Table 4. Detection of Xylella by TaqMan quantitative real-time PCR (qPCR) and conventional PCR (cPCR) with different probes and primers.
| qPCR with XrD-Pf/XrDf1 and | qPCR-Hpc | qPCR-L | cPCR with | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Species | Strain | D-XrDr2 | XrDr2 | D-XrDr9 | RST31/33 | XF1/6 | X.fas-0838S/1439A | XrDf1/D-XrDr2 | ||
| X. fastidiosa subsp. fastidiosa | ALS-BC | 10.6 ± 0.1a | 21.1 ± 0.1**/ 10.5b | 9.5 ± 0.0**/ -1.1 | nt | 13.9 ± 0.1**/ 3.3 | nt/ nt | nt/ nt | nt/ nt | +/ nt |
| ELM-1 | 12.7 ± 0.0 | 12.6 ± 0.1 | 12.9 ± 0.0**/ 0.2 | 13.8 ± 0.1**/ 1.1 | 13.5 ± 0.0**/ 0.8 | +/ ntd | +/ nt | +/ nt | +/ nt | |
| PCE-RR | 19.7 ± 0.1 | 19.4 ± 0.2 | 19.8 ± 0.1 | 19.8 ± 0.0 | 19.8 ± 0.0 | +/ + | +/ + | +/ nt | +/ + | |
| PD5-2 | 23.8 ± 0.0 | 24.0 ± 0.1*/ 0.2 | 24.7 ± 0.0**/ 0.9 | 24.6 ± 0.1**/ 0.9 | 24.6 ± 0.0**/ 0.9 | +/ + | +/ nt | +/ nt | +/ nt | |
| OAK | 15.0 ± 0.1 | nt | 15.0 ± 0.1 | 15.5 ± 0.1*/ 0.5 | 15.4 ± 0.2 | +/ nt | +/ nt | +/ nt | +/ nt | |
| Ann 1 | 13.9 ± 0.1 | nt | 14.0 ± 0.1 | 14.9 ± 0.1**/ 1.0 | 14.4 ± 0.1*/ 0.6 | +/ nt | +/ nt | +/ nt | +/ nt | |
| subsp. multiplex | PLM G83 | 13.7 ± 0.0 | 13.8 ± 0.1 | 14.2 ± 0.0**/ 0.5 | 14.6 ± 0.1**/ 1.0 | 14.2 ± 0.1**/ 0.6 | +/ nt | +/ nt | +/ nt | +/ nt |
| subsp. pauca | 9a5c | 11.1 ± 0.0 | 11.4 ± 0.0**/ 0.3 | 11.3 ± 0.1 | nt | 9.6 ± 0.0**/ -1.5 | nt/ nt | nt/ nt | nt/ nt | +/ nt |
| CM1 | 17.3 ± 0.1 | 21.1 ± 0.1**/ 3.8 | 14.7 ± 0.0**/ -2.6 | nt | 10.4 ± 0.0**/ -6.9 | nt/ nt | nt/ nt | nt/ nt | +/ nt | |
| Xfp01 | 21.0 ± 0.1 | 21.1 ± 0.1 | 20.9 ± 0.0 | 20.9 ± 0.2 | 20.8 ± 0.1 | +/ + | +/ nt | +/ nt | +/ + | |
| X. taiwanensis | PLS235 | 24.4 ± 0.1 | 17.8 ± 0.1**/ -6.6 | 20.0 ± 0.1**/ -4.4 | (50 ± 0.0)**/ 25.6 | (40.0 ± 0.4)**/ 15.7 | –/ – | –/ – | +/ nt | +/ nt |
| (Other bacteria) | ||||||||||
| B. gladioli | BRA 1 | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | nt/ nt | nt/ nt | –/ nt | nt/ – |
| B. gladioli pv. gladioli | AZ 87108 | (48.7 ± 1.3) | nt | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | nt/ nt | nt/ nt | –/ nt | nt/ – |
| E. coli | BL21(DE3) pLysS | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | nt/ nt | nt/ nt | –/ nt | nt/ – |
| R. pickettii | C-176 | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | nt/ nt | nt/ nt | –/ nt | nt/ – |
| S. maltophilia | K-14 (6I11) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | 38.5 ± 0.7**/ -11.5 | nt/ nt | nt/ nt | +/ nt | nt/ – |
| X. albilineans | T161 | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | 15.8 ± 0.0**/ -34.2 | nt/ nt | nt /nt | +/ nt | nt/ – |
| X. arboricola | C1 | (50.0 ± 0.0) | (50.0 ± 0.0) | (42.2 ± 7.8) | (50.0 ± 0.0) | 37.6 ± 0.3**/ -12.4 | –/ nt | –/ nt | +/ nt | nt/ – |
| X. campestris pv. citri | N6101 | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (46.1 ± 3.5) | –/ – | –/ – | +/ nt | nt/ – |
| X. oryzae pv. oryzae | H-9101 | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | 38.9 ± 1.3**/ -11.1 | nt/ nt | nt/ nt | +/ nt | nt/ – |
| Diagnostic sensitivity (%) | 100 | 100 | 100 | 87.5 | 97 | |||||
| Diagnostic specificity (%) | 100 | 100 | 96.3 | 100 | 55.6 | |||||
a Mean ± standard error (n = 3) of the threshold cycle (Ct) values at an arbitrary threshold of 0.02. Undetermined Ct values within 50 cycles were temporarily calculated as 50; nt: not tested. Mean values over 40 in parentheses were considered negatives.
b A Student’s t-test was performed to compare the Ct values with those of`the qPCR with XrD-Pf/XrDf1/D-XrDr2. The same DNA preparations were used for evaluating the assays. Significant values are shown as: * P < 0.05, ** P < 0.01. Significantly superior and inferior Ct values are highlighted in orange and gray, respectively, indicating differences in the values after slashes.
d +: positive,–: negative at 35/ 43 cycles.