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 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
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 2 1,000 copies/ml 3 100 copies/ml 3 30 copies/ml
2
300 copies/ml
3
Negative
Randomly Assorted
Semi blinded
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 runs Run Control for Blood Screening Assays
Accurun 865 WNV Negative Run Control
* NY99 isolate, Lineage 2
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
WNV RNA Standards-RNA Transcripts
WNV RNA Quantitation
10,000
Standard
1,000 300 30 Neg.
Copies/ml
Ultra
10,000 1,000 300 100 30
Neg.
Reproducibility of Ultrasensitive Assay
50
(n = 12)
45
Ct Values
40 35 30 25 0.5
100
32.2 0.43 1.35
1.5 RNA Copies/ml
30
35.0 0.8 2.3
2.5
Ave. Ct STD %CV
QWN702 Panel Quantitation
5’-LTR 140uL Panel Member QWN 701. 03 QWN 701. 12 QWN 701. 05 QWN 701. 10 QWN 701. 06 QWN 701. 15 QWN 701. 01 QWN 701. 07 QWN 701. 11 QWN 701. 04 QWN 701. 09 QWN 701. 14 QWN 701. 02 QWN 701. 08 Target 10,000 10,000 1,000 1,000 300 300 100 100 100 30 30 30 0 0 Ct 33.36 33.43 36.66 37.35 38.04 37.94 39.49 40.60 40.50 50 50 50 50 50 Env Primer 140uL Ct 29.24 29.10 32.96 32.76 32.92 34.12 33.21 35.97 35.43 36.14 50 50 50 50 Env CDC Ct 29.25 29.05 32.00 31.55 33.80 33.10 36.05 36.30 36.40 39.15 45.90 44.65 50 50 BBI Ultra Assay Ct 26.32 26.00 30.09 29.91 30.36 30.50 32.69 32.21 32.88 35.47 34.61 35.33 50 50
QWN 701. 13
0
50
50
50
50
Comparison of Assays
WNV TaqMan Assay Ct vs. Copy number
50
Ct Value
5'-LTR 140uL Env-140uL Env-CDC BBI Ultra
45 40 35 30 25 10,000 1,000 300 100 30
RNA Quant (Copies/mL)
Negative Specimens
Material Serum Plasma Basematrix Source of materials
American Red Cross
No. of samples
128 100 92
Ct
50 50 50
Results
Negative Negative Negative
BBI Diagnostics BBI Diagnostics
WNV Positive Samples
Target
108
107
2.0x 107 5.7x 106 28.5% 13
106
2.1x 106 6.5x 105 31.3% 12
105
2.1x 105 3.9x 104 18.6% 18
104
2.9x 104 2.2x 104 78% 14
103
3.4x 103 2.6x 103 76.2% 10
Average 1.1x108 (Copy/mL) STDEV CV #Samples
2.1x 107 19.0% 8
WNV Titers in Avian and Mosquito Tissues
Avian Brains
ID ZL02-249 ZL02-251 ZL02-255 ZL02-257 ZL02-265 ZL02-754 ZL02-755 ZL02-756 ZL02-760 ZL02-761 copies/g 0 0 0 0 0 136,000,000 37,800,000 52,200,000 10,400,000 190,000,000 ID SU02-01735 SL02-01742 SL02-01782 EM02-04183 NF02-07432 NF02-07465 NF02-07483 SU02-10395 SU02-10402 SU02-10466
Mosquito Pools
copies/ml 0 0 1,360 0 155,000 559,000 4,200,000 0 0 465,000
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
Acknowledgements
BBI Biotech Xiuli Chen, Ph.D. Jienli Wu Ron Meixell Boston Biomedica Inc. Alan Doty Barbara Weiblen Harmesh Sharma 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.