An ELISA-like sensitive and visual detection system targeting Yersinia pestis based on CRISPR/Cas12a and DNAzyme

Abstract

Yersinia pestis is the causative agent of plague, a human disease with potentially devastating consequences. Here, we developed an enzyme-linked immunosorbent assay-like visual detection method based on clustered regularly interspaced short palindromic repeats (CRISPR) detection and DNAzyme for the cost-effective and highly sensitive detection of Y. pestis. A novel specific gene sequence (CH57_3927) was screened for the detection target of Y. pestis. The recombinase-aided amplification (RAA) assay, CRISPR/Cas12a detection assay, and G-quadruplex (G4) DNAzyme-based color development assay were separately established and optimized. These three optimized assays were integrated into an advanced ELISA-like visual detection method-RAA-CRISPR/Cas12a-DNAzyme (RCCD)-by further optimization of their components to improve the compatibility between them. The amplified target sequence binds to crRNA and activates the Cas12a nucleases for trans-cleave G4. As a result, the cleaved G4 is unable to bind with hemin to exert peroxidase activity, thus impeding the catalysis of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^2-) colorimetric reaction. Consequently, negative samples exhibit a dark green coloration, while the positive products appear nearly colorless, facilitating visual differentiation with the naked eye. In addition, the RCCD detection platform effectively distinguished Y. pestis from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using Y. pestis DNA-spiked blood samples and uninfected samples, both sensitivity and specificity were 100%. The method shows significant potential for detecting targets in clinical samples and is well-suited for use in resource-limited environments. It offers advantages such as visual detection, batch detection, and low cost.IMPORTANCEWe utilized Mauve software to screen Yersinia pestis specific genes and integrated CRISPR-Cas12a, RAA amplification, and G-quadruplex DNAzyme technology to establish an advanced ELISA-like visual detection method. The visual detection method offers a more cost-effective alternative compared to the conventional CRISPR detection method that relies on fluorescence-labeled ssDNA reporter or lateral flow (LF) test strips. With only one thermostatic device required, it enhances the convenience of rapid on-site screening of Y. pestis outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.

Keywords: CRISPR-Cas12a; G-quadruplex; Yersinia pestis; diagnosis; nucleic acid detection; recombinase-aided amplification.

Fig 1

Schematic illustration of the visual detection system of Y. pestis combining RAA, CRISPR/Cas12a, and G-quadruplex DNAzyme.

Fig 2

crRNA selection in CRISPR experiments. Each crRNA was individually tested with the CH57_3927 DNA. The negative control was treated with DEPC water instead of CH57_3927 DNA, with the same other components. All experimental data are represented as mean ± standard deviation (SD) of two technical replicates.

Fig 3

Optimization of the Cas12a-G4 visual detection system. (A) UV-visible absorption curve of G4 catalyzed ABTS²⁻-H₂O₂ color reaction. (B) Impact of 250 nM crRNA, 111 nM Cas12a, and 2 nM CH57_3927 (corresponding to the 20 µL CRISPR reaction system) on color development. The color reaction was conducted in a 100 µL solution containing 5 pmol G4. The control group lacked any CRISPR system components. (C and D) Various concentrations of Cas12a (223 nM in C and 111 nM in D) and G4 (0.25 µM, 0.5 µM, and 1 µM) on color development. (E) UV-visible absorbance values of G4 under various reaction conditions. (F and G) The influence of different concentrations of hemin (2.5 µM, 5 µM, and 10 µM) and types of color reagents (TMB in F and ABTS in G) on color development. (H) Impact of G4 with different sequences on color development. (I) Fluorescence intensity generated by the CRISPR/Cas12a system cleaving FAM-G-rich ssDNA-4-BHQ1. The negative control was treated with DEPC water instead of CH57_3927 DNA, with the same other components. Data are represented as the mean ± SD of two biological replicates. (J) Detection of a series of gradient dilutions of CH57_3927 using the CRISPR/Cas12a-G4 system. All experimental data in B-H are represented as mean ± standard deviation (SD) of two technical replicates. Differences among groups in B were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Differences among groups in C, D, F, J, and H were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4

Optimization of the RAA and RCCD visual detection system. (A and B) Real-time fluorescence curves of 14 RAA primer combinations in RAA nucleic acid amplification experiments. (C) Reversal of color inhibition caused by DTT with H₂O₂. The colorimetric reaction was performed in a 100 µL solution comprising 50 nM G4, 500 nM hemin, and 2.5 mM DTT. (D) Color reaction of hemin with varied concentrations. Conduct gradient dilutions of DNA for RAA amplification and subsequently employ the amplification products for color reaction with diverse hemin concentrations. (E through G) Effect of different volumes of RAA amplification products (1 µL, 0.5 µL, and 0.25 µL) on color development. All experimental data in A through G are represented as mean ± standard deviation (SD) of two technical replicates. Data in E through G were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 5

Limit of detection and specificity of Y. pestis detection by the RCCD visualization system. (A) LOD of the RCCD visualization system in detecting Y. pestis genomic DNA. (B) Specificity of the RCCD visualization system in detecting Y. pestis genomic DNA. Mix bacteria is a mixture of the genomes from eight bacteria, excluding Y. pestis. All experimental data in A and B are represented as mean ± standard deviation (SD) of two technical replicates. Differences among groups in A were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. ****, P < 0.0001.

Fig 6

Detection of Y. pestis in simulated clinical samples by RCCD detection assay. Tested samples, Y. pestis DNA-spiked blood samples; NTCs, uninfected blood samples as no-template controls. Each test was performed in duplicate.