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

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