TMA Assay for Detection of West Nile Virus

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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
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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.

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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 gt 99.5
• Internal Control to validate each reaction
• Completely compatible with Procleix eSAS
  (semi-automated) and TIGRISTM (fully automated)
  instrument platforms

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Procleix Semi-Automated System Assay Protocol
Sample Processing Viral Lysis, RNA Capture, and
Washes TCS
Detection (HPA) Probe Hybridization Selection W
ater bath
Amplification (TMA) Amp Rgt., Oil, and
Enzyme Water baths
Pipetting Calibrators, Specimens, and TCR TECAN
Results Automatic RLU Reading Luminometer
TECAN
Target Capture System (TCS)
Luminometer
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Specimen Processing Target 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

TTTTTTTTTTTTTT
TTTTTTTTTTTTTT
Magnetic Microparticle
TTTTTTTTTTTTTT
Target RNA or DNA
Magnet
5 GUAGAUUGGCA 3
3 AAAAAAAAACAUCUAACCGU 5
TTTTTTTTTTTTTT
TTTTTTTTTTTTTT
Capture Oligo
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Amplification Transcription-Mediated
Amplification (TMA)

• Utilizes two enzymes Reverse
  Transcriptase
• T7 RNA Polymerase
• Amplifies RNA or DNA
• Produces RNA amplicon
• Exponential amplification(gt billion fold
  amplification in less than one hour)
• Isothermal, simplifies automation

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Detection Hybridization Protection Assay (HPA)

• Utilizes Acridinium Ester (AE) labeled probes
• Reaction Steps
• 1. Hybridization
• AE-labeled probe hybridizes to target RNA in
  solution
• 2. Selection
• Label on unhybridized probe is hydrolyzed, label
  on hybridized probe is protected
• 3. Detection
• Label on protected hybridized probe is detected
  by chemiluminescence

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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
• 2Methyl Acridinium Ester labeled probes Glower
  probes, hybridize to West Nile virus amplicon
• ETF Algorithm deconvolutes light-off and
  calculates each signal

RLU
Background
Flasher
Glower
1
4
7
10
13
16
19
22
25
28
31
34
37
40
43
46
49
Interval
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Current Performance of WNV Assay

• Specificity in normal blood donor specimens
• Specificity in the presence of other blood borne
  viruses
• Analytical sensitivity
• Clinical sensitivity

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Specificity in normal blood donor specimens
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Specificity in the presence of other blood borne
viruses
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Analytical SensitivityLimit of detection using
in vitro transcript
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Analytical SensitivityDetection of WNV in CDC
viral panel
Provided by Dr. Robert Lanciotti, CDC-DVBID
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Analytical SensitivityDetection of WNV in
Qualification Panel QWN701 from BBI
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Analytical SensitivityLimit of detection in
Lineage 2 Panel
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Clinical SensitivityDetection of WNV in
Specimens from CDC Case Investigations
Reactive in either plasma or RBC segment
IgM positive Reactive with increased sample
volume
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Summary

• Specificity
• No cross-reactivity with other blood borne
  viruses
• Initial reactive rate in normal blood donor
  population was 0 n575, for 100 specificity

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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

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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

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Conclusions

• Performance to date meets design goals for
  specificity and sensitivity
• Results support feasibility of pool testing

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Acknowledgments
This project is funded in part with federal funds
from the National Heart, Lung and Blood Institute
under Contract