Accurate pathogen detection is vital in clinical laboratories for timely diagnosis and disease management strategies. This study aims to transform research findings into robust clinical protocols through method development, validation, and application. The study focused on two fungal pathogen targets.
The first objective focused on detecting Batrachochytrium dendrobatidis, the cause of chytridiomycosis in amphibians, in the environment . This was achieved by developing and optimising a workflow that included methods for collecting eDNA, preserving samples , and sample processing with multiple DNA extraction methods. A standard qPCR assay was used to determine the efficiency of multiple variables at each step. The most effective workflow delivered comparable results to the current gold standard results for t he detection of B. dendrobatidis .
The second objective was to amplify the target DNA. Two molecular detection assays were developed and optimised: a LAMP assay to detect B. dendrobatidis from eDNA and a quantitative real-time assay to detect a different target , Aphanomyces invadans, the agent responsible for Epizootic Ulcerative Syndrome (EUS) in fish. A primer set consisting of four primers was designed using the nucleotide sequence of the ITS1-5.8S-ITS2 region of B. dendrobatidis , which amplified a fragment of 201 bp. Similarly, a TaqMan primer-probe set was designed for A. invadans . This set targets the same gene and amplified a 93 bp fragment of the ITS2 region. The analytical specificity of both sets was validated and confirmed in silico and against a panel of single spore cultures of phylogenetically closely related oomycete species and environmental fungi. The performance of the assay was determined to aid in future interpretation of the results. This study addressed real-world challenges encountered during validation, highlighting the intricate balance between sensitivity, specificity, and practical applicability.
In conclusion, this study worked through the transformative process of bringing novel detection protocols to clinical laboratories. It highlights the importance of following the World Organisation for Animal Health (WOAH) guidelines, which emphasise the alignment of protocols with worldwide standards. Our results showcase protocol validation, demonstrating their use in the broader research context.