Establishment of a degenerate primer-based multiplex qPCR method for sensitive detection of Gas-Producing Bacillus...

Current quality control measures for thermally processed foods predominantly rely on reactive post-spoilage analyses rather than proactive monitoring of specific spoilage organisms. In this study, 64 gas-producing strains were isolated from swollen bagged soy sauce and identified as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis. Notably, B. velezensis was associated with gas spoilage in soy sauce for the first time. Inoculation experiments conducted directly in soy sauce confirmed species-specific gas production within 1-3 days. Among the tested species, B. licheniformis posed the highest risk due to the earliest onset (1 day). To trace contamination, critical control points in soy sauce processing were analyzed. High-throughput sequencing showed elevated levels of Bacillus spp. Under high-heat conditions, primarily B. velezensis (38.11%), B. subtilis (6.13%), and B. amyloliquefaciens (4.83%) in group JY100, whereas B. licheniformis was primarily detected in equipment pipelines. For proactive monitoring, a multiplex qPCR assay was developed. A degenerate gyrB primer set enabled simultaneous detection of B. subtilis, B. amyloliquefaciens, and B. velezensis, while B. licheniformis was specifically detected using an adk-based set. The method showed no cross-reactivity with 15 non-target strains. The limits of detection (LOD) were 110, 105, 108, and 102 CFU/mL for each species in pure culture and soy sauce, with amplification efficiencies between 92.71% and 100.92%. When applied to 84 commercial samples, the positive detection rate was 15.48%. This integrated approach enables a transition from reactive post-spoilage analysis to proactive surveillance, providing a replicable framework for managing microbial spoilage risks in thermally processed foods.