A genus-specific TaqMan real-time polymerase chain reaction assay for the rapid detection of Wallemia in bakery production environments.

The xerophilic fungal genus Wallemia is an important cause of fungal contamination in bread manufacturing environments, but its detection by conventional culture-based methods is often difficult because of slow growth and competitive overgrowth by other fungi. Here, we report the development of a genus-specific TaqMan real-time polymerase chain reaction assay targeting the internal transcribed spacer (ITS) region for rapid detection of Wallemia. Primer and probe sequences were designed based on comparative ITS analysis of all nine recognized Wallemia species and non-target fungi commonly encountered in bakery environments. The probe-binding region was fully conserved among all Wallemia ITS reference sequences evaluated. Analytical specificity was confirmed using synthetic DNA fragments representing predicted amplicon groups, with amplification observed exclusively in Wallemia. The assay demonstrated excellent linearity over 10²-10⁶ copies per reaction (R² = 0.9988), with an amplification efficiency of 102.7%. Analytical sensitivity evaluation yielded an estimated LOD95 of 9.46 copies per reaction. Application to environmental swab samples from an operational bakery production line enabled culture-independent detection of Wallemia, including in samples in which culture-based identification was obscured by fungal overgrowth. This assay provides a rapid and specific molecular tool for culture-independent monitoring of Wallemia contamination and may support improved hygienic control in bakery manufacturing environments.