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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 controlsTranscribed RNA Ct < 29Negative control – RNease free water
Check background fluorescenceCheck 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-linearbaseline
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 PCRFluorescence derived from unbound probe, free dye, non-specific cleavage of probe or sample auto-fluorescenceRepresents 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 driftCalculates the average background signal and subtracts this from each data point for each specimenBetween Bkgnd Min and Max Cycle After a cycle threshold is detected there is no further background subtractionBackground fluoresce should not exceed 500 FU
Results interpretation
Following run evaluationValid positive and negative controlSpecimen has a normal curve
Record the cycle threshold (Ct) valuesIf a sample has no cycle threshold values (0.00) it is negative
Determine if there are any suspect samplesWeak positives- Ct values >35
Suspect samplesFor AIV or NDV a farm or premise is never considered positive based on one positive rRT-PCR result
Epidemiology- dangerous contactClinical conditionOther positive diagnostic test
Flu Detect (AIV) Virus isolation A second rRT-PCR test for a different target
AIV – H5 or H7NDV- 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 MatrixrRT-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 isolatesVirulent NDVAvirulent vaccine strainsPPMV
vNDV - VFP-1primer/probeTarget: fusion gene cleavage site
Designed to detect the CA 2002/03 strain of vNDVWill detect most velogens and mesogens.Will not detect vaccine strainsWill detect some PPMV
RRT-PCR for AIV
MatrixPrimers/probe
Will detect all 16 H subtypes (H1-16) of AIVDetects both HPAI and LPAIDetects Asian H5N1
H5 Primers/probeDetects most North American strains of H5 AIVDetects Asian H5N1Detects both HPAI & LPAI
H7 Primers/probeDetects most North Americans strains of H7 AIVDetects both HPAI & LPAI
Evaluation of H5 Subtype rRT-PCR Test for Asian H5N1
H5 test was originally designed primarily for North American isolatesCan identify Asian H5N1 viruses with lower sensitivitySequence analysis of Asian isolates showed good conservation with reverse primer and probe, but 4 mismatches with forward primerRedesigned H5 test to include forward primers optimized for both Asian and North American viruses
NA H5F TGACTATCCACAATACTCAEA 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 ICUses the same primer sites as viral targetAI matrix reagent beads - Cepheid
Non-competitiveMultiplex – completely different target and PCR in the same tubeSpiked positive control – duplicate well with diagnostic specimen and spiked +
Instrument Equivalency Evaluations
1st studyCepheid SmartCycler 2.0
StratageneMX3005P
BioRad iQ5
2nd studyCepheid SmartCycler 2.0
StratageneMX3005P
ABI 7500
Real-time Instrument Evaluation
Interpretation of results was conducted with automatic baseline settings and background subtractionThermal cycling times were adjusted as needed for instrument ramp speed and collection of fluorescenceThermal 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 chemistrySignificant (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)
EODCepheid - 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-PCR7500 – 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
1520253035404550
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-PCROne-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 CepheidSignificant 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 chemistryAmbion 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
Background Subtraction
Thank Your For Your Attention