Development and validation of a PCR-based detection assay for xerophilic Wallemia species informed by comparative mitogenomics.

Abstract

Wallemia species, filamentous fungi known as foodborne pathogens, pose significant health risks to humans and animals. Despite their ecological significance, robust detection methodologies for these fungi remain underdeveloped. The polymorphic, multi-copy nature of mitochondrial DNA serves as a reliable genetic marker for identifying Wallemia species. In the present study, we report the first complete assembly of mitochondrial genomes (mitogenomes) from five Wallemia species: Wallemia hederae, Wallemia mellicola, Wallemia muriae, Wallemia sebi, and Wallemia tropicalis. These mitogenomes exhibit circular configurations, varying in size from 26,178 bp to 44,887 bp, with AT content consistently above 72%. Each mitogenome encompasses a conserved core set of mitochondrial genes; however, interspecific variation was noted in intron composition, non-conserved ORF distribution, and gene order. By targeting mitochondrial genes nad2, nad3, nad5, and rns, we designed three primer pairs for the specific detection of Wallemia. These primers showed no cross-reactivity with non-target fungi and successfully detected Wallemia in both pure cultures and naturally contaminated food samples. The assay demonstrated a detection sensitivity threshold of 5 × 10-3 ng/μL, underscoring its potential utility in food safety surveillance. This integration of comparative mitogenomic analysis with PCR assay development represents a promising strategy for precise, in situ detection of Wallemia fungi, addressing a critical gap in food safety diagnostics.