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

Transcription

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

2 Instrumentation Cepheid Smartcycler Training Diagnostic testing Equivalency testing for 96 well platforms ABI 7900HT and 7500 Stratagene MX3005P BioRad iq5

3 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

4 Primary Growth Curve Plateau baseline Baseline Log-linear Log-linear

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

6 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

7 Software Growth Curve Artifacts

8 Software Artifacts Correction

9 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

10 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.

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

12 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

13 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

14 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?

15 Surveillance for AIV by rrt-pcr AIV Matrix rrt-pcr Positive H5 & H7 rrt-pcr Negative No further testing Negative Report to NVSL for Confirmation with VI Positive Report to NVSL for Confirmation with VI and rrt-pcr

16 Surveillance for APMV-1 by rrt-pcr APMV-1 Matrix rrt-pcr Negative No further testing Positive Negative vndv rrt-pcr Report to NVSL for Confirmation with VI and B1 rrt-pcr (vaccine) Positive Report to NVSL for Confirmation with VI and rrt-pcr

17 APMV-1 RRT-PCR Assay APMV-1 primer/probe vndv - VFP-1 primer/probe Target: Matrix gene Target: fusion gene cleavage site Will detect most APMV-1 isolates Virulent NDV Avirulent vaccine strains PPMV 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

18 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

19 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

20 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 +

21 Instrument Equivalency Evaluations 1 st study Cepheid SmartCycler 2.0 Stratagene MX3005P BioRad iq5 2 nd study Cepheid SmartCycler 2.0 Stratagene MX3005P ABI 7500

22 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

23 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) AI Matrix EOD Cepheid BioRad 10-7 Stratagene Ceph BioR Strat Linear (Ceph) Linear (BioR) Linear (Strat)

24 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 EOD Cepheid BioRad 10-8 Stratagene Ceph BioR Strat Linear (Ceph) Linear (BioR) Linear (Strat)

25 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

26 ABI 7500 Comparison EOD ABI 10-7, Cepheid 10-6 EOD ABI, Stratagene and Cepheid 10-8 AI H5 AI H5 Qiagen Ceph ABI Linear (Ceph) Linear (ABI) AI H5 AI H5 Ambion Ag-Path Ceph Strat ABI Linear (Ceph) Linear (Strat) 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

27 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

28 Chemistry Comparison with Cepheid Significant difference in sensitivity between each of the One-Step RT-PCR chemistry kits Invitrogen significant decrease in sensitivity AI H5 Cepheid Endpoint Of Detection Qiagen 10-6 Ambion Ag-Path 10-7 ABI One-Step 10-5 Invitrogen Qiagen Ambion Applied Invitrogen Linear (Qiagen) Linear (Ambion) Linear (Applied) Linear (Invitrogen)

29 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 Endpoint Of Detection Qiagen 10-8 Ambion Ag-Path 10-8 ABI One-Step 10-6 Invitrogen Qiagen Ambion Applied Invitrogen Linear (Qiagen) Linear (Ambion) Linear (Applied) Linear (Invitrogen)

30 Background Subtraction

31 Thank Your For Your Attention