P-195 - Optimizing Endogenous Plant Internal Controls for Molecular Detection of Plant Pathogens

Real time PCR is widely used for detecting, quantifying, and identifying plant pathogens, with internal controls essential for reliable results. Host DNA can be targeted to serve as a Plant Internal Control (PIC) to verify DNA quality and identify presence of PCR inhibitors. When choosing a PIC, it is important to ensure specificity, effective amplification, and no interference with target amplification. While several “universal” PICs exist, their performance across various plant taxa needs to be explored. Our goal was to provide guidelines for incorporating PICs into pathogen diagnostic real time PCR assays. We selected 10 PICs from the literature based on their suitability and broad applicability, conducting a preliminary screening to confirm their performance. Seven PICs were successful, and their sensitivity and specificity were tested in singleplex with 64 plant species. Two PICs, ‘18S-uni’ and ‘FMPI (cox1)’, showed the highest sensitivity and broadest plant coverage. While 18S-uni covered all plants, FMPI did not amplify gymnosperm taxa consistently. Both PICs were tested in multiplex with a published Phytophthora spp. assay. 18S-uni high sensitivity caused water control contamination, affecting pathogen detection. FMPI was more stable, leading to further evaluation under various conditions. Sequence alignments revealed why FMPI failed to amplify gymnosperms and alternative primer, and probe sets were designed for improved amplification for those taxa. This research provides a foundation for standardizing PICs in diagnostic PCR assays for plant pathogen detection.