Genome-Informed Real-Time PCR Assay for Detection of ‘Candidatus Phytoplasma Prunorum,’ Which Is Associated with European Stone Fruit Yellows

'<i>Candidatus</i> Phytoplasma prunorum' has been associated with severe disease in <i>Prunus</i> spp., which are commodities of economic importance in the USA. The introduction and establishment of '<i>Ca.</i> P. prunorum' in the USA could result in huge economic losses, thus creating a need for validated diagnostic tools, which are the cornerstone of successful surveillance, quarantine, and eradication measures. Whole-genome comparisons led to the identification of a diagnostic marker gene specific to '<i>Ca.</i> P. prunorum' (PE639). The PE639 assay was duplexed with an 18S rDNA plant internal control and compared to modified 23S (phytoplasmas) and <i>imp</i> ('<i>Ca.</i> P. mali') assays. The PE639 assay produced congruent results to 23S and <i>imp</i> assays for all metrics, demonstrating high linearity, repeatability, intermediate precision, and reproducibility. The limit of detection was comparable for all assays tested, and all demonstrated 100% analytical specificity, selectivity, and diagnostic specificity for their respective target species. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™ OPUS. A synthetic gBlocks™ control was designed and validated to work with all assays, as well as conventional PCR assays targeting 16S rDNA and <i>tuf</i> genes. These validated assays and synthetic control represent beneficial tools that support efforts to protect USA agriculture and facilitate safe trade.