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

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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

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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

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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

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