 16,261. No discordant results were observed in any specimen category, indicating consistent performance across swabs, crusts, and lesion fluids. Per-target analysis confirmed that both the F3L and B6R assays individually achieved 100 percent sensitivity and 100 percent specificity, underscoring the robustness of the primer design and the dual-target interpretation algorithm.

The assay also demonstrated consistent detection across a broad range of viral loads. Among the 50 qPCR-positive specimens, cycle threshold values ranged from 18.55 to 37.21, with a median of 26.11. The LAMP assay detected every qPCR-positive specimen across this entire range, including the highest Ct specimen evaluated, and maintained 100 percent sensitivity within each Ct category examined, spanning values below 20, between 20 and 25, between 25 and 30, and at or above 30. The authors caution that these Ct-stratified findings should not be interpreted as a formal analytical limit of detection, because quantified monkeypox virus reference material was not available for standardized serial dilution testing. A formal limit of detection assessment will require quantified reference material, replicate testing, and predefined confidence thresholds in future validation studies.

Analytical specificity testing provided further evidence of the assay’s discriminatory power. A dedicated panel of 64 specimens included samples positive for Vaccinia virus, Buffalopox virus, Herpes simplex virus, and Varicella zoster virus, each represented by 16 specimens. Herpes simplex virus and Varicella zoster virus were included because they are clinically relevant causes of vesicular or pustular lesions that overlap with mpox in the differential diagnosis. As expected, the non-orthopox DNA viruses produced no amplification in either target, while Vaccinia and Buffalopox viruses yielded the characteristic B6R-positive, F3L-negative pattern, confirming that the assay can reliably distinguish monkeypox virus from genetically related orthopoxviruses while showing no cross-reactivity with unrelated viral pathogens.

The study’s authors note that several LAMP-based assays for mpox detection have been reported previously, but those efforts focused primarily on analytical sensitivity using synthetic templates or limited clinical evaluation sets. In contrast, the present work emphasizes clinical validation using a structured evaluation panel and comprehensive diagnostic accuracy metrics, including likelihood ratios and agreement statistics, in line with current recommendations for reporting diagnostic accuracy studies. The incorporation of uracil-DNA glycosylase in the commercial master mix further guards against carryover contamination, improving reliability in routine diagnostic settings.

Nevertheless, the researchers acknowledge several limitations. The clinical evaluation was retrospective, used archived specimens from a single reference laboratory, and included a limited sample size of 50 positive and 80 negative specimens, so the performance estimates should not be overgeneralized. A formal analytical limit of detection was not determined, the specificity panel did not include all infectious and non-infectious causes of vesiculopustular lesions, and formal reproducibility testing across multiple days, operators, reagent lots, and low-positive specimens was not performed because of limited specimen volume. Future studies incorporating quantified standards, broader specificity panels, multi-site validation, reproducibility assessment, and prospective field evaluations will be needed before wider clinical implementation.

From a public health perspective, the simplicity of the colorimetric LAMP assay, combined with its minimal equipment requirements, rapid turnaround time, and visual readout, makes it a promising tool for expanding mpox diagnostic capacity beyond centralized laboratories. The diagnostic method is covered by an Indian patent application filed in October 2022 and published in April 2024, held by the Indian Council of Medical Research. While qPCR will continue to play a central role in reference and confirmatory testing, the new assay is particularly relevant for outbreak response, surveillance activities, and diagnostic decentralization in resource-limited settings, aligning with recent WHO mpox preparedness and response frameworks. As mpox transmission continues across multiple continents, accessible tools of this kind may prove decisive in closing the diagnostic gap that has long hampered containment efforts.

Subject of Research: Development and clinical validation of a colorimetric loop-mediated isothermal amplification assay for rapid detection of monkeypox virus

Article Title: Development and clinical validation of a colorimetric isothermal assay for rapid detection of monkeypox virus

Article References: Nandi, S. S., Sawant, S. A., Yadav, P., Shete-Aich, A., Majumdar, T. D., Bavkar, K. C., Mukherjee, A., & Deshpande, J. M. (2026). Development and clinical validation of a colorimetric isothermal assay for rapid detection of monkeypox virus. New Microbes and New Infections, 73, Article 101848. https://doi.org/10.1016/j.nmni.2026.101848

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101848

Keywords: mpox, monkeypox virus, LAMP assay, colorimetric detection, isothermal amplification, qPCR, diagnostic validation, orthopoxvirus, B6R gene, F3L gene, ICMR-NIV, point-of-care testing