SOLID PHASE MICROPLATE ASSAYS FOR TTV

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A specific and reliable plate format for testing large numbers of clinical samples for the genotypes of TTV, a newly described human transfusion hepatitis virus, will be developed. The conditions for sample processing will be optimized to ensure the detection of the virus and the sensitivity and reproducibility using multiple internal and external controls. The viral genome will be PCR-amplified with biotin labeled primers and the PCR products are then hybridized to viral-specific oligonucleotide probes bound covalently on 96- well microtiter wells. The presence of viral specific sequences will be determined with a colorimetric reaction using streptavidin conjugated alkaline phosphatase and substrate. The MOD at 450 nm will be recorded using a microplate reader. This assay will be used to screen and genotype plasma samples from individuals at high- or low- risk for hepatitis infection. TTV positive plasma will be selected, further characterized, and used for assay standardization and proficiency panels in research and clinical laboratories. In the course of Phase II, a semi- automated solid phase microplate (SPM) assay will be developed for TTV quantitation and multi-hepatitis viral screening and genotyping kits, and evaluated for suitability for use in a clinical or research laboratory setting. PROPOSED COMMERCIAL APPLICATIONS: The system will be used to detect and genotype TTV in serum and plasma samples of high risk individuals with hepatitis of unknown etiology, and to prepare and characterize quality controls for TTV which could be used to evaluate test kit performance, to generate results for comparative analysis, to develop TTV quality controls for use in proficiency and training programs in clinical and research laboratories.