Interpretation of Results

1. Interpretation of Results Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010

2. Instrumentation • Cepheid Smartcycler • Training • Diagnostic testing • Equivalency testing for 96 well • platforms • ABI 7900HT and 7500 • Stratagene MX3005P • BioRad iQ5

3. Results interpretation • Check the controls • Transcribed RNA • Ct < 29 • Negative control – RNease free water • Check background fluorescence • Check each sample individually • Does the primary growth curve have a flat baseline and log linear phase? • Growth curve artifacts are part of rRT-PCR

4. Primary Growth Curve Plateau Log-linear Log-linear baseline Baseline

5. Evaluation of Growth Curve Threshold set too low Log-linear baseline Threshold set appropriately Curve entering Log-linear baseline

6. Results Table FAM Ct - cycle threshold or PCR cycle number at which the specimen tested positive Status – functional status of instrument for individual test site – OK, Warning, or Error

7. Software Growth Curve Artifacts

8. Software Artifacts Correction

9. Background Fluorescence • Is a normal property of Real Time PCR • Fluorescence derived from unbound probe, free dye, non-specific cleavage of probe or sample auto-fluorescence • Represents the baseline phase • Log-linear phase represents background + fluorescence from amplified DNA Total FU – background FU = specific FU

10. Background Fluorescence Represents the Baseline of a Real Time PCR Growth Curve Background Fluorescence Off Raw fluorescence data provides essential information about the magnitude of the background signal and the shape of the growth curve without drift correction.

11. Source of Background Fluorescence Background Fluorescence ON • Background fluorescence is from unbound probe • Free dye • Non-specific cleavage of probe • Sample auto-fluorescence

12. Background Subtraction • Corrects for any positive or negative drift • Calculates the average background signal and subtracts this from each data point for each specimen • Between Bkgnd Min and Max Cycle • After a cycle threshold is detected there is no further background subtraction • Background fluoresce should not exceed 500 FU

13. Results interpretation • Following run evaluation • Valid positive and negative control • Specimen has a normal curve • Record the cycle threshold (Ct) values • If a sample has no cycle threshold values (0.00) it is negative • Determine if there are any suspect samples • Weak positives- Ct values >35

14. Suspect samples • For AIV or NDV a farm or premise is never considered positive based on one positive rRT-PCR result • Epidemiology- dangerous contact • Clinical condition • Other positive diagnostic test • Flu Detect (AIV) • Virus isolation • A second rRT-PCR test for a different target • AIV – H5 or H7 • NDV- vNDV or vaccine virus specific • Are other samples from the same farm positive? • Are there enough samples from the farm?

15. Surveillance for AIV by rRT-PCR AIVMatrix rRT-PCR Negative No further testing Positive H5 & H7rRT-PCR Positive Negative Report to NVSL for Confirmation with VI and rRT-PCR Report to NVSL for Confirmation with VI

16. Surveillance for APMV-1 by rRT-PCR APMV-1MatrixrRT-PCR No further testing Negative Positive vNDV rRT-PCR Positive Negative Report to NVSL for Confirmation with VI and rRT-PCR Report to NVSL for Confirmation with VI and B1 rRT-PCR (vaccine)

17. APMV-1 primer/probe Target: Matrix gene Will detect most APMV-1 isolates Virulent NDV Avirulent vaccine strains PPMV vNDV - VFP-1primer/probe Target: fusion gene cleavage site Designed to detect the CA 2002/03 strain of vNDV Will detect most velogens and mesogens. Will not detect vaccine strains Will detect some PPMV APMV-1 RRT-PCR Assay

18. Matrix Primers/probe Will detect all 16 H subtypes (H1-16) of AIV Detects both HPAI and LPAI Detects Asian H5N1 H5 Primers/probe Detects most North American strains of H5 AIV Detects Asian H5N1 Detects both HPAI & LPAI H7 Primers/probe Detects most North Americans strains of H7 AIV Detects both HPAI & LPAI RRT-PCR for AIV

19. Evaluation of H5 Subtype rRT-PCR Test for Asian H5N1 • H5 test was originally designed primarily for North American isolates • Can identify Asian H5N1 viruses with lower sensitivity • Sequence analysis of Asian isolates showed good conservation with reverse primer and probe, but 4 mismatches with forward primer • Redesigned H5 test to include forward primers optimized for both Asian and North American viruses • NA H5F TGACTATCCACAATACTCA • EA H5F TGACTACCCGCAGTATTCA • H5 reagent bead increases sensitivity of detection for the Asian H5 lineage of AI

20. Internal Control for Detection of False Negative Results • Competitive IC • Uses the same primer sites as viral target • AI matrix reagent beads - Cepheid • Non-competitive • Multiplex – completely different target and PCR in the same tube • Spiked positive control – duplicate well with diagnostic specimen and spiked +

21. 1st study Cepheid SmartCycler 2.0 Stratagene MX3005P BioRad iQ5 2nd study Cepheid SmartCycler 2.0 Stratagene MX3005P ABI 7500 Instrument Equivalency Evaluations

22. Real-time Instrument Evaluation • Interpretation of results was conducted with automatic baseline settings and background subtraction • Thermal cycling times were adjusted as needed for instrument ramp speed and collection of fluorescence • Thermal cycling temperatures remained the same as official NVSL protocol • ABI – adjustment in PCR steps for 3 step PCR

23. Stratagene, BioRad and Cepheid Comparison with Matrix Assay • Qiagen One-Step RT-PCR chemistry (gold standard) • Significant (p<0.01) difference in detection between Cepheid and BioRad as compared to Stratagene • Ct values • Endpoint of Detection (EOD) • EOD • Cepheid - 10-7 • BioRad – 10-7 • Stratagene – 10-8

24. Stratagene, BioRad and Cepheid Comparison with H5 Assay • Qiagen One-Step RT-PCR chemistry • Significant (p<0.01) difference in detection between Stratagene and BioRad as compared to Cepheid with • Ct values • Endpoint of Detection (EOD) EOD Cepheid - 10-6 BioRad – 10-8 Stratagene – 10-8

25. ABI 7900 and 7500 Equivalency Evaluation • Separate equivalency validation studies • 7900 – Laser excitation with scanning head, detection via spectrograph and CDC camera • 7500 – Tungsten-halogen lamp, detection via CDC camera • 7900 – Previously compared to Cepheid system using Qiagen One-Step RT-PCR • 7500 – compared to Cepheid and Stratagene using 4 different One-Step kits

26. ABI 7500 Comparison EOD ABI 10-7 , Cepheid 10-6 EOD ABI, Stratagene and Cepheid 10-8 AI H5 Qiagen AI H5 Ambion Ag-Path • Significant difference in detection (p<0.01) between Cepheid and ABI 7500 with Qiagen chemistry • Similar sensitivity and EOD between Cepheid, Stratagene and ABI 7500 with Ambion Ag-Path chemistry

27. Chemistry Equivalency Evaluation • Chemistries compared • Qiagen One-Step RT-PCR kit • Ambion Ag-Path chemistry • ABI One-Step RT-PCR kit • Invitrogen Ultrasense One-Step RT-PCR • One-Step RT-PCR kits were compared with Cepheid, ABI 7500, and Stragagene instruments

28. Chemistry Comparison with Cepheid • Significant difference in sensitivity between each of the One-Step RT-PCR chemistry kits • Invitrogen – significant decrease in sensitivity Endpoint Of Detection Qiagen 10-6 Ambion Ag-Path 10-7 ABI One-Step – 10-5 Invitrogen – 10-3

29. Chemistry Comparison with ABI 7500 • ABI 7900 was previously shown to be equivalent to the Cepheid using Qiagen chemistry • Ambion Ag-Path kit out performed Qiagen, ABI and Invitrogen One-Step RT-PCR kits with ABI 7500, Cepheid and Stratagene instruments Endpoint Of Detection Qiagen 10-8 Ambion Ag-Path 10-8 ABI One-Step – 10-6 Invitrogen – 10-7

30. Thank Your For Your Attention