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TMA Assay for Detection of West Nile Virus
BPAC Meeting - March 13, 2003
Cristina Giachetti, Ph.D.J.Linnen; A.Broulik; W. Wu; J.Cline; M.Lewis;
G.Dennis; M.Cass; M.Alden; V.Casas; and S.Miller
Gen-Probe Incorporated, San Diego, CA
Objectives of the West Nile Virus Assay Development Program
To develop and manufacture a TMA-based assay for detection of West Nile virus in blood, plasma and organ/tissue donor specimens Phase 1 clinical study to determine RNA reactivity in
archived linked samples collected in areas of potentially high WNV incidence rate during 2002
Phase 2 clinical protocol to allow for widespread donor screening. It is anticipated that this trial will be initiated July 2003.
WNV Assay Development Goals
Analytical sensitivity: at least 95% detection at 50 copies/mL
Detection of genetic variants of WNV (e.g Lineage 1, including Kunjin virus, and Lineage 2 strains) with similar sensitivity
Analytical specificity: > 99.5 %
Internal Control to validate each reaction
Completely compatible with Procleix® eSAS (semi-automated) and TIGRISTM (fully automated) instrument platforms
Amplification(TMA)
Amp Rgt.,Oil,
and Enzyme
Water baths
Procleix® Semi-Automated System: Assay Protocol
Pipetting
Calibrators,Specimens,
and TCR
TECAN
SampleProcessing
Viral Lysis,RNA Capture, and Washes
TCS
Detection(HPA)
ProbeHybridization
Selection
Water bath
Results
AutomaticRLU
Reading
Luminometer
TECAN Target Capture System (TCS) Luminometer
Specimen ProcessingTarget Capture/Magnetic Microparticle Separation
Viral Lysis Treat specimens with heat and detergent Release nucleic acid
Nucleic Acid Capture Hybridize target sequence to capture probes Hybridize capture probe to oligomer sequence bind to magnetic particle
Removal of unwanted specimen Apply magnetic field to separate target from residual sample Remove residual specimen by washing
Magnetic Microparticle
TTTTTTTTTTTTTT
TTTTTTTTTTTTTT
TTTTTTTTTTTTTT
Ma
gn
e t
3’ AAAAAA…AAACAUCUAAC…CGU 5’
5’ GUAGAUUG…GCA 3’
Capture Oligo
Target RNA orDNA
TT
TT
TT
TT
TT
TT
TT
TT
TT
TT
TT
TT
TT
TT
AmplificationTranscription-Mediated Amplification (TMA)
Utilizes two enzymes: Reverse
Transcriptase
T7 RNA Polymerase
Amplifies RNA or DNA
Produces RNA amplicon
Exponential amplification(> billion fold amplification in less than one hour)
Isothermal, simplifies automation
DetectionHybridization Protection Assay (HPA)
Utilizes Acridinium Ester (AE) labeled probes
Reaction Steps:
1. HybridizationAE-labeled probe hybridizes to target RNA in solution
2. SelectionLabel on unhybridized probe is hydrolyzed, label on hybridized probe is protected
3. DetectionLabel on protected hybridized probe is detected by chemiluminescence
Detection (cont.)Dual Kinetics Analysis (DKA)
Used to differentiate Internal Control (IC) signal from target signal
Utilizes Acridinium Ester (AE) labeled probes with differential kinetics of light-off
Ortho Fluoro Acridinium Ester labeled probe = flasher probe, hybridizes to IC
2’Methyl Acridinium Ester labeled probes = Glower probes, hybridize to West Nile virus amplicon
ETF Algorithm deconvolutes light-off and calculates each signal
0
2000
4000
6000
8000
10000
12000
14000
16000
1 4 7 10
13
16
19
22
25
28
31
34
37
40
43
46
49
Interval
RLU
Flasher
Background
Glower
Current Performance of WNV Assay
Specificity in normal blood donor specimens Specificity in the presence of other blood
borne viruses Analytical sensitivity Clinical sensitivity
Specificity in normal blood donor specimens
Frozenplasmas
Freshplasmas
Combinedresults
Number tested 475 100 575Initial reactive rate (%) 0 0 0
Repeat reactive rate (%) 0 0 0
Initial IC failures 4 0 4
Initial IC failure rate (%) 0.84 0 0.69
Repeat IC failure rate 0 0 0
Specificity in the presence of other blood borne viruses
Blood borne viruses Number tested Result
HAV 5 Non-reactiveCMV 7 Non-reactiveEBV 7 Non-reactiveHBV 4 Non-reactiveHCV 2 Non-reactiveHIV-1 1 Non-reactiveHTLV I/II 7 Non-reactive
Normal populationn=575
Blood borne virusesN=33
Average s/co IC 1.99 2.1Average s/co analyte 0.01 0.00Initial/repeat reactive rate 0 0Specificity 100% 100%
Analytical SensitivityLimit of detection using in vitro transcript
WNV RNA(copies/mL)
AnalyteAve. s/co
% positiven=40
95% detectionlimit [95% CI]
50% detectionlimit [95% CI]
300 31.8 100100 31.1 100
30 26.0 100
10 18.1 97.5
3 12.1 55.0
1 11.7 35.0
0.3 2.9 10.8
0.1 0.08 0
0 0.08 0
8.2[6.6-11.5]
copies/mL
2.9[2.2-3.9]
copies/mL
Analytical SensitivityDetection of WNV in CDC viral panel*
WNV PFU/mL Positivity (%)N= 20
95% DetectionLimit [95% CI]
50% DetectionLimit [95% CI]
1.1 100
0.37 100
0.12 100
0.04 100
0.013 100
0.004 70
0.0015 55
0 0
0.008[0.005-0.042]
PFU/mL
0.001[-0.009-0.003]
PFU/mL
* Provided by Dr. Robert Lanciotti, CDC-DVBID
Analytical SensitivityDetection of WNV in Qualification Panel QWN701 from BBI
Panel Member WNV copies/mL % Positive (n=4)
QWN701.01 100 100QWN701.02 0 0QWN701.03 30 100QWN701.04 1,000 100QWN701.05 300 100QWN701.06 100 100QWN701.07 0 0QWN701.08 30 100QWN701.09 1,000 100QWN701.10 100 100QWN701.11 0 0QWN701.12 30 100QWN701.13 300 100
Analytical SensitivityLimit of detection in Lineage 2 Panel
WNV RNA(copies/mL)
AnalyteAve. s/co
% positiven=20
95% detectionlimit [95% CI]
50% detectionlimit [95% CI]
300 31.2 100100 24.7 100
30 16.4 100
10 7.57 95.0
3 8.45 45.0
1 1.59 5.0
0.3 0.2 0
0.1 0.02 0
0 0.02 0
9.0[7.1-12.8]
copies/mL
4.5[3.5-6.1]
copies/mL
Clinical SensitivityDetection of WNV in Specimens from CDC
Case Investigations
* Reactive in either plasma or RBC segment; ** IgM positive; *** Reactive with increased sample volume
Procleix WNV Assay TMA Confirmatory AssayCase IDNeat 1:8 1:16 Neat 1:8 1:16
CDC TaqManAssay*
IA-1 ++ +++ +++ ++ +++ +++ +IN-3 ++ +++ +++ ++ +++ +++ +IN-6 ++ +++ +++ ++ +++ +++ +MI-11 ++ +++ +++ ++ +++ +++ +MI-1 ++ +++ +++ ++ +++ +++ +MI-4 ++ +++ +++ ++ +++ +++ +OH-3 ++ +++ +++ ++ +++ +++ +IN-4 ++ +++ +++ ++ +++ +++ +GA-1 ++ +++ +++ ++ +++ +++ -/+***IL-4 ++ +++ +++ ++ +++ +++ -/+***NE-1** ++ -----+ +++--- ++ --- +-- -
Summary
Specificity
No cross-reactivity with other blood borne viruses
Initial reactive rate in normal blood donor population was 0% [n=575], for 100% specificity
Summary
Analytical Sensitivity 95% Detection rate at
6.6-11.5 copies/mL Lineage 1 transcript 7.1-12.8 copies/mL Lineage 2 virus 0.005-0.042 pfu/mL CDC virus panel
50% Detection rate at 2.2-3.9 copies/mL Lineage 1 transcript 3.5-6.1 copies/mL Lineage 2 virus 0.001-0.003 PFU/mL CDC virus panel
Summary
Clinical Sensitivity
Procleix WNV assay has a clinical sensitivity equal or better than CDC Taqman Assay
All reactive results with Procleix WNV Assay were confirmed with TMA assay using an alternative amplification region
Conclusions
Performance to date meets design goals for specificity and sensitivity
Results support feasibility of pool testing