Corinne Byrdsong Spring 2014 Seminar
Relevance Regulation Effects of exposure Method of action Detection methods PCR analysis
1978 - Bulgarian dissident Georgi Markov assassinated in London April 2013 – Letters addressed to the US President March 2014 – Pennsylvania teen March 2014 – ricin in Georgetown dorm Both a criminal and a public health issue
Ricin is considered a biological and a chemical weapon Classified as Category B biological threat by the CDC
Prohibited by international treaties Biological Weapons Anti-Terrorism Act of 1989 Antiterrorism and Effective Death Penalty Act of 1996 USA PATRIOT Act of 2001 Public Health Security and Bioterrorism Preparedness Act of 2002
• Ricinus communis – Castor beans
• A major protein in castor beans
• Comes from the waste of castor oil production
Image from: http://www.printmag.com/design-inspiration/botany-blueprint-the-castor-bean/
Image from: http://dsonggonplant.blogspot.com/2012/02/ricinus-communis-usually-name-called.html
• Many uses for the oil: – Cosmetics – Paints, varnishes – Organic fertilizer – Nylon – Synthetic flavors – Potential biofuel
Image from: http://beautyeditor.ca/2010/11/15/my-brief-traumatic-foray-into-the-oil-cleansing-method-or-how-overreaching-can-sometimes-backfire-on-your-skin/
R. communis grows in most tropical and temperate regions throughout the world The Anarchist’s Cookbook The Poisoner’s Handbook The Internet Easily and inexpensively obtained
Image from: http://en.wikipedia.org/wiki/The_Anarchist_Cookbook
A plant toxin A lectin Ribosome inactivating protein (RIP)
Image from: http://www.wiley.com/college/boyer/0470003790/cutting_edge/molecular_recognition/ricin.gif
Inhibits protein synthesis This leads to the death of eukaryotic cells
Image from: http://ibbio.pbworks.com/w/page/28624940/Nucleic%20Acids%20and%20Proteins
Lethal dose depends on route of exposure Generally, 1 – 20 mg of ricin/kg body weight
Death takes place 36 – 72 hours after exposure, depending on the amount and route of exposure Accidental exposure unlikely
Depends on route of exposure Non-specific but severe
Will ultimately see respiratory, organ failure Ricin poisoning not contagious
No antidote Treat symptoms Try to get ricin out of body
Recent ingestion – flush stomach with activated charcoal
Know if a person has been exposed Decontamination measures Show ricin to be biologically active Presumptive screening
Rapid results Confirmatory analysis
Environmental Sample
Immunological assays
Mass spectrometry
Detection of ricin protein
Biological Assays
Detection of residual DNA Real-time PCR
Environmental Sample
Immunological assays
Mass spectrometry
Detection of ricin protein
Biological Assays
Detection of residual DNA Real-time PCR
Rapid screening Specific High sensitivity Quantitative
Image from: http://classconnection.s3.amazonaws.com/826/flashcards/512826/png/picture131323331655437.png
Amplification of specific sequence Ricin-specific primers Ricin-specific probes ‘Real-time’ data collection
Point when amplification first detected during cycle
Important for: Risk assessment Medical treatment
Results in 4-5 hours
Detection of R. communis DNA Detect presence of DNA encoding the gene for ricin A-chain Specific nucleic acid sequences
• R. communis DNA as residual contaminant from ricin preparation
Small amounts detected High detection from various samples
Castor bean suspension Spiked powders
Amount of R. communis DNA Get idea of how much toxin present
Determined using exponential phase Doubling of products
Image from: http://www.biotechniques.com/BiotechniquesJournal/specialissues/2008/April/Twenty-five-years-of-quantitative-PCR-for-gene-expression-analysis/biotechniques-45217.html?pageNum=2
Direct investigators to next step Confirmatory assays
Agricultural plant-breeding programs Low levels of ricin
Ricin Biological and chemical agent Easily processed
Detection Methods Presumptive
PCR Analysis Confirmatory
Future studies
Dr. Staton Dr. Fenger Darcie Winkler Kel Daniel AJ Montañez KariAnna Baber
Bundowle, B., Schutzer, S. E., Breeze, R. G., Keim, P. S., & Morse, S. A. (Eds.). (2010). Microbial forensics. (2nd ed.). Burlington, MA: Elsevier/Academic Press. Hutchins, A. S., Astwood, M. J., Saah, J. R., Michel, P. A., Newton, B. R., & Dauphin, L. A. (2014). Evaluation of automated and manual dna purification methods for detecting ricinus communis dna during ricin investigations. Forensic Science International, 236, 10-15. doi: 10.1016/j.forsciint.2013.12.011. Melchior, W.B., Jr., & Tolleson, W.H. (2010). A functional quantitative polymerase chain reaction assay for ricin, Shiga toxin, and related ribosome-inactivating proteins. Analytical Biochemistry, 396, 204-211. doi: 10.1016/j.ab.2009.09.024 Puri, P., & Kumar, O. (2011). Integrating immunobased detection and identification methods for ricin analysis: An overview. Journal of Bioterrorism & Biodefense, S2:003. doi: Integrating Immunobased Detection and Identification Methods for Ricin Analysis: An Overview Schieltz, D.M., McGrath, S.C., McWilliams, L.G., Rees, J., Bowen, M.D., Kools, J.J., Dauphin, L.A., Gomez-Saladin, E., Newton, B.N., Stang, H.L., Vick, M.J., Thomas, J. Pirkle, J.L., & Barr, J.R. (2011). Analysis of active ricin and castor bean proteins in a ricin preparation castor bean extract, and surface swabs from a public health investigation. Forensic Science International, 209, 70-79. doi: 10.1016/j.forsciint.2010.12.013 Shea, D. A., & Gottron, F. (2010, December). Ricin: technical background and potential role in terrorism. Library of Congress Washington D.C. Congressional Research Service.
Image from: http://www.picstopin.com/1832/cloud-question-mark/http:%7C%7Cwww*giacom*com%7Cblog%7Cwp-content%7Cuploads%7C2013%7C03%7CQuestion-Clouds*jpg/