Rapid, sensitive, and highly specific detection of monkeypox virus by CRISPR-based diagnostic platform

Abstract

Background: Monkeypox (MPX), caused by the Monkeypox virus (MPXV), has incurred global attention since it broke out in many countries in recent times, which highlights the need for rapid and reliable diagnosis of MPXV infection.

Methods: We combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based detection to devise a diagnostic test for detection of MPXV and differentiation of its two clades [Central Africa clade (MPXV-CA) and West Africa clade (MPXV-WA)], and called it MPXV-RCC. The sensitivity, specificity and practicability of this method have been analyzed.

Results: The optimal conditions of MPXV-RCC assay include two RPA reactions at 38°C for 25 min and a CRISPR/Cas12a-gRNA detection at 37°C for 10 min. The results of MPXV-RCC assay were indicated by a real-time fluorescence analysis software. Thus, the whole detection process, including rapid template preparation (20 min), RPA reaction (25 min) and CRISPR-based detection (10 min), could be finished within 1 hour. The sensitivity of MPXV-RCC for MPXV-CA and MPXV-WA detection was down to 5~10 copies of recombination plasmids and pseudovirus per reaction. Particularly, MPXV-RCC assay could clearly differentiate MPXV-CA from MPXV-WA, and had no cross-reactivity with other pathogens. In addition, the feasibility of MPXV-RCC assay was further validated by using spiked clinical samples.

Conclusion: The MPXV-RCC assay developed here is a promising tool for quick and reliable diagnosis of MPXV infection.

Keywords: CRISPR/Cas12a; MPXV-RCC; RPA; monkeypox; monkeypox virus.

Figure 1

Schematic illustration of the MPXV-RCC technique. (A) Outline of RPA amplification for MPXV. Step 1: recombinase–primer complex specifically recognizes the target sequence containing a PAM site. Step 2: a D-loop is formed by the combination of SSB and DNA chain, and then, DNA polymerase activates the isothermal amplification. Step 3: displaced D-loop keeps the DNA open when the amplification continues. Step 4: products are generated exponentially. (B) Schematic illustration of CRISPR/Cas12a-gRNA detection. Step 1: Cas12a enzyme couples with gRNA to constitute CRISPR/Cas12a-gRNA complex. Step 2: gRNA sequence combines with target DNA near the PAM site. Step 3: Cas12a enzyme is activated to cut the template chain. Step 4: ssDNA is cleaved non-specifically by the Cas12a enzyme, and it could be available as a fluorescent probe. (C) Primer and gRNA sequence alignment on the MPXV segment. Nucleotide sequences of MPXV-CA and MPXV-WA are indicated. The locations of primers and gRNA are underlined, and the arrowheads show the amplified direction of primers. (D) Outline of MPXV-RCC assay workflow. MPXV-RCC diagnosis platform employs three steps: DNA preparation, RPA amplification, and CRISPR/Cas12a-gRNA cut and signal detection.

Figure 2

Confirmation of the MPXV-RCC assay. Agarose gel electrophoresis (AAG) indicated the RPA results, and fluorescence signal (FS) detection showed further CRISPR results. AAG (A) and FS (B) exhibited the detection of MPXV-CA. AAG (A)/FS (B) 1–4 represented MPXV-CA plasmid, MPXV-WA plasmid, enterovirus (JACDC-1), and distilled water, respectively. AAG (C) and FS (D) exhibited the detection of MPXV-WA. AAG (C)/FS (D) 1–4 singly represented MPXV-WA plasmid, MPXV-CA plasmid, enterovirus (JACDC-1), and distilled water.

Figure 3

Sensitivity of the MPXV-RCC assay in recombinant plasmids. (A, C) Indicate the fluorescence results of the MPXV-RCC assay for MPXV-CA and MPXV-WA detection with recombinant plasmids, respectively. (B, D) Show the statistical results of fluorescence intensity for two clades. Lines/bars 1–9 represent the results with plasmid levels of 1.0 × 10³, 1.0 × 10², 50, 25, 10, 5, 2.5, 1 copies/reaction, and blank control, respectively.

Figure 4

Specificity of the MPXV-RCC assay for MPXV-CA detection. The results of two repeated tests are shown in the figure. Graphs 1–5: MPXV-CA agents (spiked clinical samples). Graphs 6–21: influenza A virus, enterovirus, adenovirus, coronavirus, dengue virus, Epstein-Barr virus, hepatitis B virus, human rhinovirus, herpes simplex virus-1, influenza B virus, measles virus, parainfluenza virus, rubella virus, respiratory syncytial virus, Visna virus, and vesicular stomatitis virus, separately. Graph 22, blank control.