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Neil leblanc f7 rapid methods call amsterdam may 2010
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Neil leblanc f7 rapid methods call amsterdam may 2010

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  • Microarrays, including SNP arrays,
  • Open architecture allows for different biomolecules to be conjugated to beads to create assays
  • In the samples shown, results correlate with ELISA data and vaccine information, Positive for IBV and IBDV, Negative for Reo and NDV. SVA will test several hundred samples with Alpha development kits.

Transcript

  • 1. Strategic Meeting, EU FP7-KBBE-2011-5 call Amsterdam, May 20, 2010 KBBE.2011.1.3-02 Field and simple laboratory methods Neil LeBlanc
  • 2. SVA’s interest in penside testing
    • EU project Lab-on-site coordinator
      • Both molecular and protein tests developed, including a commercial lateral flow device for FMDV
    FMDV
  • 3. Epizone WP Leader:
  • 4. What is a Luminex Suspension Microarray?
    • Uses flow cytometry, microspheres, lasers, digital signal processing
    • Resulting platform allows for multiplexing of 100 unique assays within a single sample
  • 5. Luminex Laboratory at SVA Luminex 200 system and Tecan automated wash station
  • 6. Points of Interest
    • Open architecture for development of multiplex assays
    • Nucleic acid, protein detection and other biomolecules may be used, eg. microarrays, multiplex ELISA
    • All types of ELISA can be used; competitive, sandwich, etc…
    • Work at FoU
      • Phosphoprotein to study affect of viral infection on cellualr pathways
      • Develop and testing antigens and antibodies for multiplex assays
      • Subtyping and pathotyping microarrays
      • Combine with real-time PCR
  • 7. Molecular assays
  • 8.
    • biotin on PCR product
    • catches conjugate
    • reporter SA-PE
  • 9. Combining Luminex and Real-time PCR
    • Objective:
    • Develop a system which combines real-time PCR detection amenable to use in routine diagnostics along with the multiplex capability of a microarray format.
    Veterinary Microbiology , Volume 142, Issues 1-2 , 21 April 2010 , Pages 81-86
  • 10. Pestiviruses
    • Flaviviruses, enveloped, positive sense, single-stranded RNA
    • Genome approx. 12 kb, one large ORF, flanked by highly conserved 5’ and 3’ non-translated regions
    • 4 recognized species: CSFV, BVDV1, BVDV2, BDV
    • Viruses are closely related and not species specific
    • Antigenically cross-reactive, which can diagnostic confusion
  • 11. New RT Real-Time PCR Primers: Biotin and Phos modified; Deregt at al., 2006 Probe: SVA routine (Thoren) Epizone ring trial in duplicate 100 genome copies Use Lambda exonuclease, instead of asymmetric PCR
  • 12. Ct values and Median Fluorescence Intensity Virus Strain name Strain No. Ct CSFV A CSFV B BVDV1 BVDV2 BVDV Com BDV Comm A Comm B TKK CSFV C-Stamm 1.1 28.98 4175 4999 69 59 61 77 467 1197 59 CSFV Eystrup91 1.1 29.41 4269 5521 53 57 45 71 1415 2789 41 CSFV Alfort187 1.1 30.34 4199 5775 53 41 31 37 737 2721 31 CSFV Koslov1128 1.2 29.70 4459 5849 39 37 11 61 1707 2939 15 CSFV Brescia 1.2 28.86 4179 6453 13 27 45 61 627 1677 39 CSFV Schweiz II 2.1 29.75 2577 53 27 11 15 23 573 1471 5 CSFV Pader 2.1 30.03 2231 49 27 43 17 29 1447 2591 43 CSFV Bergen 2.2 29.81 1607 115 19 47 11 25 1609 3119 45 CSFV D4886/82/Ro 2.2 30.52 1287 19 39 29 25 15 541 1677 47 CSFV Uelzen 2.3 29.47 1437 79 19 33 25 27 1669 2931 33 CSFV Spante 2.3 30.26 1571 847 27 57 29 17 993 2977 41 CSFV Congenital Tremor 3.1 32.62 2693 5979 35 57 21 47 1177 2647 17 CSFV Kanagawa 3.4 29.53 2371 5059 43 37 19 45 217 1755 39 BDV Gifhorn 3 31.20 61 35 49 147 23 3207 1121 3215 27 BDV 137/4 2 30.84 59 183 31 43 51 3673 151 1969 15 BDV Rudolph 1 30.28 15 31 91 19 25 2217 199 2475 0 BDV Isard 4 30.93 27 65 31 59 29 285 141 1151 45 BVDV1 cp7 1 29.91 49 55 1353 85 3487 33 523 2765 49 BVDV1 NADL 1 31.17 85 45 4827 281 4351 43 1551 3573 37 BVDV1 Grub 1 30.75 35 35 2825 61 3193 37 535 1969 21 BVDV2 München2 2 32.90 103 35 45 1623 877 29 1293 1257 53 BVDV2 CS8644 2 32.00 67 29 51 1523 1213 39 1157 1277 49 BVDV2 Bure 2 31.40 63 33 115 999 759 21 925 883 23 NTC NTC n/a Neg 31 25 55 31 37 27 33 53 55
  • 13. Other Results
    • Was used in a clinical setting, when BVDV screening program became contaminated
    • Detected BVDV in FCS when other methods failed.
    • atypical BVDV and BVDV 1 were detected in FCS and from other experiments using contaminated FCS
    • These results were subsequently were confirmed by sequencing.
    Sample CSFV A CSFV B BVDV 1 BVDV 2 BVDV C BDV Comm A Comm B TKK/HoBi TKK1100 59 91 69 77 49 53 797 481 1057 1 FCS 103 97 127 97 619 99 987 1295 405 2 FCS 71 67 1289 87 739 89 353 723 55 BCV CC 43 47 913 51 1485 43 513 1739 57
  • 14. Results Summary
    • All ring trial samples were detected with 100% concordance between real-time and Luminex.
    • Sensitivity was tested for three CSFV strains and found to be six viral genome copies/reaction. Correspondingly, the microarray signals at six genome copies were above the threshold of 500 MFI.
    • No cross-reactivity among the array probes.
    • Heterologous pathogens and clinical negatives did not produce a signal.
    • Veterinary Microbiology, 2010, 142:81-86
  • 15. New Data – New Primers to help detect atypical pestiviruses PCR seems to be improved by combining primers from Hoffman and Deregt
  • 16. Luminex Results Luminex results tended to be Modulated by Combining primers Hoffman Fwd Primer cuts out BDV and BVDV Com probes Mix Sample Atypical BVDV (36) BDV (29) BVDV Com (28) BVDV1 (26) BVDV2 (27) Com A (30) Com B (31) CSFV A (24) CSFV B (25) Hoffman Panpesti (23) 1 CSFV Eystrup 59 122 58 95 51 236 1271 2037 2438. 118 2 59 148 194 218 79 2132 5154 2780 3301. 1861 3 51 131 90 133 41 732 2824 2706 3452. 162 1 BDV Rudolph 53 619 40 155 49 56 876 39 150 123 2 70 87 117 198 40 1710 4449 42 141 1812 3 46 827 71 210 43 76 3165 19 138 135 1 BVDV 1 Egbert 88 103 1132 544 52 76 1069 50 187 125 2 64 111 131 1659 88 1262 4594 36 147 726 3 53 160 170 1054 49 212 2833 40 138 107 1 Epi 30 Hobi 96 151 98 106 81 60 316 53 233 114 2 2389 122 178 303 129 1498 4373 46 135 662 3 1753 129 99 156 63 405 3041 49 168 138
  • 17. Conclusion
    • The system allows for identification of a pestivirus using a sensitive and specific TaqMan RT-PCR, thus suitable to integration into a routine molecular laboratory. The added benefit is that positive samples can be species typed rapidly, providing crucial information and verification of the real-time PCR result.
  • 18. Luminex Collaboration with LLNL: AIV subtyping
    • Assay developed at Lawrence Livermore National Laboratory
    • Tested at SVA
    • 41-plex primers incl. controls, 45 tags
    • Allow the identification of the most common human H1,2,3 and many common avian H3,5,7,9 subtypes
  • 19. Results from testing at SVA
    • 41 avian samples: 40/41 detected, 34/40 subtyped correctly, no incorrect subtyping
      • also swine and human samples tested
    • 58/68 total samples called correct 85%
      • 3 detection failures, 1 mistype, 6 untyped
    • 7 human clinical samples: 4 H3, 3 H1s detected but 2 not subtyped (incl. “swine flu”)
    • Heterologous pathogens: Negative
  • 20. ASFV genotyping
    • SNP based approach using direct hybridization of 15-19 mer probes to 470 bp PCR product from p72 region
    • Total of 38 probes tested so far.
    • System currently designed and functioning for genotypes 1, 2, 5, 6, 8, 9, and 10.
  • 21. AIV Pathotyping
    • Expansion of system developed for real-time PCR
    • The pathotyping assay is also suitable for use in portable molcular systems
  • 22. Magnetic Bead Microarray
    • Problem: Access to multiplex readout in laboratories with limited resources
    • Current technology: Requires high-tech, expensive equipment
    • State of the art technology developed at SLU/SVA (with partners): Simple technique for visualization on microarray using biotinylated PCR product and streptavidin coated magnetic beads.
    • Benefits: Slides produced on a standard microarray spotter can be used in modestly equipped labs to produce high quality, robust results.
    40x magnification Slide: 16 pestivirus arrays with Epizone ring trial samples. Picture taken with handheld digital camera (dark spots are fingers). * Manuscript accepted in JVM Journal of Virological Methods 155 (2009) 1–9
  • 23. Protein assays
  • 24. Cytokine Capture/Sandwich IL-2 IL-4 TNF  GmCSF
  • 25. Luminex 4-plex poultry Kit –Alpha testing Designed for Vaccine control, so includes standards and controls for all targets Tests for antibodies for IBV, IBDV, Reovirus, NDV Automated wash station (for polystyrene or magnetic beads) Allows for good throughput and consistent treatment of samples In the sample shown, results correlate with ELISA data and vaccine information, Positive for IBV and IBDV, Negative for Reo and NDV. SVA will test several hundred samples with Alpha development kits. Sample IBDV IBV Reo NDV Low control 2443 819 1584 718 Low control 2599 767 1844 736 High control 10608 7973 9393 9592 High control 15054 11224 11973 12018 Standard5 10896 12474 14950 13634 Standard5 10218 12396 15450 14582 Standard4 7125 5832 8923 9066 Standard4 8039 5544 10638 8964 Standard3 6057 2782 5226 5527 Standard3 4689 2509 4828 5042 Standard2 3059 1160 2701 3222 Standard2 2995 1004 2554 2941 Standard1 1859 423 935 1507 Standard1 1978 380 968 1669 Sample: Poultry Serum (vaccinated for IBV and IBDV) 3053 6836 619 36
  • 26. Other work Commercial phosphoprotein assays have been used to study effect of EAV infection on cell pathways Some work on pestiviruses, including development of an antibody detection assay for BVDV using whole virus antigen Multiplex cytokine assays for swine and cattle
  • 27. SAMPLE CAMPYLO VIBRIO GIARDIA CRYPTO SALM. EPEC ROTA ETEC C C C C C C C C ‘ SUNFLOWER’ PLATFORM SHIG. ENTAMOEBA C C Multiplex immmunochromatography assay to detect the 10 globally most important enteric pathogens
  • 28. Test strips for individual antigens plus control Pad for samples Absorbent pad Plastic casette holding test strips
  • 29. Large and diverse pathogen groups without a single common target antigen might necessitate the addition of further strips or additional test line(s) on the same strip!!!! Just a reminder on the structure of colloidal gold based immunochromatography assays
  • 30. To be able to potentially increase the sensitivity of the colloidal gold (CG) technology, the CG particles may be attached to a larger particle (latex beads?)  this is a theoretic application of ‘ signal amplification systems ’ Traditional approach of CG based assay: Modified approach: Latex beads are sensitised with analyte specific antibodies and CG is attached to the surface. Theoretically this may enhance the sensitivity, although reduced motility or steric hindrance due to larger particle size might interfere with assay performance
  • 31. SAMPLE CAMPYLO VIBRIO GIARDIA CRYPTO SALM. EPEC ROTA ETEC C C C C C C C C ‘ SUNFLOWER’ PLATFORM SHIG. ENTAMOEBA C C The test depicted here is positive for Salmonella