Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus

Figures

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  Figure 1. Regression equation, correlation coefficient (R2), and amplification efficiency of standard curves for BEV, BCoV, and BRV qPCR assays. (A) Standard curve of BEV with a primer concentration of 200 nM and a probe concentration of 400 nM. (B) Standard curve of BCoV with a primer concentration of 300 nM and a probe concentration of 150 nM. (C) Standard curve of BRV with a primer concentration of 300 nM and a probe concentration of 500 nM. Horizontal coordinate: number of copies (A,B) or logarithm of the number of copies (C); vertical coordinate: cycle threshold (A,B); equation: standard curve equation; R2: correlation coefficient; E: amplification efficiency.

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  Figure 2. Sensitivity of the ddPCR assay. The standards of BEV, BCoV, and BRV were diluted to 1 × 109–1 × 102 copies/μL in a 10-fold gradient and assayed by the constructed ddPCR method. (A) Sensitivity assay of the BEV ddPCR method. (B) Sensitivity assay of the BCoV ddPCR method. (C) Sensitivity assay of the BRV ddPCR method. Horizontal coordinate: plasmid copy number; vertical coordinate: fluorescence amplitude. Ch1 amplitude: FAM, respectively.

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  Figure 3. Specificity detection results of ddPCR assays. Specificity detection of BEV, BCoV, BRV, and H2O using established ddPCR assays. (A) Specificity detection of BEV using ddPCR. (B) Specificity detection of BCoV using ddPCR. (C) Specificity detection for BRV ddPCR. Horizontal coordinate: set experimental group; vertical coordinate: fluorescence amplitude. Ch1 amplitude: FAM, respectively.

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  Figure 4. Establishment of the BCoV and BRV dual ddPCR detection systems. 2D plot of BCoV and BRV dual ddPCR results; blue: BCoV-positive droplets; green: BRV-positive droplets; orange: BCoV and BRV double-positive droplets; gray: negative. The horizontal and vertical coordinates are the fluorescence amplitudes of HEX and FAM, respectively. Channel 1 amplitude: FAM, respectively; channel 2 amplitude: HEX, respectively.

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  Figure 5. Establishment and optimization results of multiplex ddPCR assays for BEV, BCoV, and BRV. Multiplex ddPCR results for probe concentrations: BCoV FAM 200 nM, BRV FAM 100 nM, HEX 100 nM, and BEV HEX 200 nM; at an annealing temperature of 58°C and a primer concentration of 500 nM. Red: BCoV-positive droplets; yellow: BRV-positive droplets; purple: BEV-positive droplets; orange red: BRV and BEV double-positive droplets; brown: BCoV and BRV double-positive droplets; blue: BCoV and BEV double-positive droplets; orange: BEV, BCoV, and BRV triple-positive droplets; gray: negative. The horizontal and vertical coordinates are the fluorescence amplitudes of HEX and FAM, respectively. Channel 1 amplitude: FAM, respectively; channel 2 amplitude: HEX, respectively.

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  Figure 6. Results of clinical samples tested by duplex and multiplex ddPCR. (A) Results of co-infection in clinical samples tested by duplex ddPCR for BCoV and BRV; blue: BRV-positive droplets; green: BCoV-positive droplets; red: BRV and BCoV double-positive droplets. (B,C) Results of clinical tests by multiplex ddPCR for BCoV, BEV, and BRV. (B) Test results of 68 samples collected in Bole, Xinjiang, China. (C) Test results of 93 samples collected in Ürümqi County, Xinjiang, China. Red: BCoV-positive droplets; yellow: BRV-positive droplets; purple: BEV-positive droplets; orange: BRV and BEV double-positive droplets; blue: BCoV and BEV double-positive droplets; gray: negative. The horizontal and vertical coordinates are the fluorescence amplitudes of HEX and FAM, respectively. Channel 1 amplitude: FAM, respectively; channel 2 amplitude: HEX, respectively.