An advanced rapid-visual CRISPR assay for detecting porcine reproductive and respiratory syndrome virus

Figures

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  Fig. 1. Screening of crRNAs for the PRRSV M gene using a CRISPR/Cas13a-based detection method. (a) Schematic representation of the PRRSV WUH3 M gene (top) and the specific locations for each of the twelve crRNA spacer regions (bottom). (b) Screening crRNAs for the CRISPR/Cas13a assay, demonstrating the relative fluorescence (y-axis) against time (x-axis) of various crRNAs during the 90-min CRISPR/Cas13a reaction. Data are presented as the mean ± SEM (n = 3).

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  Fig. 2. Optimizing CRISPR/Cas13a-based detection of the PRRSV M gene. (a) Evaluation of fluorescence intensity and index using different concentrations (0, 12.5, 25, 50, 100 and 200 nM) of crRNA362 against target and non-target RNA over 90 min. (b) Evaluation of RQ-5U reporter concentrations at 0, 50, 100, 200, 400, 500, 800, 1000 and 2000 nM. (c) Evaluation of LwCas13a concentrations at 0, 5, 10, 15, 25, 35, 45 and 55 nM. (d) Evaluation of CRISPR/Cas13a reaction temperatures at 32 °C, 34 °C, 36 °C, 37 °C, 38 °C, 40 °C and 42 °C. (e) Fluorescence values of the CRISPR/Cas13a detection system preincubated at room temperature. Data are presented as the mean ± SEM (n = 3).

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  Fig. 3. Evaluation of the sensitivity of the CRISPR/Cas13a-based PRRSV M gene detection assay using multiple crRNAs. (a) Schematic representation of the CRISPR/Cas13a-based PRRSV M gene detection assay utilizing multiple crRNAs to improve the fluorescence signal intensity. (b) The CRISPR/Cas13a system with crRNA362 detected the PRRSV M gene at concentrations ranging from 6.0 × 107 to 6.0 × 109 copies/μL within 90 min. (c) The CRISPR/Cas13a system using crRNA362 and crRNA203 detected the PRRSV M gene at concentrations ranging from 6.0 × 107 to 6.0 × 109 copies/μL within 90 min. (d) The CRISPR/Cas13a system with crRNA362 and crRNA208 detected the PRRSV M gene at concentrations ranging from 6.0 × 107 to 6.0 × 109 copies/μL within 90 min. (e) The CRISPR/Cas13a system using crRNA362, crRNA208, and crRNA283, detected the PRRSV M gene at concentrations ranging from 6.0 × 107 to 6.0 × 109 copies/μL within 90 min. (f) Optimization of the crRNA cocktail (crRNA362 + crRNA208 + crRNA283) concentrations ranging from 0 to 200 nM including 12.5, 25, 50, and 100 nM. (g) Optimization of LwCas13a concentrations from 0, 5, 10, 15, 25, 35, 45, and 55 nM. (h) Optimization of RQ-5U reporter concentrations…

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  Fig. 4. Evaluation of the sensitivity of the RT-RPA-CRISPR/Cas13a assay. (a) The RT-RPA-CRISPR/Cas13a detection system was visually inspected under blue LED transilluminator (G600312-0001, Sangon Biotech), and the image was acquired via smartphone photography. (b) The fluorescence intensity of the RT-RPA-CRISPR/Cas13a assay was quantified using ImageJ analysis with background subtraction. Samples 1–8 represent final concentrations ranging from 6.0 × 107 to 6.0 × 100 copies/μL. (c) Agarose gel electrophoresis was used to identify the RT-RPA amplification products of PRRSV, PEDV, and TGEV RNA. Uncropped gel image is shown in Fig. S4. (d) The detection results of the RT-RPA-CRISPR/Cas13a assay for PRRSV, PEDV, and TGEV RNA were visualized under blue LED transilluminator (G600312-0001, Sangon Biotech) and recorded with a smartphone camera. (e) Fluorescence intensity of the RT-RPA-CRISPR/Cas13a assay for PRRSV, PEDV, and TGEV RNA was quantified by ImageJ analysis with background subtraction. Data are presented as the mean ± SEM (n = 3).

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  Fig. 5. Evaluation of the reliability of RT-RPA-CRISPR/Cas13a for detecting the PRRSV M gene in porcine sera. (a) Schematic of RT-RPA-Cas13a detection for PRRSV technique. (b) Detection of twelve PRRSV-infected and uninfected samples using RT-RPA-CRISPR/Cas13a with fluorescence intensity quantification via ImageJ software. (c) Confirmation of the twelve PRRSV-infected and uninfected samples by RT-qPCR. Data are presented as the mean ± SEM (n = 3).