A Sensitive Real-Time PCR-Based Assay for the Identification of Colletotrichum in Phytosanitary and Clinical Applications

<i>Colletotrichum</i> species are major plant pathogens and emerging opportunistic human pathogens. Due to their vast genetic diversity, existing diagnostic tools often suffer from narrow specificity or labor-intensive workflows. In this study, we developed a rapid, universal, and highly sensitive genus-specific real-time PCR assay utilizing a TaqMan MGB probe targeting the conserved 28S rDNA region. The assay demonstrated exceptional specificity, with no cross-reactivity against closely related fungal taxa or common co-occurring pathogens. The method exhibited high sensitivity, achieving a limit of detection (LOD) of 680 fg of genomic DNA. Furthermore, the assay was successfully validated using simulated environmental samples, where it accurately identified <i>Colletotrichum</i> within complex fungal communities. By providing a robust platform for genus-level screening, this methodology significantly enhances the efficiency of phytosanitary inspections and clinical diagnostics, facilitating timely biosecurity interventions and therapeutic decisions.