Abstract

Thermophilic spore-forming Caldibacillus thermoamylovorans is a major spoiler of processed milk, as its thermophilic spores would survive ultra-high temperature (UHT) treatment and form viable cells in suitable conditions. However, conventional methods could not accurately detect the extremely low level of contamination, and existing PCR-based assays fail to distinguish viable from non-viable cells, leading to inaccurate risk assessment. Herein, we developed a highly specific propidium monoazide-qPCR (PMA-qPCR) assay for selective detection of viable C. thermoamylovorans . A species-specific molecular target was identified via pan-genome analysis of 47 Bacillus genomes, and the optimized primers/probe (primer 7-2/Probe 3) exhibited no cross-reactivity with 6 closely related Bacillus species. The assay was further optimized for PMA treatment (10 μM PMA, 15-min dark incubation) and reaction conditions (58 ℃ annealing temperature, 3 mM Mg²⁺, 2 μM primers, 1 μM probe), achieving excellent linearity (R² = 0.9944) over 10¹–10⁵ CFU/mL and a detection limit of 10¹ CFU/mL in milk matrices. Validation with artificially contaminated commercial milk confirmed high sensitivity, specificity, and reproducibility. When applied to 120 retail milk samples, the assay detected 1 positive sample, indicating the ability to identify infinitesimal contamination. Collectively, this pan-genome-guided PMA-qPCR platform provides a rapid, reliable, and biologically relevant tool for early surveillance of C. thermoamylovorans in dairy production, supporting quality control and spoilage risk mitigation.