Detection of Bovine High Consequence Agents on an Electronic Microarray Platform - PowerPoint PPT Presentation

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Detection of Bovine High Consequence Agents on an Electronic Microarray Platform

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  1. Detection of Bovine High Consequence Agentson an Electronic Microarray Platform Kimberley Burton Hughes Canadian Food Inspection Agency Lethbridge Laboratory

  2. Bovine High Consequence Pathogen Assays • Component 1: • Foot and Mouth Disease Virus • Component 2: • FMD Differentials • Vesicular Stomatitis • Exotic Malignant Catarrhal Fever • Rinderpest • Blue Tongue • BVD • BHV • Parapox complex Clinical Symptoms: Fever Blisters in mouth Excessive salivation Blisters on feet Weight loss

  3. Bovine HC Pathogen Assays: Overview Suspect case Detect and Serotype FMD FMD Negative Bovine HC Multiplex Testing for 7 FMD differentials + FMD Strain Identification (with strain specific probes) - Vaccine selection - Epidemiology FMD Positive Heat map depicting fluorescent intensities of 2 detection (A) and 12 serotyping (B) probes against 23 isolates that represent all 7 serotypes.

  4. Example Unknown strain Site 1 Site 2 Site 3 Probes Reference strains Site 1 Site 2 Site 3 A C E F B D Reference Strain FMD Strain ID Strategy - 3 highly variable sites were identified within VP 1 coding region VP 1 1 2 3 - Probes were designed at the 3 sites for reference strains of each serotype Reference strains selected: - WRLFMD recommended vaccine strains - major genetic lineages or topotypes (genetic lineages that fall within geographical boundries)

  5. Strains Tested Probes FMD O Strain Identification • *WRLFMD recommended vaccine strains • **Samuel and Knowles, J. Gen Virol. 2001

  6. Bovine High Consequence Pathogen Assays • Component 1: • Foot and Mouth Disease Virus • Component 2: • FMD Differentials • Vesicular Stomatitis • Exotic Malignant Catarrhal Fever • Rinderpest • Blue Tongue • BVD • BHV • Parapox complex

  7. Bovine HC Multiplex PCR BVD VSV (NJ) BHV VSV (IND) BTV RPV MCFV ORF Clinical Neg Oral BTV Entero NTC

  8. Bovine HC Electronic Microarray Results (PN) Virus Strains BVDV VSV BTV RPV 2 BHV NJ IND 1 Entero MCFV ORFV NTC Probes

  9. Sensitivity Bovine HC Assay TCID50/ml: 104 103 102 101 100 10-1 10-2 10-3 Limit of Detection (TCID50/ml) RPV 235 bp NC400 Results

  10. Clinical Samples Tested

  11. Clinical Sample Testing Results Strong, specific reaction starting at 2 dpi. No cross reaction to other probes in assay.

  12. -5 - - - - 1 - - - - + + + 2 - + + + + 3 + + 4 - - inc + + + 5 + + + 6 - 7 - - - - 8 - - - - inc + + + 9 10 - - - - 11 - - - - DPI Advantages of electronic microarray: - highly multiplexed, type and subtype - robust platform - faster - higher throughput - simplifed data analysis PCR NC400 RT-PCR* Slide Array * NCFAD completed RT-PCR

  13. Current Work • Adapt assays to new format • Fully automated, integrated system • magnetic sample preparation • amplification • electronic microarray • Portable, compact unit for on-site testing • Fully bio-contained cartridge • Rapid • Low cost (~$10)

  14. CFIA, Lethbridge Oliver Lung Dirk Deregt Tara Furukawa-Stoffer Mathew Fisher Samuel Ohene-Adjei Kristen Hahn* Anne Beeston* Jennifer Geddes* Billy Mauro* Nexogen, Inc., San Diego, USA Dalibor Hodko CFIA, NCFAD, Winnipeg John Pasick Yohannes Berhane Alfonso Clavijo* Animal and Technical Staff IAH, Pirbright, UK Donald King Scott Reid Funding: CRTI (The Chemical, Biological, Radiological or Nuclear Research and Technology Initiative) * Former members CFIA Lethbridge Laboratory

  15. Bovine HC Multiplex PCR Development

  16. Specificity across serotypes

  17. PN Values for heat map

  18. Bovine HC Pathogen Assays: Overview - FMD Negative + FMD Positive Bovine HC assay Testing for 7 other HC agents and differentials FMD Strain Identification (with strain specific probes) - Vaccine selection - Epidemiology Sample from suspect FMD case Simultaneous detection and serotyping of FMD

  19. Bovine High Consequence Agents Foot and Mouth Disease Virus • Detection (polymerase gene) • Serotyping (VP3 gene) • Strain Identification Previously presented

  20. Clinical Signs: Fever Blisters in mouth Excessive salivation Blisters on feet Weightloss Foot-and-Mouth Disease • Highly infectious, expensive to control, and economically devastating • 7 serotypes: A, O, C, Asia 1, SAT 1, 2, 3 • Confirmation requires laboratory diagnosis • Last cases in Canada:1953 (US 1929)

  21. Current Work

  22. DPI

  23. reporter probe Amplicon Biotinylated capture probe + + Positive Charge Positive Charge • RNA isolation • RT-PCR amplification of target • NC400 • 1. Print biotinylated capture probes • 2. Add amplicon • 3. Add reporter probe Printing Hybridization Reporting • 4. Measure fluorescence at test site Biotinylated Capture Probe Amplicon Wash

  24. Electronic Microarray 50 m diameter test site Array Platinum Microelectrode Hydrogel Permeation Layer Containing Streptavidin CMOS Chip • Electrophoretically driven printing • and hybridization • 400 independently activated test sites (Movie here) Time (seconds) Time (hours) Electronic Passive

  25. reporter probe • NC400 • 1. Print biotinylated capture probes Amplicon Biotinylated capture probe • 2. Add amplicon • 3. Add reporter probe + + Positive Charge Positive Charge • RNA isolation • RT-PCR amplification of target Printing Hybridization Reporting • 4. Measure fluorescence at test site Biotinylated Capture Probe Amplicon Wash