vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-68vavilov-3477Research ArticleГЕНЕТИКА МИКРООРГАНИЗМОВMICROBIAL GENETICSДифференциация штаммов Bacillus anthracis на основе SNP- и VNTR-полиморфизма геномовBacillus anthracis strain differentiation based on SNP and VNTR locihttps://orcid.org/0000-0002-0416-6193АнисимоваЕ. А.AnisimovaE. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

elizaveta-real@mail.ruhttps://orcid.org/0000-0002-6522-7930ФахрутдиновН. А.FakhrutdinovN. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0002-4709-314XМиргазовД. А.MirgazovD. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0002-6919-4064ДодоноваЕ. А.DodonovaE. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0003-0802-435XЕлизароваИ. А.ElizarovaI. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0002-0707-2117ГорбуноваМ. Е.GorbunovaM. E.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0001-5669-1486ХаммадовН. И.KhammadovN. I.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0002-3086-4921ЗайнуллинЛ. И.ZainullinL. I.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

https://orcid.org/0000-0003-2763-8605ОсянинК. А.OsyaninK. A.

Научный городок-2, Казань

Nauchny Gorodok-2, Kazan

Федеральный центр токсикологической, радиационной и биологической безопасностиРоссияFederal Center for Toxicological, Radiation and Biological SafetyRussian Federation202209102022266560567Copyright © Анисимова Е.А., Фахрутдинов Н.А., Миргазов Д.А., Додонова Е.А., Елизарова И.А., Горбунова М.Е., Хаммадов Н.И., Зайнуллин Л.И., Осянин К.А., 20222022Анисимова Е.А., Фахрутдинов Н.А., Миргазов Д.А., Додонова Е.А., Елизарова И.А., Горбунова М.Е., Хаммадов Н.И., Зайнуллин Л.И., Осянин К.А.Anisimova E.A., Fakhrutdinov N.A., Mirgazov D.A., Dodonova E.A., Elizarova I.A., Gorbunova M.E., Khammadov N.I., Zainullin L.I., Osyanin K.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3477

Bacillus anthracis являются возбудителем сибирской язвы. Для эпидемиологии этой инфек- ции имеет значение не только идентификация этиологического агента, но и выяснение закономерности его эволюции и распространения. Современные методы молекулярной биологии позволяют определить ряд генетических маркеров, пригодных для индикации и дифференциации штаммов B. anthracis. К таким маркерам относят VNTR-локусы – последовательности, организованные в геноме в виде тандемных повторов, а также SNP – отличия в последовательности ДНК в сравниваемых локусах размером в один нуклеотид. Целью настоящей работы была оценка эффективности совместного применения SNP-анализа и ПЦР-амплификации VNTR-локусов с анализом температуры плавления ампликонов высокого разрешения для идентификации и дифференциации штаммов возбудителя сибирской язвы. Исследовали семь штаммов B. anthracis, полученных из образцов почвы и трупов животных, в качестве референс-микроорганизма был вакцинный штамм B. anthracis СТИ-1. Для молекулярно-генетической характеристики данных бактерий проведен анализ 12 однонуклеотидных полиморфизмов, а также вариабельности восьми VNTR-локусов, для определения различий в которых был впервые использован метод определения температур плавления ПЦР-продуктов в присутствии интеркалирующего красителя EvaGreen (ЗАО «Синтол», Россия). Для детекции SNP применен метод полимеразной цепной реакции (ПЦР) с использованием двойных TaqMan-зондов. Обнаружено, что все изучаемые вирулентные штаммы, кроме B. anthracis № 1 и 3, по SNP-профилю не могут быть отнесены к какой-либо филогенетической подгруппе возбудителя сибирской язвы. Методический подход, включающий в себя анализ SNP- и VNTR-последовательностей, позволил дифференцировать между собой штаммы B. anthracis № 1–4, в то время как бактерии B. anthracis № 5–7 демонстрируют одинаковые SNP- и HRM-профили и, как следствие, формируют один кластер. Таким образом, показана принци- пиальная возможность использования рассмотренной в этой работе методики для предварительного анализа эпизоотической ситуации при вспышках сибирской язвы.

Bacillus anthracis is the anthrax causative agent. For its epidemiology, it is important not only to identify the etiological agent but also to determine the patterns of its evolution and spread. Modern methods of molecular biology make it possible to detect a number of genetic markers suitable for indicating and differentiating the strains of B. anthracis, including the loci arranged as variable number tandem repeats (VNTRs) and SNPs, one nucleotide-sized differences in the DNA sequence of the loci being compared. The objective of the present study was to examine the effectiveness of SNP analysis and PCR amplif ication of VNTR loci combined with the high-resolution amplicon melting analysis for identif ication and differentiation of the anthrax agent strains. In the study, seven strains of B. anthracis obtained from soil samples and animal carcasses were investigated using vaccine strain STI-1 as a reference. For molecular genetic characterization of these bacteria, analysis of 12 SNPs and variability analysis of eight VNTR loci were carried out. To detect the differences between the strains, their PCR product melting points were measured in the presence of the EvaGreen (Sintol, Russia) intercalating dye. For SNP detection, a PCR assay with double TaqMan probes was applied. It was found that the studied virulent strains, except for B. anthracis No. 1 and 3, could not be attributed to any phylogenetic subgroup of the anthrax agents. The proposed method made it possible to differentiate four out of the seven investigated strains. Strains No. 5–7 had identical SNP and HRM prof iles and, as a result, formed a single cluster. Our investigation has conf irmed that the proposed method can be successfully used for preliminary analysis of an epizootic situation in the case of anthrax.

Bacillus anthracisгенотипированиеVNTRSNPHRM-анализэпидемиология сибирской язвыBacillus anthracisgenotypingVNTRSNPHRM-analysisepidemiology of anthraxThe research was carried out with the support of the Federal Center for Toxicological, Radiation, and Biological Safety.ReferencesAchtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu. Rev. Microbiol. 2008;62:53-70. DOI 10.1146/annurev.micro.62.081307.162832.Achtman M. Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. Annu. Rev. Microbiol. 2008;62:53-70. DOI 10.1146/annurev.micro.62.081307.162832.Afanas’ev M.V., Kravets E.V., Dugarzhapova Z.F., Takaishvili V.E., Polovinkina V.S., Balakhonov S.V. Comparative multilocus VNTR and SNP analysis of Bacillus anthracis vaccine strains. Mol. Genet. Microbiol. Virol. 2014;29(2):86-92. DOI 10.3103/S0891416814020025.Afanas’ev M.V., Kravets E.V., Dugarzhapova Z.F., Takaishvili V.E., Polovinkina V.S., Balakhonov S.V. Comparative multilocus VNTR and SNP analysis of Bacillus anthracis vaccine strains. Mol. Genet. Microbiol. Virol. 2014;29(2):86-92. DOI 10.3103/S0891416814020025.Birdsell D.N., Pearson T., Price E.P., Hornstra H.M., Nera R.D., Stone N., Gruendike J., Kaufman E.L., Pettus A.H., Hurbon A.N., Buchhagen J.L., Harms N.J., Chanturia G., Gyuranecz M., Wagner D.M., Keim P.S. Melt analysis of mismatch amplification mutation assays Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS One. 2012;7(3):e32866. DOI 10.1371/journal.pone.0032866.Birdsell D.N., Pearson T., Price E.P., Hornstra H.M., Nera R.D., Stone N., Gruendike J., Kaufman E.L., Pettus A.H., Hurbon A.N., Buchhagen J.L., Harms N.J., Chanturia G., Gyuranecz M., Wagner D.M., Keim P.S. Melt analysis of mismatch amplification mutation assays Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS One. 2012;7(3):e32866. DOI 10.1371/journal.pone.0032866.Bondareva O.S., Savchenko S.S., Tkachenko G.A., Abueva A.I., Muratova Yu.O., Antonov V.A. Modern approaches to the genotyping of causative agents of particularly dangerous infections. Epidemiologiya i Infektsionnye Bolezni = Epidemiology and Infectious Diseases. 2014;1:34-44. (in Russian)Bondareva O.S., Savchenko S.S., Tkachenko G.A., Abueva A.I., Muratova Yu.O., Antonov V.A. Modern approaches to the genotyping of causative agents of particularly dangerous infections. Epidemiologiya i Infektsionnye Bolezni = Epidemiology and Infectious Diseases. 2014;1:34-44. (in Russian)Chang C.H., Chang Y.C., Underwood A. VNTRDB: a bacterial variable number tandem repeat locus database. Nucleic Acids Res. 2007; 35(Database issue):D416-D421. DOI 10.1093/nar/gkl872.Chang C.H., Chang Y.C., Underwood A. VNTRDB: a bacterial variable number tandem repeat locus database. Nucleic Acids Res. 2007; 35(Database issue):D416-D421. DOI 10.1093/nar/gkl872.Derzelle S., Laroche S., Le Flèche P., Hauck Y., Thierry S., Vergnaud G., Madani N. Characterization of genetic diversity of Bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis. J. Clin. Microbiol. 2011;49(12):4286-4292. DOI 10.1128/JCM.05439-11.Derzelle S., Laroche S., Le Flèche P., Hauck Y., Thierry S., Vergnaud G., Madani N. Characterization of genetic diversity of Bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis. J. Clin. Microbiol. 2011;49(12):4286-4292. DOI 10.1128/JCM.05439-11.Eremenko E.I., Pisarenko S.V., Aksenova L.Yu., Ryazanova A.G., Zhirov A.M., Semenova O.V., Bobrysheva O.V., Kovalev D.A., Kulichenko A.N. Phylogenetics, evolution, and phylogeography of Bacillus anthracis. Bakteriologiya = Bacteriology. 2018;3(2):57-63. DOI 10.20953/2500-1027-2018-2-57-63. (in Russian)Eremenko E.I., Pisarenko S.V., Aksenova L.Yu., Ryazanova A.G., Zhirov A.M., Semenova O.V., Bobrysheva O.V., Kovalev D.A., Kulichenko A.N. Phylogenetics, evolution, and phylogeography of Bacillus anthracis. Bakteriologiya = Bacteriology. 2018;3(2):57-63. DOI 10.20953/2500-1027-2018-2-57-63. (in Russian)Eremenko E.I., Ryazanova A.G., Tsygankova O.I., Tsygankova E.A., Buravtseva N.P., Kulitchenko A.N. Genotype diversity of Bacillus anthracis strains isolated from the Сaucasus region. Mol. Genet. Microbiol. Virol. 2012;27(2):74-78. DOI 10.3103/S0891416812020024.Eremenko E.I., Ryazanova A.G., Tsygankova O.I., Tsygankova E.A., Buravtseva N.P., Kulitchenko A.N. Genotype diversity of Bacillus anthracis strains isolated from the Сaucasus region. Mol. Genet. Microbiol. Virol. 2012;27(2):74-78. DOI 10.3103/S0891416812020024.Gierczynski R., Jackubczak A., Jagielski M. Extended multiple-locus variable-number tandem-repeat analysis of Bacillus anthracis strains isolated in Poland. Pol. J. Microbiol. 2009;58(1):3-7.Gierczynski R., Jackubczak A., Jagielski M. Extended multiple-locus variable-number tandem-repeat analysis of Bacillus anthracis strains isolated in Poland. Pol. J. Microbiol. 2009;58(1):3-7.Graham R., Liew M., Meadows C., Lyon E., Wittwer C.T. Distinguishing different DNA heterozygotes by high-resolution melting. Clin. Chem. 2005;51(7):1295-1298. DOI 10.1373/clinchem.2005.051516.Graham R., Liew M., Meadows C., Lyon E., Wittwer C.T. Distinguishing different DNA heterozygotes by high-resolution melting. Clin. Chem. 2005;51(7):1295-1298. DOI 10.1373/clinchem.2005.051516.Hugh-Jones M., Blackburn J. The ecology of Bacillus anthracis. Mol. Aspects Med. 2009;30(6):356-367. DOI 10.1016/j.mam.2009.08.03.Hugh-Jones M., Blackburn J. The ecology of Bacillus anthracis. Mol. Aspects Med. 2009;30(6):356-367. DOI 10.1016/j.mam.2009.08.03.Jackson P.J., Hill K.K., Laker M.T., Ticknor L.O., Keim P. Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis. J. Appl. Microbiol. 1999;87(2):263-269. DOI 10.1046/j.1365-2672.1999.00884.x.Jackson P.J., Hill K.K., Laker M.T., Ticknor L.O., Keim P. Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis. J. Appl. Microbiol. 1999;87(2):263-269. DOI 10.1046/j.1365-2672.1999.00884.x.Jackson P.J., Walthers E.A., Kalif A.S., Richmond K.L., Adair D.M., Hill K.K., Kuske C.R., Andersen G.L., Wilson K.H., Hugh-Jones M., Keim P. Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates. Appl. Environ. Microbiol. 1997;63(4):1400-1405. DOI 10.1128/aem.63.4.1400-1405.1997.Jackson P.J., Walthers E.A., Kalif A.S., Richmond K.L., Adair D.M., Hill K.K., Kuske C.R., Andersen G.L., Wilson K.H., Hugh-Jones M., Keim P. Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates. Appl. Environ. Microbiol. 1997;63(4):1400-1405. DOI 10.1128/aem.63.4.1400-1405.1997.Keim P., Gruendike J.M., Klevytska A.M., Schupp J., Challacombe J., Okinaka R. The genome and variation of Bacillus anthracis. Mol. Aspects Med. 2009;30(6):397-405. DOI 10.1016/j.mam.2009.08.005.Keim P., Gruendike J.M., Klevytska A.M., Schupp J., Challacombe J., Okinaka R. The genome and variation of Bacillus anthracis. Mol. Aspects Med. 2009;30(6):397-405. DOI 10.1016/j.mam.2009.08.005.Keim P., Price L.B., Klevytska A.M., Smith K.L., Schupp J.M., Okinaka R., Jackson P.J., Hugh-Jones M.E. Multiple-locus variablenumber tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 2000;182(10):2928-2936. DOI 10.1128/JB.182.10.2928-2936.2000.Keim P., Price L.B., Klevytska A.M., Smith K.L., Schupp J.M., Okinaka R., Jackson P.J., Hugh-Jones M.E. Multiple-locus variablenumber tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 2000;182(10):2928-2936. DOI 10.1128/JB.182.10.2928-2936.2000.Keim P., Van Ert M.N., Pearson T., Vogler A.J., Huynh L.Y., Wagner D.M. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect. Genet. Evol. 2004;4(3):205-213. DOI 10.1016/j.meegid.2004.02.005.Keim P., Van Ert M.N., Pearson T., Vogler A.J., Huynh L.Y., Wagner D.M. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect. Genet. Evol. 2004;4(3):205-213. DOI 10.1016/j.meegid.2004.02.005.Kenefic L., Pearson T., Okinaka R., Schupp J., Wagner D., Ravel J., Hoffmaster A., Trim C.P., Chung W.-K., Beaudry J.A., Foster J., Mead J., Keim P. Pre-Columbian origins for North American anthrax. PLoS One. 2009;4(3):e4813. DOI 10.1371/journal.pone.0004813.Kenefic L., Pearson T., Okinaka R., Schupp J., Wagner D., Ravel J., Hoffmaster A., Trim C.P., Chung W.-K., Beaudry J.A., Foster J., Mead J., Keim P. Pre-Columbian origins for North American anthrax. PLoS One. 2009;4(3):e4813. DOI 10.1371/journal.pone.0004813.Koteneva E.A., Tsygankova O.I., Kalinin A.V., Abramovich A.V. The spectrum of canSNP genotypes as an indication of intraspecific genetic and phenotypic diversity of Bacillus anthracis strains isolated in North Caucasus and in neighboring areas. Meditsinskiy Vestnik Severnogo Kavkaza = Medical News of North Caucasus. 2019; 14(4):580-582. DOI 10.14300/mnnc.2019.14144. (in Russian)Koteneva E.A., Tsygankova O.I., Kalinin A.V., Abramovich A.V. The spectrum of canSNP genotypes as an indication of intraspecific genetic and phenotypic diversity of Bacillus anthracis strains isolated in North Caucasus and in neighboring areas. Meditsinskiy Vestnik Severnogo Kavkaza = Medical News of North Caucasus. 2019; 14(4):580-582. DOI 10.14300/mnnc.2019.14144. (in Russian)Kravets E.V., Dugarzhapova Z.F., Takaishvili V.E., Ivanova T.A., Chesnokova M.V., Balakhonov S.V. Biological and molecular properties of Bacillus anthracis strains isolated in Siberia and the Russian Far East (1959–2013). Problemy Osobo Opasnykh Infektsiy = Problems of Particularly Dangerous Infections. 2018;3:54-59. DOI 10.21055/0370-1069-2018-3-54-59. (in Russian)Kravets E.V., Dugarzhapova Z.F., Takaishvili V.E., Ivanova T.A., Chesnokova M.V., Balakhonov S.V. Biological and molecular properties of Bacillus anthracis strains isolated in Siberia and the Russian Far East (1959–2013). Problemy Osobo Opasnykh Infektsiy = Problems of Particularly Dangerous Infections. 2018;3:54-59. DOI 10.21055/0370-1069-2018-3-54-59. (in Russian)Le Flèche P., Hauck Y., Onteniente L., Prieur A., Denoeud F., Ramisse V., Sylvestre P., Benson G., Ramisse F., Vergnaud G. A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis. BMC Microbiol. 2001;1:2. DOI 10.1186/1471-2180-1-2.Le Flèche P., Hauck Y., Onteniente L., Prieur A., Denoeud F., Ramisse V., Sylvestre P., Benson G., Ramisse F., Vergnaud G. A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis. BMC Microbiol. 2001;1:2. DOI 10.1186/1471-2180-1-2.Logvin F.V., Kondratenko T.A., Vodyanitskya S.Y. Anthrax in the world, CIS, and Russian Federation (literature review). Meditsinskiy Vestnik Yuga Rossii = Medical Herald of the South of Russia. 2017; 8(3):17-22. DOI 10.21886/2219-8075-2017-8-3-17-22.Logvin F.V., Kondratenko T.A., Vodyanitskya S.Y. Anthrax in the world, CIS, and Russian Federation (literature review). Meditsinskiy Vestnik Yuga Rossii = Medical Herald of the South of Russia. 2017; 8(3):17-22. DOI 10.21886/2219-8075-2017-8-3-17-22.Margraf R.L., Mao R., Highsmith W.E., Holtegaard L.M., Wittwer C.T. Mutation scanning of the RET protooncogene using high-resolution melting analysis. Clin. Chem. 2006;52(1):138-141. DOI 10.1373/clinchem.2005.052951.Margraf R.L., Mao R., Highsmith W.E., Holtegaard L.M., Wittwer C.T. Mutation scanning of the RET protooncogene using high-resolution melting analysis. Clin. Chem. 2006;52(1):138-141. DOI 10.1373/clinchem.2005.052951.MUK 4.2.2941-11 Organization and Protocol of Anthrax Laboratory Diagnostics in Municipal, Regional, and Federal Laboratories: Methodological Guidelines. Moscow: Rospotrebnadzor Federal Hygiene and Epidemiology Center, 2011. (in Russian)MUK 4.2.2941-11 Organization and Protocol of Anthrax Laboratory Diagnostics in Municipal, Regional, and Federal Laboratories: Methodological Guidelines. Moscow: Rospotrebnadzor Federal Hygiene and Epidemiology Center, 2011. (in Russian)Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Yu., Dugarzhapova Z.F., Evchenko A.Yu., Kravets E.V., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Volynkina A.S., Balakhonov S.V., Kulichenko A.N. Phylogenetic analysis of Bacillus anthracis strains from Western Siberia reveals a new genetic cluster in the global population of the species. BMC Genomics. 2019;20(1):692. DOI 10.1186/s12864-019-6060-z.Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Yu., Dugarzhapova Z.F., Evchenko A.Yu., Kravets E.V., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Volynkina A.S., Balakhonov S.V., Kulichenko A.N. Phylogenetic analysis of Bacillus anthracis strains from Western Siberia reveals a new genetic cluster in the global population of the species. BMC Genomics. 2019;20(1):692. DOI 10.1186/s12864-019-6060-z.Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 1989.Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 1989.Selander R.K., Caugant D.A., Ochman H., Musser J.M., Gilmour M.N., Whittam T.S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 1986;51(5):873-884. DOI 10.1128/aem.51.5.873-884.1986.Selander R.K., Caugant D.A., Ochman H., Musser J.M., Gilmour M.N., Whittam T.S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 1986;51(5):873-884. DOI 10.1128/aem.51.5.873-884.1986.Thierry S., Tourterel C., Le Flèche P., Derzelle S., Dekhil N., Mendy Ch., Colaneri C., Vergnaud G., Madani N. Genotyping of french Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database. PLoS One. 2014;9(6):e95131. DOI 10.1371/journal.pone.0095131.Thierry S., Tourterel C., Le Flèche P., Derzelle S., Dekhil N., Mendy Ch., Colaneri C., Vergnaud G., Madani N. Genotyping of french Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database. PLoS One. 2014;9(6):e95131. DOI 10.1371/journal.pone.0095131.Timofeev V.S., Bakhteeva I.V., Dyatlov I.A. Genotyping of Bacillus anthracis and closely related microorganisms. Russ. J. Genet. 2018;54(1):1-11. DOI 10.1134/S1022795418010118.Timofeev V.S., Bakhteeva I.V., Dyatlov I.A. Genotyping of Bacillus anthracis and closely related microorganisms. Russ. J. Genet. 2018;54(1):1-11. DOI 10.1134/S1022795418010118.Van Ert M.N., Easterday W.R., Huynh L.Y., Okinaka R.T., Hugh-Jones M.E., Ravel J., Zanecki S.R., Pearson T., Simonson T.S., U’Ren J.M., Kachur S.M., Leadem-Dougherty R.R., Rhoton S.D., Zinser G., Farlow J., Coker P.R., Smith K.L., Wang B., Kenefic L.J., Fraser-Liggett C.M., Wagner D.M., Keim P. Global genetic population structure of Bacillus anthracis. PLoS One. 2007;2(5):e461. DOI 10.1371/journal.pone.0000461.Van Ert M.N., Easterday W.R., Huynh L.Y., Okinaka R.T., Hugh-Jones M.E., Ravel J., Zanecki S.R., Pearson T., Simonson T.S., U’Ren J.M., Kachur S.M., Leadem-Dougherty R.R., Rhoton S.D., Zinser G., Farlow J., Coker P.R., Smith K.L., Wang B., Kenefic L.J., Fraser-Liggett C.M., Wagner D.M., Keim P. Global genetic population structure of Bacillus anthracis. PLoS One. 2007;2(5):e461. DOI 10.1371/journal.pone.0000461.Wang D., Wang B., Zhu L., Wu S., Lyu Y., Feng E., Pan C., Jiao L., Cui Y., Liu X., Wang H. Genotyping and population diversity of Bacillus anthracis in China based on MLVA and canSNP analysis. Microbiol. Res. 2020;233:126414. DOI 10.1016/j.micres.2020.126414.Wang D., Wang B., Zhu L., Wu S., Lyu Y., Feng E., Pan C., Jiao L., Cui Y., Liu X., Wang H. Genotyping and population diversity of Bacillus anthracis in China based on MLVA and canSNP analysis. Microbiol. Res. 2020;233:126414. DOI 10.1016/j.micres.2020.126414.Winchell J.M., Wolff B.J., Tiller R., Bowen M.D., Hoffmaster A.R. Rapid identification and discrimination of Brucella isolates by use of real-time PCR and high-resolution melt analysis. J. Clin. Microbiol. 2010;48(3):697-702. DOI 10.1128/JCM.02021-09.Winchell J.M., Wolff B.J., Tiller R., Bowen M.D., Hoffmaster A.R. Rapid identification and discrimination of Brucella isolates by use of real-time PCR and high-resolution melt analysis. J. Clin. Microbiol. 2010;48(3):697-702. DOI 10.1128/JCM.02021-09.

The authors declare that there are no conflicts of interest present.