Streamlined Probe-Melt PCR for Multiplex Detection of Wheat Fungal Pathogens

Fungal diseases in wheat contribute to significant yield losses, with annual reductions of up to 160 million tons in the US alone. Molecular diagnostic tools, particularly polymerase chain reaction (PCR), offer rapid, sensitive, and specific detection of fungal pathogens and have the potential to support more timely and targeted disease management. High-resolution melt PCR, widely employed for this purpose, differentiates DNA sequences based on melting curve variations. However, it often encounters difficulty in distinguishing closely related species with minor sequence differences. While TaqMan PCR offers high specificity, it is limited by high costs and restricted multiplexing capabilities. To address these challenges, we developed a simplified probe-based melt curve analysis method coupled with asymmetric PCR for multiplexed detection of three common wheat fungal pathogens─<i>Zymoseptoria tritici</i>, <i>Puccinia triticina</i>, and <i>Puccinia striiformis f. sp. tritici</i>─using a universal primer and probe. This approach enabled clear differentiation of each fungal species by distinct melting temperatures using a single fluorescent dye. Validation studies confirmed the assay's sensitivity down to 3 copies/μL, demonstrated reproducibility across multiple isolates, and verified its ability to detect multitarget mixtures. This method offers a promising approach for rapid, accurate, and multiplexed detection of wheat fungal pathogens, paving the way for more timely and cost-effective disease management strategies.