Development of a rapid comQ-targeted quantitative polymerase chain reaction assay for specific identification and quantification of Bacillus subtilis subsp. natto

Bacillus subtilis subsp. natto is a specialized functional group essential for fermented soybean production. Despite its industrial importance, genetic differentiation of this lineage remains challenging because of its high genomic similarity within the B. subtilis species complex. Here, we developed and validated a high-precision quantitative real-time polymerase chain reaction (qPCR) assay targeting the comQ gene-a core component of the quorum-sensing system. Comparative genomic analysis of 70 bacterial genomes, including 9 B. subtilis strains newly sequenced in this study, revealed that the comQ gene is under intense purifying selection (dN/dS = 0.10) and exhibits near-absolute conservation within the natto lineage (42 strains). While screening of these 70 genomes revealed a total of 15 atypical isolates (such as Miz-8 and PN1236) with reduced sequence identity (≤99%) or deletions in traditional metabolic (bio and fliF) and insertion sequence (IS) markers, their comQ loci remained 100% structurally intact. Phylogenetic analysis further confirmed that comQ forms a distinct monophyletic cluster for subsp. natto, providing superior diagnostic reliability over conventional markers, such as bioF or IS elements. By leveraging this highly conserved target, the established qPCR platform achieved an exceptionally wide, linear quantification framework (R2 = 0.9973, E = 94.2%) that completely bypasses peripheral genomic instability, maintaining high fidelity even under a 10,000-fold excess of non-target DNA. Direct validation on commercial fermented soybean matrices demonstrated a seamless correlation between molecular comQ copy number and viable cell count, underscoring its resilience against complex sample-derived enzymatic inhibition without the need for culture expansion. This assay transcends application in conventional industrial quality control-by providing a useful diagnostic and quantitative proxy, it provides a practical tool for tracking potentially opportunistic natto lineages linked to severe clinical bacteremia. Overall, we have established a reliable molecular surveillance method applicable to both industrial biotechnology and high-resolution clinical epidemiology.