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
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 FluorescenceON
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
AIV Matrix
rRT-PCR
Negative
No further testing
Positive
H5 & H7 rRT-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-1 Matrix rRT-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 RRT-PCR Assay APMV-1 primer/probe Target: Matrix gene
Will detect most APMV-1 isolates
Virulent NDV
Avirulent vaccine strains
PPMV
vNDV - VFP-1 primer/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

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

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

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
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
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, Stratagene and Cepheid 10-8
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. Background Subtraction

31. Thank Your For Your Attention