Biopolym. Cell. 2025; 41(3):171.
http://dx.doi.org/10.7124/bc.000B24
Reviews
Challenges in PCR detection of Bacillus anthracis
1Skorobogatov O. Yu., 1Melnichuk N. S., 1Tkachuk Z. Yu.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

Bacillus anthracis is large Gram-positive bacteria which causes anthrax – one of the deadliest known diseases. Different approaches have been developed to detect it, from more precise, time-consuming culture-based methods to more rapid PCR-based methods. The latter rely on the identification of specific regions in the B. anthracis genome, which is composed of two plasmids, pXO1 and pXO2, and the chromosome. Targeting only plasmid markers may provide unreliable results due to high similarities between B. anthracis and other genetically related species of Bacillus cereus sensu lato group, which necessitates the use of additional chromosomal targets. This article aims to provide a brief non- exhaustive overview on the relevant genetic markers used as targets for PCR-based detection of B. anthracis.
Keywords: Bacillus anthracis, genetic markers, PCR
Full text: (PDF, in English)

References

[1] Bradford H. Anthrax: blains upon man. Antimicrob Infect Dis Newsl. 2000; 18(9):65-71.
[2] Dirckx JH. Virgil on anthrax. Am J Dermatopathol. 1981; 3(2):191-5.
[3] Cieslak TJ, Eitzen EM Jr. Clinical and Epidemiologic Principles of Anthrax. Emerg Infect Dis. 1999; 5(4):552-5.
[4] Berry D. Pierre-François Olive Rayer: Biography. Med Hist Suppl. 2005;(24):7-13.
[5] Barker AE. On Some Points regarding the Distribution of Bacillus Anthracis in the Human Skin in Malignant Pustule. Med Chir Trans. 1886; 69:127-134.1.
[6] Tomkins H. Note on the Cultivation of Bacillus Anthracis. Br Med J. 1887; 1(1363):328-9.
[7] Hardy WB, Keng LB. On the Changes in the Number and Character of the Wandering Cells of the Frog induced by the presence of Urari or of Bacillus Anthracis. J Physiol. 1893; 15(4):361-400.1.
[8] Burke V, Skinner H. Resistance of bacterial spores to the triphenylmethane dyes. J Exp Med. 1925; 41(4):471-7.
[9] Zozaya J. A serological study of the polysaccharides of meningococcus, B. anthracis, B. proteus, B. subtilis and B. mesentericus. J Exp Med. 1931; 54(5):725-32.
[10] Stein CD, Rogers H. Observations on the resistance of anthrax spores to heat. Vet Med. 1945; 40:406-10.
[11] Ravel J, Jiang L, Stanley ST, Wilson MR, Decker RS, Read TD, Worsham P, Keim PS, Salzberg SL, Fraser-Liggett CM, Rasko DA. The complete genome sequence of Bacillus anthracis Ames "Ancestor". J Bacteriol. 2009; 191(1):445-6.
[12] Okinaka RT, Cloud K, Hampton O, Hoffmaster AR, Hill KK, Keim P, Koehler TM, Lamke G, Kumano S, Mahillon J, Manter D, Martinez Y, Ricke D, Svensson R, Jackson PJ. Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol. 1999; 181(20):6509-15.
[13] Okinaka RT, Cloud K, Hampton O, Hoffmaster A, Hill K, Keim P, Koehler T, Lamke G, Kumano S, Manter D, Martinez Y, Ricke D, Svensson R, Jackson P. Sequence, assembly and analysis of pX01 and pX02. J Appl Microbiol. 1999; 87(2):261-2.
[14] Zasada AA. Detection and Identification of Bacillus anthracis: From Conventional to Molecular Microbiology Methods. Microorganisms. 2020; 8(1):125.
[15] Zhu H, Zhang H, Xu Y, Laššáková S, Korabečná M, Neužil P. PCR past, present and future. Biotechniques. 2020; 69(4):317-25.
[16] Metzker M, Caskey C. Polymerase Chain Reaction (PCR). eLS, John Wiley & Sons, Ltd (Ed). 2009.
[17] Turnbull PC. Definitive identification of Bacillus anthracis--a review. J Appl Microbiol. 1999; 87(2):237-40.
[18] Marston CK, Beesley C, Helsel L, Hoffmaster AR. Evaluation of two selective media for the isolation of Bacillus anthracis. Lett Appl Microbiol. 2008; 47(1):25-30.
[19] Mechaly A, Vitner E, Levy H, Weiss S, Bar-David E, Gur D, Koren M, Cohen H, Cohen O, Mamroud E, Fisher M. Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays. J Clin Microbiol. 2018; 56(4):e01479-17.
[20] Kuehn A, Kovác P, Saksena R, Bannert N, Klee SR, Ranisch H, Grunow R. Development of antibodies against anthrose tetrasaccharide for specific detection of Bacillus anthracis spores. Clin Vaccine Immunol. 2009; 16(12):1728-37.
[21] Morawska K, Sikora T, Nakonieczna A, Tyśkiewicz R, Wiśnik-Sawka M, Osuchowski Ł, Osuchowska P, Grabka M, Witkiewicz Z. Electrochemical Immunodetection of Bacillus anthracis Spores. Sensors. 2025; 25(19):5948.
[22] Brukner I, Oughton M. Direct PCR for Rapid and Safe Pathogen Detection: Laboratory Evaluation Supporting Field Use in Infectious Disease Outbreak. LabMed. 2025; 2(3):12.
[23] Carl M, Hawkins R, Coulson N, Lowe J, Robertson DL, Nelson WM, Titball RW, Woody JN. Detection of spores of Bacillus anthracis using the polymerase chain reaction. J Infect Dis. 1992; 165(6):1145-8.
[24] Makino SI, Iinuma-Okada Y, Maruyama T, Ezaki T, Sasakawa C, Yoshikawa M. Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction. J Clin Microbiol. 1993; 31(3):547-51.
[25] Reif TC, Johns M, Pillai SD, Carl M. Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format. Appl Environ Microbiol. 1994; 60(5):1622-5.
[26] Johns M, Harrington L, Titball RW, Leslie DL. Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction. Lett Appl Microbiol. 1994; 18(4):236-8.
[27] Beyer W, Glöckner P, Otto J, Böhm R. A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites. Microbiol Res. 1995; 150(2):179-86.
[28] Patra G, Sylvestre P, Ramisse V, Thérasse J, Guesdon JL. Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis. FEMS Immunol Med Microbiol. 1996; 15(4):223-31.
[29] Ramisse V, Patra G, Garrigue H, Guesdon JL, Mock M. Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA. FEMS Microbiol Lett. 1996; 145(1):9-16.
[30] Ramisse V, Patra G, Vaissaire J, Mock M. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community. J Appl Microbiol. 1999; 87(2):224-8.
[31] Jackson PJ, Hugh-Jones ME, Adair DM, Green G, Hill KK, Kuske CR, Grinberg LM, Abramova FA, Keim P. PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The presence of multiple Bacillus anthracis strains in different victims. Proc Natl Acad Sci U S A. 1998; 95(3):1224-9.
[32] Patra G, Vaissaire J, Weber-Levy M, Le Doujet C, Mock M. Molecular Characterization of BacillusStrains Involved in Outbreaks of Anthrax in France in 1997. J Clin Microbiol. 1998; 36(11):3412-4.
[33] Ash C, Farrow JA, Dorsch M, Stackebrandt E, Collins MD. Comparative Analysis of Bacillus anthracis, Bacillus cereus, and Related Species on the Basis of Reverse Transcriptase Sequencing of 16S rRNA. Int J Syst Bacteriol. 1991; 41(3):343-6.
[34] Jackson PJ, Walthers EA, Kalif AS, Richmond KL, Adair DM, Hill KK, Kuske CR, Andersen GL, Wilson KH, 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):1400-5.
[35] Ko KS, Kim JM, Kim JW, Jung BY, Kim W, Kim IJ, Kook YH. Identification of Bacillus anthracis by rpoB Sequence Analysis and Multiplex PCR. J Clin Microbiol. 2003; 41(7):2908-14.
[36] Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, Weyant RS, Popovic T. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis. Emerg Infect Dis. 2002; 8(10):1117-23.
[37] Qi Y, Patra G, Liang X, Williams LE, Rose S, Redkar RJ, DelVecchio VG. Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis. Appl Environ Microbiol. 2001; 67(8):3720-7.
[38] Brumlik MJ, Szymajda U, Zakowska D, Liang X, Redkar RJ, Patra G, Del Vecchio VG. Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracis Strains. Appl Environ Microbiol. 2001; 67(7):3021-8.
[39] Oggioni MR, Meacci F, Carattoli A, Ciervo A, Orru G, Cassone A, Pozzi G. Protocol for Real-Time PCR Identification of Anthrax Spores from Nasal Swabs after Broth Enrichment. J Clin Microbiol. 2002; 40(11):3956-63.
[40] Ellerbrok H, Nattermann H, Özel M, Beutin L, Appel B, Pauli G. Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol Lett. 2002; 214(1):51-9.
[41] Shin S, Ryu C, Oh H, Song C, Seong K. Evaluation of gyrB as Chromosomal Marker in Bacillus anthracis. J Bacteriol Virol. 2004; 34(3):191-200.
[42] La Duc T, Satomi M, Agata N, Venkateswaran K. gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group. J Microbiol Methods. 2004; 56(3):383-94.
[43] Hurtle W, Bode E, Kulesh DA, Kaplan RS, Garrison J, Bridge D, House M, Frye MS, Loveless B, Norwood D. Detection of the Bacillus anthracis gyrA Gene by Using a Minor Groove Binder Probe. J Clin Microbiol. 2004; 42(1):179-85.
[44] Turnbull PC, Hutson RA, Ward MJ, Jones MN, Quinn CP, Finnie NJ, Duggleby CJ, Kramer JM, Melling J. Bacillus anthracis but not always anthrax. J Appl Bacteriol. 1992; 72(1):21-8.
[45] Koehler TM. Bacillus anthracis genetics and virulence gene regulation. Curr Top Microbiol Immunol. 2002; 271:143-64.
[46] Bode E, Hurtle W, Norwood D. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis. J Clin Microbiol. 2004; 42(12):5825-31.
[47] Antwerpen MH, Zimmermann P, Bewley K, Frangoulidis D, Meyer H. Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis. Mol Cell Probes. 2008; 22(5-6):313-5.
[48] Radnedge L, Agron PG, Hill KK, Jackson PJ, Ticknor LO, Keim P, Andersen GL. Genome Differences That Distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol. 2003; 69(5):2755-64.
[49] Cieślik P, Knap J, Kolodziej M, Mirski T, Joniec J, Graniak G, Zakowska D, Winnicka I, Bielawska-Drózd A. Real-Time PCR Identification of Unique Bacillus anthracis Sequences. Folia Biol (Praha). 2015; 61(5):178-83.
[50] Wielinga PR, Hamidjaja RA, Ågren J, Knutsson R, Segerman B, Fricker M, Ehling-Schulz M, de Groot A, Burton J, Brooks T, Janse I, van Rotterdam B. A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types. Int J Food Microbiol. 2011; 145(Suppl 1):137-44.
[51] Aminu OR, Lembo T, Zadoks RN, Biek R, Lewis S, Kiwelu I, Mmbaga BT, Mshanga D, Shirima G, Denwood M, Forde TL. Practical and effective diagnosis of animal anthrax in endemic low-resource settings. PLoS Negl Trop Dis. 2020; 14(9):e0008655.
[52] Ågren J, Hamidjaja RA, Hansen T, Ruuls R, Thierry S, Vigre H, Janse I, Sundström A, Segerman B, Koene M, Löfström C, Van Rotterdam B, Derzelle S. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. Virulence. 2013; 4(8):671-85.
[53] Lekota KE, Hassim A, Mafofo J, Rees J, Muchadeyi FC, Van Heerden H, Madoroba E. Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho. J Infect Dev Ctries. 2016; 10(8):814-23.
[54] Zorigt T, Furuta Y, Paudel A, Kamboyi HK, Shawa M, Chuluun M, Sugawara M, Enkhtsetseg N, Enkhtuya J, Battsetseg B, Munyeme M, Hang'ombe BM, Higashi H. Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis. BMC Infect Dis. 2024; 24(1):942.
[55] Uhl JR, Bell CA, Sloan LM, Espy MJ, Smith TF, Rosenblatt JE, Cockerill FR 3rd. Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid Anthrax Test. Mayo Clin Proc. 2002; 77(7):673-80.
[56] King D, Luna V, Cannons A, Cattani J, Amuso P. Performance assessment of three commercial assays for direct detection of Bacillus anthracis spores. J Clin Microbiol. 2003; 41(7):3454-5.
[57] Fasanella A, Losito S, Trotta T, Adone R, Massa S, Ciuchini F, Chiocco D. Detection of anthrax vaccine virulence factors by polymerase chain reaction. Vaccine. 2001; 19(30):4214-8.
[58] Shangkuan YH, Chang YH, Yang JF, Lin HC, Shaio MF. Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD. Lett Appl Microbiol. 2001; 32(3):139-45.
[59] Moser MJ, Christensen DR, Norwood D, Prudent JR. Multiplexed detection of anthrax-related toxin genes. J Mol Diagn. 2006; 8(1):89-96.
[60] Kumar S, Tuteja U. Detection of virulence-associated genes in clinical isolates of bacillus anthracis by multiplex PCR and DNA probes. J Microbiol Biotechnol. 2009; 19(11):1475-81.
[61] Liang X, Zhang H, Zhang E, Wei J, Li W, Wang B, Dong S, Zhu J. Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains. Virulence. 2016; 7(5):578-86.
[62] Banger S, Pal V, Tripathi NK, Goel AK. Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax. Mol Biotechnol. 2021; 63(8):702-9.
[63] Riojas MA, Kiss K, McKee ML, Hazbón MH. Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids. Health Secur. 2015; 13(2):122-9.
[64] Bell CA, Uhl JR, Hadfield TL, David JC, Meyer RF, Smith TF, Cockerill FR 3rd. Detection of Bacillus anthracis DNA by LightCycler PCR. J Clin Microbiol. 2002; 40(8):2897-902.
[65] Cote CK, Rossi CA, Kang AS, Morrow PR, Lee JS, Welkos SL. The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb Pathog. 2005; 38(5-6):209-25.
[66] Elzi MV, Mallard K, Droz S, Bodmer T. Polyphasic Approach for Identifying Bacillus spp. J Clin Microbiol. 2005; 43(2):1010.
[67] Makino S, Cheun HI. Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores. J Microbiol Methods. 2003; 53(2):141-7.
[68] Wang SH, Wen JK, Zhou YF, Zhang ZP, Yang RF, Zhang JB, Chen J, Zhang XE. Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip. Biosens Bioelectron. 2004; 20(4):807-13.
[69] Olani A, Galante D, Lakew M, Wakjira BS, Mekonnen GA, Rufael T, Teklemariam T, Kumilachew W, Dejene S, Woldemeskel A, Wakjira A, Abichu G, Ashenafi B, Kebede N, Feleke Haile A, Bari FD, Del Sambro L, Eguale T. Identification of Bacillus anthracis Strains from Animal Cases in Ethiopia and Genetic Characterization by Whole-Genome Sequencing. Pathogens. 2025; 14(1):39.
[70] Ochai SO, Hassim A, Dekker EH, Magome T, Lekota KE, Makgabo SM, de Klerk-Loris LM, van Schalkwyk LO, Kamath PL, Turner WC, van Heerden H. Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax. PLoS Negl Trop Dis. 2024; 18(11):e0012122.
[71] Wang S, Suluku R, Jalloh MB, Samba AF, Jiang B, Xie Y, Harding D, Zhang M, Sahr F, Sesay ME, Squire JS, Vandi MA, Kallon MN, Zhang S, Hu R, Zhao Y, Mi Z. Molecular characterization of an outbreak-involved Bacillus anthracis strain confirms the spillover of anthrax from West Africa. Infect Dis Poverty. 2024; 13(1):6.
[72] Kędrak-Jabłońska A, Budniak S, Szczawińska A, Reksa M, Krupa M, Szulowski K. Evaluation of Real-time PCR Based on SYBR Green I Fluorescent Dye for Detection of Bacillus Anthracis Strains in Biological Samples. J Vet Res. 2018; 62(4):549-54.
[73] Makino SI, Cheun HI, Watarai M, Uchida I, Takeshi K. Detection of anthrax spores from the air by real-time PCR. Lett Appl Microbiol. 2001; 33(3):237-40.
[74] Sjöstedt A, Eriksson U, Ramisse V, Garrigue H. Detection of Bacillus anthracis spores in soil by PCR. FEMS Microbiol Ecol. 1997; 23(2):159-68.
[75] Zincke D, Norris MH, Cruz O, Kurmanov B, McGraw WS, Daegling DJ, Krigbaum J, Hoang TTH, Khanipov K, Golovko G, Hadfield T, Blackburn JK. TaqMan Assays for Simultaneous Detection of Bacillus anthracis and Bacillus cereus biovar anthracis. Pathogens. 2020; 9(12):1074.
[76] Kim K, Seo J, Wheeler K, Park C, Kim D, Park S, Kim W, Chung SI, Leighton T. Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis. FEMS Immunol Med Microbiol. 2005; 43(2):301-10.
[77] Berg T, Suddes H, Morrice G, Hornitzky M. Comparison of PCR, culture and microscopy of blood smears for the diagnosis of anthrax in sheep and cattle. Lett Appl Microbiol. 2006; 43(2):181-6.
[78] Levine SM, Perez-Perez G, Olivares A, Yee H, Hanna BA, Blaser MJ. PCR-Based Detection of Bacillus anthracis in Formalin-Fixed Tissue from a Patient Receiving Ciprofloxacin. J Clin Microbiol. 2002; 40(11):4360-2.
[79] Brightwell G, Pearce M, Leslie D. Development of internal controls for PCR detection of Bacillus anthracis. Mol Cell Probes. 1998; 12(6):367-77.
[80] Lee MA, Brightwell G, Leslie D, Bird H, Hamilton A. Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR. J Appl Microbiol. 1999; 87(2):218-23.
[81] Levi K, Higham JL, Coates D, Hamlyn PF. Molecular detection of anthrax spores on animal fibres. Lett Appl Microbiol. 2003; 36(6):418-22.
[82] Shannon JG, Ross CL, Koehler TM, Rest RF. Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin. Infect Immun. 2003; 71(6):3183-9.
[83] Braun P, Nguyen MD, Walter MC, Grass G. Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts. Int J Mol Sci. 2021; 22(22):12224.
[84] Wang Y, Qian PY. Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies. PLoS One. 2009; 4(10):e7401.
[85] Turnbull P. Guidelines for the Surveillance and Control of Anthrax in Human and Animals, 3rd ed.; World Health Organization: Geneva, Switzerland, 2015.
[86] Bassy O, Jiménez-Mateo O, Ortega MV, Granja C, Cabria JC. Rapid identification of Bacillus anthracis by real-time PCR with dual hybridization probes in environmental swabs. Mol Cell Probes. 2018; 37:22-7.