A CRISPR/Cas12a-based reverse transcription multiple cross displacement amplification technique for sensitive, rapid, and highly specific detection of human enterovirus A71 in clinical application

Figures

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  Figure 1. Schematic illustration of the principle of the EVA71-RT-MCDA-CRISPR assay. (A) EVA71 RNA was reverse-transcribed into cDNA first. In the RT-MCDA reaction, CP1* primers with PAM sequence (TTTC) modified in the linker region were used to amplify specific products containing PAM sites. (B) After the Cas12a-gRNA complex specifically recognizes the target DNA, Cas12a exhibits activated trans-cleavage activity to non-specifically cleave 5’-FAM and 3’-BHQ1 dual-labeled ssDNA reporter probes, releasing fluorescent signals. (C) Schematic workflow of the EVA71-RT-MCDA-CRISPR assay. The entire detection system consists of four steps: RNA extraction (15 min), RT-MCDA pre-amplification (40 min), CRISPR/Cas12a trans-cleavage with fluorescence monitoring (15 min), and the total procedure can be completed within 70 min.

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  Figure 2. Verification of the EVA71-RT-MCDA-CRISPR assay. (A) The VDR method and (B) Agarose gel electrophoresis were employed for the EVA71 RT-MCDA pre-amplification products. (C) The target amplicons were cleaved by the CRISPR/Cas12a system, and the fluorescence signals were monitored via a real-time PCR system. (D) Visual observation was conducted under blue light. The EVA71 RNA standard and EVA71 isolate RNA were used as positive controls, the virus RNA of CVA16 was used as a negative control, and RNase–free water served as the blank control (BC).

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  Figure 3. Optimization of the conditions for the EVA71-RT-MCDA-CRISPR assay. (A) Results of varying volumes of the Cas12a-gRNA complex. (B) Effects of varying volumes of the ssDNA reporter probe. (C) Varying volumes of MCDA pre-amplified products. (D–F) Demonstration of differential fluorescence intensity corresponding to varying volumes of the Cas12a-gRNA complex, the probe, and the products in the EVA71-RT-MCDA-CRISPR assay. (G–I) Results of varying volumes of the Cas12a-gRNA complex, the probe, and the products in the EVA71-RT-MCDA-CRISPR assay under blue–light illumination.

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  Figure 4. Sensitivity evaluation of the EVA71-RT-MCDA-CRISPR assay. Analytical sensitivity was assessed using serially diluted EVA71 RNA templates. (A) The fluorescence signals of serially diluted EVA71 RNA samples were recorded using a real-time fluorescence detector. (B) A two-tailed Student’s t test was conducted. Error bars represented mean ±S.E.M. (n = 3). S.E.M., standard error of mean. **** p < 0.0001; *** p < 0.001; ns, no significant. (C) It was visualized with the naked eye under blue light. Each assay was carried out in triplicate to guarantee reproducibility.

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  Figure 5. Specificity evaluation of the EVA71-RT-MCDA-CRISPR assay. (A) All eight EVA71 strains exhibited positive reaction curves, while no amplification was detected for non-EVA71 pathogens. (B) The fluorescence signals were monitored and analyzed under blue-light illumination. (C) The fluorescence intensity of 8 EVA71 strains and 16 non-EVA71 pathogens. 1–8: EVA71 strains; 9–24: non-EVA71 pathogens.

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  Figure 6. Application of the EVA71-RT-MCDA-CRISPR assay in clinical samples. (A) EVA71-RT-MCDA-CRISPR assay. 1–22: the EVA71 positive samples, 23–96: the EVA71 negative samples. (B) Heatmap generated based on fluorescence endpoint values using the EVA71-RT-MCDA-CRISPR assay. A1–A44: anal swabs, T1–T52: throat swabs.