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TRANSCRIPT
NDV Technical roundup:
Update on NDV/APMV-1 pathotyping assay
(“Pathstat”) and testing algorithm for APMV-1
David Sutton, Brandon Löndt & Ian Brown
22nd Joint Annual Meetings of the National Reference
Laboratories for Avian Influenza and Newcastle Disease
Copenhagen 18-21 April 2016
Original Collins/Aldous method
~780bp
374bp for phylogenetic analyses
NDV conventional RT-PCR re-design
• Improved sensitivity
• Potential for enhanced turnaround times in NDV detection
• * alignment to consistent approach for APMV-1 phylogenetic analyses
200bp for F cleavage site
Full F gene for phylogenetic analyses*
Specificity • Panel of contemporary isolates comprising
57 APMV-1 isolates covering all lineages* and regions
18 negative - 1 each of APMV-2 to 9, 1 each of AmPV-A to C, 3 x
IBV & 4 x AIV
RNA extracted from cultured virus
positives tested from 1:100 dilution, non-APMV1 virus tested
undiluted
200bp for F cleavage site
Sequence data for 22 isolates confirmed that
Pathstat primers matched sequences derived from
original primer set
Test To Exclude (TTE) scheme
• Clinical samples - swabs/tissues
Fuller 2009
New “Pathstat”
Original and Pathstat primers on BPL
inactivated lyophilized EU panel samples • Sample 2 and 6 – strong and
weak titre of a mixed AI/NDV
isolate (Israel lin. 5d)
- Existing primers fail with
weaker dilution
- Pathstat primers (proposed
new) amplify both strong and
weaker dilutions
• Sample 9 NDV isolate
(Romania 5d)
- Failure with existing primers
- Pathstat primers success
• Lineage 6 is lineage 6 specific
forward pathstat primer
Pathstat primers 200bp product
Original primers 780bp productl
Case 3
Current status and proposed approaches for
APMV1 diagnosis
Molecular Classical
Screening: MGB L gene Isolation in Eggs/Cells
Virulence: ‘Pathotyping’ F PCR HA /antibody typing
Pathostat
avirulent?
ICPI
Green font ISO17025 acceditation in application at EURL
Black font ISO17025 acceditation at EURL
Acknowledgements
• Scott Reid
• Steve Essen