The 2022 monkeypox (Mpox) outbreak, declared a Public Health Emergency of International Concern by the WHO, underscored the urgent need for rapid, sensitive, and accessible diagnostic tools. Mpox, caused by MPXV infection, presents with symptoms including fever, headache, muscle aches, back pain, swollen lymph nodes, and a characteristic rash. While most patients recover, some experience severe illness or death. Two main MPXV strains exist—Central African and West African—with the former associated with higher mortality. The current outbreak primarily involves the West African strain, which is less severe. MPXV transmission occurs through contact with infected animals, respiratory droplets, or skin lesions. The ongoing outbreak highlights the need for improved diagnostics to enable timely intervention and control. Existing diagnostic methods, primarily real-time PCR (qPCR), often require specialized equipment, trained personnel, and significant time, hindering their application in resource-limited settings. Therefore, the development of point-of-care testing (POCT) platforms for rapid and sensitive MPXV detection is crucial for effective public health response. This research aims to address this need by developing a novel CRISPR-based diagnostic platform with enhanced sensitivity, speed, and portability.

Several studies have explored the use of CRISPR-based diagnostic techniques for infectious disease detection, including monkeypox. However, these methods often involve multiple steps, complex procedures, and require specialized equipment, limiting their applicability in resource-constrained settings. Existing qPCR assays, while sensitive, are time-consuming and require specialized equipment and personnel, posing limitations in point-of-care applications. Previous CRISPR-based methods for MPXV detection have also faced challenges related to sensitivity and ease of use. This study leverages the advancements in CRISPR technology and aims to overcome these limitations by developing a single-step, rapid, and field-deployable diagnostic platform for MPXV detection.

The researchers developed SCOPE (Streamlined CRISPR On Pod Evaluation platform), a single-step CRISPR-based diagnostic system for MPXV detection. The workflow involves several key steps: 1. **Sample Preparation:** The assay utilizes readily available samples like rash fluid swabs, saliva, and urine. A streamlined, extraction-free viral lysis protocol is employed to release viral nucleic acids within 2 minutes at 80°C. The optimal lysis buffer ratio for each sample type was determined through optimization experiments. 2. **Single-step RPA-CRISPR/Cas13a Reaction:** The released viral DNA is then subjected to a single-step RPA-CRISPR/Cas13a reaction. This combined reaction incorporates recombinase polymerase amplification (RPA) for rapid DNA amplification, T7 transcription for RNA generation, and Cas13a-mediated trans-cleavage for signal generation. The researchers optimized the reaction conditions, including the concentrations of dNTPs, T7 RNA polymerase, primers, Cas13a, crRNA, and RNase H, to achieve optimal sensitivity. Primer and crRNA sequences were carefully selected and optimized through various screening experiments. The researchers also explored the use of Cas13a instead of Cas12a in the single-step reaction, citing its superior collateral activity and reduced interference with the RPA amplification process. 3. **Reagent Lyophilization:** The reaction reagents were lyophilized with the addition of 0.1% bovine serum albumin (BSA) to enhance stability during storage and transportation. 4. **Vest-pocket Device (CPod):** The entire process is conducted within a custom-designed, portable, and cost-effective vest-pocket device called CPod. The device integrates heating, fluorescence detection using an AS7341 spectral sensor, and result interpretation. The device offers two working modes: a standalone mode with visual indicator lights and a wireless mode enabling real-time data readout via a smartphone application. The researchers performed extensive optimization experiments to determine the optimal reaction conditions for each step, including lysis temperature and time, reagent concentrations, and primer and crRNA selection. The analytical performance of SCOPE, including limit of detection (LoD), specificity, and reproducibility, was rigorously evaluated using both MPXV pseudovirus and clinical samples.

The key findings of the study demonstrate SCOPE's superior performance: 1. **High Sensitivity and Specificity:** SCOPE achieved a LoD of 0.5 copies/µL (2.5 copies/reaction), demonstrating single-molecule level sensitivity. This sensitivity was validated using both a dilution series of MPXV pseudovirus and a panel of clinical samples. The assay showed no cross-reactivity with other orthopoxviruses or viruses with similar symptoms. 2. **Rapid Turnaround Time:** The entire detection process, from sample input to result interpretation, was completed within 15 minutes. 3. **Clinical Validation:** SCOPE was validated using 102 clinical samples (35 MPXV positive, 48 negative, and 19 HSV positive samples). The results showed 100% concordance with qPCR results, demonstrating its clinical applicability. 4. **Portability and Ease of Use:** The vest-pocket device, CPod, makes SCOPE highly portable and user-friendly, requiring minimal training and specialized equipment. This feature is particularly relevant for point-of-care applications in resource-limited settings. 5. **Cost-Effectiveness:** The use of a simplified workflow and a low-cost portable device makes SCOPE cost-effective compared to existing methods.

SCOPE addresses the limitations of existing MPXV diagnostic methods by combining the high sensitivity of CRISPR-based detection with the speed and portability of a miniaturized device. The single-step RPA-CRISPR/Cas13a assay significantly simplifies the workflow, reducing the time and complexity associated with conventional methods. The extraction-free lysis protocol further enhances the ease of use and reduces the need for specialized equipment or reagents. The results from clinical sample validation demonstrate the excellent performance and reliability of SCOPE, paving the way for its deployment in diverse settings. The use of Cas13a over Cas12a in the single-step reaction is a key innovation that enhances the overall sensitivity. The development of a user-friendly, portable device makes SCOPE readily adaptable for point-of-care use, particularly in resource-limited settings where rapid and reliable diagnostics are crucial for effective disease management. The high sensitivity and specificity, rapid turnaround time, and ease of use make SCOPE a promising tool for combating future outbreaks of MPXV and similar viral infections.

This study successfully developed and validated SCOPE, a highly sensitive, rapid, and user-friendly CRISPR-based diagnostic platform for MPXV detection. SCOPE's ability to detect MPXV at single-molecule levels within 15 minutes, its 100% concordance with qPCR in clinical samples, and its portability make it ideal for point-of-care settings. Future studies could focus on expanding the assay's multiplex capabilities to detect other pathogens simultaneously, optimizing the lysis procedure for diverse sample types, and further miniaturizing the device for increased portability.

While SCOPE demonstrated excellent performance, several limitations should be acknowledged. The current study focused on male patients, potentially limiting the generalizability of the results to other populations. Further studies are needed to assess the performance of SCOPE across diverse demographic groups. The optimization of the lysis process was performed with specific sample types and may need further adjustment for other types of clinical samples. Finally, while the CPod device is portable, its availability and affordability may be a constraint in certain resource-limited settings.