Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.

Collection of Animal Pathogenic Microorganisms Department of Bacteriology Veterinary Research Institute Hudcova 296 70 621 00 Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Kotlarska 2 611 37 Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

Department of Meat Hygiene and Technology Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackeho tr 1946 1 612 42 Brno Czech Republic

Department of Microbiology and Antimicrobial Resistance Veterinary Research Institute Hudcova 296 70 621 00 Brno Czech Republic

Military Health Institute Military Medical Agency Tychonova 1 160 01 Prague 6 Czech Republic

Military Veterinary Institute Opavska 29 748 01 Hlucin Czech Republic

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Arun Kumar R., Nishanth T., Ravi Teja Y., Sathish Kumar D. Bio threat s bacterial warfare agents. J. Bioterr. Biodef. 2011;2:2.

Christopher L.G.W., Cieslak L.T.J., Pavlin J.A., Eitzen E.M. Biological warfare: A historical perspective. JAMA. 1997;278:412–417. doi: 10.1001/jama.1997.03550050074036. PubMed DOI

Shah J., Wilkins E. Electrochemical biosensors for detection of biological warfare agents. Electroanal. Int. J. Devoted Fundam. Pract. Asp. Electroanal. 2003;15:157–167. doi: 10.1002/elan.200390019. DOI

Flora S., Pachauri V. Handbook on Biological Warfare Preparedness. Academic Press; Cambridge, MA, USA: 2019.

Mölsä M., Hemmilä H., Katz A., Niemimaa J., Forbes K.M., Huitu O., Stuart P., Henttonen H., Nikkari S. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument. J. Microbiol. Methods. 2015;115:89–93. doi: 10.1016/j.mimet.2015.05.026. PubMed DOI

Peculi A., Campese E., Serrecchia L., Marino L., Boci J., Bijo B. Genotyping of Bacillus anthracis strains circulating in Albania. J. Bioterror. Biodef. 2015;6 doi: 10.4127/2157-2526.1000131. DOI

Carlson C.J., Kracalik I.T., Ross N., Alexander K.A., Hugh-Jones M.E., Fegan M., Elkin B.T., Epp T., Shury T.K., Zhang W. The global distribution of Bacillus anthracis and associated anthrax risk to humans, livestock and wildlife. Nat. Microbiol. 2019;4:1337–1343. doi: 10.1038/s41564-019-0435-4. PubMed DOI

Wilson W.J., Erler A.M., Nasarabadi S.L., Skowronski E.W., Imbro P.M. A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents. Mol. Cell. Probes. 2005;19:137–144. doi: 10.1016/j.mcp.2004.10.005. PubMed DOI

Fennelly K.P., Davidow A.L., Miller S.L., Connell N., Ellner J.J. Airborne infection with Bacillus anthracis—From mills to mail. Emerg. Infect. Dis. 2004;10:996. doi: 10.3201/eid1006.020738. PubMed DOI PMC

World Health Organization . International Health Regulations (2005) World Health Organization; Geneva, Switzerland: 2008.

Kool J.L., Weinstein R.A. Risk of person-to-person transmission of pneumonic plague. Clin. Infect. Dis. 2005;40:1166–1172. doi: 10.1086/428617. PubMed DOI

Gur D., Glinert I., Aftalion M., Vagima Y., Levy Y., Rotem S., Zauberman A., Tidhar A., Tal A., Maoz S. Inhalational gentamicin treatment is effective against pneumonic plague in a mouse model. Front. Microbiol. 2018;9:741. doi: 10.3389/fmicb.2018.00741. PubMed DOI PMC

Perry R.D., Fetherston J.D. Yersinia pestis—Etiologic agent of plague. Clin. Microbiol. Rev. 1997;10:35–66. doi: 10.1128/CMR.10.1.35. PubMed DOI PMC

Kolodziejek M.A., Hovde C.J., Minnich S.A. Yersinia pestis Ail: Multiple roles of a single protein. Front. Cell. Infect. Microbiol. 2012;2:103. doi: 10.3389/fcimb.2012.00103. PubMed DOI PMC

Skládal P., Pohanka M., Kupská E., Šafář B. Biosensors. INTECH; Vienna, Austria: 2010. Biosensors for Detection of Francisella tularensis and Diagnosis of Tularemia; pp. 115–126.

Gillard J.J., Laws T.R., Lythe G., Molina-Paris C. Modeling early events in Francisella tularensis pathogenesis. Front. Cell. Infect. Microbiol. 2014;4:169. doi: 10.3389/fcimb.2014.00169. PubMed DOI PMC

Soares C.d.P.O.C., Teles J.A.A., dos Santos A.F., Silva S.O.F., Cruz M.V.R.A., da Silva-Júnior F.F. Prevalence of Brucella spp in humans. Rev. Latino-Am. Enferm. 2015;23:919–926. doi: 10.1590/0104-1169.0350.2632. PubMed DOI PMC

Scholz H.C., Nöckler K., Göllner C., Bahn P., Vergnaud G., Tomaso H., Al Dahouk S., Kämpfer P., Cloeckaert A., Maquart M. Brucella inopinata sp. nov., isolated from a breast implant infection. Int. J. Syst. Evolut. Microbiol. 2010;60:801–808. doi: 10.1099/ijs.0.011148-0. PubMed DOI

Wang Q., Zhao S., Wureli H., Xie S., Chen C., Wie Q., Cui B., Tu C., Wang Y. Brucella melitensis and B. abortus in eggs, larvae and engorged females of Dermacentor marginatus. Ticks Tick-Borne Dis. 2018;9:1045–1048. doi: 10.1016/j.ttbdis.2018.03.021. PubMed DOI

Abou Zaki N., Salloum N.T., Osman M., Rafei R., Hamze M., Tokajian S. Typing and comparative genome analysis of Brucella melitensis isolated from Lebanon. FEMS Microbiol. Lett. 2017;364 doi: 10.1093/femsle/fnx199. PubMed DOI

Deng H., Zhou H.J., Gong B., Xiao M., Zhang M., Pang Q., Zhang X., Zhao B., Zhou X. Screening and identification of a human domain antibody against Brucella abortus VirB5. Acta Trop. 2019;197:105026. doi: 10.1016/j.actatropica.2019.05.017. PubMed DOI

Yang Y., Wang J., Wen H., Liu H. Comparison of Two Suspension Arrays for Simultaneous Detection of Five Biothreat Bacterial in Powder Samples. J. Biomed. Biotechnol. 2012;2012:831052. doi: 10.1155/2012/831052. PubMed DOI PMC

Saiki R.K., Scharf S., Faloona F., Mullis K.B., Horn G.T., Erlich H.A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:1350–1354. doi: 10.1126/science.2999980. PubMed DOI

Reslova N., Michna V., Kasny M., Mikel P., Kralik P. xMAP technology: Applications in detection of pathogens. Front. Microbiol. 2017;8:55. doi: 10.3389/fmicb.2017.00055. PubMed DOI PMC

Reslova N., Huvarova V., Hrdy J., Kasny M., Kralik P. A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay. Sci. Rep. 2019;9:13. PubMed PMC

Deshpande A., Gans J., Graves S.W., Green L., Taylor L., Kim H.B., Kunde Y.A., Leonard P.M., Li P.-E., Mark J., et al. A rapid multiplex assay for nucleic acid-based diagnostics. J. Microbiol. Methods. 2010;80:155–163. doi: 10.1016/j.mimet.2009.12.001. PubMed DOI

Stucki D., Mall B., Hostettler S., Huna T., Feldmann J., Yeboah-Manu D., Borrell S., Fenner L., Comas I., Coscolla M. Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages. PLoS ONE. 2012;7:e41253. doi: 10.1371/journal.pone.0041253. PubMed DOI PMC

Wuyts V., Mattheus W., Roosens N.H., Marchal K., Bertrand S., De Keersmaecker S.C. A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium. Appl. Microbiol. Biotechnol. 2015;99:8137–8149. doi: 10.1007/s00253-015-6831-7. PubMed DOI PMC

Ceyssens P.-J., Garcia-Graells C., Fux F., Botteldoorn N., Mattheus W., Wuyts V., De Keersmaecker S., Dierick K., Bertrand S. Development of a Luminex xTAG® assay for cost-effective multiplex detection of β-lactamases in Gram-negative bacteria. J. Antimicrob. Chemother. 2016;71:2479–2483. doi: 10.1093/jac/dkw201. PubMed DOI

Woods T.A., Mendez H.M., Ortega S., Shi X., Marx D., Bai J., Moxley R.A., Nagaraja T.G., Graves S.W., Deshpande A. Development of 11-Plex MOL-PCR assay for the rapid screening of samples for Shiga toxin-producing Escherichia coli. Front. Cell. Infect. Microbiol. 2016;6:92. doi: 10.3389/fcimb.2016.00092. PubMed DOI PMC

Wang H.-Y., Wu S.Q., Jiang L., Xiao R.H., Li T., Mei L., Lv J.Z., Liu J.J., Lin X.M., Han X.Q. Establishment and optimization of a liquid bead array for the simultaneous detection of ten insect-borne pathogens. Parasites Vectors. 2018;11:442. doi: 10.1186/s13071-018-2996-0. PubMed DOI PMC

US Food & Drug Administration Office . Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds. US Food & Drug Administration Office of Foods and Veterinary Medicine; Silver Spring, MD, USA: 2015.

Slana I., Kaevska M., Kralik P., Horvathova A., Pavlik I. Distribution of Mycobacterium avium subsp. avium and M. a. hominissuis in artificially infected pigs studied by culture and IS901 and IS1245 quantitative real time PCR. Vet. Microbiol. 2010;144:437–443. doi: 10.1016/j.vetmic.2010.02.024. PubMed DOI

Vasickova P., Slany M., Chalupa P., Holub M., Svoboda R., Pavlik I. Detection and phylogenetic characterization of human hepatitis E virus strains, Czech Republic. Emerg. Infect. Dis. 2011;17:917. doi: 10.3201/eid1705.101205. PubMed DOI PMC

Van Tongeren S.P., Roest H.I.J., Degener J.E., Harmsen J.M. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station: A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis. PLoS ONE. 2014;9:e98871. doi: 10.1371/journal.pone.0098871. PubMed DOI PMC

Bell C.A., Uhl J.R., Hadfield T.L., David J.C., Meyer R.F., Smith T.F., Cockerill F.R. Detection of Bacillus anthracis DNA by LightCycler PCR. J. Clin. Microbiol. 2002;40:2897–2902. doi: 10.1128/JCM.40.8.2897-2902.2002. PubMed DOI PMC

Tomaso H., Reisinger E.C., Al Dahouk S., Frangoulidis D., Rakin A., Landt O., Neubauer H. Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes. FEMS Immunol. Med. Microbiol. 2003;38:117–126. doi: 10.1016/S0928-8244(03)00184-6. PubMed DOI

Hatkoff M., Runco L.M., Pujol C., Jayatilaka I., Furie M.B., Bliska J.B., Thanassi D.G. Roles of chaperone/usher pathways of Yersinia pestis in a murine model of plague and adhesion to host cells. Infect. Immun. 2012;80:3490–3500. doi: 10.1128/IAI.00434-12. PubMed DOI PMC

Versage J.L., Severin D.D.M., Chu M.C., Petersen J.M. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J. Clin. Microbiol. 2003;41:5492–5499. doi: 10.1128/JCM.41.12.5492-5499.2003. PubMed DOI PMC

Fujita O., Tatsumi M., Tanabayashi K., Yamada A. Development of a real-time PCR assay for detection and quantification of Francisella tularensis. Jpn. J. Infect. Dis. 2006;59:46–51. PubMed

Probert W.S., Schrader K.N., Khuong N.Y., Bystrom S.L., Graves M.H. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J. Clin. Microbiol. 2004;42:1290–1293. doi: 10.1128/JCM.42.3.1290-1293.2004. PubMed DOI PMC

Bounaadja L., Albert D., Chénais B., Hénault S., Zygmunt M.S., Poliak S., Garin-Bastuji B. Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes. Vet. Microbiol. 2009;137:156–164. doi: 10.1016/j.vetmic.2008.12.023. PubMed DOI

Thierry S., Hamidjaja R.A., Girault G., Löfström C., Ruuls R., Sylviane D. A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis. J. Microbiol. Methods. 2013;95:357–365. doi: 10.1016/j.mimet.2013.10.004. PubMed DOI

Kralik P., Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front. Microbiol. 2017;8:108. doi: 10.3389/fmicb.2017.00108. PubMed DOI PMC

Longo C.M., Berninger M.S., Hartley J.L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125–128. doi: 10.1016/0378-1119(90)90145-H. PubMed DOI

Wuyts V., Roosens N.H.C., Bertrand S., Marchal K., De Keersmaecker S.C.J. Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array. BioMed Res. Int. 2015;2015:790170. doi: 10.1155/2015/790170. PubMed DOI PMC

US Food and Drug Administration . Guidelines for the Validation of Microbiological Methods for the FDA Foods Program. 3rd ed. US Food and Drug Administration; Silver Spring, MA, USA: 2019.

Yan Y., Luo J.Y., Chen Y., Wang H.H., Zhu G.Y., He P.Y., Guo J.L., Lei Y.L., Chen Z.W. A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses. Oncotarget. 2017;8:96913. doi: 10.18632/oncotarget.18533. PubMed DOI PMC

Nguyen T., Chidambara V.A., Andreasen S.Z., Golabi M., Huynh V.N., Linh Q.T., Bang D.D., Wolff A. Point-of-care devices for pathogen detections: The three most important factors to realise towards commercialization. TrAC Trends Anal. Chem. 2020;131:116004. doi: 10.1016/j.trac.2020.116004. DOI