Real-time PCR
Quantitative PCR (qPCR) was performed with the Bio-Rad CFX96 Thermal
Cycler (Bio-Rad Laboratories GmbH, Munich, Germany) using primer pair
and probe of a TaqMan assay developed by Christensen et al. (2004) for
the generic detection of phytoplasmas. The amplification of a part of
the 16S rDNA gene was performed in 25 µl reactions containing 1 µl of
DNA extraction, 0.625 U of FastGene Taq DNA Polymerase (Nippon Genetics
Europe GmbH, Düren, Germany) with provided 10 x reaction buffer A (with
1.5 mM MgCl2), 0.5 µl of dNTPs (10 mM each, Steinbrenner
Laborsysteme GmbH, Wiesenbach, Germany), 1 µl of each primer (10 µM,
Eurofins Genomics Germany GmbH, Ebersberg, Germany), 0.5 µl of TaqMan
probe (10 µM, Eurofins Genomics Germany GmbH), and HPLC water (VWR
International GmbH). Amplification parameters were 15 min at 95 °C
followed by 46 cycles at 95 °C for 15 s and 60 °C for 1 min. Data
analysis was performed with the BioRad CFX Manager 3.0 software (Bio-Rad
Laboratories GmbH, Munich, Germany).