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Qualification Panels for West Nile Virus RNA Tests

Qualification Panels for West Nile Virus RNA Tests . Mark Manak, Ph.D. Boston Biomedica, Inc. West Bridgewater, MA. SoGAT Langen, Germany July 3, 2003. WNV Controls/Panels. Panel/Controls Design Designed to closely mimic a true serum sample Cultured Virus from an infected individual

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Qualification Panels for West Nile Virus RNA Tests

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  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

  2. 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 • Standardization of WNV RNA concentration • Useful for quantitation • Monitoring assay performance

  3. 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

  4. 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

  5. RT-PCR Quantitative WNV Assay • Specific Primers for WNV • 5’UTR: 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

  6. WNV RNA Standards-RNA Transcripts

  7. 10,000 1,000 300 100 30 WNV RNA Quantitation 10,000 Standard 1,000 300 30 Neg. Copies/ml Ultra Neg.

  8. Reproducibility of Ultrasensitive Assay (n = 12) 100 30 Ave. Ct 32.2 35.0 STD 0.43 0.8 %CV 1.35 2.3

  9. QWN702 Panel Quantitation

  10. Comparison of Assays

  11. Negative Specimens

  12. WNV Positive Samples

  13. Avian Brains                 Mosquito PoolsID      copies/g        ID       copies/ml      ZL02-249       0SU02-017350ZL02-251       0       SL02-01742      0      ZL02-255       0       SL02-01782      1,360  ZL02-257        0       EM02-04183     0      ZL02-265        0       NF02-07432      155,000ZL02-754        136,000,000      NF02-07465      559,000ZL02-755         37,800,000       NF02-07483      4,200,000       ZL02-756        52,200,000       SU02-10395     0      ZL02-760        10,400,000       SU02-10402      0      ZL02-761        190,000,000       SU02-10466      465,000 WNV Titers in Avian and Mosquito Tissues

  14. 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

  15. Boston Biomedica Inc. Alan Doty Barbara Weiblen Harmesh Sharma American Red Cross Chyang Fang, Ph.D. MA State HealthDepart. Barbara Werner, Ph.D. FDA Maria Rios, Ph.D. CDC Robert Lanciotti, Ph.D. Acknowledgements BBI Biotech Xiuli Chen, Ph.D. Jienli Wu Ron Meixell

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