Baric Sanja
Faculty of Science and Technology, Free University of Bozen-Bolzano, Universitätsplatz 5, Bozen-Bolzano, BZ, Italy.
Methods Mol Biol. 2019;1875:131-141. doi: 10.1007/978-1-4939-8837-2_10.
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 C values of the two target genes (ΔC). 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.
