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 –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

Primers anneal to template 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.html 2.Department of Defense AVIP (Anthrax Immunization Program) website http://www.anthrax.osd.mil/and http://www.anthrax.mil/media/pdf/enclosure4.pdf 3.Anthrax BTAtmand Plague BTAtmProduct Literature, Tetracore, Inc., 11 Firstfield Road, Suite C, Gaithersburg, MD 20878 4.IVDT archive, Concurrent Engineering for Lateral-Flow Diagnostics, Medical Devicelink website, Nov 1999, http://www.devicelink.com/ivdt/archive/99/11/009.html 5.Millipore Inc. Technical Publications, Rapid Lateral Flow Test Strips –Considerations for Product Development, http://www.millipore.com/publications.nsf/docs/tb500en00 6.“Purchase of Anthrax Detection Technologies”, Memorandum for Federal Mail Managers and First Responders to Federal Mail Centers, John Marburger, 19 July 2002 7.“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