Biological Weapons Detection Methods * Adapted from US Dept. of Homeland Security – Advanced Chemical/Biological Integrated Response
Mar 29, 2015
Biological Weapons Detection Methods
* Adapted from US Dept. of Homeland Security – Advanced Chemical/Biological Integrated Response
Objectives
• Outline biological detection methods• Define methodology• Discuss capabilities and limitations• Discuss future technology
Rapid Detection Methods
• Antibody-Antigen based• DNA-based • Visual examination • Mobile Laboratory Detection Equipment
Antibodies and Antigens
• Antibodies –high sensitivity and specificity –produced in response to an antigen• Antigens –recognized as foreign by the body –stimulate an immune response
Hand Held Assay Detection
• Biological Agent Detection Device (BADD)• Sensitive Membrane Antigen Rapid Test
(SMART tickets)• Biothreat(TetraCore)• RAMP BiowarfareDetection and Guardian
Reader System (hand held assay and reader)• Many others
Hand Held Assay (HHA) Components
Possible Results
Limitations
1.Detection Limit
2.Matrix Interference
3.Cross-Reactivity
4.Hook Effect
HHA Detection Limit
•Not enough agent present
•Different for each agent
•Infectious dose usually lower than detection limit – negative test does not always mean no agent present
Cross-Reactivity
•Occurs when close relatives share common antigens
–Bacillus anthracis–Bacillus thuringiensis–Bacillus cereus
Hook Effect
• Occurs when there is a higher ratio of agent to antibodies
–More often with toxins–Result is a false negative–The solution is dilution
Serial Dilution Example
HHA Capabilities
• Rapid field test! Although presumptive, it allows decision makers to:
–Take protective actions–Treat potential infections –Involve other authorities
(Health, Law Enforcement, etc.)
What is PCR?
• Method to increase the copies of a specific DNA sequence
• Small amounts of DNA are needed. Sample matrix may include:
–Soil–Blood–Powder–Skin
Deoxyribonucleic Acid (DNA)
• Hereditary information found in almost every cell
• Composed of four bases attached to a sugar-phosphate backbone
-Adenine (A)-Thymine (T)-Guanine (G)-Cytosine (C)
PCR Components
• DNA template (sample)• Primer• dNTPs• TaqDNA polymerase
Basic PCR Reaction
1.Denaturation (heat)2.Annealing (primers)3.Extension (DNA polymerase)
Result: Amplification of DNA
Denaturation
Annealing
Primers anneal to template
Extension
Completion of First Cycle
Note – 2 strands of DNA
PCR Equipment - Thermocyclers
Thermocyclers
Real-Time PCR
• Monitor PCR reaction in “real time”–Uses fluorescent probe–Quantify products–Rapid result–Decreased turn-around time
Taq technique and fluorescence resonance energy transfer are two ways of achieving “real time” PCR
Capabilities of PCR
• Billions of copies of DNA target produced in a relatively short time
• Efficient method to identify an agent• Equipment automates process
Limitations of PCR
• DNA sequence must be known– Need a sequence to compare
• Specialized equipment and reagents needed– Equipment is expensive– Need to verify in lab anyway
• Specialized training required
Visual Examination
Light Microscopy
• Bright field microscopy –Stained preparation• Phase contrast
microscopy –Wet mount preparation
Microscopic Identification
Fluorescence Microscope
Mobile Lab Detection Equipment
Future Technology
• Reusable Biological Detectors• Faster PCR• Environmental Air Samplers
References
1.US Dept of Health & Human Services Presentation: Update on Biodetection: Problems and Prospects, Michael S. Ascher; http://www.hhs.gov/ophp/presentations/ascher.html2.Department of Defense AVIP (Anthrax Immunization Program) website http://www.anthrax.osd.mil/and http://www.anthrax.mil/media/pdf/enclosure4.pdf3.Anthrax BTAtmand Plague BTAtmProduct Literature, Tetracore, Inc., 11 Firstfield Road, Suite C, Gaithersburg, MD 208784.IVDT archive, Concurrent Engineering for Lateral-Flow Diagnostics, Medical Devicelink website, Nov 1999, http://www.devicelink.com/ivdt/archive/99/11/009.html5.Millipore Inc. Technical Publications, Rapid Lateral Flow Test Strips –Considerations for Product Development, http://www.millipore.com/publications.nsf/docs/tb500en006.“Purchase of Anthrax Detection Technologies”, Memorandum for Federal Mail Managers and First Responders to Federal Mail Centers, John Marburger, 19 July 20027.“Guidelines for Federal Mail Centers in the Washington DC Metropolitan Area for Managing Possible Anthrax Contamination”, GSA Policy Advisory, GSA Office of Governmentwide Policy, 22 July 2002