DEVELOPING A HYBRIDIZATION ASSAY FOR HTLV-I AND HTLV-II

开发 HTLV-I 和 HTLV-II 的杂交检测方法

• 批准号: 3492852
• 负责人: DAVID A FUCCILLO
• 金额: $ 5万
• 依托单位: SRA TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-28 至 1991-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3492852
• 关键词: AIDS; AIDS /HIV diagnosis; AIDS /HIV test; HIV infections; RNA directed DNA polymerase; autoradiography; diagnosis design /evaluation; early diagnosis; human T cell lymphotropic virus type 1; human T cell lymphotropic virus type 2; immunologic techniques; noninvasive diagnosis; nucleic acid hybridization; nucleic acid probes; scintillation counter; tissue /cell culture; virus DNA

The human retroviruses that have been extensively characterized (HTLV-I, HTLV-II, HIV-1, and HIV-2) all have a tropism for the peripheral blood mononuclear cells (PBMC). Serological assays have been used quite successfully to identify persons with prior exposure to these viruses but fail to determine a current infection. virus isolation assays require prolonged co-cultivation of patients' PBMC with a susceptible cell line and have been shown to lack sensitivity. A newly described DNA amplification technique, the polymerase chain reaction (PCR) appears to have a great potential for detection of viral DNA. The PCR however, requires highly trained personnel, expensive equipment and about three days to perform an assay. Microbiological Associates Inc. (MBA) proposes to develop a simple, rapid, hybridization assay to detect current infections by these retroviruses. The method employs a plastic capillary as a solid phase to perform the hybridization assay. The capillary provides a very large reactive surface relative to sample size and also forces the close proximity of reactants. This environment produces increased sensitivity with shorter incubation times and temperature requirements. A non-isotopic, enzyme immunoassay amplification detection system is used to indicate hybridization with a known DNA probe.

已广泛表征的人类逆转录病毒（htlv-i， HTLV-II，HIV-1和HIV-2）都对周围血具有向往 单核细胞（PBMC）。血清学测定已被大量使用 成功地识别事先接触这些病毒的人，但 无法确定当前的感染。病毒隔离测定需要 患者PBMC与易感细胞系的长期共培养和 已证明缺乏灵敏度。新描述的DNA扩增 技术，聚合酶链反应（PCR）似乎具有很棒的 检测病毒DNA的潜力。但是，PCR需要高度 训练有素的人员，昂贵的设备和大约三天 测定。 Microbiological Associates Inc.（MBA）建议开发一个简单的， 快速的杂交测定法以检测这些测定 逆转录病毒。该方法采用塑料毛细管作为固相 执行杂交测定。毛细管提供了很大的 反应性表面相对于样品量，也迫使接近 反应物的接近。这种环境产生提高的灵敏度 随着较短的孵化时间和温度要求。一个非同位素， 酶免疫测定放大检测系统用于指示 与已知的DNA探针的杂交。