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
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
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
Raw fluorescence data provides essential information about the magnitude
of the background signal and the shape of the growth curve without drift
correction.
Background
Fluorescence
Off
11. Source of Background Fluorescence
Background fluorescence is from unbound probe
•Free dye
•Non-specific cleavage of probe
•Sample auto-fluorescence
Background
Fluorescence
ON
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. AIV Matrix
rRT-PCR
H5 & H7 rRT-PCR
Positive
No further
testing
Positive
Report to NVSL for
Confirmation with VI
Report to NVSL for
Confirmation with VI and
rRT-PCR
Negative
Negative
Surveillance for AIV by rRT-PCR
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
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
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 +
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. AI Matrix
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
0 2 4 6 8 10
Ceph BioR Strat
Linear (Ceph) Linear (BioR) Linear (Strat)
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)
AIV H5
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
0 2 4 6 8 10
Ceph BioR Strat
Linear (Ceph) Linear (BioR) Linear (Strat)
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
AI H5 Ambion
15
20
25
30
35
40
45
50
0 2 4 6 8 10
Ceph Strat ABI
Linear (Ceph) Linear (Strat) Linear (ABI)
AI H5 Qiagen
15
20
25
30
35
40
45
50
0 2 4 6 8 10
Ceph ABI Linear (Ceph) Linear (ABI)
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
EOD
ABI 10-7 , Cepheid 10-6
EOD
ABI, Stratagene and Cepheid 10-8
AI H5 Qiagen AI H5 Ambion Ag-Path
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. AI H5 Cepheid
15
20
25
30
35
40
45
50
0 2 4 6 8 10
Qiagen Ambion Applied
Invitrogen Linear (Qiagen) Linear (Ambion)
Linear (Applied) Linear (Invitrogen)
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
AI H5 ABI
15
20
25
30
35
40
45
50
0 2 4 6 8 10
Qiagen Ambion Applied
Invitrogen Linear (Qiagen) Linear (Ambion)
Linear (Applied) Linear (Invitrogen)
Endpoint Of Detection
Qiagen 10-8
Ambion Ag-Path 10-8
ABI One-Step – 10-6
Invitrogen – 10-7
