1. Biological Weapons Detection Methods
* Adapted from US Dept. of Homeland Security – Advanced Chemical/Biological Integrated Response

2. Objectives Outline biological detection methods Define methodology
Discuss capabilities and limitations
Discuss future technology

3. Rapid Detection Methods
Antibody-Antigen based
DNA-based
Visual examination
Mobile Laboratory Detection Equipment

4. Antibodies and Antigens
–high sensitivity and
specificity
–produced in response
to an antigen
Antigens
–recognized as foreign by
the body
–stimulate an immune
response

5. 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

6. Hand Held Assay (HHA) Components

10. Possible Results

11. Limitations 1.Detection Limit 2.Matrix Interference 3.Cross-Reactivity
4.Hook Effect

12. 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

13. Cross-Reactivity •Occurs when close relatives share common antigens
–Bacillus anthracis
–Bacillus thuringiensis
–Bacillus cereus

14. 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

15. Serial Dilution Example

16. 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.)

18. 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

19. 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)

20. PCR Components
DNA template (sample)
Primer
dNTPs
TaqDNA polymerase

21. Basic PCR Reaction 1.Denaturation (heat) 2.Annealing (primers)
3.Extension (DNA polymerase)
Result: Amplification of DNA

22. Denaturation

23. Primers anneal to template
Annealing
Primers anneal to template

24. Extension

25. Completion of First Cycle
Note – 2 strands of DNA

26. PCR Equipment - Thermocyclers

27. Thermocyclers

28. 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

29. Capabilities of PCR
Billions of copies of DNA target produced in a relatively short time
Efficient method to identify an agent
Equipment automates process

30. 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

31. Visual Examination

32. Light Microscopy Bright field microscopy –Stained preparation
Phase contrast microscopy
–Wet mount preparation

33. Microscopic Identification

34. Fluorescence Microscope

35. Mobile Lab Detection Equipment

36. Future Technology Reusable Biological Detectors Faster PCR
Environmental Air Samplers

37. References
1.US Dept of Health & Human Services Presentation: Update on Biodetection: Problems and Prospects, Michael S. Ascher;
2.Department of Defense AVIP (Anthrax Immunization Program) website
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,
5.Millipore Inc. Technical Publications, Rapid Lateral Flow Test Strips –Considerations for Product Development,
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