Fluorescence amplitude plotted against annealing temperature gradient.

<p>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.</p

Correction of ddPCR and qPCR measurements.

<p>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.</p

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).

<p>The estimated Pearson correlation coefficient of the bacterial suspension regression curve (y = 1.9902x—283.81) is 0.995 (<i>R</i>² = 0.995, <i>P</i>< 0.0001) and that of the plasmid DNA regression curve (y = 24.607x—74.083) is 1.0 (<i>R</i>² = 1.0, <i>P</i>< 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.</p

Influence of samples spiked with serial dilutions of an inhibitor on quantification by the qPCR and ddPCR assays.

<p>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.</p

Diagnostic performance of <i>Xcc</i> quantification by the ddPCR and qPCR assays.

<p>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 <i>Xcc</i>-infected citrus samples.</p

Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of <i>Xanthomonas citri</i> Subsp. <i>citri</i>

<div><p>Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of <i>Xanthomonas citri</i> subsp. <i>citri</i>, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible <i>Citrus</i> species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; <i>P</i><0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of <i>X</i>. <i>citri</i> subsp. <i>citri</i> with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.</p></div

Correlation of infected citrus samples between ddPCR and qPCR assay.

<p>Correlation of infected citrus samples between ddPCR and qPCR assay.</p

Performance of ddPCR and qPCR assay for detection of <i>Xcc</i> in symptomatic and asymptomatic infected citrus samples.

<p>Performance of ddPCR and qPCR assay for detection of <i>Xcc</i> in symptomatic and asymptomatic infected citrus samples.</p

Calibration curves of qPCR assays run with positive plasmid DNA (unbroken line) and bacterial suspension (broken line).

<p>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% (<i>R</i>² = 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% (<i>R</i>² = 0.9955), indicating PCR inhibition probably caused by the residual medium matrix.</p

Inter-assay CV% of the qPCR and ddPCR assays.

<p>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 <i>Xcc</i>, especially for low target concentrations (numerical data supporting Fig 5 are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159004#pone.0159004.s004" target="_blank">S3 Table</a>).</p