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Interpretation of Results
Jan PedersenUSDA, APHIS, VS,
National Veterinary Services Laboratories, Ames, IA 50010
Instrumentation
Cepheid Smartcycler•Training•Diagnostic testing
Equivalency testing for 96 wellplatforms•ABI 7900HT and 7500•Stratagene MX3005P•BioRad iQ5
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
Log-linear
baselin
e
Primary Growth Curve
Baseline
Log-linear
Plateau
Log-linear
Curve entering Log-linear
baseline
baseline
Threshold set appropriately
Threshold set too low
Evaluation of Growth Curve
Results Table
FAM Ct - cycle threshold or PCR cycle number at which the specimen tested positiveStatus – functional status of instrument for individual test site – OK, Warning, or Error
Software Growth Curve Artifacts
Software Artifacts Correction
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
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.
BackgroundFluorescenceOff
Source of Background Fluorescence
Background fluorescence is from unbound probe
•Free dye•Non-specific cleavage of probe•Sample auto-fluorescence
BackgroundFluorescenceON
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
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
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?
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
APMV-1 Matrix rRT-PCR
vNDV
rRT-PCR
Positive
No further testing
Positive
Report to NVSL for Confirmation with VI and B1 rRT-PCR (vaccine)
Report to NVSL for Confirmation with VI and
rRT-PCR
Negative
Negative
Surveillance for APMV-1 by rRT-PCR
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
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
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
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 +
Instrument Equivalency Evaluations
1st study Cepheid SmartCycler 2.0
Stratagene MX3005P
BioRad iQ5
2nd study Cepheid
SmartCycler 2.0
Stratagene MX3005P
ABI 7500
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
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
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
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
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
EODABI 10-7 , Cepheid 10-6
EODABI, Stratagene and Cepheid 10-8
AI H5 Qiagen AI H5 Ambion Ag-Path
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
AI H5 Cepheid
15
20
25
30
35
40
45
50
0 2 4 6 8 10Qiagen 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 DetectionQiagen 10-6
Ambion Ag-Path 10-7
ABI One-Step – 10-5
Invitrogen – 10-3
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 10Qiagen Ambion Applied
Invitrogen Linear (Qiagen) Linear (Ambion)
Linear (Applied) Linear (Invitrogen)
Endpoint Of DetectionQiagen 10-8Ambion Ag-Path 10-8ABI One-Step – 10-6Invitrogen – 10-7
Thank Your For Your Attention