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The real-time PCR detection system for grapevine yellows phytoplasmas described here is composed of two assays for group-specific detection of flavescence dorée (FD) and bois noir (BN) phytoplasmas and a universal phytoplasma assay. It uses hydrolysis minor groove binder probes (TaqMan-MGB). The addition of an assay for amplification of plant DNA co-extracted with phytoplasma DNA provides a further quality control for the DNA extraction and PCR amplification for each sample. The detection system described is reliable, specific, sensitive, and easily applicable to fast, high-throughput diagnosis of grapevine yellows phytoplasmas. In addition to the detection system, an approach for the quantification of phytoplasmas in the sample is described.