Development of duplex crystal digital PCR (dPCR) assay for detection and differentiation of NDRV and MDRV

Figures

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  Fig. 1. Workflow of duplex crystal dPCR: it shows a series of steps for sample collection, reverse transcription, PCR system preparation, addition to the well to form a water-oil mixture system, addition of microdroplet generation and amplification, collection and analysis. Finally, positive samples can be quantified by microtubule backtracking accurately to individual microtubule images.

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  Fig. 2. Optimization of annealing temperature for duplex crystal digital PCR: The bar graph illustrates the number of positive droplets for NDRV (green), MDRV (blue), and total droplets (gray) across a temperature gradient of 56°C, 57°C, 58°C, 59°C, 60°C, and 61°C. These data were used to determine the optimal annealing temperature for simultaneous detection of both viruses.

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  Fig. 3. Optimization of primer and probe concentrations for duplex crystal dPCR: Panels (A) and (B) show the fluorescence amplitude distributions for NDRV (VIC channel) and MDRV (FAM channel), respectively, across 12 primer/probe concentration combinations. Each combination was tested to identify the optimal concentrations for maximum assay sensitivity and specificity. NTC: no template control.

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  Fig. 4. Specificity analysis of the duplex crystal dPCR for detection of NDRV (A) and MDRV (B). The fluorescence amplitudes of NDRV, MDRV, DTMUV, H5, H7, H9, NDV, IBV, EDSV, IBDV and DEV were showed. NTC: no template control.

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  Fig. 5. Sensitivity comparison of duplex crystal dPCR and duplex qPCR(A-B): digital PCR sensitivity test, lane 1-6 is the digital PCR sensitivity dynamic curve of pNDRV and pMDRV with plasmid concentration of 1.5 × 104 to 1.5 × 10-1, lane 7 is NTC; (C-D): Standard curves for duplex qPCR and duplex crystal dPCR, which were prepared by 10-fold serial dilutions of pNDRV and pMDRV standard plasmids from 1.5 × 104 copies /µl to 1.5 × 100 copies /µl.

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  Fig. 6. Spatial distribution and fluorescence imaging of positive microdroplets: The top panels show the specific localization of MDRV (blue) and NDRV (green) positive droplets within a single sapphire chip. The bottom left panel displays a 2D scatter plot of fluorescence amplitudes, distinguishing between NDRV-positive (green), MDRV-positive (blue), and co-infected (cyan) droplets. The right panel provides quantitative data on droplet analysis parameters, including the number of analyzable and positive droplets for each channel.