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.

Keywords: CRISPR/Cas12b; gold nanoparticle-based lateral flow biosensor; loop-mediated isothermal amplification; monkeypox; point-of-care testing.

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).

FIG 2

Outline of the CRISPR-MPXV assay workflow. (A) CRISPR-MPXV real-time fluorescence assay. The entire workflow employs the following closely linked steps: rapid DNA extraction (step 1), LAMP amplification (step 2), CRISPR/Cas12b cleavage, and real-time fluorescence readout (step 3). (B) CRISPR-MPXV-AuNP-LFB assay. The entire workflow employs four closely linked steps: rapid DNA extraction (step 1), LAMP amplification (step 2), CRISPR/Cas12b cleavage (step 3), and AuNP-LFB interpretation (step 4).

FIG 3

Schematic diagram showing AuNP-LFB principles for the visual interpretation of CRISPR-MPXV outcomes. (A) CRISPR-MPXV reaction products (2.0 μL) and running buffer (100 μL) were simultaneously deposited on the sample pad. (B) Due to capillary action, the running buffer containing CRISPR-MPXV products moved forward onto the conjugate pad and nitrocellulose (NC) membrane. Meanwhile, the dye streptavidin-coated gold nanoparticles (SA-AuNP) were rehydrated in the conjugate region. Then, the biotin-labeled probes were combined with SA-AuNP at the conjugate pad. (C) For positive results, the ssDNA probes (5′-FAM-TTTTTT-Biotin-3′) were trans-cleaved by activated-CRISPR/Cas12b nuclease, and the FAM and biotin were separated. Hence, the biotin-SA-AuNP complexes were captured by biotin-BSA at the test line (TL). In the negative outcome, the ssDNA probes were not cleaved and were specifically arrested by anti-FAM at the control line (CL). Therefore, the biotin of ssDNA probes combined SA-AuNP for visualization at CL. (D) Interpretation of the CRISPR-MPXV assay results. For positive outcomes, CL and TL appeared simultaneously on the AuNP-LFB. For negative results, only the CL line was observed on the AuNP-LFB.

FIG 4

Sensitivity of the CRISPR-MPXV assay. AuNP-LFB and real-time fluorescence (RTF) approaches were simultaneously used to readout the CRISPR-MPXV outcomes. (A and B) AuNP-LFB (A) and RTF (B) biosensor samples 1 to 8 represent the MPXV Congo Basin clade D14L plasmid concentrations of 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰, and 1 × 10⁻¹ copies per reaction and negative control (distilled water [DW]), respectively. (C and D) AuNP-LFB (C) and RTF (D) biosensor samples 1 to 8 represent MPXV West African clade ATI plasmid concentrations of 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰, and 1 × 10⁻¹ copies per reaction and negative control (DW), respectively. The limit of detection of the CRISPR-MPXV assay was 10 copies per reaction. +, positive; −, negative; CL, control line; TL, test line.

FIG 5

Specificity of the CRISPR-MPXV assay. (A) Specificity of the CRISPR-MPXV-AuNP-LFB assay for MPXV Congo Basin clade detection. Biosensor 1, MPXV Congo Basin-D14L-plasmid; biosensor 2, MPXV Congo Basin pseudovirus; biosensor 3, herpes simplex virus type 2 (HSV-2); biosensor 4, cytomegalovirus; biosensor 5, Epstein-Barr virus; biosensor 6, measles virus; biosensor 7, Sendai virus; biosensor 8, hepatitis B virus; biosensor 9, parvovirus; biosensor 10, parainfluenza virus type 3; biosensor 11, human enterovirus EV71; biosensor 12, coxsackievirus CAV16; biosensor 13, Adenoviridae; biosensor 14, influenza B virus; biosensor 15, influenza A virus; biosensor 16, herpes zoster virus; biosensor 17, human papillomavirus; biosensor 18, M. tuberculosis; biosensor 19, Neisseria gonorrhoeae; biosensor 20, negative control (distilled water, DW). CL, control line; TL, test line; +, positive; −, negative. (B) Specificity of the CRISPR-MPXV-AuNP-LFB assay for MPXV West African clade detection. biosensor 1, MPXV West African-ATI-plasmid; biosensor 2, MPXV West African pseudovirus; biosensor 3, herpes simplex virus type 2 (HSV-2); biosensor 4, cytomegalovirus; biosensor 5, Epstein-Barr virus; biosensor 6, measles virus; biosensor 7, Sendai virus; biosensor 8, hepatitis B virus; biosensor 9, parvovirus; biosensor 10, parainfluenza virus type 3; biosensor 11, human enterovirus EV71; biosensor 12, coxsackievirus CAV16; biosensor 13, Adenoviridae; biosensor 14, influenza B virus; biosensor 15, influenza A virus; biosensor 16, herpes zoster virus; biosensor 17, human papillomavirus; biosensor 18, M. tuberculosis; biosensor 19, N. gonorrhoeae; biosensor 20, negative control (distilled water [DW]). CL, control line; TL, test line; +, positive; −, negative.