SOLID PHASE MICROPLATE ASSAYS FOR TTV

将开发一种用于检测大量临床样本的TTV（一种新描述的人输血性肝炎病毒）基因型的特异性和可靠的平板格式。将优化样本处理条件，以确保使用多个内部和外部质控品检测病毒以及灵敏度和重现性。用生物素标记的引物对病毒基因组进行PCR扩增，然后将PCR产物与96孔微量滴定威尔斯孔上共价结合的病毒特异性寡核苷酸探针杂交。将使用链霉抗生物素蛋白缀合的碱性磷酸酶和底物通过比色反应确定病毒特异性序列的存在。使用酶标仪记录450 nm处的MOD。该检测试剂盒将用于筛查肝炎感染高风险或低风险个体的血浆样本并进行基因分型。将选择TTV阳性血浆，进一步表征，并用于研究和临床实验室的测定标准化和熟练度小组。在II期研究过程中，将开发用于TTV定量和多种肝炎病毒筛查和基因分型试剂盒的半自动固相微板（SPM）检测试剂盒，并评价其在临床或研究实验室环境中使用的适用性。拟议的商业应用：该系统将用于检测和基因型TTV在血清和血浆样本的高风险个人与不明病因的肝炎，并准备和表征TTV的质量控制，可用于评估检测试剂盒的性能，生成结果进行比较分析，开发TTV质量控制用于熟练和培训计划在临床和研究实验室。

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.