Novel Application of Nanofluidic Chip Digital PCR for Detection of African Swine Fever Virus

Figures

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  Figure 1. The strategy for the standard plasmid construction. (A) The recombination process of objective gene ASFV VP72 (B646L) and vector pFastBacI. (B) The size of target gene, ASFV B646L, was 1,941 bp on 1% agarose gel electrophoresis. (C) The cleavage sites of the recombination process is EcoR I/Xba I.

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  Figure 2. Identification of target gene. (A) Target nucleotide sequences of MGB probe and primers for cdPCR within the conserved region of ASFV B646L gene. Forward primer was marked in orange, reverse primer was marked in red and the probe was marked in purple. (B) Amplification products were analyzed by agarose gel electrophoresis.

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  Figure 3. The optimum reaction condition for qPCR. (A) Influence of different concentrations PCR primers and probe with use of 108 copies/μl ASFV standard plasmid. (B) The optimum reaction system of qPCR. (C) The operational procedure of qPCR.

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  Figure 4. The standard curve of cdPCR and qPCR. (A) The standard curve of cdPCR. The slope of this linear fitting equation is 1.029 and the Y-intercept is 0.1048. (B) The standard curve of qPCR. The slope of this linear fitting equation is 0.9944 and the Y-intercept is 0.03865.

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  Figure 5. The LOD95% of cdPCR and qPCR assay. Logit analysis plots of the cdPCR (A) and qPCR (B) used in the study show the LOD95%, which are the minimum amounts of DNA detectable with a 95% probably. The amplification curve of cdPCR (C) and qPCR (D) is obtained with 10-fold diluted standard plasmids.

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  Figure 6. The specificity of cdPCR assay. Only the ASFV pFastBacI-p72 standard plasmid was positive, i.e., nucleic acid templates of other pathogens were negative.

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  Figure 7. Repeatability analysis. (A) Trend line comparing coefficient of variation (CV%) for cdPCR and qPCR at different concentration points. The square with the full line stands for CV % of cdPCR and the triangle with the dotted line indicated as the CV% of qPCR. (B) Table shows that average CV% value of cdPCR (9.25%) is lower than that of qPCR (12.67%).

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  Figure 8. Statistical analysis for cdPCR in testing clinical blood samples. Pearson correlation and linear regression analysis showed well-consistency between qPCR approved by OIE (R2 = 0.864) (A) as well as between cdPCR and commercial qPCR kit (R2 = 0.864) (D). The standard deviation of cdPCR is lower than that of qPCR approved by OIE (B) and that of commercial qPCR kit (E). (C) Bland and Altman analyses plots for cdPCR and qPCR approved by OIE demonstrated that 5.797% (4/69) dots were outside the region between 95% lower limit of agreement and 95% upper limit of agreement, and the bias value for this agreement's range was 1,381 copies/ml (P < 0.05) by Graphpad Prism 7.04. (F) Bland and Altman analyses plots for cdPCR and the kit demonstrated that 1.449% (1/69) dots were outside the region between 95% lower limit of agreement and 95% upper limit of agreement with that of the bias value.