Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection

Figures

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  Figure 1. Schematic diagram of CRISPR-Cas12a coupled with a reverse transcription-enzymatic recombinase amplification assay for enhancement of fluorescence and visualization.

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  Figure 2. Feasibility analysis of the Cas12a platform for plasmid detection, Specific validation and screening of five GAstV crRNAs designed for the ORF2 gene. (A) Design of the crRNA sequence based on the ORF2 gene of GAstV. (B,C) ORF2 gene was recognized by corresponding five pairs of crRNAs. The fluorescent images and intensities at 30 min of the reaction were shown. (D) Fluorescence analysis of the feasibility of the Cas12a platform for nucleic acids detection.

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  Figure 3. RT-ERA coupled with Cas12a at optimization of timing of the Cas12/crRNA reaction.

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  Figure 4. Validation of the specificity and sensitivity of Cas12a/crRNA for the detection of GAstV. (A) Validation of crRNA sensitivity reacts with the target gene. Sensitivity assay using crRNA to detect gradient ORF2 gene plasmid from 2×107 to 2×100 copies. (B) The fluorescence intensity of different dilution copy numbers at 30 min was shown. (C) Validation of crRNA specifically reacts with the target gene. (D) GAstV and other virus were detected Fluorescence intensity at every 5 min.

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  Figure 5. Detection of GAstV in 30 clinical samples by the conventional culture method (qPCR) and Cas12a/crRNA assay. The RT-ERA-based Cas12a heatmap represents normalized mean fluorescence values. Lanes 1–30: the clinical sample number.