Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(
Keywords: citrus bacterial canker; Citrus lemon; Citrus sinensis; diagnostics; DNA quality
Citrus bacterial canker (CBC), caused by Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), is one of the most serious diseases of several host species of the Rutaceae family, among which is included Citrus, Fortunella, Poncirus and their hybrids [[
Moreover, Annex IV, part A, Section 1, of the Directive 2000/29/EC [[
The purpose of this intra-laboratory study was to compare several DEMs to assess their possible influence on the detection of Xcc in association to several plant matrices. This evaluation was performed following these approaches: (i) a comparison by real-time PCR, Cubero and Graham (2005) [[
The evaluation of homogeneity was performed for samples SET1 and SET2 (see material methods for details). The lower DNA (1 fg/reaction, SET1) and bacterial (10
The optimal annealing/elongation temperature was identified at 58 °C, either with 5 or 8 µL of DNA per reaction. At this temperature, the three following parameters were satisfied: (i) positive droplets with the highest fluorescence amplitude; (ii) a better separation between positive/negative droplets; and (iii) less rain (i.e., droplets ranging between the positive and negative ones) (data not shown). The optimal selected volume was 8 µL, as the increase in DNA volume resulted in a higher number of positive droplets with respect to 5 µL. The detection was successful testing the SET1 and SET2 samples, for all DEM/plant matrices combinations (Table 1 and Table 2) and samples of bacterial DNA from strain Xcc NCPPB 3234 (Table 3). Figure 1 shows the results obtained on samples of SET2 from 10
The raw data of real-time PCR and ddPCR results for SET1 are reported in Table 1, according to the DEM and the different plant matrices. The Table 3 shows the result of the standard curve obtained by real-time PCR on bacterial DNA from a pure culture. The amplification efficiency of DNA from the Xcc pure culture showed an adequate value (E = 98.17%); this was comparable to the efficiency shown by most of the combinations of DEM/plant matrices of SET1, which give acceptable values between 90–105%. The exceptions were orange fruit/Mericon (E = 88%), orange fruit/Quick (E = 89%), and lemon leaf/Ctab (E = 111%), whose E values slightly deviate from the acceptable values.
The comparison between the standard curves of bacterial DNA and SET1 (Figure 2), highlight an overlapping for all plant species/matrices, that was particularly evident at the higher bacterial concentration. On the other hand, at lower bacterial concentration some differences were observed due to a higher difference among the Ct (in particular at 10 fg/real-time PCR reaction) (Figure 2). This was predictable at low target concentrations due to the greater variability between technical replicates.
The range of Ct values for SET1 samples and for bacterial DNA are graphically represented in Figure 3. The range of Ct were not particularly variable among the different combinations of DEM and plant matrices, showing comparable results with bacterial DNA.
The Bland–Altman plot reported in Figure 4 shows the percentage variation of the average of sample concentrations of SET1 (represented by the black line of Figure 4) with respect to a null variation (represented by the red line in Figure 4). The percentage variation indicates whether the method underestimates or overestimates the overall Ct values. A good performance of the method implies that the black line approaches the red one, indicating a low variability. All DEM/plant matrices combinations were within the 95% of confidence interval included in the graph by black dashed lines. In particular, the Plant DEM gave the best performance with all plant matrices, except for the orange fruit.
In order to assess the effect of inhibitors related to the different DEM/plant matrices combination, the SET1 was analyzed by ddPCR, a technique known to be less affected by inhibitors with respect to real-time PCR [[
The analysis by real-time PCR [[
The results of the real-time PCR and ddPCR on SET2 samples are reported in Table 2. The homogeneity showed an acceptable range of variation within bacterial concentrations from 10
The analytical sensitivity (ASE) was evaluated on SET2 considering different DEM/plant matrices. In particular, ASE was estimated for real-time PCR [[
The diagnosis of plant pathogenic bacteria represents one of the more important challenges aimed to prevent the dissemination of world-wide diseases. The adoption of a reliable detection method is crucial to correctly determine the presence of a pest before its establishment in a pest-free area and/or its spread. To standardize the use of diagnostics, in plant pathology it is suggested that their validation follow UNI EN ISO 16140 [[
Furthermore, the EPPO protocols for diagnostics generally do not require DNA quality control as a mandatory step, therefore the DNA is not subjected to control before performing a test. Neither of the majority of diagnostic methods for phytopathogenic bacteria foresee the use of internal DNA amplification controls in each sample (i.e., the mitochondrial cytochrome oxidase, cox gene; the region coding for the 5.8S rRNA, 5.8S rDNA gene), rather, additional samples (PIC, positive isolation) are provided to guarantee the success of the DNA extraction. Moreover, many commercial kits based on standardized procedures have been developed which can ensure the successful outcome of the analysis, and today many kits based on different principles or systems (i.e., columns, beads, manual, automatized) are available. However, the choice of the most appropriate extraction kit is related to various factors ranging from technical aspects (e.g., type of plant matrix) to the economic availability of the laboratory. Taking this last aspect into account, still today many designated laboratories that carry out official analyses use home-made DEM based on the use of Ctab, as for the detection of Xylella fastidiosa [[
Xcc, along with Xca, are listed by the EU as a priority pest. Both pathogens were included in the EURL-BAC work program in 2019–2020 with the aim of standardizing, inventorying test protocols, developing and validating detection tests, and providing reference material to standardize detection/identification and facilitate disclosure to national reference laboratories (NRLs). In the case of Xcc, a TPS validation was recently organized within the Valitest Project (XCC-1) (GA n°773139), aimed at validating several diagnostic methods for Xcc detection. The obtained results provided useful indications on the validated diagnostic methods, highlighting their performance. An adequate availability of diagnostic tools for the detection of Xcc emerged from the report by the Valitest Project (XCC-1) [[
At the lowest concentrations (10 fg/PCR reaction-SET1 and 10 cfu/mL-SET2), most of the samples did not show an acceptable homogeneity or a reliable result by real-time PCR, which resulted in variable results, frequently non-consistent, depending on the plant matrices /methods combination. This evidence shows that at this condition the DNA extraction methodology can affect the reliability of the detection tests. The evaluation of the ASE of SET2 (spiked samples) by the real-time PCR of Cubero and Graham [[
The ddPCR is notoriously less affected by inhibitors [[
The overall results indicate that the use of commercial kits is adequate for a reliable DNA extraction for all the tested plant matrices at a high concentration of the target. Particular attention must be paid to the choice of the most appropriate extraction method when processing samples with an expected low bacterial load, as in the case of asymptomatic samples. In this case, the use of Quick and Plant seems the most reliable DEM. An adequate choice of the DNA extraction procedure allows to improve the sensitivity of the method by one decimal factor. In this regard, the adaptation of a ddPCR system according to the Cubero and Graham test [[
Finally, in our own laboratory conditions late Ct values occurred by real-time PCR on healthy plant matrices, with a high incidence for Ctab and low for Quick, supporting the argument that Quickis the most suitable method for DNA extraction. A ddPCR was performed to evaluate if the late Ct were due to background contaminations and the obtained negative results confirmed that these late Ct values were false signals. We suggest establishing a cut-off for real-time PCR [[
Although molecular diagnostic tests allow for reaching the levels of sensitivity adequate for the detection of Xcc in low concentrations, the validation data produced in this study show that the wrong choice of the extraction method can affect the outcome of official analyses, especially in critical samples (i.e., low concentration of the pathogen, pest-free areas). Conversely, applying reliable diagnostics will help protect pest-free areas from accidental introduction of this quarantine pathogen.
The strain NCPPB 3234 (Xcc) was used for the samples' preparation. This strain, belonging to the National Collection of Plant Pathogenic Bacteria (NCPPB Fera Science Ltd - UK), was isolated in 1982 in Japan from Citrus spp. and belong to pathotype A. The lyophilized strain was grown in NGA (nutrient agar added with 0.25% d-glucose) at 28 °C. Bacterial suspensions were prepared in phosphate saline buffer (PBS 10 mM, pH = 7.2) and spectrophotometrically (DS-11 Fx+, Spectrophotometer-Fluorometer Denovix Inc., Wilmington, DE, USA) measured at a concentration corresponding to about 10
Plant material of the host plants was collected from plants in open fields (leaves) or bought in trade markets (fruits) located in the Latium and Campania Regions (Italy) (Table 7).
The plant extract was prepared as reported in [[
Samples prepared for the intra-laboratory study were grouped in SET1 and SET2. SET1 was prepared by amending the DNA of the plant extract (orange fruit, lemon leaves and lemon fruit) obtained with the four different DEM, with a ten-fold dilution of Xcc DNA (from 1 ng to 1 fg/real-time PCR or ddPCR reaction). SET2 consisted of spiked samples prepared by adding bacterial suspensions at known concentrations (CFU/mL) to healthy plant extracts (orange fruit, lemon leaves and lemon fruit). The obtained spiked samples (bacterial suspension + plant extract) were extracted by DEM. The bacterial suspension was added in the different plant matrices at a final concentration of Xcc 10
Total bacterial DNA was extracted from 1 mL of bacterial cultures of Xcc NCPPB 3234 using Gentra Puregene Yeast/Bact. Kit (Qiagen, Venlo, The Netherlands). The DNA concentration was evaluated by Qubit (dsDNA HS Assay kit, Invitrogen, Waltham, MA, USA). Plant DNA extraction was performed following the manufacturers' instructions of the commercial kits, i.e., DNeasy Plant Mini kit (Qiagen, Venlo, The Netherlands), DNeasy Mericon TM Food Kit (Qiagen, Venlo, The Netherlands), and QuickPick™ SML Plant DNA kit (QRET Technologies Ltd., Turku, Finland), manual version, from 500 mL of plant extract. The Ctab DEM was performed as described for Xylella fastidiosa [[
The Cubero and Graham [[
The ddPCR was developed adapting the method of Cubero and Graham [[
Following Biorad ddPCR
The samples of SET1 were run in one biological replicate. The samples of SET 2 were run in three biological replicates (for each plant matrix) (n = 3). In particular, there were tested samples from 100 pg to 10 fg/ddPCR for SET1 and samples from 10
The homogeneity was evaluated for samples of SET1 and SET2 by considering the (SD) of Ct values obtained for each sample by real-time PCR. The replicates were considered sufficiently homogeneous with SD < 1.
The following performance criteria, diagnostic specificity, diagnostic sensitivity, accuracy, repeatability, and analytical sensitivity were evaluated on samples of SET2 according to the EPPO PM 7/98 (
The sample mean ± the standard deviation (SD) was calculated for all data of the real-time PCR and ddPCR. Excel Microsoft 365® was employed as the statistic package. The analysis of qualitative data was performed by the calculation of performance criteria as reported in the PM 7/122 (
Graph: Figure 1 Graphical representation of the results obtained on SET2 samples (from 103 to 10 cfu/mL) by ddPCR. Blue dots indicate positive droplets with amplification while black dots indicate negative dots without amplification. Purple line indicates the threshold separating positive from negative dots; h.p.m. = healthy plant matrix.
Graph: Figure 2 Standard curves represented as linear regression of the quantitation cycle (Ct) values (Y axis) of SET1 samples versus the DNA bacterial concentration of Xcc (X axis); the r2 values are reported in Table 1 and Table 3. Different colors indicate the standard curves generated using different DEM (Plant = DNeasy Plant Mini kit, Mericon = DNeasy Mericon Food Kit, Ctab = CTAB extraction method, Quick = QuickPick SML Plant DNA kit) in comparison with the standard curve of Xcc bacterial DNA (Citri STD). DEM = DNA extraction method.
Graph: Figure 3 Range of Ct values (y-axis) of SET1 considering the different combination of DEM/plant matrices (x-axis) in comparison with bacterial DNA. (Plant = DNeasy Plant Mini kit, Mericon = DNeasy Mericon Food Kit, Ctab = CTAB extraction method, Quick = QuickPick SML Plant DNA kit) Lem = lemon, Ora = Orange; LF = leaf; FR = fruit. DEM = DNA extraction method.
Graph: Figure 4 Bland–Altman plot: X axis reports the Ct of Xcc bacterial DNA standard curves, Y axis reports the percentage variation of the DEM with respect to the Xcc bacterial DNA standard. The black horizontal line represents the average (considering all concentrations together) of the percentage variation and indicates whether the method underestimates or overestimates the overall Ct. The red line represents the zero-percentage variation. The black dashed lines define the 95% confidence interval with respect to the black line. DEM = DNA extraction method.
Table 1 Results obtained by real-time PCR and droplet digital PCR in samples of SET1. Samples of SET1 were prepared on different plant matrices (lemon leaves and fruits, orange fruit), extracted with the four different DEM (Plant = DNeasy Plant Mini kit, Mericon = DNeasy Mericon Food Kit, CTAB = CTAB-based method, Quick = QuickPick SML Plant DNA kit). For real-time PCR are reported the average of Ct values and the number of positive wells on the total assessed; for ddPCR are reported copies/mL and positive droplets. Grey boxes indicate inconsistent results between the replicates (ΔCt less than 3 cycles between ten-decimal dilutions) and SD > 1. The efficiency, r
ng of Bacterial DNA per Reaction Plant Real Time PCR Cubero et al. (2005) ddPCR Adapted from Cubero et al. (2005) Lemon Fruit Orange Fruit Lemon Leaf Lemon Fruit Orange Fruit Lemon Leaf N° pos Wells Ct Mean St.dev N° pos Wells Ct Mean St.dev N° pos Wells Ct Mean St.dev Copies/μL Positive Droplets Copies/μL Positive Droplets Copies/μL Positive Droplets 1 ng 3/3 17.10 0.19 3/3 16.27 0.11 3/3 17.03 0.36 - - - - - - 100 pg 3/3 20.29 0.08 3/3 19.57 0.05 3/3 20.66 0.22 - - - - - - 10 pg 3/3 23.81 0.66 3/3 22.31 0.10 3/3 24.2 0.05 164 1190 342 2407 152 1635 1 pg 3/3 27.33 0.22 3/3 25.82 3/3 27.65 0.33 15.2 157 43 467 14.8 169 100 fg 3/3 30.20 3/3 28.64 0.24 3/3 31.04 0.49 1.1 7 4.7 54 3.2 33 10 fg 3/3 35.30 0.92 3/3 32.99 0.73 3/3 0.42 4 0.53 6 0.34 3 1 fg 3/3 35.92 3/3 36.7 0 0 0.18 2 0.09 1 Std curve parameters E: 93.60%; r2: 0.991; E: 103.90%; r2: 0.996; E: 101.80%; r2: 0.994; 1 ng 3/3 16.9 0.14 3/3 16.86 0.19 3/3 16.82 0.11 - - - - - - 100 pg 3/3 20.32 0.11 3/3 20.46 0.07 3/3 20.14 0.33 - - - - - - 10 pg 3/3 23.74 0.41 3/3 24.14 0.04 3/3 23.6 0.12 60.5 589 138 1073 114 1118 1 pg 3/3 27.30 0.09 3/3 27.58 0.66 3/3 26.83 0.36 11 114 15.1 111 7.1 80 100 fg 3/3 30.88 0.31 3/3 31.7 0.87 3/3 30.55 0.64 1.1 12 1.6 14 0.51 5 10 fg 3/3 35.64 0.40 3/3 36.22 3/3 34.35 0 0 0 0 1 fg 3/3 3/3 0 0 0 0 Std curve parameters E: 99.70%; r2: 0.975; E: 88.10%; r2: 0.997; E: 93.20%; r2: 0.99; 1 ng 3/3 17.27 0.16 3/3 17.17 0.09 3/3 16.98 0.36 - - - - - - 100 pg 3/3 20.78 0.17 3/3 20.33 0.16 3/3 20.33 0.22 - - - - - - 10 pg 3/3 24.16 0.74 3/3 24.18 0.12 3/3 23.67 0.05 159 1402 145 1522 214 2257 1 pg 3/3 27.84 0.34 3/3 27.38 0.41 3/3 27.03 0.33 17.5 170 19.4 201 27.4 298 100 fg 3/3 31.20 0.33 3/3 31.02 0.30 3/3 30.18 0.49 1 11 1.4 13 1.6 18 10 fg 3/3 34.93 3/3 34.66 3/3 32.59 0.1 0.11 1 0.54 6 1 fg 1/3 3/3 0 0 0.38 4 Std curve parameters E: 92.20%; r2: 0.996; E: 92.50%; r2: 0.994; E: 111.4%; r2: 0.993; 1 ng 3/3 17.41 0.36 3/3 17.18 0.09 3/3 17.15 0.15 - - - - - - 100 pg 3/3 21.40 0.32 3/3 20.35 0.35 3/3 21.2 0.16 - - - - - - 10 pg 3/3 24.42 0.22 3/3 24.29 0.24 3/3 24.3 0.17 125 1197 147 1328 127 1163 1 pg 3/3 27.40 0.13 3/3 27.43 0.01 3/3 27.59 0.07 2.1 112 17.8 177 14.4 149 100 fg 3/3 31.54 0.59 3/3 31.68 0.85 3/3 31.23 0.48 0.24 23 1.9 19 2.5 28 10 fg 3/3 34 0.86 3/3 36.21 0.98 3/3 33.94 0.17 3 0 0.19 2 1 fg 2/3 0.21 0 2 0 0 Std curve parameters E: 93.50%; r2: 0.987; E: 89.30%; r2: 0.997; E: 100.40%; r2: 0.99;
Table 2 Results obtained by real-time PCR and ddPCR in spiked samples of lemon fruit and leaves and orange fruit of SET2 extracted with the four different DEM (Plant = DNeasy Plant Mini kit, Mericon = DNeasy Mericon Food Kit, CTAB = CTAB-based method, Quick = QuickPick SML Plant DNA kit). For real-time PCR are reported the Ct values mean ± SD and the number of positive replicates/number of replicates analyzed; for ddPCR are reported the positive droplets mean, the copies/μL mean ± SD and the number of positive replicates/number of replicates analyzed. Grey boxes indicate inconsistent results between the replicates (ΔCt less than 3 cycles between ten-decimal dilutions) and SD > 1. The number of total droplets in dd-PCR was >10.000 for all data considered for the analyses. Not tested samples are indicate as follows (-). DEM = DNA extraction method.
Real Time PCR Cubero et al. (2005) ddPCR Adapted from Cubero et al. (2005) Lemon Fruit Orange Fruit Lemon Leaf Lemon Fruit Orange Fruit Lemon Leaf Bacterial CFU per Reaction Plant N° pos Wells Ct Mean St.dev N° pos Wells CT MEAN St.dev N° pos Wells Ct Mean St.dev N° pos Wells Positive Droplets Mean Copies/μL Mean St.dev N° pos Wells Positive droplets Mean Copies/μL Mean St.dev N° pos Wells Positive Droplets Mean Copies/μL Mean St.dev 107 9/9 16.40 0.64 9/9 18.4 0.35 9/9 18.5 0.71 - - - - - - - - - - - - 106 9/9 19.93 0.16 9/9 20.3 0.18 9/9 22.9 - - - - - - - - - - - - 105 9/9 23.06 0.46 9/9 24.2 1 9/9 25.5 0.66 - - - - - - - - - - - - 104 9/9 26.48 0.26 9/9 27.2 0.3 9/9 29.7 1.79 - - - - - - - - - - - - 103 9/9 29.77 0.20 9/9 29.5 0.49 9/9 31.9 0.94 3/3 128 10.3 1.15 3/3 149 12.8 2.51 3/3 20.7 2.3 2.51 102 9/9 32.96 0.48 33.2 9/9 34.1 0.56 3/3 16.3 1.22 0.68 3/3 13.7 1.18 0.90 3/3 4.66 0.45 0.32 101 36.10 9/9 34.8 36.52 0.08 0.08 2/3 5.66 0.45 0.64 2/3 2.66 0.19 0.12 Bacterial CFU per reaction Mericon N° pos wells Ct mean St.dev N° pos wells Ct mean St.dev N° pos wells Ct mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev 107 9/9 17.29 0.16 9/9 19.3 0.19 9/9 17.5 0.78 - - - - - - - - - - - - 106 9/9 19.92 0.29 9/9 21.7 0.8 9/9 20.9 0.33 - - - - - - - - - - - - 105 9/9 22.56 0.35 9/9 24.7 0.28 9/9 24 0.2 - - - - - - - - - - - - 104 9/9 26.09 0.36 9/9 27.9 0.51 9/9 28.2 0.8 - - - - - - - - - - - - 103 9/9 28.95 0.41 9/9 30.2 0.32 9/9 31.6 0.74 2/3 177 11.4 6.57 3/3 206 18.9 2.90 3/3 22 2.13 0.76 102 9/9 32.08 9/9 33.1 9/9 33.3 3/3 33 2.75 2.92 3/3 22.7 2.06 1.19 2/3 5.5 0.53 0.24 101 9/9 36.1 3/3 11.7 1.1 0.34 2/3 0.13 0.05 3.66 0.36 0.63 Bacterial CFU per reaction Ctab N° pos wells Ct mean St.dev N° pos wells Ct mean St.dev N°pos wells Ct mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev 107 9/9 16.53 0.56 9/9 20.1 0.23 9/9 18.3 0.36 - - - - - - - - - - - - 106 9/9 20.79 0.17 9/9 22.2 0.74 9/9 22.3 0.16 - - - - - - - - - - - - 105 9/9 23.850 0.52 9/9 25.1 0.22 9/9 25.7 0.19 - - - - - - - - - - - - 104 9/9 28.75 9/9 29.2 0.99 9/9 28.8 0.61 - - - - - - - - - - - - 103 9/9 30.24 0.45 9/9 30.1 0.66 9/9 31.6 0.67 2/3 103 8 0.28 3/3 79.7 6.9 2.38 3/3 46 4.13 2.22 102 9/9 32.7 9/9 34.3 9/9 33.5 0.78 2/3 23.5 1.32 1.94 2/3 6 0.57 0.41 3/3 10.7 1.03 0.92 101 9/9 37.6 2/3 9.5 0.75 1.06 0.09 0.09 2/3 3.5 1.51 0.10 Bacterial CFU per Reaction Quick N° pos wells Ct mean St.dev N° pos wells Ct mean St.dev N°pos wells Ct mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev N° pos wells Positive droplets mean Copies/μL mean St.dev 107 9/9 17.3 0.16 9/9 18.8 0.17 9/9 17.3 0.69 - - - - - - - - - - - - 106 9/9 19.6 0.8 9/9 20.9 0.33 9/9 19.4 0.36 - - - - - - - - - - - - 105 9/9 22.4 0.78 9/9 24.2 0.44 9/9 23 0.17 - - - - - - - - - - - - 104 9/9 25.4 0.15 9/9 27.2 0.37 9/9 26.9 0.46 - - - - - - - - - - - - 103 9/9 29.8 0.74 9/9 29.4 0.34 9/9 30.8 0.66 2/3 99 8.9 7.35 3/3 177 17.5 2.46 3/3 50.3 4.46 1.70 102 9/9 9/9 32.8 0.51 9/9 32.8 0.53 2/3 67.6 5.36 7.23 2/3 16 1.55 0.49 2/3 15 1.35 0.49 101 35.20 0.97 3/3 16.7 1.74 2.65 2/3 3.5 0.3 0.04 3/3 11 0.82 0.68
Table 3 Results obtained by real-time PCR and droplet digital PCR in samples of ten-fold dilution of bacterial DNA from strain Xcc NCPPB 3234, pathotype A were prepared as a standard reference curve. For real-time PCR are reported the average of Ct values and the number of positive wells on the total assessed; for ddPCR are reported copies/mL and positive droplets. Grey boxes indicate inconsistent results between the replicates (ΔCt less than 3 cycles between ten-decimal dilutions) and SD > 1. The efficiency, r
Bacterial Culture (NCPPB 3234) Real Time PCR Cubero et al. (2005) ddPCR Adapted from Cubero et al. (2005) ng per Reaction N° pos Wells Ct Mean St.dev Copies/µL Positive Droplets 1ng 3/3 17.15 0.23 - - 100pg 3/3 20.77 0.44 - - 10pg 3/3 24.26 0.47 163 1971 1pg 3/3 27.86 0.39 13.30 135 100fg 3/3 31.88 0.81 1.80 22 10fg 3/3 0.60 0.16 2 1fg 3/3 37.77 0.42 0.09 Std curve parameters E: 98.7%; r2: 0.984;
Table 4 Percentage of healthy samples which yielded high Ct in duplicates or in one out of two replicates (i.e., Ct value/NA) for each DEM/plant matrices combinations (Plant = DNeasy Plant Mini kit; Mericon = DNeasy Mericon Food Kit, Ctab = CTAB extraction method, Quick = QuickPick SML Plant DNA kit). The raw data are reported in Table S1. DEM = DNA extraction method.
Mericon Ctab Quick Plant 30% 50% 5% 20% 20% 40% 25% 10% 35% 40% 5% 45% 28% 43% 11% 25%
Table 5 Values of performance criteria obtained by real-time PCR diagnostic sensitivity, diagnostic specificity and accuracy. Positive agreement (PA), negative agreement (NA), positive deviation (PD) and negative deviation (ND) obtained by testing spiked samples contaminated from 10
Performance Criteria Lemon Fruit Orange Fruit Lemon Leaves Plant Mericon Ctab Quick Plant Mericon Ctab Quick Plant Mericon Ctab Quick 45 45 45 45 45 45 45 45 45 45 45 45 0 0 0 0 0 0 0 0 0 0 0 0 20 20 20 20 20 20 20 20 20 20 20 20 0 0 0 0 0 0 0 0 0 0 0 0 45 45 45 45 45 45 45 45 45 45 45 45 20 20 20 20 20 20 20 20 20 20 20 20 65 65 65 65 65 65 65 65 65 65 65 65 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
Table 6 Analytical sensitivity (ASE) obtained by using different DNA extraction method (DEM) on spiked samples (SET2).
Plant Matrices DEM Detection Method Lemon Fruit Orange Fruit Lemon Leaf 102 103 102 103 103 103 103 103 102 103 102 102 102 10 10 10 102 10 10 102 10 10 10 10
Table 7 Plant species, matrix, place and year of sampling.
Plant Specie Matrix Place of Sampling Year of Sampling Orange Fruit Rome (Italy) 2020 Lemon Leaves Latina (Italy) 2020 Lemon Fruit Naples (Italy) 2020
Conceptualization, S.L. (Stefania Loreti), N.P. and V.S.; data curation, S.L. (Stefania Loreti) and N.P.; methodology, A.L., G.T., S.L. (Simone Lucchesi).; funding acquisition S.L. (Stefania Loreti); project administration, S.L. (Stefania Loreti); statistical analysis, M.S.; writing—Original draft, S.L. (Stefania Loreti); writing—Review and editing, S.L. (Stefania Loreti), N.P. and V.S. All authors have read and agreed to the published version of the manuscript.
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Informed consent was obtained from all subjects involved in the study.
Not applicable.
The authors hereby declare to have no conflict of interest regarding this article.
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By Nicoletta Pucci; Valeria Scala; Giuseppe Tatulli; Alessia L'Aurora; Simone Lucchesi; Manuel Salustri and Stefania Loreti
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