Ultrasensitive and Specific Identification of Monkeypox Virus Congo Basin and West African Strains Using a CRISPR/Cas12b-Based Platform

ABSTRACT Human monkeypox (MPX) is a severe and reemerging infectious disease caused by monkeypox virus (MPXV) and forms two distinct lineages, including Congo Basin and West African clades. Due to the absence of specific vaccines and antiviral drugs, developing a point-of-care (POC) testing system to identify MPXV is critical for preventing and controlling MPX transmission. Here, a CRISPR/Cas12b diagnostic platform was integrated with loop-mediated isothermal amplification (LAMP) to devise a novel CRISPR-MPXV approach for ultrasensitive, highly specific, rapid, and simple detection of MPXV Congo Basin and West African strains, and the detection results were interpreted with real-time fluorescence and a gold nanoparticle-based lateral flow biosensor (AuNP-LFB). The optimal detection process, including genomic DNA extraction (15 min), LAMP preamplification (35 min at 66°C), CRISPR/Cas12b-based detection (5 min at 45°C), and AuNP-LFB readout (~2 min), can be completed within 60 min without expensive instruments. Our assay has a limit of detection of 10 copies per test and produces no cross-reaction with any other types of pathogens. Hence, our CRISPR-MPXV assay exhibited considerable potential for POC testing for identifying and distinguishing MPXV Congo Basin and West African strains, especially in regions with resource shortages. IMPORTANCE Monkeypox (MPX), a reemerging zoonotic disease caused by monkeypox virus (MPXV), causes a smallpox-like disease in humans. Early diagnosis is critical to prevent MPX epidemics. Here, CRISPR/Cas12b was integrated with LAMP amplification to devise a novel CRISPR-MPXV approach to achieve highly specific, ultrasensitive, rapid, and visual identification of MPXV Congo Basin and West African strains.

For AuNP-LFB identification, CRISPR-MPXV products (2.0 mL) and 100 mL of running buffer (100 mM phosphate-buffered saline [PBS; pH 7.4]) were concurrently added to an AuNP-LFB sample pad (Fig. 3A). The CRISPR-MPXV product-containing flowing buffer moved along the AuNP-LFB through capillary action, and the crimson red dye streptavidin-AuNP was rehydrated on the conjugate pad (Fig. 3B). For a positive result, the ssDNA probe molecules were trans-cleaved with activated CRISPR/Cas12b nuclease, and FAM and biotin were separated. The biotin-streptavidin-AuNP complexes were then seized by using biotinylated bovine serum albumin (biotin-BSA) at the test line (TL) (Fig. 3C). For a negative outcome, the ssDNA probe molecules were not cleaved, and the crimson red dye streptavidin-AuNP-ssDNA probes were specifically captured by anti-FAM at the control line (CL) (Fig. 3C). The interpretation of the CRISPR-MPXV assay using AuNP-LFB is outlined in Fig. 3D, as well as Fig. S2 in the supplemental material.
Optimal reaction conditions for the CRISPR-MPXV assay. The incubation temperature is critical for LAMP amplification, and the reaction temperature for the LAMP preamplification stage was optimized to range from 61 to 69°C with D14L and ATI plasmids (1.0 Â 10 4 copies). The outcomes of the LAMP reaction were tracked with real-time turbidity using a real-time turbidity monitoring device (LA-500). The results indicated that the robust amplification of D14L-LAMP and ATI-LAMP occurred at 66°C (see Fig. S3A and B).
Multiple assay reaction times (1, 2, 5, 10, and 20 min) were tested to confirm the optimal reaction time for CRISPR/Cas12b detection. Real-time fluorescence and AuNP-LFB were simultaneously used to track the outcomes. The results showed that a stable visual signal of AuNP-LFB and fluorescent signal were detected within 5 min (see Fig.  S4A to D).
Sensitivity and specificity of the CRISPR-MPXV assay. Serial dilutions of nucleic acid templates (D14L and ATI plasmids) ranging from 5.0 Â 10 4 to 5.0 Â 10 21 copies were used to identify the limitation of detection of our assay. CRISPR-MPXV assays were carried out as described above, and the outcomes were monitored by real-time fluorescence and AuNP-LFB. As shown in Fig. 4, the sensitivity of the assay was 10 copies per test for both the D14L plasmid and ATI plasmid, and the visual AuNP-LFB readouts were consistent with the real-time fluorescence detections.
The specificity of the CRISPR-MPXV assay was verified by testing D14L plasmid, ATI plasmid, MPXV-D14L pseudovirus, MPXV-ATI pseudovirus, and 17 other microbes FIG 1 Schematic illustration of the LAMP-CRISPR/Cas12b assay for MPXV detection. The target gene containing a PMA site (TTC) is specifically amplified by the LAMP reaction (step 1). The target amplicons were captured with the gRNA-CRISPR/Cas12b complex through specific gRNA (step 2). Upon recognition of the matching target sequence, the gRNA-CRISPR/Cas12b complex was induced to nonspecifically cleave single-strand DNA reporter molecules (step 3).
CRISPR/Cas12b Platform for Identification of MPXV Microbiology Spectrum (Table 1). The CRISPR-MPXV assay protocol was performed as described above, and the results were read with real-time fluorescence and AuNP-LFB. The positive outcomes appeared only for the templates extracted from the MPXV Congo Basin or West African clade, while non-MPXV microbes and the blank control presented negative outcomes ( Fig. 5; see also Fig. S5 and S6), and no cross-reactions were observed from the CRISPR-MPXV assay. Therefore, our assay accurately detected and distinguish MPXV Congo Basin and West African clades. Evaluating the feasibility of the CRISPR-MPXV assay using simulated clinical samples. Next, the feasibility of the CRISPR-MPXV assay to accurately identify and discriminate MPXV Congo Basin and MPXV West African strains was confirmed. Sixty simulated clinical samples (30 skin swabs and 30 throat swabs) were simultaneously detected using DNA sequencing and our assay. The DNA sequencing results showed that 12 skin swabs and 9 throat swabs were verified as belonging to the MPXV Congo Basin clade, and 8 skin swabs and 16 throat swabs were verified as belonging to the MPXV West African clade. These results were consistent with our CRISPR-MPXV assay outcomes (Table 2). Thus, these data indicated that the CRISPR-MPXV assay functioned as an advanced diagnostic tool to identify and discriminate MPXV Congo Basin and West African strains.

DISCUSSION
In our study, a novel CRISPR-MPXV diagnosis platform that combined CRISPRR/Cas12bbased testing with LAMP preamplification was successfully devised for the ultrasensitive, highly specific, rapid and visual identification and discrimination of MPXV Congo Basin and West African strains. The feasibility of our platform was verified using simulated clinical samples, and the results were compared to DNA sequencing. Multiple laboratory diagnostic approaches have been reported to detect monkeypox virus, such as cultivation, electron microscopy, and nucleic acid amplification technologies (NAATs) (27)(28)(29). Culture and electron microscopy were not recommended since high containment facilities and specific facilities were needed. With respect to high specificity and sensitivity, real-time PCR is currently the gold standard for diagnosing monkeypox virus (29). Li et al. used real-time PCR to specifically identify West African and Congo Basin monkeypox virus strains and detected 3.5 and 40.4 copies, respectively (29). However, high-precision thermal cyclers and wellestablished laboratory infrastructures are needed, which limits accessibility in resource- poor settings. Here, our CRISPR-MPXV diagnostic system can perform isothermally without specialized instruments, and then the results can be visually readout using AuNP-LFB. The CRISPR-MPXV detection procedure, including nucleic acid extraction (15 min), LAMP preamplification (35 min), CRISPR/Cas12b detection (5 min), and AuNP-LFB visual result interpretation (within 2 min), can be completed within 60 min.
CRISPR/Cas systems were initially discovered in the adaptive immunity of bacteria and archaea for defense against invasion nucleic acids (30,31). Over the last few years, many CRISPR/Cas systems, such as CRISPR/Cas12, CRISPR/Cas9, and CRISPR/Cas13, have been widely applied in biomedical fields for gene editing (30,32). The RNA-guided cleavage of Cas effectors is flexibly utilized through a simple redesign of spacer sequences for different target genes (32). Recently, CRISPR/Cas systems have been repurposed and show promise for the development of robust diagnostic tools to detect nucleic acids, these systems exhibit unique characteristic, including collateral cleavage for target genes and nonspecific single-stranded nucleic acids (13,17). The ultrasensitivity, precision, and specificity of CRISPR/Cas-based detection could be due to target-dependent gRNA. In the present study, specific gRNAs for MPXV West African and Congo Basin strains were successfully devised, and the gRNAs efficiently navigated Cas12b effectors to each of the target sequences. The specificity of the CRISPR-MPXV assay was verified with MPXV strains and other pathogens. Owing to the lack of reference strains for the MPXV West African and Congo Basin clades in our laboratory, the full-length Congo Basin clade D14L gene and the West African clade ATI gene were extracted from synthetically produced MPXV-D14L pseudovirus and MPXV-ATI pseudovirus, respectively. Each of them acted as a positive control. As expected, the CRISPR-MPXV detection system clearly distinguished MPXV West African and Congo Basin strains and showed no cross-reaction with other To test the sensitivity of the CRISPR-MPXV assay, each of the synthesized plasmids was diluted from 1.0 Â 10 5 to 1.0 Â 10 21 copies/test. The results confirmed that our novel CRISPR-MPXV assay can detect as few as 10 copies of genomic DNA per test. More importantly, we also successfully applied the CRISPR-MPXV assay to test simulated clinical samples. The artificial samples were simultaneously detected with CRISPR-MPXV and DNA sequencing methods, and concordance results were obtained between the former and latter assays. A shortcoming of our study is that monkeypox-positive clinical samples were not collected to further confirm the feasibility of our assay, as monkeypox cases are lacking in China to date. Fortunately, pseudoviruses can verify the feasibility of nucleic acid detection technologies (33,34).
In the present study, loop-mediated isothermal amplification (LAMP) was applied to preamplify the target sequences (MPXV Congo Basin clade D14L gene and West African clade ATI gene). LAMP is a novel nucleic acid isothermal amplification approach that can robustly amplify the target sequence at a constant temperature (58 to 69°C) for 30 to 60 min using Bacillus stearothermophilus DNA polymerase with four or six primer sets that span six or eight unique sections of the target gene (35)(36)(37). The primer set contained forward and reverse outer (F3 and B3) and inner (FIP and BIP) primers, and the four primers were usually sufficient for LAMP amplification. The two extra loop primers (LF and LB) are usually incorporated into the LAMP reaction to improve the reaction's efficiency and specificity. Here, two sets of LAMP primers were successfully designed to target the D14L and ATI genes (Table 3; see also Fig. S1). The optimal LAMP reaction conditions were determined to be 66°C for 35 min (see Fig. S3).
For a visual readout of CRISPR-MPXV results, AuNP-LFB was used in our diagnosis system. AuNP-LFB is a paper-based detection platform and is promising for POC testing because it is easy to manufacture, exhibits specificity and sensitivity, is inexpensive and simple to operate, and provides a visual readout (38,39). The AuNP-LFB can visually interpret the CRISPR-MPXV outcomes for labeling with anti-FAM and BSA-biotin on the nitrocellulose membrane. For positive outcomes, the ssDNA probes (59-FAM-TTTTTT-Biotin-39) were trans-cleaved by the activated CRISPR/Cas12b nuclease, the FAM and biotin probes were separated, and the biotin-streptavidin-AuNP complexes were captured by biotin-BSA and visually read at the test line (TL). For negative outcomes, the ssDNA probes were not cleaved, and the FAM/biotin-labeled probe-streptavidin-AuNP complexes were arrested by anti-FAM at the control line (CL) (Fig. 3). In the present study, a real-time fluorescence technique was also applied to interpret the CRISPR-MPXV results. However, interpreting CRISPR-MPXV results with real-time fluorescence techniques requires expensive devices, which may not be available in many resource-limited regions.
In conclusion, CRISPRR/Cas12b was successfully integrated with LAMP amplification to devise a novel CRISPR-MPXV approach for highly specific, ultrasensitive, rapid, and visual identification and discrimination of MPXV Congo Basin and West African strains. Our assay  CRISPR/Cas12b Platform for Identification of MPXV Microbiology Spectrum exhibited a 10-copy limit of identification and showed no cross-reaction with any other pathogens. The entire testing process can be completed within 60 min and does not necessitate any sophisticated devices. Hence, these characteristics matched the WHO-recommended POC testing requirements (low cost, sensitive, specific, user-friendly, robust, equipment-free, and attainable).
Target DNA and artificial MPXV virus preparation. Full-length Congo Basin clade D14L (accession no. KP849471.1) and West African clade ATI (accession no. MT903346.1) sequences were synthetically produced and cloned into the pUC57 vector (Tsingke Biotech, China). The initial concentration of each plasmid was 1 Â 10 8 copies, and the two constructed plasmids acted as positive controls.
Two pseudoviruses (MPXV-D14L pseudovirus and MPXV-ATI pseudovirus) were constructed by Sangon Biotech Co., Ltd. (Shanghai, China), which were made with 293A cell cultures and included segments of the D14L gene (GenBank accession no. KP849471.1, genome coordinates 19294 to 19944) and ATI gene (GenBank accession no. MT903346.1, genome coordinates 135527 to 135918), respectively. Genomic DNA was extracted using viral qEx-DNA/RNA extraction kits (Xi9an Tianlong Science & Technology Co., Ltd., Xian, China) in accordance with the manufacturer's instructions. The concentrations of genomic DNA were measured using a   Table 1. LAMP primer and gRNA design. The MPXV Congo Basin and West African clade LAMP primers were designed using software programs Primer Explorer V5 (http://primerexplorer.jp/e/) and PRIMER PREMIER 5.0 in accordance with the principle of LAMP reaction based on the MPXV Congo Basin-specific D14L gene and MPXV West African-specific partial ATI gene, respectively (27). The specificity of each LAMP primer was verified with the BLAST analysis tool. Two gRNAs for the MPXV Congo Basin D14L gene and West African ATI gene were designed according to the CRISPR/Cas12b detection mechanism. The locations of each LAMP primer and gRNA are shown in Fig. S1. The principle of LAMP preamplification and CRISPR/Cas12b-based assay is shown in Fig. 1. The sequences of LAMP primers and gRNAs are shown in Table 3. All the oligonucleotides were synthesized and purified by GenScript Biotech Co., Ltd. (Nanjin, China), with high-performance liquid chromatography purification grade. CRISPR/Cas12b-based identification. In this study, AacCas12b (C2c1) was used for CRISPR/Casbased trans-cleavage identification. In brief, the CRISPR/Cas12b-gRNA complexes were preassembled in a 50-mL reaction volume, which included 2 mL of AacCas12b (100 pM), 2 mL of gRNA (10 mM), and 25 mL of 2 Â HDX buffer, adding distilled water (DW) up to 50 mL, and then the mixtures were preincubated at 37°C for 10 min. The CRISPR/Cas12b-gRNA complexes were used immediately or stored at low temperatures (0 to 4°C) for no more than 24 h before use.
AuNP-LFB preparation and assay. The gold nanoparticle-based lateral flow biosensor (AuNP-LFB; size, 60 Â 4 mm) is illustrated in Fig. 3. In brief, the AuNP-LFB was composed of four sections, including nitrocellulose membrane, sample, conjugate, and absorption pads. Crimson red dye streptavidin-gold nanoparticles (SA-GNPs) were deposited onto the conjugate pad. Anti-FAM (4 mg mL 21 ) and biotin-BSA (0.2 mg mL 21 ) were immobilized onto the nitrocellulose membrane for the control line (CL) and test line (TL), respectively, with 5 mm separating each line. For the AuNP-LFB assay, 2.0 mL of CRISPR/Cas12b trans-cleavage products and 100 mL of running buffer (100 mM PBS, 1% Tween 20 [pH 7.4]) were simultaneously added to the sample pad, and the running buffer containing ssDNA reporter molecule solution flowed along the AuNP-LFB via capillary action. Finally, the results were generated on the nitrocellulose membrane (red line) within 2 min.
Sensitivity and specificity of the CRISPR-MPXV assay. Two standard plasmids, the D14L and ATI plasmids, were serially diluted by 10-fold ranging from 1.0 Â 10 5 to 1.0 Â 10 21 copies. CRISPR-MPXV reactions were performed as previously described, and the results were monitored with real-time fluorescence and AuNP-LFB. Each dilution was tested at least three times.
To verify the specificity of our assay, pseudoviruses of MPXV and other microbes (Table 1) were used for CRISPR-MPXV detection, and all of the results were read simultaneously with real-time fluorescence and AuNP-LFB. All examinations were tested at least three times.
Verifying the feasibility of the CRISPR-MPXV assay using simulated clinical samples. Owing to the lack of MPXV clinical samples, two kinds of pseudoviruses, MPXV-D14L pseudovirus and MPXV-ATI pseudovirus, were used to verify the feasibility of our assay. Briefly, 30 human skin swabs and 30 throat swabs were collected from healthy volunteers, and each skin swab or throat swab was fully immersed in 200 mL of sterile virus preservation medium (DaAn Gene Co., Ltd., China). Then, 10 to 500 copies of each pseudovirus were randomly added to each solution. In compliance with the producer's guidelines, genomic DNA templates were obtained through viral qEx-DNA/RNA extraction kits (Xi9an Tianlong Science & Technology Co.). The CRISPR-MPXV operation was performed as described above. In addition, all artificial samples were tested using DNA sequencing (Tsingke Biotech). Finally, the results of the CRISPR-MPXV assay were compared to those from DNA sequencing.
Ethics statement. This study was approved by the Human Ethics Committee of the Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine (approval KYW2022009), and complied with the Declaration of Helsinki. Before our team obtained healthy-person samples and conducted this study, all personal patient identifiers were removed. Patient informed consent was waived by the ethics committee.
Data availability. The data sets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1.5 MB.