Qualification Panels for West Nile Virus RNA Tests

1
Qualification Panels for West Nile Virus
RNA Tests
Mark Manak, Ph.D. Boston Biomedica, Inc. West
Bridgewater, MA
SoGAT Langen, Germany July 3, 2003
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WNV Controls/Panels
Panel/Controls Design Designed to closely mimic a
true serum sample Cultured Virus from an
infected individual Heat Treated Diluted in
Defibrinated Plasma (Basematrix) Panel/Controls
Uses Method optimization and comparison Standardiz
ation of WNV RNA concentration Useful for
quantitation Monitoring assay performance
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West Nile Virus Qualification Panels
QWN702 - WNV Lineage 1, NY 1999 (flamingo
CDC) QWN701 - WNV Lineage 2, Uganda 1937 15
Member Panel 2 10,000 copies/ml 3
100 copies/ml 2 1,000 copies/ml 3
30 copies/ml 2 300 copies/ml
3 Negative Randomly Assorted Semi blinded
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WNV Run Control Accurun 365
Accurun 365 WNV Positive Run Control
Manufactured from cultured West Nile Virus
Target level of 300 copies/ml Ideally suited to
monitor assay precision Validation of
analytical runsRun Control for Blood Screening
Assays Accurun 865 WNV Negative Run Control
NY99 isolate, Lineage 2
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RT-PCR Quantitative WNV Assay
Specific Primers for WNV 5UTR Detects all
known isolates of WNV Env Lineage 1 only, more
sensitive Closed-tube, Real-time fluorescent
assay Quantified with TaqMan probe Broad linear
dynamic range Between 300 10,000,000
copies/ml Ultrasensitive assay Sensitivity to
30 copies/ml
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WNV RNA Standards-RNA Transcripts
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WNV RNA Quantitation
10,000
Standard
1,000
300
30 Neg.
Copies/ml
Ultra
Neg.
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Reproducibility of Ultrasensitive Assay
(n 12)
100 30
Ave. Ct 32.2 35.0 STD
0.43 0.8 CV
1.35
2.3
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QWN702 Panel Quantitation
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Comparison of Assays
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Negative Specimens
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WNV Positive Samples
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WNV Titers in Avian and Mosquito Tissues
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BBI WNV Panel and Controls

• BBI Panels and Controls
• ACCURUN WNV RNA Positive Control  A365    (NY99
  Strain)
• ACCURUN WNV RNA Negative Control  A865
•  
• QWN702  WNV Qualification  Panel (Lineage 1)
  (NY99 Strain)
• QWN701  WNV Qualification  Panel (Lineage 2) 
  (Uganda Strain)
• WNV Characterized Samples                  
              (US Source)
• Panels and controls Uses
• Assay Development
• Comparison/standardization of Assays
• Training of Laboratory Personnel
• Assay Validation
• Monitoring Assay Performance

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Acknowledgements

• American Red Cross
• Chyang Fang, Ph.D.
• MA State Health Depart.
• Barbara Werner, Ph.D.
• FDA
• Maria Rios, Ph.D.
• CDC
• Robert Lanciotti, Ph.D.

BBI Biotech Xiuli Chen, Ph.D. Jienli Wu Ron
Meixell

• Boston Biomedica Inc.
• Alan Doty
• Barbara Weiblen
• Harmesh Sharma