Interpretation of Results Jan Pedersen USDA APHIS VS

Interpretation of Results Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010

Instrumentation Cepheid Smartcycler • Training • Diagnostic testing Equivalency testing for 96 well platforms • ABI 7900 HT and 7500 • Stratagene MX 3005 P • Bio. Rad i. Q 5

Results interpretation v Check the controls § Transcribed RNA § Ct < 29 § Negative control – RNease free water Check background fluorescence v Check each sample individually v § Does the primary growth curve have a flat baseline and log linear phase? § Growth curve artifacts are part of r. RT-PCR

Primary Growth Curve Plateau baseline Baseline Log-linear

Evaluation of Growth Curve Threshold set too low Log-linear baseline Threshold set appropriately Curve entering Log-linear baseline

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

Software Growth Curve Artifacts

Software Artifacts Correction

Background Fluorescence v v 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 Background Fluorescence Off Raw fluorescence data provides essential information about the magnitude of the background signal and the shape of the growth curve without drift correction.

Source of Background Fluorescence ON Background fluorescence is from unbound probe • Free dye • Non-specific cleavage of probe • Sample auto-fluorescence

Background Subtraction v v v 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 v Following run evaluation v v v 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 v Determine if there any suspect samples § Weak positives- Ct values >35

Suspect samples v For AIV or NDV a farm or premise is never considered positive based on one positive r. RT-PCR result § Epidemiology- dangerous contact § Clinical condition § Other positive diagnostic test n n n Flu Detect (AIV) Virus isolation A second r. RT-PCR test for a different target n n v v AIV – H 5 or H 7 NDV- v. NDV or vaccine virus specific Are other samples from the same farm positive? Are there enough samples from the farm?

Surveillance for AIV by r. RT-PCR AIV Matrix r. RT-PCR Negative No further testing Positive H 5 & H 7 r. RT-PCR Negative Report to NVSL for Confirmation with VI Positive Report to NVSL for Confirmation with VI and r. RT-PCR

Surveillance for APMV-1 by r. RT-PCR APMV-1 Matrix r. RT-PCR Negative No further testing Positive v. NDV r. RT-PCR Positive Negative Report to NVSL for Confirmation with VI and B 1 r. RT-PCR (vaccine) Report to NVSL for Confirmation with VI and r. RT-PCR

APMV-1 RRT-PCR Assay v APMV-1 primer/probe v v Target: Matrix gene v n n Will detect most APMV-1 isolates Virulent NDV Avirulent vaccine strains PPMV v. NDV - VFP-1 primer/probe Target: fusion gene cleavage site n n Designed to detect the CA 2002/03 strain of v. NDV Will detect most velogens and mesogens. Will not detect vaccine strains Will detect some PPMV

RRT-PCR for AIV v Matrix Primers/probe n n n Will detect all 16 H subtypes (H 1 -16) of AIV Detects both HPAI and LPAI Detects Asian H 5 N 1 v H 5 Primers/probe n n n v Detects most North American strains of H 5 AIV Detects Asian H 5 N 1 Detects both HPAI & LPAI H 7 Primers/probe n n Detects most North Americans strains of H 7 AIV Detects both HPAI & LPAI

Evaluation of H 5 Subtype r. RT-PCR Test for Asian H 5 N 1 v v v H 5 test was originally designed primarily for North American isolates Can identify Asian H 5 N 1 viruses with lower sensitivity Sequence analysis of Asian isolates showed good conservation with reverse primer and probe, but 4 mismatches with forward primer Redesigned H 5 test to include forward primers optimized for both Asian and North American viruses n NA H 5 F TGACTATCCACAATACTCA n EA H 5 F TGACTACCCGCAGTATTCA H 5 reagent bead increases sensitivity of detection for the Asian H 5 lineage of AI

Internal Control for Detection of False Negative Results v Competitive IC n n v Uses the same primer sites as viral target AI matrix reagent beads - Cepheid Non-competitive n n Multiplex – completely different target and PCR in the same tube Spiked positive control – duplicate well with diagnostic specimen and spiked +

Instrument Equivalency Evaluations 1 st study Ø Cepheid Smart. Cycler 2. 0 2 nd study Ø Cepheid Smart. Cycler 2. 0 Ø Stratagene MX 3005 P Ø Bio. Rad i. Q 5 Ø ABI 7500

Real-time Instrument Evaluation Ø Ø 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

Stratagene, Bio. Rad and Cepheid Ø Qiagen. Comparisonchemistry. Matrix Assay One-Step RT-PCR with (gold standard) Ø Significant (p<0. 01) difference in detection between Cepheid and Bio. Rad as compared to Stratagene • • Ct values Endpoint of Detection (EOD) EOD • Cepheid - 10 -7 • Bio. Rad – 10 -7 • Stratagene – 10 -8

Stratagene, Bio. Rad and Cepheid Comparison with H 5 Assay Ø Qiagen One-Step RT-PCR chemistry Ø Significant (p<0. 01) difference in detection between Stratagene and Bio. Rad as compared to Cepheid with • • Ct values Endpoint of Detection (EOD) EOD Cepheid - 10 -6 Bio. Rad – 10 -8 Stratagene – 10 -8

ABI 7900 and 7500 Equivalency Evaluation Ø Separate equivalency validation studies • • 7900 – Laser excitation with scanning head, detection via spectrograph and CDC camera 7500 – Tungsten-halogen lamp, detection via CDC camera Ø 7900 – Previously compared to Cepheid system using Qiagen One-Step RT-PCR Ø 7500 – compared to Cepheid and Stratagene using 4 different One-Step kits

ABI 7500 Comparison EOD ABI 10 -7 , Cepheid 10 -6 AI H 5 Qiagen EOD ABI, Stratagene and Cepheid 10 -8 AI H 5 Ambion Ag-Path Ø Significant difference in detection (p<0. 01) between Cepheid and ABI 7500 with Qiagen chemistry Ø Similar sensitivity and EOD between Cepheid, Stratagene and ABI 7500 with Ambion Ag-Path chemistry

Chemistry Equivalency Evaluation Ø Chemistries compared • • Qiagen One-Step RT-PCR kit Ambion Ag-Path chemistry ABI One-Step RT-PCR kit Invitrogen Ultrasense One-Step RT-PCR Ø One-Step RT-PCR kits were compared with Cepheid, ABI 7500, and Stragagene instruments

Chemistry Comparison with Cepheid Ø Significant difference in sensitivity between each of the One-Step RT-PCR chemistry kits Ø Invitrogen – significant decrease in sensitivity Endpoint Of Detection Qiagen 10 -6 Ambion Ag-Path 10 -7 ABI One-Step – 10 -5 Invitrogen – 10 -3

Chemistry Comparison with ABI 7500 Ø ABI 7900 was previously shown to be equivalent to the Cepheid using Qiagen chemistry Ø Ambion Ag-Path kit out performed Qiagen, ABI and Invitrogen One-Step RT-PCR kits with ABI 7500, Cepheid and Stratagene instruments Endpoint Of Detection Qiagen 10 -8 Ambion Ag-Path 10 -8 ABI One-Step – 10 -6 Invitrogen – 10 -7

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