One‐Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point‐of‐Care Testing

Figures

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  Figure 1. Construction of a CRISPR/Cas13a Detection System: HEV RNA. Data are representative of at least three independent experiments. (A) Schematic design of crRNAs, RAA primers, and PCR primers. The designed region for this sequence (5′ to 3′) is the ORF3 region of the HEV (figure created via FigDraw). (B) Screening of RAA primers. HEV‐positive plasmids (103 copies/μL) were amplified by RAA, and the amplified products were subjected to DNA agarose gel electrophoresis. ImageJ was used to analyze the bands in grayscale and GraphPad Prism 8 software was used to present the analysis results in a heat map. (C) Screening of crRNA. The selected RAA primers amplified the HEV‐positive plasmid (103 copies/μL) and were used as templates for the CRISPR/Cas13a fluorescence assay to validate the crRNAs. (D) Conservation analysis of selected crRNA and RAA primers by WebLogo.

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  Figure 2. Performance of the CRISPR/Cas13a Detection System: HEV RNA. Data are representative of at least three independent experiments (negative: healthy individuals). (A) Alignment of the conserved HEV regions with other common clinical viruses (HAV, HBV, HCV, HDV, and HIV). The RAA primer and crRNA regions are highlighted in purple and yellow, respectively. (B) Specificity assessment of CRISPR/Cas13a detection system using clinical samples (HAV, HBV, HCV, HDV, and HIV). (C) Sensitivity assessment of the CRISPR/Cas13a detection system using plasmids (gradient concentration dilutions of HEV plasmids). (D) Assessment of the repeatability of the CRISPR/Cas13a detection system using plasmids (Std. dev., standard deviation).

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  Figure 3. Optimization of the CRISPR/Cas13a Detection System: HEV RNA. Data are representative of at least three independent experiments (*< 0.05, NC: negative control). (A) Optimization of RAA template volume (1, 5, and 10 μL), reaction time (5, 15, 30, 45, and 60 min), and reaction temperature (22°C, 32°C, 42°C, and 52°C). (B) Optimization of PCR template volume (1, 5, and 10 μL) and number of reaction cycles (25, 35, and 45 cycles). (C) Comparison of sample storage durations (0, 1, 5, 10, and 15 days). (D) Optimization of CRISPR reaction temperature (25°C, 31°C, 37°C, and 43°C) and reaction time (10, 20, and 30 min). K1, K2, and K3 represent the slopes of the fluorescence curves at the time points of 10, 20, and 30 min of the CRISPR reaction, respectively, reflecting the amplification efficiency. (E) Comparison of results from plasma and serum samples.

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  Figure 4. Assays of HEV RNA‐based CRISPR/Cas13a System: Samples from HEV‐infected patients. Data are representative of at least three independent experiments. (A) Endpoint fluorescence values of 154 HEV‐infected patients were determined using the RT‐PCR + CRISPR/Cas13a assay. The first 77 cases are illustrated in the figure, and the others are listed in Supporting Information: Table S5. (negative: healthy individuals, red line: positive detection lines). (B) The endpoint fluorescence values of the 154 HEV‐infected cases were determined using the RT‐RAA + CRISPR/Cas13a assay. The first 77 cases are illustrated in the figure, and the others are listed in Supporting Information: Table S5 (negative: healthy individuals, red line: positive detection lines). (C) The results of RT‐PCR + CRISPR/Cas13a strips assay in HEV‐infected patients with different viral loads (100–104 copies/μL, NC: healthy individuals). (D) The results of RT‐RAA + CRISPR/Cas13a strips assay in HEV‐infected patients with different viral loads (100–104 copies/μL, NC: healthy individuals). (E) Analysis of sensitivity, specificity, PPA, and NPA in the CRISPR/Cas13a assay for the detection of HEV RNA in HEV‐infected patients.

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  Figure 5. Assays of HEV RNA based CRISPR/Cas13a System in HEV‐infected animals. Data are representative of at least three independent experiments. (A) Details of 104 HEV‐infected animal samples, including species, sample type, number, and HEV genotyping. (B) Endpoint fluorescence values of 104 HEV‐infected animal samples using the RT‐PCR + CRISPR/Cas13a and RT‐RAA + CRISPR/Cas13a assays. GraphPad Prism 8 software was used to present the results on a heat map (negative: noninfected samples). (C) Results of RT‐PCR + CRISPR/Cas13a and RT‐RAA + CRISPR/Cas13a strips assay in HEV‐infected animal samples with different viral loads (100–104 copies/μL, NC: noninfected samples). (D) Analysis of sensitivity, specificity, PPA, and NPA in the CRISPR/Cas13a assay for the detection of HEV RNA in HEV‐infected animal samples.

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  Figure 6. Application of sample rapid lysate for HEV RNA‐based CRISPR/Cas13a System. Data are representative of at least three independent experiments. (A) Schematic diagram of the process of HEV RNA detection using sample rapid lysate (Figure Draw). (B) Results of RT‐PCR + CRISPR/Cas13a and RT‐RAA + CRISPR/Cas13a strips assay for rapidly processed samples from HEV‐infected animals with different viral loads and HEV‐infected patients (100–104 copies/μL, NC: noninfected samples). (C) Comparison of rapid sample lysis and RNA extraction in terms of sensitivity (NC: noninfected samples).

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  Figure 7. One‐Pot assays of HEV RNA‐based CRISPR/Cas13a System in HEV‐infected animals. Data are representative of at least three independent experiments. (A) Schematic diagram of the operation of the One‐Pot device for the detection of HEV RNA, with detailed procedures described in the Supporting Information (MicroDiag Biomedicine). (B) Flowchart showing the operation of the One‐Pot method for HEV virus detection. (C) Results of One‐Pot assay for HEV‐infected patients and animals with different viral loads (100–104 copies/μL, NC: noninfected samples). (D) The comparative analysis of positive/negative detection rate of HEV RNA in HEV‐infected patient and animal samples. (E) Comparison and percentage analysis of RNA extraction assay time, sample rapid lysis assay time, and One‐Pot assay time. (F) The comparison between routine CRISPR assay and One‐Pot assay.