{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T08:35:26Z","timestamp":1773390926605,"version":"3.50.1"},"reference-count":49,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2021,11,12]],"date-time":"2021-11-12T00:00:00Z","timestamp":1636675200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Bundeswehr Joint Medical Service","award":["Medical Biological Defense Research Program of the Bundeswehr Joint Medical Service"],"award-info":[{"award-number":["Medical Biological Defense Research Program of the Bundeswehr Joint Medical Service"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium\u2019s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2\u20135 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies\/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis\u2013specific 16S rRNA gene alleles afforded \u22642 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies\/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.<\/jats:p>","DOI":"10.3390\/ijms222212224","type":"journal-article","created":{"date-parts":[[2021,11,12]],"date-time":"2021-11-12T08:10:22Z","timestamp":1636704622000},"page":"12224","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts"],"prefix":"10.3390","volume":"22","author":[{"given":"Peter","family":"Braun","sequence":"first","affiliation":[{"name":"Bundeswehr Institute of Microbiology (IMB), 80937 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin Duy-Thanh","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Bundeswehr Institute of Microbiology (IMB), 80937 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3012-2626","authenticated-orcid":false,"given":"Mathias C.","family":"Walter","sequence":"additional","affiliation":[{"name":"Bundeswehr Institute of Microbiology (IMB), 80937 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8002-7718","authenticated-orcid":false,"given":"Gregor","family":"Grass","sequence":"additional","affiliation":[{"name":"Bundeswehr Institute of Microbiology (IMB), 80937 Munich, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Turnbull, P. (2008). Anthrax in Humans and Animals, WHO Press.","DOI":"10.1007\/978-1-4419-1108-7_4"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Ehling-Schulz, M., Lereclus, D., and Koehler, T.M. (2019). The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol. Spectr., 7.","DOI":"10.1128\/microbiolspec.GPP3-0032-2018"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1146\/annurev.micro.55.1.647","article-title":"Anthrax","volume":"55","author":"Mock","year":"2001","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3405","DOI":"10.1128\/IAI.01857-14","article-title":"Exposure to Bacillus anthracis capsule results in suppression of human monocyte-derived dendritic cells","volume":"82","author":"Jelacic","year":"2014","journal-title":"Infect. Immun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/j.resmic.2016.07.002","article-title":"Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria","volume":"168","author":"Sanchis","year":"2017","journal-title":"Res. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"S137","DOI":"10.1016\/j.ijfoodmicro.2010.07.039","article-title":"A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types","volume":"145","author":"Wielinga","year":"2011","journal-title":"Int. J. Food Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1111\/j.1574-6968.2002.tb11324.x","article-title":"Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR","volume":"214","author":"Ellerbrok","year":"2002","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1111\/j.1574-6968.1996.tb08548.x","article-title":"Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA","volume":"145","author":"Ramisse","year":"1996","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1016\/j.mcp.2008.06.001","article-title":"Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis","volume":"22","author":"Antwerpen","year":"2008","journal-title":"Mol. Cell. Probes"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1995","DOI":"10.1128\/JCM.43.4.1995-1997.2005","article-title":"Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis","volume":"43","author":"Easterday","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"671","DOI":"10.4161\/viru.26288","article-title":"In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences","volume":"4","author":"Hamidjaja","year":"2013","journal-title":"Virulence"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.mimet.2012.10.013","article-title":"Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR","volume":"92","author":"Straub","year":"2013","journal-title":"J. Microbiol. Methods"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e00065-18","DOI":"10.1128\/mSystems.00065-18","article-title":"Genomic characterization and copy number variation of Bacillus anthracis plasmids pXO1 and pXO2 in a historical collection of 412 strains","volume":"3","author":"Marston","year":"2018","journal-title":"mSystems"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4747","DOI":"10.1128\/JB.183.16.4747-4751.2001","article-title":"Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels","volume":"183","author":"Hansen","year":"2001","journal-title":"J. Bacteriol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Bremer, H., and Dennis, P.P. (2008). Modulation of chemical composition and other parameters of the cell at different exponential growth rates. EcoSal Plus, 3.","DOI":"10.1128\/ecosal.5.2.3"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1016\/j.jprot.2014.07.012","article-title":"Multi-enzyme digestion FASP and the \u2018Total Protein Approach\u2019-based absolute quantification of the Escherichia coli proteome","volume":"109","author":"Rakus","year":"2014","journal-title":"J. Proteom."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1128\/jb.105.1.20-27.1971","article-title":"Rate of ribosome production in Bacillus licheniformis","volume":"105","author":"Planta","year":"1971","journal-title":"J. Bacteriol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1261\/rna.5163104","article-title":"Interaction of the Bacillus subtilis RNase P with the 30S ribosomal subunit","volume":"10","author":"Barrera","year":"2004","journal-title":"RNA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1128\/AEM.01224-06","article-title":"Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR","volume":"73","author":"Matsuda","year":"2007","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Beissner, M., Symank, D., Phillips, R.O., Amoako, Y.A., Awua-Boateng, N.Y., Sarfo, F.S., Jansson, M., Huber, K.L., Herbinger, K.H., and Battke, F. (2012). Detection of viable Mycobacterium ulcerans in clinical samples by a novel combined 16S rRNA reverse transcriptase\/IS2404 real-time qPCR assay. PLoS Negl. Trop. Dis., 6.","DOI":"10.1371\/journal.pntd.0001756"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Beissner, M., Woestemeier, A., Saar, M., Badziklou, K., Maman, I., Amedifou, C., Wagner, M., Wiedemann, F.X., Amekuse, K., and Kobara, B. (2019). Development of a combined RLEP\/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples. BMC Infect. Dis., 19.","DOI":"10.1186\/s12879-019-4349-9"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1099\/00207713-41-3-343","article-title":"Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA","volume":"41","author":"Ash","year":"1991","journal-title":"Int. J. Syst. Bacteriol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1099\/mic.0.26870-0","article-title":"Two distinct types of rRNA operons in the Bacillus cereus group","volume":"150","author":"Candelon","year":"2004","journal-title":"Microbiology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2749","DOI":"10.1128\/JCM.01267-16","article-title":"Identification and analysis of informative single nucleotide polymorphisms in 16S rRNA gene sequences of the Bacillus cereus group","volume":"54","author":"Hakovirta","year":"2016","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Braun, P., Zimmermann, F., Walter, M.C., Mantel, S., Aistleitner, K., St\u00fcrz, I., Grass, G., and Stoecker, K. (2021). In-depth analysis of Bacillus anthracis 16S rRNA genes and transcripts reveals intra- and intergenomic diversity and facilitates anthrax detection. bioRxiv.","DOI":"10.1101\/2021.08.02.454851"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1007\/s00253-010-2848-0","article-title":"Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples","volume":"88","author":"Irenge","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e01501-16","DOI":"10.1128\/mBio.01501-16","article-title":"A Bacillus anthracis genome sequence from the Sverdlovsk 1979 autopsy specimens","volume":"7","author":"Sahl","year":"2016","journal-title":"mBio"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Antwerpen, M., Beyer, W., Bassy, O., Ortega-Garc\u00eda, M.V., Cabria-Ramos, J.C., Grass, G., and W\u00f6lfel, R. (2019). Phylogenetic placement of isolates within the Trans-Eurasian clade A.Br.008\/009 of Bacillus anthracis. Microorganisms, 7.","DOI":"10.3390\/microorganisms7120689"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2542","DOI":"10.1099\/ijs.0.000298","article-title":"Sphingomonas zeae sp. nov., isolated from the stem of Zea mays","volume":"65","author":"Busse","year":"2015","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_30","first-page":"1869","article-title":"Bacterial and fungal genome detection PCR\/NAT: Comprehensive discussion of the June 2018 distribution for external quality assessment of nucleic acid-based protocols in diagnostic medical microbiology by INSTAND e.V., GMS Z Forder Qualitatssich","volume":"10","author":"Reischl","year":"2019","journal-title":"Med. Lab."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Braun, P., Grass, G., Aceti, A., Serrecchia, L., Affuso, A., Marino, L., Grimaldi, S., Pagano, S., Hanczaruk, M., and Georgi, E. (2015). Microevolution of anthrax from a young ancestor (M.A.Y.A.) suggests a soil-borne life cycle of Bacillus anthracis. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0135346"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3607","DOI":"10.1016\/S0040-4020(98)00094-5","article-title":"LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition","volume":"54","author":"Koshkin","year":"1998","journal-title":"Tetrahedron"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.mcp.2003.12.004","article-title":"Detection by 5\u2019-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world\u2019s most frequent clinical cases","volume":"18","author":"Perelle","year":"2004","journal-title":"Mol. Cell. Probes"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"12947","DOI":"10.1073\/pnas.93.23.12947","article-title":"Efficiency of DNA replication in the polymerase chain reaction","volume":"93","author":"Stolovitzky","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.bdq.2017.04.001","article-title":"Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR)","volume":"12","author":"Forootan","year":"2017","journal-title":"Biomol. Detect. Quantif."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.femsle.2004.09.042","article-title":"Oligonucleotide microarray for identification of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA","volume":"240","author":"Schmidt","year":"2004","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1128\/AEM.72.2.1295-1301.2006","article-title":"Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis","volume":"72","author":"Daffonchio","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1128\/AEM.69.1.33-40.2003","article-title":"Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S-23S ribosomal DNA intergenic transcribed spacers containing tRNA genes","volume":"69","author":"Cherif","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.mimet.2009.04.005","article-title":"Use of molecular beacons and multi-allelic real-time PCR for detection of and discrimination between virulent Bacillus anthracis and other Bacillus isolates","volume":"78","author":"Hadjinicolaou","year":"2009","journal-title":"J. Microbiol. Methods"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3317","DOI":"10.1128\/AEM.69.6.3317-3326.2003","article-title":"Fluorescent heteroduplex assay for monitoring Bacillus anthracis and close relatives in environmental samples","volume":"69","author":"Merrill","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"731","DOI":"10.2144\/05385ST03","article-title":"Specific detection of Bacillus anthracis using a TaqMan mismatch amplification mutation assay","volume":"38","author":"Easterday","year":"2005","journal-title":"Biotechniques"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1111\/j.1365-2672.2005.02764.x","article-title":"Bacillus cereus is common in the environment but emetic toxin producing isolates are rare","volume":"100","author":"Altayar","year":"2006","journal-title":"J. Appl. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Mancilla, M., Ulloa, M., L\u00f3pez-Go\u00f1i, I., Moriy\u00f3n, I., and Mar\u00eda Z\u00e1rraga, A. (2011). Identification of new IS711 insertion sites in Brucella abortus field isolates. BMC Microbiol., 11.","DOI":"10.1186\/1471-2180-11-176"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.vetmic.2008.12.023","article-title":"Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes","volume":"137","author":"Bounaadja","year":"2009","journal-title":"Veter- Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Klee, S.R., Tyczka, J., Ellerbrok, H., Franz, T., Linke, S., Baljer, G., and Appel, B. (2006). Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii. BMC Microbiol., 6.","DOI":"10.1186\/1471-2180-6-2"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Backstedt, B.T., Buyuktanir, O., Lindow, J., Wunder, E.A., Reis, M.G., Usmani-Brown, S., Ledizet, M., Ko, A., and Pal, U. (2015). Efficient detection of pathogenic Leptospires using 16S ribosomal RNA. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0128913"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"5440","DOI":"10.1128\/AEM.03132-09","article-title":"Detection of human intestinal catalase-negative, Gram-positive cocci by rRNA-targeted reverse transcription-PCR","volume":"76","author":"Kubota","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.3389\/fmicb.2018.01417","article-title":"Counting the countless: Bacterial quantification by targeting rRNA molecules to explore the human gut microbiota in health and disease","volume":"9","author":"Tsuji","year":"2018","journal-title":"Front. Microbiol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/22\/12224\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:29:00Z","timestamp":1760167740000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/22\/12224"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,12]]},"references-count":49,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["ijms222212224"],"URL":"https:\/\/doi.org\/10.3390\/ijms222212224","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.09.20.21263746","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,12]]}}}