Table 7. TaqMan multiplex real-time quantitative real-time PCR (qPCR) to detect three targets from various templates.
| Samples | Extraction methodsa | Multiplex qPCR | ||
|---|---|---|---|---|
| Phytoplasma | Xylella | Plants | ||
| The phytoplasma strain TF19C57 | Pure extraction | 23.5 ± 0.0b | (50.0 ± 0.0) | 20.2 ± 0.1 |
| Crude extraction | 32.0 ± 0.1**/ 8.5c | (50.0 ± 0.0) | 27.1 ± 0.2**/ 6.9 | |
| Crude extraction/ isopropanol | 26.2 ± 0.2**/ 2.7 | (50.0 ± 0.0) | 21.5 ± 0.3**/ 1.3 | |
| The X. fastidiosa strain PCE-RR | Pure extraction | (50.0 ± 0.0) | 18.7 ± 0.1 | 19.8 ± 0.1 |
| Crude extraction | (50.0 ± 0.0) | 26.2 ± 0.0**/ 7.5 | 27.0 ± 0.2**/ 7.2 | |
| Crude extraction/ isopropanol | (50.0 ± 0.0) | 21.6 ± 0.1**/ 2.9 | 22.6 ± 0.1**/ 2.8 | |
| (Healthy plants) | ||||
| Vitis spp. cv. Aurora Black | Pure extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 22.3 ± 0.1 |
| Vitis vinifera cv. Ruby Okuyama | Pure extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 23.1 ± 0.1 |
| Citrus jambhiri | Pure extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 21.7 ± 0.0 |
| Malus domestica | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 27.9 ± 0.2 |
| Actinidia deliciosa cv. Hayward | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 30.5 ± 0.1 |
| Olea europaea cv. Manzanillo | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 28.5 ± 0.1 |
| Ficus carica cv. Masui Dauphine | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 27.1 ± 0.2 |
| Prunus persica cv. Akatsuki | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 23.5 ± 0.0 |
| Pyrus pyrifolia | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 32.2 ± 0.1 |
| Hydrangea macrophylla | Crude extraction | (50.0 ± 0.0) | (50.0 ± 0.0) | 34.1 ± 0.1 |
| TE buffer | (50.0 ± 0.0) | (50.0 ± 0.0) | (50.0 ± 0.0) | |
a Pure extraction was obtained using a commercial kit. Crude extraction was obtained according to the method described by Nakaune and Nakano [23]. Additional isopropanol-precipitation was performed to concentrate crude extracts.
b Mean ± standard error (n = 3) of threshold cycle (Ct) values at an arbitrary threshold of 0.02. Undetermined Ct values within 50 cycles were temporally calculated as 50. Mean values in parentheses were considered negatives: those over 45 for detection of phytoplasmas and Xylella spp. and those over 40 for detection of plants.
c A Student’s t-test was performed to compare the Ct values with those from pure extracts in the same sample. Significantly inferior Ct values (** P < 0.01) are highlighted in gray, indicating differences in the values after slashes.