{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T17:55:26Z","timestamp":1785779726361,"version":"3.56.0"},"reference-count":41,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    <jats:italic>Bacillus subtilis<\/jats:italic>\n                    subsp.\n                    <jats:italic>natto<\/jats:italic>\n                    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\n                    <jats:italic>B. subtilis<\/jats:italic>\n                    species complex. Here, we developed and validated a high-precision quantitative real-time polymerase chain reaction (qPCR) assay targeting the\n                    <jats:italic>comQ<\/jats:italic>\n                    gene\u2014a core component of the quorum-sensing system. Comparative genomic analysis of 70 bacterial genomes, including 9\n                    <jats:italic>B. subtilis<\/jats:italic>\n                    strains newly sequenced in this study, revealed that the\n                    <jats:italic>comQ<\/jats:italic>\n                    gene is under intense purifying selection (\n                    <jats:italic>dN\/dS<\/jats:italic>\n                    \u2009=\u20090.10) and exhibits near-absolute conservation within the\n                    <jats:italic>natto<\/jats:italic>\n                    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 (\u226499%) or deletions in traditional metabolic (\n                    <jats:italic>bio<\/jats:italic>\n                    and\n                    <jats:italic>fliF<\/jats:italic>\n                    ) and insertion sequence (IS) markers, their\n                    <jats:italic>comQ<\/jats:italic>\n                    loci remained 100% structurally intact. Phylogenetic analysis further confirmed that\n                    <jats:italic>comQ<\/jats:italic>\n                    forms a distinct monophyletic cluster for subsp.\n                    <jats:italic>natto<\/jats:italic>\n                    , providing superior diagnostic reliability over conventional markers, such as\n                    <jats:italic>bioF<\/jats:italic>\n                    or IS elements. By leveraging this highly conserved target, the established qPCR platform achieved an exceptionally wide, linear quantification framework (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u2009=\u20090.9973,\n                    <jats:italic>E<\/jats:italic>\n                    \u2009=\u200994.2%) 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\n                    <jats:italic>comQ<\/jats:italic>\n                    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\u2014by providing a useful diagnostic and quantitative proxy, it provides a practical tool for tracking potentially opportunistic\n                    <jats:italic>natto<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0355394","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T17:26:58Z","timestamp":1785778018000},"page":"e0355394","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a rapid comQ-targeted quantitative polymerase chain reaction assay for specific identification and quantification of Bacillus subtilis subsp. natto"],"prefix":"10.1371","volume":"21","author":[{"given":"Mayu","family":"Takizawa","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daisuke","family":"Kyoui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rika","family":"Horiguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayano","family":"Sakurai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taketo","family":"Kawarai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5932-497X","authenticated-orcid":true,"given":"Naoki","family":"Narisawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"pone.0355394.ref001","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1186\/1471-2164-11-243","article-title":"Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data","volume":"11","author":"Y Nishito","year":"2010","journal-title":"BMC Genomics"},{"issue":"3","key":"pone.0355394.ref002","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1111\/j.1472-765X.2006.01966.x","article-title":"Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand","volume":"43","author":"Y Inatsu","year":"2006","journal-title":"Lett Appl Microbiol"},{"key":"pone.0355394.ref003","doi-asserted-by":"crossref","first-page":"1348411","DOI":"10.3389\/fmicb.2024.1348411","article-title":"Poly (\u03b3) glutamic acid: a unique microbial biopolymer with diverse commercial applicability","volume":"15","author":"K Elbanna","year":"2024","journal-title":"Front Microbiol"},{"key":"pone.0355394.ref004","doi-asserted-by":"crossref","first-page":"5863887","DOI":"10.1155\/2022\/5863887","article-title":"Nutritional Health 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Natto Strains on Antiviral Responses in Resiquimod-Stimulated Human M1-Phenotype Macrophages","volume":"12","author":"K Fujii","year":"2023","journal-title":"Foods"},{"key":"pone.0355394.ref008","article-title":"The majority of Bacillus subtilis strains isolated from blood cultures were derived from traditional Japanese fermented soybeans natto: A single-center retrospective study","author":"RM Sada","year":"2025","journal-title":"Open Forum Infect Dis"},{"issue":"18","key":"pone.0355394.ref009","doi-asserted-by":"crossref","first-page":"6463","DOI":"10.1128\/AEM.00448-11","article-title":"Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production","volume":"77","author":"Y Kubo","year":"2011","journal-title":"Appl Environ Microbiol"},{"issue":"10","key":"pone.0355394.ref010","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0109999","article-title":"Whole genome complete resequencing of Bacillus subtilis natto by combining long reads 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Kimijima","year":"2022","journal-title":"FSTR"},{"issue":"3","key":"pone.0355394.ref014","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1271\/bbb.68.739","article-title":"Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2","volume":"68","author":"M Sasaki","year":"2004","journal-title":"Biosci Biotechnol Biochem"},{"issue":"10","key":"pone.0355394.ref015","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0141369","article-title":"Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods","volume":"10","author":"M Kamada","year":"2015","journal-title":"PLoS One"},{"issue":"1","key":"pone.0355394.ref016","doi-asserted-by":"crossref","first-page":"43097","DOI":"10.1038\/s41598-025-29683-y","article-title":"Conserved accessory genes link a phylogenetically distinct Bacillus subtilis strain from Indian bekang to the Japanese natto 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