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Use of Molecular Methods in Biothreat Preparedness National Defence University Series 1: Research Publications No. 13 USE OF MOLECULAR METHODS IN BIOTHREAT PREPAREDNESS Katja Koskela KATJA KOSKELA USE OF MOLECULAR METHODS IN BIOTHREAT PREPAREDNESS Research and Development Department Centre for Military Medicine and Department of Bacteriology and Immunology University of Helsinki and Doctoral Programme in Microbiology and Biotechnology University of Helsinki ACADEMIC DISSERTATION To be presented for public examination, with the permission of the Faculty of Medicine, in Lecture Hall 5, University Main Building, (Fabianinkatu 33) on 5th of May 2017, at 12 o´clock. HELSINKI 2017 NATIONAL DEFENCE UNIVERSITY SERIES 1: RESEARCH PUBLICATIONS NO. 13 MAANPUOLUSTUSKORKEAKOULU JULKAISUSARJA 1: TUTKIMUKSIA NRO 13 USE OF MOLECULAR METHODS IN BIOTHREAT PREPAREDNESS KATJA KOSKELA NATIONAL DEFENCE UNIVERSITY HELSINKI 2017 Katja Koskela: Use of Molecular Methods in Biothreat Preparedness National Defence University Series 1: Research Publications No. 13 Doctoral dissertation Supervisors: Professor Simo Nikkari, MD, PhD Research and Development Department Centre for Military Medicine Helsinki, Finland Professor Mikael Skurnik, PhD Department of Bacteriology and Immunology Medicum University of Helsinki Helsinki, Finland Reviewers: Docent Kaisu Rantakokko-Jalava, MD, PhD Turku University Hospital, Turku, Finland Docent Susanna Lukinmaa-Åberg, PhD Päijät-Häme Central Hospital, Lahti, Finland Custos: Professor Mikael Skurnik, PhD University of Helsinki Opponent: Florent Sebbane, PhD INSERM Research Director Center for Infection and Immunity of Lille University of Lille, Institut Pasteur de Lille Lille, France Most recent publications in pdf-format: http://www.doria.fi ©Katja Koskela & National Defence University ISBN 978-951-25-2902-5 (Pbk.) ISBN 978-951-25-2903-2 (PDF) ISSN 2342-9992 (Print) ISSN 2343-0001 (Web) Maanpuolustuskorkeakoulu National Defence University Juvenes Print Tampere 2017 “The more I learn, the more I realize how much I don't know” Albert Einstein TABLE OF CONTENTS ABSTRACT ............................................................................................................... iii TIIVISTELMÄ (SUMMARY IN FINNISH) ........................................................... v LIST OF ORIGINAL PUBLICATIONS ................................................................ vii ABBREVIATIONS ................................................................................................. viii 1. INTRODUCTION ............................................................................................. 1 2. REVIEW OF THE LITERATURE ................................................................... 3 2.1. History....................................................................................................................................3 2.1.1. Early use of biological warfare..................................................................................3 2.1.2. Biological weapon (BW) programs...........................................................................3 2.1.3. Biological and Toxin Weapons Convention (BTWC)..........................................4 2.1.4. Biological warfare in the 20th century.....................................................................4 2.1.5. Amerithrax and powder letters..................................................................................5 2.1.6. Laboratory accidents....................................................................................................5 2.2. Potential and probable biothreats today..........................................................................6 2.2.1. Select agents and toxins..............................................................................................6 2.2.2. Anthrax – Bacillus anthracis bacteria...........................................................................7 2.2.3. Botulism – Clostridium botulinum bacteria..................................................................8 2.2.4. Plague – Yersinia pestis bacteria...................................................................................8 2.2.4.1 Yersinia pseudotuberculosis...........................................................................................9 2.2.5. Smallpox – Variola major virus...............................................................................10 2.2.6. Tularemia – Francisella tularensis bacteria................................................................10 2.2.7. Ebola virus...................................................................................................................11 2.2.8. Brucellosis – Brucella species bacteria.....................................................................11 2.2.9. Q-fever – Coxiella burnetii bacteria...........................................................................12 2.2.10. Ricin toxin – Ricinus communis plant........................................................................12 2.2.11. Vibrio cholerae................................................................................................................13 2.3. Methods to detect and type biological agents..............................................................13 2.3.1. Culture..........................................................................................................................14 2.3.2. Lateral flow devices....................................................................................................14 2.3.3. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry................................................................................................................................15 2.3.4. Polymerase chain reaction........................................................................................15 2.3.5. 16S rRNA gene-based detection methods............................................................17 2.3.6. Clustered regularly interspaced short palindromic repeats (CRISPR).............18 i 2.3.7. Sequencing and next-generation sequencing (NGS)...........................................19 2.3.8. Forensic investigations and typing strains using NGS.......................................20 3. AIMS OF THE STUDY .................................................................................... 21 4. MATERIALS AND METHODS ...................................................................... 22 4.1. Samples and data used in this study...............................................................................22 4.1.1. Ethical permission......................................................................................................22 4.1.2. Human samples..........................................................................................................22 4.1.3. Bacterial samples........................................................................................................22 4.1.4. Vole samples................................................................................................................22 4.1.5. Sequence data..............................................................................................................24 4.2. Bacterial culture and nucleic acid extraction.................................................................24 4.3. PCR Methods......................................................................................................................24 4.4. PCR controls.......................................................................................................................26 4.5. Instruments..........................................................................................................................26 4.6. Methods based on the 16S rRNA gene.........................................................................27 4.6.1. Broad-range PCR.......................................................................................................27 4.6.2. Pyrosequencing (454 Sequencing)..........................................................................28 4.7. CRISPR-based typing........................................................................................................29 5. RESULTS .......................................................................................................... 30 5.1. Vibrio cholerae detection (I)................................................................................................30 5.2. Utility of the 16S rRNA method (II, III)......................................................................30 5.3. Yersinia pseudotuberculosis typing with CRISPR method (IV).......................................32 6. DISCUSSION .................................................................................................... 34 6.1. Detecting biological agents using molecular methods................................................34 6.2. Detecting biothreat agents with 16S rRNA gene-based methods............................36 6.3. Typing biological agents....................................................................................................38 7. CONCLUDING REMARKS ............................................................................ 40 8. ACKNOWLEDGEMENTS .............................................................................. 41 9. REFERENCES ................................................................................................. 42 ii ABSTRACT A biological threat is an epidemic or its threat
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