Qualification Panels for West Nile Virus RNA Tests

Mark Manak, Ph.D.Boston Biomedica, Inc.West Bridgewater, MA

SoGATLangen, Germany

July 3, 2003

WNV Controls/Panels

Panel/Controls DesignDesigned to closely mimic a true serum sample Cultured Virus from an infected individualHeat TreatedDiluted in Defibrinated Plasma (Basematrix)

Panel/Controls UsesMethod optimization and comparisonStandardization of WNV RNA concentrationUseful for quantitationMonitoring 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 3 100 copies/ml

2 1,000 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 runsRun Control for Blood Screening Assays

Accurun 865 WNV Negative Run Control

* NY99 isolate, Lineage 2

RT-PCR Quantitative WNV Assay

Specific Primers for WNV5’UTR: Detects all known isolates of WNVEnv: Lineage 1 only, more sensitive

Closed-tube, Real-time fluorescent assayQuantified with TaqMan probe

Broad linear dynamic rangeBetween 300 – 10,000,000 copies/ml

Ultrasensitive assay Sensitivity to 30 copies/ml

WNV RNA Standards-RNA Transcripts

WNV RNA Quantitation

Standard

Ultra

1,000300

10,000

10,0001,000

30

300100

Copies/ml30Neg.

Neg.

Reproducibility of Ultrasensitive Assay

25

30

35

40

45

50

0.5 1.5 2.5

RNA Copies/ml

Ct

Val

ues

100 30

Ave. Ct 32.2 35.0STD 0.43 0.8%CV 1.35 2.3

(n = 12)

QWN702 Panel Quantitation

Panel Member Target

5’-LTR140uL

Env Primer140uL

EnvCDC

BBI Ultra Assay

Ct Ct Ct Ct

QWN 701. 03 10,000 33.36 29.24 29.25 26.32

QWN 701. 12 10,000 33.43 29.10 29.05 26.00

QWN 701. 05 1,000 36.66 32.96 32.00 30.09

QWN 701. 10 1,000 37.35 32.76 31.55 29.91

QWN 701. 06 300 38.04 32.92 33.80 30.36

QWN 701. 15 300 37.94 34.12 33.10 30.50

QWN 701. 01 100 39.49 33.21 36.05 32.69

QWN 701. 07 100 40.60 35.97 36.30 32.21

QWN 701. 11 100 40.50 35.43 36.40 32.88

QWN 701. 04 30 50 36.14 39.15 35.47

QWN 701. 09 30 50 50 45.90 34.61

QWN 701. 14 30 50 50 44.65 35.33

QWN 701. 02 0 50 50 50 50

QWN 701. 08 0 50 50 50 50

QWN 701. 13 0 50 50 50 50

Comparison of Assays

WNV TaqMan Assay Ct vs. Copy number

25

30

35

40

45

50

10,000 1,000 300 100 30

RNA Quant (Copies/mL)

Ct V

alue

5'-LTR 140uL

Env-140uL

Env-CDC

BBI Ultra

Negative Specimens

MaterialSource of materials

No. of samples

Ct Results

SerumAmerican Red Cross

128 50 Negative

PlasmaBBI Diagnostics

100 50 Negative

BasematrixBBI Diagnostics

92 50 Negative

WNV Positive Samples

Target 108 107 106 105 104 103

Average (Copy/mL)

1.1x108 2.0x 107 2.1x 106 2.1x 105 2.9x 104 3.4x 103

STDEV 2.1x 107 5.7x 106 6.5x 105 3.9x 104 2.2x 104 2.6x 103

CV 19.0% 28.5% 31.3% 18.6% 78% 76.2%

#Samples 8 13 12 18 14 10

WNV Titers in Avian and Mosquito Tissues

Avian Brains                 Mosquito Pools ID      copies/g        ID       copies/ml                                     ZL02-249       0       SU02-01735    0      ZL02-251       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

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

Boston Biomedica Inc.Alan Doty

Barbara Weiblen Harmesh Sharma

American Red CrossChyang Fang, Ph.D.

MA State Health Depart.Barbara Werner, Ph.D.

FDAMaria Rios, Ph.D.

CDCRobert Lanciotti, Ph.D.

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