RealReal--Time PCR Diagnostics for Time PCR Diagnostics for Detecting and Identifying Potential Detecting and Identifying Potential
BioweaponsBioweaponsDavid Norwood, Ph.D.David Norwood, Ph.D.
Diagnostic Systems DivisionDiagnostic Systems Division
USAMRIID USAMRIID -- Ft. DetrickFt. Detrick
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1. REPORT DATE 18 NOV 2003
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4. TITLE AND SUBTITLE Real-Time PCR Diagnostics for Detecting and Identifying Potential Bioweapons
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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Diagnostic Systems Division Diagnostic Systems Division USAMRIIDUSAMRIID - Ft. Detrick Ft. Detrick, MD
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13. SUPPLEMENTARY NOTES See also ADM001851, Proceedings of the 2003 Joint Service Scientific Conference on Chemical &Biological Defense Research, 17-20 November 2003. , The original document contains color images.
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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18
USAMRIID
OverviewOverview
!! DSD Overview DSD Overview
!! Determine gene target(s)Determine gene target(s)
!! Design specific probesDesign specific probes
!! Design specific primersDesign specific primers
!! Optimize assay conditionsOptimize assay conditions
!! Determine limits of detectionDetermine limits of detection
!! Test for cross reactivity, interferenceTest for cross reactivity, interference
!! MultiplexingMultiplexing
USAMRIID
Diagnostics Systems DivisionDiagnostics Systems Division
!! Primary mission: research & development to Primary mission: research & development to advance diagnostic technologies for detecting advance diagnostic technologies for detecting biological agentsbiological agents
!! Five branchesFive branches–– Applied Diagnostics (Immunoassay development)Applied Diagnostics (Immunoassay development)–– Systems Development (Nucleic acid assay Systems Development (Nucleic acid assay
development)development)–– Field Operations & Training (transition lab assays to Field Operations & Training (transition lab assays to
field environments)field environments)–– Clinical Pathology (USAMRIID clinical lab)Clinical Pathology (USAMRIID clinical lab)–– Special Pathogens Sample Testing Laboratory (SPSTL)Special Pathogens Sample Testing Laboratory (SPSTL)
USAMRIID
Diagnostics and Patient Care: GoalsDiagnostics and Patient Care: Goals
Provide Quality Healthcare
Reduce/Prevent Morbidity
Vaccination/Prophylaxis
Detect/Diagnose to Treat
USAMRIID
Diagnostic EssentialsDiagnostic Essentials
!! SpeedSpeed
!! AccuracyAccuracy–– SensitivitySensitivity
–– SpecificitySpecificity
USAMRIID
Impact of Diagnostics on Patient CareImpact of Diagnostics on Patient Care
• Immediate postexposure (up to 24 h)" very low concentration of agent" IMPACT
• Acutely ill" low concentration of agent" IMPACT
• Critically ill" High concentration" IMPACT
USAMRIID
Diagnostic Time ConstraintDiagnostic Time Constraint
!! Usable results in under 24 hrsUsable results in under 24 hrs
!! IssuesIssues–– Detection of an eventDetection of an event
–– Transport of samplesTransport of samples
–– Diagnostic testingDiagnostic testing
–– Reporting resultsReporting results
USAMRIID
Classical Methods for Identifying Classical Methods for Identifying Biological AgentsBiological Agents
Method Time Sensitivity Specificity
Culture Isolation 1 - 30 days high high
Animal Inoculation 2 - 30 days high high
Antigen Detection 4 - 18 hrs low to high high*
Antibody Detection 4 hr - 10 days high high*
Nucleic Acid Detection 3-8 hrs high high*
* reagent dependent
USAMRIID
Diagnostic Sensitivity RequirementsDiagnostic Sensitivity Requirements
Anthrax 8,000 to 50,000 spores
Smallpox 10-100 organisms
Brucellosis 10-100 organisms
VEE 10-100 organisms
Plague 100-500 organisms
Viral Hemorrhagic Fevers
1-10 organisms
Q-fever 1-10 organisms Botulinum Toxins
~70 ng
Tularemia 10-50 organisms
Staph Enterotoxin B
~30 ng
Agent Infective Dose Agent Infective Dose
USAMRIID
Integrated Identification and Diagnostic System
Other
" Definitive biological agent diagnosisrequires integrated identification technologies
Immuno-diagnostics
NucleicAcid Detection
w/sampleprocessing
Classical Virology/
Microbiology
ClinicalDiagnosis
Medical Intelligence
USAMRIID
RealReal--Time PCRTime PCR
!! RapidRapid–– < 30 minutes for DNA targets< 30 minutes for DNA targets
–– < 45 minutes for RNA targets< 45 minutes for RNA targets
!! SensitiveSensitive–– Potential for single copy gene detectionPotential for single copy gene detection
!! SpecificSpecific–– Added specificity of probeAdded specificity of probe
USAMRIID
Real Time PCRReal Time PCR
USAMRIID
Quantitative PCRQuantitative PCR
USAMRIID
Sample TypesSample Types
" Each matrix may require a unique processing protocol.
Medical specimens• swabs• whole blood and serum• urine• feces• sputum• lesion exudate• tissues
Environmental samples• air samplers/collectors• swabs• water• soil
USAMRIID
BiomarkersBiomarkers
Host Response Markers
Common pathogenic markers& antibiotic resistance
Genus and species markers
Specific virulence markers
Div
ersi
ty &
Dep
th
USAMRIID
Determine Gene Target(s)Determine Gene Target(s)
!! Targets that are specific to agent of interestTargets that are specific to agent of interest–– Specific virulence (toxin genes)Specific virulence (toxin genes)
–– Specific function (accessory genes)Specific function (accessory genes)
!! Genes that code for protein productGenes that code for protein product
!! Exploit sequence variability for allelic Exploit sequence variability for allelic discriminationdiscrimination
USAMRIID
TaqmanTaqman®® ChemistryChemistry
Reverse Primer 5’
5’3’
5’ 3’
Forward Primer R Q5’
5’ 3’3’ 5’
5’
5’ QR5’
3’
Fluorescent reporter and quencher dyescovalently linked to oligonucleotide probe
Nucleic Acid Template
Reporter dye released during amplification.
Reverse Primer
3’5’
Forward Primer
USAMRIID
Common RealCommon Real--Time PCR Chemistry: Time PCR Chemistry: TaqManTaqMan --MGBMGB
Reverse Primer 5’5’ 3’3’ 5’
5’ 3’Forward Primer R
5’
5’ 3’3’ 5’
RP5’
FP5’
R
5’
3’
Fluorescent reporter and quencher dyescovalently linked to olignucleotide probe
Nucleic Acid Template
Reporter dye released during amplification.
MGBQ
MGBQ
USAMRIID
Primer Design GuidelinesPrimer Design Guidelines
!! Length of 18Length of 18--25 nucleotides25 nucleotides!! Primer Tms of 59Primer Tms of 5900--60600 0 C, both primer Tms within 2C, both primer Tms within 200CC!! No more than 2 Gs or Cs within the 5 terminal No more than 2 Gs or Cs within the 5 terminal
nucleotides at the 3’ endnucleotides at the 3’ end!! The primers corresponding to the same strand as the The primers corresponding to the same strand as the
probe should be within 30 nucleotides of the probeprobe should be within 30 nucleotides of the probe!! Avoid long runs of single nucleotidesAvoid long runs of single nucleotides!! 4040--60% GC content 60% GC content !! Amplicon of 80Amplicon of 80--150 base pairs150 base pairs
USAMRIID
Probe Design GuidelinesProbe Design Guidelines
!! Length of Length of ≤≤ 30 oligonucleotides30 oligonucleotides!! Probe Tm 7Probe Tm 700--10100 0 C higher than primer Tms (67C higher than primer Tms (67--707000C)C)!! No 5’ terminal G residuesNo 5’ terminal G residues!! Less than 30 nucleotides from corresponding strand’s Less than 30 nucleotides from corresponding strand’s
primerprimer!! Avoid long runs of single nucleotidesAvoid long runs of single nucleotides!! Select strand which gives probe more C than G Select strand which gives probe more C than G
residuesresidues!! GC content 40GC content 40--60%60%
USAMRIID
TaqmanTaqman®® MGB ProbesMGB Probes
!! Licensed to Applied Biosystems by Epoch biosciencesLicensed to Applied Biosystems by Epoch biosciences!! Protein which binds double stranded DNA within the Protein which binds double stranded DNA within the
minor grooveminor groove–– Attached to 3’ end of probeAttached to 3’ end of probe–– Folds back and stabilizes the duplex after hybridizationFolds back and stabilizes the duplex after hybridization–– Stabilization raises the Tm of the probe by 10Stabilization raises the Tm of the probe by 10--121200CC
»» Shortens probe lengthShortens probe length»» Improves allelic discriminationImproves allelic discrimination»» Provides greater flexibility in probe designProvides greater flexibility in probe design
!! Probes are coupled with a nonProbes are coupled with a non--fluorescent quencher at the fluorescent quencher at the 3’ end3’ end
USAMRIID
TaqmanTaqman®® MGB Probes for MGB Probes for Allelic DiscriminationAllelic Discrimination
USAMRIID
Design SoftwareDesign Software
!! Primer Express 2.0 Primer Express 2.0 –– Applied BiosystemsApplied Biosystems
!! LightCycler Primer and Probe DesignLightCycler Primer and Probe Design
!! NetPrimerNetPrimer (Premier (Premier BiosoftBiosoft))
!! OMIGA 2.0OMIGA 2.0
!! OligoOligo
USAMRIID
TaqmanTaqman®® OptimizationOptimization
!! Optimize the following parametersOptimize the following parameters–– MgClMgCl2 2 concentration (3mMconcentration (3mM--7mM)7mM)
–– Primer concentration (0.1Primer concentration (0.1µµM M -- 1.01.0µµM)M)
–– Temperature (Smart Temperature (Smart CyclerCycler))
–– ProbeProbe
!! Criteria for optimal conditionsCriteria for optimal conditions–– Conditions which result in the earliest crossing threshold (Ct)Conditions which result in the earliest crossing threshold (Ct)
–– Conditions which produce the most fluorescence (endpoint Conditions which produce the most fluorescence (endpoint fluorescence)fluorescence)
–– All conditions assayed in triplicate All conditions assayed in triplicate -- average of triplicates are used average of triplicates are used for comparisonsfor comparisons
USAMRIID
ChemistryChemistry
!! Buffers from Idaho TechnologiesBuffers from Idaho Technologies–– dNTPs and 10X buffer with MgCldNTPs and 10X buffer with MgCl22
!! Smart CyclerSmart Cycler™™ Additive Reagent (SCAR Additive Reagent (SCAR buffer), Cepheid technical notebuffer), Cepheid technical note
!! Platinum Taq DNA Polymerase (Invitrogen)Platinum Taq DNA Polymerase (Invitrogen)
USAMRIID
Typical Cycling ParametersTypical Cycling Parameters
!! TwoTwo--step PCRstep PCR–– DenatureDenature–– Extend and annealExtend and anneal
!! ParametersParameters–– 959500C for 2 minutes (activates Taq polymerase)C for 2 minutes (activates Taq polymerase)–– 959500C for 1 secondC for 1 second–– 606000C for 20 secondsC for 20 seconds
!! Total time <30 minutesTotal time <30 minutes
45 cycles
USAMRIID
Sensitivity / Optimized AssaySensitivity / Optimized Assay
-100
0
100
200
300
400
500
600
700
800
900
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Cycle Number
FAM
Flu
ores
cenc
e
100pg
10pg
1pg
100fg
10fg
NTC
100pg-opt
10pg-opt
1pg-opt
100fg-opt
10fg-opt
NTC-opt
USAMRIID
SpecificitySpecificityOrganism Organism OrganismAcineobacter baumanni Bacillus subtilis var niger Staphylococcus saprophyticusBacillus anthracis BA0068 Bacillus bronchiseptica Staphylococcus epidermidisBacillus anthracis Clostridium botulinium Staphylococcus AureusBacillus anthracis Comamonas acidivarns Streptococcus pyogenesBacillus anthracis N.H. Corynebacterium sp. Streptococcus pneumoniaeBacillus anthracis (Ames) Enterococcus durans Yersinia enterocoliticaBacillus anthracis (Sterne) Enterococcus faecalis Yersinia kristenseniiBacillus anthracis (Buffalo) Enterococcus facealis Yersinia frederikseniiBacillus anthracis (ST1) Escherichia coli Yersinia pseudotuberculosisBacillus anthracis (SPS.97.13.079) H. influenzae Yersinia ruckeriBacillus anthracis (SPS 97.13.213) Klebsiella Pneumoniae Yersinia enterocoliticaBacillus anthracis (V770-NP-1R) Neisseria lactamica Yersinia pestisBacillus anthracis (CDC 476) Proteus mirabilis Yersinia pestisBacillus anthracis (Vollum) Providencia stuartii Yersinia pestisBacillus anthracis (NH) Pseudomonas aeurgenosia Yersinia pestisBacillus cereus Ralsonia picketti Yersinia pestisBacillus cereus Salmonella enteritidis Yersinia pestisBacillus thurigiensis Serratia odorifera Yersinia pestisBacillus coagulans Shigella flexneri Yersinia pestisBacillus macerans Shigella sonnei Yersinia pestisBacillus megaterium Staphylococcus aureus Yersinia pestisBacillus popilliae Staphylococcus hominis
USAMRIID
InterferenceInterferenceCt values
no spike 5 ng Hu DNA spikeSample AVE AVE
100pg 24.05 24.2010pg 28.20 27.761pg 31.62 32.31
100fg 36.32 37.1110fg 0.00 0.00
Endpoint fluorescenceno spike 5 ng Hu DNA spike
Sample AVE AVE100pg 464.85 442.6010pg 358.03 330.981pg 218.81 128.91
100fg 27.13 9.3310fg -1.67 -5.51
USAMRIID
Assays AvailableAssays Available
!! Bacillus anthracisBacillus anthracis
!! Yersinia pestisYersinia pestis
!! Brucella sp.Brucella sp.
!! Burkholderia mallei / Burkholderia mallei / pseudomalleipseudomallei
!! Clostridium botulinum Clostridium botulinum toxinstoxins
!! CoxiellaCoxiella burnettiburnetti
!! Francisella tularensisFrancisella tularensis
!! OrthopoxOrthopox SpeciesSpecies
!! VariolaVariola
!! MonkeypoxMonkeypox
!! Staphylococcus Staphylococcus aureusaureustoxinstoxins
!! SARSSARS
!! FilovirusesFiloviruses
USAMRIID
AcknowledgmentsAcknowledgments
!! LtColLtCol David David KuleshKulesh
!! Bonnie LovelessBonnie Loveless
!! Deanna ChristensenDeanna Christensen
!! Laurie HartmanLaurie Hartman
!! Melissa FryeMelissa Frye
!! Jeff GarrisonJeff Garrison
!! Deanna BridgeDeanna Bridge
!! Rebecca KaplanRebecca Kaplan
!! DSD DSD –– USAMRIIDUSAMRIID
!! Virology Division Virology Division --USAMRIIDUSAMRIID
