Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Interpretation of Results Jan Pedersen USDA, APHIS, VS, National Veterinary Services Laboratories, Ames, IA 50010."— Presentation transcript:

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

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

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

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

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 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 Surveillance for AIV by rRT-PCR

16 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 Surveillance for APMV-1 by rRT-PCR

17 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

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) EOD Cepheid BioRad – Stratagene – 10 -8

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) EOD Cepheid BioRad – Stratagene – 10 -8

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  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 EOD ABI 10 -7, Cepheid EOD ABI, Stratagene and Cepheid AI H5 QiagenAI H5 Ambion Ag-Path

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 Endpoint Of Detection Qiagen Ambion Ag-Path ABI One-Step – Invitrogen – 10 -3

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 Endpoint Of Detection Qiagen 10-8 Ambion Ag-Path 10-8 ABI One-Step – 10-6 Invitrogen – 10-7

30 Background Subtraction

31 Thank Your For Your Attention


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

Similar presentations


Ads by Google