Nucleic Acid Approaches To Toxin Detection Nicola Chatwell, BSc Thesis submitted to the University of Nottingham for the degree of Master of Philosophy December 2013 ABSTRACT PCR is commonly used for detecting contamination of foods by toxigenic bacteria. However, it is unknown whether it is suitable for detecting toxins in samples which are unlikely to contain bacterial cells, such as purified biological weapons. Quantitative real-time PCR assays were developed for amplification of the genes encoding Clostridium botulinum neurotoxins A to F, Staphylococcal enteroxin B (SEB), ricin, and C. perfringens alpha toxin. Botulinum neurotoxins, alpha toxin, ricin and V antigen from Yersinia pestis were purified at Dstl using methods including precipitation, ion exchange, FPLC, affinity chromatography and gel filtration. Additionally, toxin samples of unknown purity were purchased from a commercial supplier. Q-PCR analysis showed that DNA was present in crudely prepared toxin samples. However, the majority of purified or commercially produced toxins were not detectable by PCR. Therefore, it is unlikely that PCR will serve as a primary toxin detection method in future. Immuno-PCR was investigated as an alternative, more direct method of toxin detection. Several iterations of the method were investigated, each using a different way of labelling the secondary antibody with DNA. It was discovered that the way in which antibodies are labelled with DNA is crucial to the success of the method, as the DNA concentration must be optimised in order to fully take advantage of signal amplification without causing excessive background noise. In general terms immuno-PCR was demonstrated to offer increased sensitivity over conventional ELISA, once fully optimised, making it particularly useful for biological weapons analysis. ii Finally, genetic methods for the differentiation of toxigenic Ricinus communis strains were examined, including SSR, RAPD, RFLP and SNP analysis using next generation sequencing. The results showed that the species has low genetic diversity, making genotyping a particularly difficult task, however SSR analysis was able to provide a degree of differentiation and 454 Sequencing™ identified six SNP targets that warrant further investigation in future. iii ACKNOWLEDGEMENTS This research was funded by the Ministry of Defence. I would like to thank my supervisor Professor Christine Dodd for her support and help in completing this thesis. I would also like to thank a number of Dstl staff for general support. Specific thanks go to Dr Martin Pearce, Dr Carol Stone and Dr Naren Modi for committing to obtaining funding for the projects included in this study. Additional support was received from the Gene Probes team in its various incarnations. Dr Steve Lonsdale, Dr Beverley Roberts and Helen Burnell from the Antibody team gave generous support to a non-antibody expert. The Protein Biochemistry team were instrumental in providing toxin samples; Sarah Hayward, Dr Julie Miller, Nicki walker and Dr Helen Jones. Immuno-phage-PCR work was made possible by kind donations of recombinant antibodies from Dr Carl Mayers. Dr Anne Tinsley-Bown, Dr David Squirrell and Dr Martin Lee provided support on the Immuno-PCR project. Finally, I would like to thank my friends and family for their continual support. This thesis is dedicated to my late father, my mum and my boys, David, Ethan and James. iv CONTENTS ABSTRACT ________________________________________________________ ii 1. INTRODUCTION _________________________________________________ 1 1.1 TOXINS AS BIOLOGICAL WEAPONS __________________________ 1 1.2 TOXINS ____________________________________________________ 2 1.3 Genetics of toxin production _____________________________________ 3 1.4 Protein Purification ___________________________________________ 19 1.5 Toxin Detection methods ______________________________________ 24 1.6 Next generation sequencing ____________________________________ 36 1.7 Project Aims ________________________________________________ 37 2 MATERIALS AND METHODS ___________________________________ 38 2.1 Bacterial strains ______________________________________________ 38 2.2 DNA extraction procedures _____________________________________ 43 2.3 PCR methodology ____________________________________________ 47 2.4 Cloning ____________________________________________________ 49 2.5 Analysis of DNA by gel electrophoresis ___________________________ 51 2.6 DNA quantification ___________________________________________ 52 2.7 Protein Purification ___________________________________________ 53 2.8 Immuno-PCR _______________________________________________ 59 2.9 Systematic Evolution of Ligands exponentially (selex) _______________ 68 2.10 Differentiation of ricinus communis strains using DNA analysis _______ 71 2.11 454™ sequencing ___________________________________________ 73 3 PCR DETECTION OF PROTEIN TOXINS _________________________ 80 3.1 Introduction _________________________________________________ 80 v 3.2 Assay design and optimisation __________________________________ 82 3.3 Clostridium botulinum neurotoxin detection _______________________ 87 3.4 Recombinant V antigen PCR assay ______________________________ 123 3.5 Ricinus communis ricin detection _______________________________ 127 3.6 Clostridium perfringens phospholipase C _________________________ 133 3.7 Detection of Sigma toxins _____________________________________ 139 3.8 Multiplex PCR _____________________________________________ 144 3.9 Chapter summary ___________________________________________ 150 3.10 Discussion ________________________________________________ 155 4 IMMUNO-PCR ________________________________________________ 161 4.1 Introduction ________________________________________________ 161 4.2 Aims _____________________________________________________ 161 4.3 Immuno-PCR assays _________________________________________ 163 4.4 Detection of ovalbumin using immuno-pCR ______________________ 166 4.5 Detection of Bacillus cereus by I-φ-PCR _________________________ 189 4.6 Immuno-PCR using anti-ovalbumin aptamers _____________________ 200 4.7 Discussion _________________________________________________ 212 5 GENETIC DIFFERENTIATION OF R. COMMUNIS ________________ 219 5.1 Introduction ________________________________________________ 219 5.2 SNP discovery by 454 Sequencing™ ____________________________ 222 5.3 Alternative approaches _______________________________________ 238 5.4 Discussion _________________________________________________ 249 5.5 CONCLUSIONS ____________________________________________ 251 6 GENERAL DISCUSSION ________________________________________ 253 6.1 OVERVIEW _______________________________________________ 253 6.2 IMMUNO-PCR _____________________________________________ 255 vi 6.3 Genetic differentiation of R. communis __________________________ 258 6.4 Concluding Remarks _________________________________________ 259 7 APPENDICES __________________________________________________ 261 8 REFERENCES __________________________________________________ 264 vii 1. INTRODUCTION 1.1 TOXINS AS BIOLOGICAL WEAPONS Bioterrorism is the intentional use of microorganisms or toxins derived from living organisms to cause death or disease in humans, animals or plants on which we depend (Ashford, 2003). Biological warfare (BW) specifically refers to the use of such weapons in the battlefield (Carter, 2000). The Biological and Toxin Weapons convention (BTWC) prohibits the development, production and testing of biological weapons (United Nations report, 1971). A number of toxins are on the Centers for Disease Control and prevention (CDC) category A, B and C biological weapons threat list (http://www.bt.cdc.gov/agent/agentlist-category.asp), including Clostridium botulinum neurotoxin, ricin, Staphylococcal enterotoxin B (SEB) and C. perfringens alpha toxin. Toxins may be produced as agents either in liquid or powdered form (Carter, 2000), with the most potent method of delivery being via the aerosol route (Hawley et al., 2001). Inhalation of the weapon may or may not be lethal. For example, SEB may evoke a quick and transient debilitation lasting less than 24 hours (Carter, 2000). Inhalation of botulinum neurotoxin is more likely to cause fatality (Madsen, 2001). Detection of a disease or intoxication outbreak is essential to aid the investigation of potential production facilities, environmental or clinical samples (Zilinskas, 1998). Antibody-based detection techniques such as ELISA were first described forty years ago (Engvall and Perlmann, 1971). The utility of ELISA for toxin detection has steadily increased since it was first used to detect cholera toxin (Holmgren and Svennerholm, 1973). However, identification of a toxin in a BTWC scenario would require confirmation using an orthologous technique. PCR is exquisitely sensitive and is routinely used for detection of trace amounts of DNA enabling specific identification of a source in humans (Jeffreys et 1 al., 1991) and similar methods exist for numerous other species including the BW agent anthrax (Hoffmaster et al., 2002). It is therefore conceivable that PCR may be suitable for detecting toxin DNA in samples where the quantity of DNA is limited. Currently, it is unknown whether DNA will be present in a toxin sample presented for BTWC analysis, since it may be subject to crude or stringent purification. To determine whether or not this is the case is the primary aim of this thesis. The secondary aim is to investigate the potential of alternative