Abstract
The chapter describes a simple quantitative approach to assess phytoplasma load in samples obtained from "Candidatus Phytoplasma mali"-infected apple plants without the use of external standard curves. The assay is based on the simultaneous detection of a gene of the pathogen and a gene of the host plant in a duplex single-tube real-time PCR reaction using TaqMan chemistry. The quantity of the phytoplasma, relative to its host plant, is determined as the difference between the CT values of the two target genes (ΔCT). A critical data analysis step, affecting the inter-assay reproducibility between different amplification runs, is the setting of the threshold level, which is achieved by the recurrent analysis of a calibrator sample. The relative quantification procedure allows analyzing 45 DNA samples in duplicates on a 96-well reaction plate, in addition to the control and calibrator samples, and thus contributes to a substantial increase of analysis throughput and decrease of reagent/consumable costs per sample