Table 1.
DNA sequences and description of the pecan (AF303825) and walnut (HE574850) primers and probes to develop a real time PCR [9].
| Detected Species | Primer and Probe |
Length (bp) | Sequence (5′ → 3′) | nM | Cycling Conditions |
|---|---|---|---|---|---|
| Walnut | WalITSdir | 20 | GACAATCGGTGGTTGAGAAA | 300 | Initial denaturation: 10 min 95 °C Amplification: 50 cycles at 95 °C for 5 s 60 °C for 30 s 72 °C for 1 s Cooling: 40 °C for 30 s. |
| WallITSinv | 20 | GTCGAGGAGCACCTTCACAG | 900 | ||
| WalITSP | 18 | 6FAM-TGACCCGTCGTGTGTTGCCC-BBQ | 2000 | ||
| Pecan | PecITSdir | 18 | ATGAAAGCTGCCCACCGC | 300 | |
| PecITSinv | 18 | CATTGTTCGACCGGGAAG | 900 | ||
| PecITSP | 19 | 6FAM-CGGGTCAGTCTCCTCGTTC-BBQ | 2000 | ||
| 18S | 18Sdir | 16 | TGGTGCCAGCAGCCGC | 300 | |
| 18Sinv | 25 | TCCAACTACGAGCTTTTTAACTGCA | 900 | ||
| 18SP | 22 | 6FAM-CGCTATTGGAGCTGGAATTACC-BBQ | 2000 |
The PCR was carried out using the LightCycler® TaqMan® Master (Roche Molecular Systems, Mannheim, Germany) in a total reaction volume of 10 µL in glass capillary tubes.