Ultrasensitve single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device

Since the 2022 Mpox outbreak, more than 90,000 cases across 116 countries led WHO to declare a public health emergency of international concern. MPXV is a zoonotic DNA virus transmitted via animal bites, respiratory droplets, direct contact with lesions, and possibly contaminated materials. Although many infections are self-limited, severe disease can occur; two clades differ in virulence, with West African strains generally causing milder disease. Rapid, sensitive, easy-to-use point-of-care diagnostics are therefore urgently needed to support surveillance and clinical management, especially outside well-equipped laboratories. This study aims to develop and validate a rapid, ultrasensitive, single-step CRISPR-based assay and companion vest-pocket device (SCOPE/CPod) for on-site MPXV detection from minimally processed clinical samples.

CRISPR-Dx platforms have shown strong potential for rapid nucleic acid testing. Many existing approaches are two-step workflows coupling preamplification with CRISPR trans-cleavage, improving sensitivity and speed but complicating workflows for point-of-care use. Cas12a has been widely used for DNA targets but can suffer from target loss due to cis-cleavage of DNA during early amplification in one-pot formats. Cas13a exhibits strong collateral activity and, when targeting transcribed RNA in single-step assays, avoids depleting DNA templates, potentially improving efficiency. Prior CRISPR methods for SARS-CoV-2 and other pathogens have demonstrated either amplification-free strategies or enhanced one-pot sensitivity with additives and clever reaction engineering, but many lack streamlined sample prep, lyophilized reagents, miniaturized instrumentation, and comprehensive clinical validation. The present work builds on these advances by integrating a Cas13a-based single-step RPA–T7–Cas assay, extraction-free lysis, reagent lyophilization, and a low-cost portable reader, and by validating performance on clinical MPXV samples with qPCR comparison and cross-reactivity testing.

Assay design and workflow: SCOPE integrates extraction-free viral lysis (80 °C, 2 min) with a single-tube, single-step RPA–T7 transcription–CRISPR/Cas13a reaction (37 °C, 10 min). The system targets the MPXV F3L gene. dsDNA amplicons generated by RPA carry a T7 promoter (on the forward primer), enabling transcription of ssRNA, which activates the Cas13a–crRNA complex to cleave a FAM-labeled RNA reporter for fluorescence readout. Primer/crRNA screening and reaction optimization: Sixteen primer pairs were screened by RPA and gel electrophoresis; multiple crRNAs were evaluated against F3L RNA. Optimal performance in the single-step context did not come from the individually best primer/crRNA sets, highlighting the need to co-screen combinations to balance amplification and Cas13a cleavage while minimizing interference. Key reaction components were iteratively optimized (one variable at a time), identifying optimal conditions such as approximately: dNTP (rNTP) mix 1 mM, T7 RNA polymerase 1 µL, primers 500 nM, LwaCas13a 50 nM, crRNA 100 nM, and RNase H 0.05 µL for robust signal within 10 minutes. Extraction-free lysis: Rash fluid swab, oral swab, saliva, and urine samples were evaluated. Lysis temperature screening (room temp to 98 °C) showed saturated nucleic acid release above ~80 °C. Lysis time at 80 °C was minimized to about 2 minutes. Lysis buffer-to-sample ratios and lysate input volumes into the CRISPR reaction were optimized empirically for each matrix; using 1 µL of lysed sample per reaction provided the best performance across specimen types. Direct lysis yielded results comparable to commercial extraction for rash fluid, oral swab, and saliva; urine showed reduced performance with direct lysis due to matrix complexity. Reagent lyophilization: The single-step reaction master mix was lyophilized to simplify logistics and on-site use. Initial lyophilization caused signal attenuation, which was mitigated by adding 0.1% BSA as a cryoprotectant. Lyophilized pellets were rehydrated immediately prior to use with RPA rehydration buffer, water, magnesium acetate, and target input. Device (CPod) fabrication and operation: A compact, low-cost device was built with an ESP32 microcontroller, a PT1000 ceramic heater for precise temperature control (room temperature to 80 °C heating/cooling), and an AS7341 spectral sensor for fluorescence detection. Excitation was provided by a blue LED; emission was captured on the FAM channel (500–520 nm). Two result modes were supported: standalone (indicator LEDs for qualitative readout) and wireless (real-time fluorescence and automated interpretation via a smartphone app). Software included baseline correction and thresholding. The device is USB-C powered. Analytical testing: Sensitivity was assessed using quantified MPXV pseudovirus DNA standards (digital PCR-calibrated) across serial dilutions. Readouts were collected on both the CPod and a commercial plate reader for comparison, with nonlinear regression used to relate fluorescence to input concentration and establish detection thresholds (mean of negative controls plus 3×SD). Cross-reactivity was tested against related orthopoxviruses and clinically similar viruses (e.g., cowpox, vaccinia, variola, HSV, coronaviruses). Clinical evaluation: A total of 102 clinical samples from male patients/volunteers (various matrices: rash fluid swab, oral swab, saliva, urine) were processed via extraction-free lysis and tested by SCOPE in parallel with qPCR. qPCR used standard TaqMan chemistry and thermal cycling on ABI-7500. SCOPE runtime was ~15 minutes (2-min lysis + 10-min CRISPR + automated interpretation). Performance metrics (sensitivity, specificity) were calculated against qPCR results.

- Speed and workflow: End-to-end sample-to-answer time ~15 minutes (2-min extraction-free lysis at 80 °C + 10-min single-step RPA–T7–Cas13a reaction + automated readout), with simple, hands-free operation on a palm-sized device. - Sensitivity: Limit of detection (LoD) determined as 0.5 copies/µL (≈2.5 copies per 5-µL input) for MPXV DNA; device performance matched a commercial plate reader. Detection rates across low concentrations supported the 0.5 copies/µL LoD criterion (>95% positive rate thresholding). - Specificity: No cross-reactivity detected with tested orthopoxviruses and other viruses with similar symptoms (e.g., cowpox, vaccinia, variola, HSV, coronaviruses). - Clinical validation: On 102 clinical samples spanning rash fluid swab, oral swab, saliva, and urine, SCOPE results were 100% concordant with qPCR, achieving 100% sensitivity and 100% specificity across a qPCR Cq range of ~15.88–39.02. - Sample preparation: Direct lysis performed comparably to commercial extraction for rash fluid, oral swab, and saliva; urine matrix showed reduced performance with direct lysis relative to extraction. - Reagent/device engineering: Optimized one-pot chemistry (including component concentrations and co-screened primer–crRNA combinations), lyophilized reagents stabilized with 0.1% BSA, and a low-cost wireless device (ESP32 + PT1000 + AS7341) enabling standalone or smartphone-controlled operation.

The study addresses a key need for rapid, sensitive, and field-deployable MPXV diagnostics by integrating extraction-free sample prep with a true single-step CRISPR assay and compact instrumentation. By choosing Cas13a and inserting a T7 transcription step between RPA and detection, the system minimizes interference between amplification and nuclease activity and avoids depletion of DNA targets, which can plague one-pot Cas12a workflows. The optimized chemistry and co-screened primer–crRNA pairs improved assay kinetics and sensitivity, enabling single-molecule-level detection claims in minutes. Analytically, SCOPE achieves a qPCR-comparable LoD of 0.5 copies/µL with excellent specificity against related viruses. Clinically, 100% concordance with qPCR across multiple specimen types demonstrates robust real-world applicability. The vest-pocket device consolidates heating, reaction control, fluorescence acquisition, and automated interpretation, supporting both lay and professional users in resource-limited settings. Together, these results suggest SCOPE can substantially enhance point-of-care MPXV testing capacity and could be adapted for other pathogens via reprogrammed primers/crRNAs.

SCOPE delivers an ultrasensitive, single-step CRISPR/Cas13a assay for MPXV with a streamlined, extraction-free workflow and a low-cost vest-pocket device, achieving 0.5 copies/µL LoD and sample-to-answer results in ~15 minutes. It demonstrated 100% concordance with qPCR on 102 clinical samples and no cross-reactivity with related viruses. The platform integrates lyophilized reagents, automated analysis, and dual-mode readouts (indicator LEDs or smartphone), making it well-suited for point-of-care deployment. Future work should further optimize lysis chemistry/time (ideally eliminating high-temperature steps), improve performance in challenging matrices such as urine, expand multiplexing capabilities leveraging the spectral sensor, and generalize the assay to other targets and settings.

- The extraction-free lysis currently requires heating to 80 °C, adding a thermal step that may limit some POC contexts; buffer composition and conditions may need further optimization to enable ambient or lower-temperature lysis. - Urine showed reduced performance with direct lysis compared to conventional extraction, indicating matrix effects and the need for matrix-specific optimization. - The reported clinical cohort comprised male patients/volunteers only, potentially limiting generalizability; broader, diverse populations and larger cohorts would strengthen clinical validation. - Some protocol parameters (e.g., optimal lysis buffer-to-sample ratios) vary by specimen and were derived empirically; standardization and robustness across sample variability will be important for deployment.