Performance of qPCR and ddPCR assays using SP1 and ORF1 primers for detection of <i>Spiroplasma citri</i> in DNA extracted from fruit columella versus leaf tissue collected from 50 trees.

<p>Performance of qPCR and ddPCR assays using SP1 and ORF1 primers for detection of <i>Spiroplasma citri</i> in DNA extracted from fruit columella versus leaf tissue collected from 50 trees.</p

Application of droplet digital PCR for quantitative detection of <i>Spiroplasma citri</i> in comparison with real time PCR

<div><p>Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the <i>Spiroplasma citri</i>, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for <i>S</i>. <i>citri</i> detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect <i>S</i>. <i>citri</i> infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the <i>S</i>. <i>citri</i> spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of <i>S</i>. <i>citri</i> compare to qPCR.</p></div

One dimensional plot of ddPCR assay showing <i>Spiroplasma citri</i> titer in fruit and leaf samples for ORF1 and SP1.

<p>The samples are divided by vertical dotted yellow lines. 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.</p

Diagnostic performance comparison between qPCR and ddPCR assays for citrus stubborn disease diagnosis.

<p>ROC curve indicates better diagnostic performance of ddPCR assays compared to the qPCR assays for differentiating between healthy and <i>S</i>. <i>citri</i> infected leaf samples with significantly (<i>P</i> <0.05) broader AUC 0.867 and 0.967 with (a) SP1 and (b) ORF1 primers, respectively.</p

Influence of citrus leaf petiole and fruit columella extract on quantification of <i>S</i>. <i>citri</i> by ddPCR and qPCR assays for SP1 and ORF1 genes and 1-D plot of ddPCR reactions.

<p>Samples spiked with different quantity of (a) citrus leaf petiole, (b) fruit columella extracts and equal amount of <i>S</i>. <i>citri</i> plasmid DNA. Error bars denote standard error of inhibition between three replicates of each reaction. Asterisks (*) above each bar denote significantly (<i>P</i> <0.05) different measurements, compare to no inhibition control. The non-significant measurements denoted by ns. The 1-D plot show only one of three replication for SP1 and ORF1 with citrus leaf petiole (c & d respectively) and fruit columella extract (e & f respectively).</p

Primer and probe sequences used for the qPCR and ddPCR assay for detection of <i>Spiroplasma citri</i>.

<p>Primer and probe sequences used for the qPCR and ddPCR assay for detection of <i>Spiroplasma citri</i>.</p

Calibration curve of qPCR assays with tenfold serially diluted SP1 and ORF1 Plasmid DNA (1.48E+07 to 1.48E+01 copies/μl and 1.80E+06 to 1.80E+01, respectively) and <i>Spiroplasma citri</i> DNA (10⁻⁹ to 10⁻¹³ for SP1 and 10⁻¹⁴ for ORF1) using SP1 (unbroken line) and ORF1 (broken line) primers.

<p>(a) The Plasmid DNA standard curve slope is -3.2628, equivalent to an efficiency of 102.53% for SP1 and -3.4034, equivalent to an efficiency of 96.71 for ORF1. (b) The <i>S</i>. <i>citri</i> DNA standard curve slope is -3.261, equivalent to an efficiency of 102.61% for SP1 and -3.3534, equivalent to an efficiency of 98.70% for ORF1.</p

Correlation between ddPCR and qPCR measurements for spiralin gene using <i>S</i>. <i>citri</i> infected fruit and leaf samples.

<p>Both the assays were significantly (R² = 0.9596, <i>P</i> <0.0001) correlated.</p

Linear regression of the ddPCR assays.

<p>The Pearson correlation coefficient are shown for the regression curves for <i>Spiroplasma citri</i> DNA from: (a) SP1 plasmid, y = 0.967x—182.68 (R² = 1.0, <i>P</i> <0.0001); and (b) ORF1 plasmid y = 0.971x—26.109 (R² = 1, <i>P</i> <0.0001); (c) SP1 <i>S</i>. <i>citri</i> DNA R² = 0.9998, <i>P</i> <0.0001, and (d) ORF1 <i>S</i>. <i>citri</i> DNA R² = 0.9992, <i>P</i> <0.0001. The inner error bars indicate the Poisson 95% confidence interval (CI) and the outer error bars show the total 95% CI of replicates.</p

Repeatability (Intra-assay variation) and reproducibility (Inter-assay variation) of ddPCR method for detection of <i>Spiroplasma citri</i>.

<p>Repeatability (Intra-assay variation) and reproducibility (Inter-assay variation) of ddPCR method for detection of <i>Spiroplasma citri</i>.</p