Table 1.
Primer and probe sequences used for the qPCR and ddPCR assay for detection of Spiroplasma citri.
Fig 1.
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 Spiroplasma citri DNA (10−9 to 10−13 for SP1 and 10−14 for ORF1) using SP1 (unbroken line) and ORF1 (broken line) primers.
(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 S. citri 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.
Fig 2.
Thermal gradient PCR for optimizing annealing temperature for (a) SP1 (b) ORF1 of Spiroplasma citri. Eight ddPCR reactions with an annealing temperature gradient ranged from 48°C to 63°C are divided by vertical dotted yellow lines. The pink line is the threshold, above which are positive droplets (blue) and below that are negative droplets (gray) without any target DNA.
Fig 3.
Linear regression of the ddPCR assays.
The Pearson correlation coefficient are shown for the regression curves for Spiroplasma citri DNA from: (a) SP1 plasmid, y = 0.967x—182.68 (R2 = 1.0, P <0.0001); and (b) ORF1 plasmid y = 0.971x—26.109 (R2 = 1, P <0.0001); (c) SP1 S. citri DNA R2 = 0.9998, P <0.0001, and (d) ORF1 S. citri DNA R2 = 0.9992, P <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.
Table 2.
Performance of qPCR and ddPCR assays using SP1 and ORF1 primers for detection of Spiroplasma citri in DNA extracted from fruit columella versus leaf tissue collected from 50 trees.
Fig 4.
One dimensional plot of ddPCR assay showing Spiroplasma citri titer in fruit and leaf samples for ORF1 and SP1.
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.
Fig 5.
Diagnostic performance comparison between qPCR and ddPCR assays for citrus stubborn disease diagnosis.
ROC curve indicates better diagnostic performance of ddPCR assays compared to the qPCR assays for differentiating between healthy and S. citri infected leaf samples with significantly (P <0.05) broader AUC 0.867 and 0.967 with (a) SP1 and (b) ORF1 primers, respectively.
Fig 6.
Correlation between ddPCR and qPCR measurements for spiralin gene using S. citri infected fruit and leaf samples.
Both the assays were significantly (R2 = 0.9596, P <0.0001) correlated.
Fig 7.
Influence of citrus leaf petiole and fruit columella extract on quantification of S. citri by ddPCR and qPCR assays for SP1 and ORF1 genes and 1-D plot of ddPCR reactions.
Samples spiked with different quantity of (a) citrus leaf petiole, (b) fruit columella extracts and equal amount of S. citri plasmid DNA. Error bars denote standard error of inhibition between three replicates of each reaction. Asterisks (*) above each bar denote significantly (P <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).
Table 3.
Repeatability (Intra-assay variation) and reproducibility (Inter-assay variation) of ddPCR method for detection of Spiroplasma citri.