Table 2. Primers and probes used in this study.
| Target | Gene | Primer/probea | Sequence (5′-3′) and modificationb | Tm (°C)c | Usaged | References |
|---|---|---|---|---|---|---|
| Pytoplasmas | 16S rDNA | UPH-F (F) | CGTACGCAAGTATGAAACTTAAAGGA | 58.9 | qPCR, cPCR | [9,10] |
| UPH-R (R) | TCTTCGAATTAAACAACATGATCCA | 58.6 | qPCR-C2004/C2013 | [9,10] | ||
| UPH-Pb (P) | FAM-TGACGGGACTCCGCACAAGCG-BHQ1 | 69.4 | qPCR | [9,10] | ||
| UPH-P (P) | FAM-TGACGGGACTCCGCACA-MGB | 69 | qPCR | [17] | ||
| UPHr2 (R) | CGACAACCATGCACCACCTG | 61.9 | qPCR | This study | ||
| D-UPHr2 (R) | CGACAACCATGCACCACCTGIIIIICTGATAACC | 76.3 | qPCR, cPCR | This study | ||
| 23S rDNA | JH-F 1 (F) | GGTCTCCGAATGGGAAAACC | 59.2 | qPCR-Hd | [11] | |
| JH-F all (F) | ATTTCCGAATGGGGCAACC | 60.5 | qPCR-Hd | [11] | ||
| JH-R (R) | CTCGTCACTACTACCRGAATCGTTATTAC | 58.7 | qPCR-Hd | [11] | ||
| JH-P uni (P) | FAM-AACTGAAATATCTAAGTAAC-MGB | 65 | qPCR-Hd | [11] | ||
| OYrDr (R) | GACAAGATTTCTCGTGTCTCGC | 57.7 | Cloning | This study | ||
| Xylella spp. | 16S rRNA processing protein | XF-F (F) | CACGGCTGGTAACGGAAGA | 58.3 | qPCR-Hp | [13] |
| XF-R (R) | GGGTTGCGTGGTGAAATCAAG | 61.2 | qPCR-Hp | [13] | ||
| XF-P (P) | FAM-TCGCATCCCGTGGCTCAGTCC-BHQ1 | 68.5 | qPCR-Hp | [13] | ||
| 16S rDNA | XF16Sf (F) | CGGCAGCACGTTGGTAGTAA | 58.5 | qPCR-L | [14] | |
| XF16Sr (R) | CCGATGTATTCCTCACCCGTC | 60.2 | qPCR-L | [14] | ||
| XF16Sp (P) | FAM-CATGGGTGGCGAGTGGC-BHQ1 | 60.6 | qPCR-L | [14] | ||
| XrDf1 (F) | GGCTCATCCAATCGCACAA | 59.2 | qPCR, cPCR | This study | ||
| XrDr2 (R) | CGGACGGCAGCACAITGGTA | 62.6 | qPCR | This study | ||
| D-XrDr2 (R) | CGGACGGCAGCACAITGGTAIIIIIACCATGG | 80.3 | qPCR, cPCR | This study | ||
| D-XrDr9 (R) | CGGACGGCAGCACAITGGTAIIIIIACCATGGG | 82.2 | qPCR | This study | ||
| XrD-Pf (P) | FAM-CCTAAGGTCCCCTGCTT-MGB | 70 | qPCR | This study | ||
| XrD-P (P) | VIC-CCTAAGGTCCCCTGCTT-MGB | 70 | qPCR | This study | ||
| XF1 (F) | CAGCACATTGGTAGTAATAC | 44.3 | cPCR | [18] | ||
| XF6 (R) | ACTAGGTATTAACCAATTGC | 45.3 | cPCR | [18] | ||
| X.fas-0838S (F) | GCAAATTGGCACTCAGTATCG | 57 | cPCR | [19] | ||
| X.fas-1439A (R) | CTCCTCGCGGTTAAGCTAC | 54 | cPCR | [19] | ||
| XylR2 | CTACGCATTTCACTGCTACAC | 52.5 | Sequencing | This study | ||
| RNA polymerase sigma factor | RST31 (F) | GCGTTAATTTTCGAAGTGATTCGATTGC | 66.1 | cPCR | [20] | |
| RST33 (R) | CACCATTCGTATCCCGGTG | 58.2 | cPCR | [20] | ||
| Bacteria | 16S rDNA | FP1 (F) | AGAGTTTGATCCTGGCTCAGG | 57.1 | Cloning | [21] |
| fD1 (F) | AGAGTTTGATCCTGGCTCAG | 53.2 | Sequencing | [22] | ||
| rD1 (R) | AAGGAGGTGATCCAGCC | 51.6 | Sequencing | [22] | ||
| Plants | 18S rDNA | C18S-F2 (F) | CAGCTCGCGTTGACTACGTC | 58.3 | qPCR | This study |
| D-C18Sr6 (R) | GATCCGAACACTTCACCGGAIIIIICAATCGGTA | 76.9 | qPCR | This study | ||
| C18S-Pt (P) | TAMRA-ACACACCGCCCGTCGCTCC-BHQ2a | 67.5 | qPCR | [9,10] |
a F: forward primer, R: reverse primer, P: probe. Those used in the final optimized TaqMan multiplex quantitative real-time PCR (qPCR) are indicated in bold.
b FAM: 6-carboxyfluorescein, VIC: 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine, MGB: minor-groove-binding non-fluorescent quencher, BHQ: black hole quencher, I: inosine.
c Melting temperature (Tm) values were predicted using the Primer Express software (Applied Biosystems, Foster City, CA, USA) or an oligonucleotide synthesis company (Fasmac Co., Ltd., Kanagawa, Japan; underlined), both of which use the nearest neighbor algorithm.