{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T11:09:31Z","timestamp":1788520171442,"version":"build-2803163510"},"reference-count":69,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T00:00:00Z","timestamp":1773360000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Fast and accurate genetic subtyping of pathogens is required to respond appropriately to biological events caused by natural outbreaks or bioattacks involving anthrax. In this study, we developed and validated a culture-free genotyping method that combines a multiplex PCR-based amplicon sequencing method on the Nanopore platform with\n                    <jats:italic>in silico<\/jats:italic>\n                    multiple-locus variable-number tandem repeat analysis (MLVA) of 31 loci to identify an unknown\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    strain directly from environmental samples. The novel method accurately identified repeat numbers for all loci in 12 different MLVA genotype\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    strains analyzed in the study, matching 100% with the reference capillary electrophoresis and Sanger sequencing results. The detection limit of the method, at which all 31 variable-number tandem repeat loci were successfully identified, was found to be 10\n                    <jats:sup>4<\/jats:sup>\n                    \u202fCFU spores\/sample for pure spore samples and at 10\n                    <jats:sup>6<\/jats:sup>\n                    \u202fCFU spores\/sample for spiked environmental samples from three matrices (soil, swab, and muddy water). Specificity tests yielded negative results for samples containing only non-\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    members of the\n                    <jats:italic>Bacillus cereus<\/jats:italic>\n                    group, which produced sequencing reads for 15 loci but were non-specific to\n                    <jats:italic>Bacillus anthracis.<\/jats:italic>\n                    To validate the method, we genotyped 11\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    strains originating from a historical collection of Hungarian isolates. The MLVA31 typing scheme classified the strains into five groups, four of which fell into the A.Br.008\/009 Trans-Eurasian (TEA) group within the clade A, and one into the B.Br.CNEVA group within the clade B. The largest group within clade A comprises six strains that are assumed to be members of the dominant\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    population in Hungary. Our results demonstrate that PCR-based amplicon sequencing using the portable MinION device is highly effective for on-site genotyping of pathogens directly from environmental samples. This establishes the NGS-based MLVA genotyping as a valuable tool for biodefense laboratories in preliminary forensic investigations of bioterrorism-related anthrax outbreaks. Furthermore, our results provide new insights into the genetic diversity of\n                    <jats:italic>Bacillus anthracis<\/jats:italic>\n                    in a region (Hungary, Central Europe) that is underrepresented in research and has limited scientific data.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2026.1771578","type":"journal-article","created":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T06:49:23Z","timestamp":1773384563000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of Nanopore amplicon sequencing method for culture-free genotyping of Bacillus anthracis strains directly from environmental samples"],"prefix":"10.3389","volume":"17","author":[{"given":"\u00c1gnes","family":"Nagy","sequence":"first","affiliation":[{"name":"Hungarian Defence Forces Medical Centre","place":["Budapest, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"G\u00e1bor Endre","family":"T\u00f3th","sequence":"additional","affiliation":[{"name":"Virus Metagenomics and Evolution Group, Bernhard Nocht Institute for Tropical Medicine","place":["Hamburg, Germany"]},{"name":"Department of Arbovirology and Entomology, Bernhard Nocht Institute for Tropical Medicine","place":["Hamburg, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P\u00e9ter","family":"S\u00e1ly","sequence":"additional","affiliation":[{"name":"HUN-REN Centre for Ecological Research, Institute of Aquatic Ecology","place":["Budapest, Hungary"]},{"name":"Department of Fisheries Research and Development, Institute of Aquaculture and Environmental Safety, Hungarian University of Agriculture and Life Sciences","place":["G\u00f6d\u00f6ll\u0151, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Csaba Istv\u00e1n","family":"Pereszl\u00e9nyi","sequence":"additional","affiliation":[{"name":"Hungarian Defence Forces Medical Centre","place":["Budapest, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gergely Csaba","family":"Babinszky","sequence":"additional","affiliation":[{"name":"Hungarian Defence Forces Medical Centre","place":["Budapest, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"L\u00e1szl\u00f3","family":"Makrai","sequence":"additional","affiliation":[{"name":"Autovakcina Kft.","place":["Budapest, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bal\u00e1zs Antal","family":"Somogyi","sequence":"additional","affiliation":[{"name":"National Laboratory of Virology, University of P\u00e9cs","place":["P\u00e9cs, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikl\u00f3s","family":"Gyuranecz","sequence":"additional","affiliation":[{"name":"HUN-REN Veterinary Medical Research Institute","place":["Budapest, Hungary"]},{"name":"National Laboratory of Health Safety","place":["Budapest, Hungary"]},{"name":"MolliScience Kft.","place":["Biatorb\u00e1gy, Hungary"]},{"name":"University of Veterinary Medicine","place":["Budapest, Hungary"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,13]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"e02889","DOI":"10.1128\/JCM.02889-20","article-title":"A whole-genome-based gene-by-gene typing system for standardized high-resolution strain typing of Bacillus anthracis","volume":"59","author":"Abdel-Glil","year":"2021","journal-title":"J. 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