J. Callahan, Pabilonia, K.L.. Presented by Nardy Robben. The world leader in serving science

Transcription

1 The effect of pooling 5 or 11 Tracheal/Oropharyngeal (TR/OP) swabs on the performance of a USDA Licensed reverse transcriptase, real-time PCR (rrt-pcr) test for avian influenza virus J. Callahan, Pabilonia, K.L.. Presented by Nardy Robben The world leader in serving science

2 Types of response/ reaction with Avian Influenza No Avian Influenza around First confirmation of Avian Influenza 2

3 Individual samples, pools of 5 or 11 samples surveillance outbreak 3

4 Agenda Individual testing, pooling Study method and results for pooling of samples Summary The study was conducted by Colorado State University, USA The samples tested in the Veterinary Laboratory of Colorado State University, USA 4

5 Study design Round 1 Round 2 60 birds 50 birds negative control group 10 birds challenged 20 birds negative control group 8 birds challenged Daily sampling from day 1 to day 9 Challenged with low pathogenic AI strain 5

6 Pooling strategy for testing Round 1 5 pools: Round 2 8 pools: 11 samples 11 samples 10 pools: 8 pools: 5 samples 5 samples Pooling composition different per round 6

7 PCR workflow used Sample extraction: Amplification MagMAX -96 Viral RNA Isolation Kit KingFisher automated nucleic acid extraction platform VetMAX -Gold AIV Detection Kit NAHLN* avian influenza virus standard operating procedure all pools: tested with both amplification methods. Acceptance criteria for pools in this study: - to be considered as positive: all ct values in the positive range according to SOP/ IFU of the methods. - pools with ct values in the suspect or negative range and which did not meet the acceptance criteria were not included in the data calculation in this study 7 *NAHLN: national animal health laboratory network, USA

8 Study and Test results Analysis of Days 0-9 post infection. individual assay interpretation for positive samples Combined Results Total Success # of pools (11 samples) 43 % Positive Pools # of pools (5 samples) 36 % Positive Pools Counted as positive when both PCR tests (according to cut off) give positive results 8

9 Analysis of Days 2-6 post infection, individual assay interpretation for positive samples Combined Results Total Success # of pools Pools of 11, 37 % Positive Pools # of pools Pools of 5, 26 % Positive Pools Counted as positive when both PCR tests (according to cut off) give positive results 9

10 Results with different cut off Analysis of Days 2-6 post infection, Pools with ct < 38 are interpreted as positive* Combined Results Total Success # of pools, Pools of % Positive Pools # of pools, Pools of 5 32 % Positive Pools * Protocol VetMAX Gold AIV detection kit 10

11 Analysis of Days 2-6 post infection, Pools with ct < 35 are interpreted as positive* Combined Results Total Success Pools of 11, # of pools 35 % Positive Pools Pools of 5, # of pools 27 % Positive Pools *following NAHLN protocol 11

12 Discussion and conclusion Sensitivity individual samples and pools: Cut off and test interpretation excellent results risk of contamination in a pool use of endogenous control vs exogenous control Shedding pattern of birds 12

13 summary Different sizes of pooling and individual testing Reliability and consistency in test results low volume testing and high volume testing VetMAX -Gold AIV detection kit Easy to use kit in handling and ordering for surveillance and outbreak scenarios USDA licensed and NAHLN approved. 13

14 For more information, visit our booth or VetMAX -Gold AIV Detection Kit The first USDA-licensed, PCR-based solution for the detection of avian influenza virus (AIV) 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. For Veterinary Use Only. For In Vitro Use Only. Regulatory requirements vary by country; products may not be available in your geographic area. 14