An ultrasensitive and specific CRISPR-Cas13a-mediated point-of-care assay for monkeypox detection and PCR-based clade detection

Abstract

Background: The rapid increase in the number of monkeypox cases poses a considerable threat to the international community, necessitating sensitive, fast, and available diagnostic methods. Therefore, the objective of this study was to develop a rapid, sensitive and simple method with high clinical applicability.

Methods: We developed a simple, rapid point-of-care assay to detect monkeypox virus (MPXV) using multienzyme isothermal rapid amplification (MIRA) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system. The detection system was optimized by synthesizing plasmids, and the detection sensitivity was explored by the continuous dilution of the plasmid. We validated the accuracy of this assay on 202 clinical MPXV samples and 104 interference samples through the kappa test. The visual interpretation of the results was realized by combining the assay with lateral flow strips. In addition, we developed a PCR-based method to identify MPXV Clades I and II, and the accuracy was tested through a kappa test on 202 clinical monkeypox samples and 104 interference samples.

Results: Our assay achieved an analytical sensitivity of 14.4 copies/ml and high selectivity, as it differentiated MPXV from three other Orthopoxvirus species. The clinical testing results for 202 monkeypox samples and 104 interference samples demonstrated 100% sensitivity and specificity. Compared with quantitative PCR (qPCR), three samples tested as positive using our assay, which showed that the performance of this assay was superior to that of the qPCR assay. Combined with lateral flow strips, its availability and simplicity provide an alternative point-of-care diagnostic method for MPXV testing in remote settings and resource-poor areas. The results of 32 clinical samples showed that lateral flow strips had a high detection sensitivity and could identify samples with Ct value of 39 as positive. The clade identification assay detected as few as 200 copies/ml within 40 min and no cross-reaction was observed between Clades I and II. The clinical samples tested were all Clade II, which was consistent with the circulating clade in the Chinese mainland.

Conclusions: The MIRA-CRISPR-Cas13a-MPXV system offers a rapid, sensitive and specific approach for monkeypox diagnosis, with significance for monitoring monkeypox epidemics. The clade identification assay based on PCR could accurately distinguish Clade I from Clade II within 40 min and can be implemented for high-throughput operation.

Keywords: CRISPR-Cas13a system; Lateral flow strip; MPXV clade detection; Monkeypox; Point-of-care assay.

Fig. 1

The principle of MIRA-CRISPR-Cas13a proposed in this study. MIRA for target template amplification; T7 transcription for dsDNA to target ssRNA; CRISPR-Cas13a for reporter cleavage; fluorescence assay and lateral flow strip assay for detection signal output

Fig. 2

Optimization of CRISPR-Cas13a system reaction conditions. a Three candidate crRNAs screening, including crRNA-1, crRNA-2 and crRNA-3. b The fluorescence curve of crRNA-3 concentration optimization, including 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L, 1 µmol/L and 1.5 µmol/L. c The fluorescence curve of LwaCas13a concentration optimization, including 0.25 µmol/L, 0.5 µmol/L, 1 µmol/L, 2 µmol/L and 4 µmol/L. d The fluorescence curve of reporter concentration optimization, including 1 µmol/L, 2 µmol/L, 3 µmol/L, 5 µmol/L, 10 µmol/L and 20 µmol/L. e Verification of the uniqueness of essential components of CRISPR-Cas13a system, including reporter, LwaCas13a, T7 enzyme, crRNA-3 and template

Fig. 3

Performances of the proposed MIRA-CRISPR-Cas13a-MPXV fluorescence assay. a The gel image of MIRA assay sensitivity exploration via 2% agarose gel electrophoresis. Lane 1, no template control, use DNase/RNase-free water instead of the template; lane 2, blank, only reaction mixture; lane 3, 1.44 × 10² copies/ml; lane 4, 1.44 × 10³ copies/ml; lane 5, 1.44 × 10⁴ copies/ml, lane 6, 1.44 × 10⁵ copies/ml; lane 7, 1.44 × 10⁶ copies/ml; lane 8, 1.44 × 10⁷ copies/ml; lane 9, 1.44 × 10⁸ copies/ml; lane 10, 1.44 × 10¹² copies/ml; lane M, Molecular weight markers (FY2000 DNA marker, Yugong Biotech). 1.44 × 10² copies/ml and above bands from weak to strong. b The analytical sensitivity of MIRA-CRISPR-Cas13a-MPXV system fluorescence assay. The plasmid serial dilution concentrations in the range of 1.44 × 10^–2 copies/ml to 10⁹ copies/ml were subjected to MIRA-CRISPR-Cas13a-MPXV system fluorescence assay. ∗ , P < 0.05; ∗ ∗ , P < 0.01; ∗ ∗ ∗ , P < 0.001; ∗ ∗ ∗ ∗ , P < 0.0001; and ns, not significant. P < 0.05 indicated statistical significance. c Graph of the reaction process of CRISPR-Cas13a system detected three other OPVs plasmids compared with MPXV

Fig. 4

Clinical samples detected with qPCR and MIRA-CRISPR-Cas13a assay. a The corresponding Ct values for 179 clinical samples tested by qPCR assay. b The corresponding fluorescence intensities for 182 clinical samples tested by MIRA-CRISPR-Cas13a system. c The corresponding fluorescence intensities of MIRA-CRISPR-Cas13a assay for 182 positive samples, 20 negative samples and 104 interference samples. d Specificity evaluation of the proposed MIRA-CRISPR-Cas13a assay. A heat map corresponded to the log values of the interference sample results

Fig. 5

The clinical validation of MIRA-CRISPR-Cas13a system lateral flow strip assay. Results of lateral flow strip corresponded to multiple Ct values of clinical samples. NTC no MPXV DNA template control; NPC no MIRA product control. The number below each strip represents the Ct value of the corresponding clinical sample tested by the qPCR assay