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

Colletotrichum 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 Colletotrichum 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.