Fig 1.
Fluorescence amplitude plotted against annealing temperature gradient.
The unbroken pink line is the threshold, above which are positive droplets (blue) with PCR amplification and below which are negative droplets (gray) without any amplification. Eight ddPCR reactions with the same amount of targets are divided by the vertical dotted yellow line. The reactions were across an annealing temperature gradient: 53, 54.1, 56, 58.9, 62.3, 65.1, 67.1 and 68.0°C. The optimal range of annealing temperatures giving the largest difference in fluorescence between negative and positive droplets was between 52°C and 55.4°C.
Fig 2.
Calibration curves of qPCR assays run with positive plasmid DNA (unbroken line) and bacterial suspension (broken line).
Plasmid DNA was tenfold diluted serially from 5.88E+6–5.88E+0 copies/μL. The slope of the plasmid DNA standard curve is –3.3154, equivalent to an efficiency of 100.3% (R2 = 0.9993). The bacterial suspension was 10-fold serially diluted from 1.78E+8–1.78E+1 CFU/μL. The slope of the bacterial suspension calibration curve is –3.0369, equivalent to an efficiency of 113.5% (R2 = 0.9955), indicating PCR inhibition probably caused by the residual medium matrix.
Fig 3.
Linear regression of the ddPCR assay for (a) bacterial suspension and(b) positive plasmid DNA constructed by the same serial dilution series tested with the qPCR assay (see Fig 2).
The estimated Pearson correlation coefficient of the bacterial suspension regression curve (y = 1.9902x—283.81) is 0.995 (R2 = 0.995, P< 0.0001) and that of the plasmid DNA regression curve (y = 24.607x—74.083) is 1.0 (R2 = 1.0, P< 0.0001). Both standards tested by ddPCR exhibited a dynamic range of five orders of magnitude. The vertical axis shows the log10-transformed copy number/μL of the ddPCR reaction mixture. The horizontal ordinate indicates (a) the log10-transformed expected concentration of CFU/μL of the ddPCR reaction mixture or (b) the log10-transformed expected copy number/μL of the ddPCR reaction mixture. The inner error bars indicate the Poisson 95% confidence interval (CI) and the outer error bars show the total 95% CI of replicates.
Fig 4.
Representative 1-D plot of ddPCR reactions.
The ordinate scales indicate fluorescent amplitude. The unbroken pink line is the threshold, above which are positive droplets (blue) containing at least one copy of target DNA and below which are negative droplets (gray) without any target DNA. Six ddPCR reactions with various serially diluted targets are divided by the vertical dotted yellow line. The leftmost ddPCR reaction was saturated by an excess target concentration and the rightmost reaction contains a single copy of the target.
Fig 5.
Inter-assay CV% of the qPCR and ddPCR assays.
Samples B-1 and B-2 are bacterial suspensions in high concentration. Samples P-1 –P-6 are positive plasmid DNA; among them, P-1 and P-2 are of high concentration and P-3 –P-6 are of low concentration. Histograms indicate the average copy number of each sample in log 10 scale. Lines show the trend of variation of CV of the qPCR and ddPCR assays with repeated tests of diverse sample concentrations. The ddPCR assay is more precise compared to the qPCR assay for quantification of Xcc, especially for low target concentrations (numerical data supporting Fig 5 are given in S3 Table).
Fig 6.
Influence of samples spiked with serial dilutions of an inhibitor on quantification by the qPCR and ddPCR assays.
The ddPCR assay exhibits superior tolerance to citrus extracts and Cu2+ compared to the qPCR assay. A 100% inhibition represents a completely suppressed reaction with no positive signal and a 0% inhibition indicates no suppression with the same target concentration as the no-inhibition control. (a) Citrus extract, (b) CuSO4 and (d) cupravit had an enhancing effect on both the ddPCR and qPCR assays at low levels of spiking. (d) A 1-D plot of ddPCR reactions spiked with different amounts of citrus extracts. Fluorescent signals of both positive and negative droplets were increased with increasing amounts of citrus extracts.
Fig 7.
Correction of ddPCR and qPCR measurements.
Measurements of citrus samples by ddPCR and qPCR assays were significantly correlated (Pearson r = 0.8633, P<0.0001). Solid line indicates fitting curve; dashed line represents 95% CI.
Table 1.
Performance of ddPCR and qPCR assay for detection of Xcc in symptomatic and asymptomatic infected citrus samples.
Table 2.
Correlation of infected citrus samples between ddPCR and qPCR assay.
Fig 8.
Diagnostic performance of Xcc quantification by the ddPCR and qPCR assays.
ROC and AUC were used to estimate the sensitivity and specificity of each method. AUC for each assay indicated its performance in differentiating CBC-infected trees from the control cohort in terms of sensitivity and specificity. (a) ROC curves of ddPCR and qPCR assays. The ddPCR assay indicates better diagnostic performance compared to the qPCR assay. (b) T-test shows both ddPCR and qPCR assays can discriminate significantly between healthy control and Xcc-infected citrus samples.