Validation of a modified quantitative PCR and synthetic DNA gBlocks control for detection of ‘Candidatus Phytoplasma mali’

A primer set targeting the imp gene was duplexed with an 18S rRNA plant internal control primer set and validated for specific detection of ‘Candidatus Phytoplasma mali.’ The assay was compared to a modified 23S/18S rRNA (23S) duplex universal phytoplasma assay that was validated previously. The imp duplex assay produced comparable results to the 23S duplex assay for all metrics, demonstrating high linearity, repeatability, and intermediate precision. The amplification efficiency and limit of detection were 97–98% and C_t=36 - 37, respectively. The assay showed 100% analytical specificity, selectivity, and diagnostic specificity. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™. A synthetic gBlocks™ control was designed and validated to work with both duplex assays and a semi-nested PCR assay. The imp duplex assay shows increased specificity over current EPPO protocols, and the addition of an internal control increases confidence in test results. Together, the assay and synthetic control can be successfully deployed to aid in quarantine and eradications efforts of ‘Ca. P. mali.’