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 phytoplasma DNA. 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).