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
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
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.
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
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
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
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
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
Test strips for individual antigens plus control Pad for samples Absorbent pad Plastic casette holding test strips
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
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
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