SOLID PHASE MICROPLATE ASSAYS FOR TTV

将开发一种特异和可靠的平板格式，用于检测大量临床样本的TTV基因型别，TTV是一种新描述的人类输血型肝炎病毒。将优化样品处理条件，以确保使用多种内部和外部控制手段检测病毒以及灵敏度和重复性。病毒基因组将用生物素标记的引物进行聚合酶链式反应扩增，然后将聚合酶链式反应产物与96孔微滴定井上共价结合的病毒特异性寡核苷酸探针杂交。病毒特异性序列的存在将通过使用链霉亲和素结合碱性磷酸酶和底物的比色反应来确定。450 nm处的MOD将使用微型平板阅读器进行记录。这项检测将用于对肝炎感染高危或低危人群的血浆样本进行筛查和分型。TTV阳性血浆将被选择，进一步鉴定，并用于研究和临床实验室的检测标准化和熟练程度小组。在第二阶段中，将开发一种用于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孔板(SPM)分析，并评估其是否适合在临床或研究实验室环境中使用。拟议的商业应用：该系统将用于检测病因不明的高危肝炎患者的血清和血浆样本中的TTV并对其进行分型，并制备和表征TTV的质量控制，这些质量控制可用于评估检测试剂盒的性能，产生用于比较分析的结果，开发用于临床和研究实验室熟练程度和培训计划的TTV质量控制。

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.