COMBINED ASSAY FOR HIV-1 AND HTLV-1 USING POLYMERASE CHA

需要对感染逆转录病毒的细胞进行直接和灵敏的检测 用于确认患者暴露和监测疾病状态，以及 治疗。我们建议首先开发一种检测感染有病毒的PBL的方法 用聚合酶链式反应(PRC)扩增HTLV-I病毒 DNA和非放射性夹心杂交分析(在ELISA法中 微孔法)以检测扩增产物。这样的化验应该 能在10‘6个未感染的细胞中检测到一个感染的淋巴细胞。 初步工作将包括亚克隆、简化样品制备 以及HTLV-I和β-珠蛋白内对照试验的发展。 接下来是测试HTLV-I组合的可行性 用我们现有的HIV-1检测在聚合酶链式反应水平上进行检测以及 检测化验水平。组合测试应该会大大降低成本 使用DNA探针确认逆转录病毒暴露，节省时间和劳动力 和DNA样本。当选择最终的正式化验时，临床样本 将进行检测，并将HTLV-I/HIV-1 DNA结果与患者相关 抗体、临床和病毒分离数据。在后面的过程中， 根据这项提议，我们将为HIV-2和HTLV-II开发类似的检测方法。后 开发了单独的检测方法，两种检测方法的组合将是 经过测试并最终选定了格式。临床样本将进行测试，使用 联合化验正式，以确定敏感性和特异性 化验的结果。

Direct and sensitive assays for retrovirus infected cells are required for confirming patient exposure and for monitoring disease status and treatment. We propose first to develop an assay for PBLs infected with HTLV-I by using the polymerase chain reaction (PRC) to amplify the viral DNA and a nonradioactive sandwich hybridization assay (in an ELISA microwell format) to detect the amplified product. Such an assay should be capable of detecting one infected lymphocyte in 10'6 uninfected cells. Initial work will entail subcloning, streamlining of sample preparation and development of an HTLV-I and a beta-globin internal control assay. This to be followed by testing the feasibility of combining the HTLV-I assay with our existing HIV-1 assay at the PCR level as well as the detection assay level. A combination test should greatly cut the cost of confirming retroviral exposure, using DNA probes, by saving time, labor and sample DNA. When the final assay formal is chose, clinical samples will be tested and the HTLV-I/HIV-1 DNA results correlated with patient antibody, clinical and viral isolation data. Later in the course of this proposal, we will develop similar assays for HIV-2 and HTLV-II. After the individual assays are developed, combinations of the two assays will be tested and a final format chosen. Clinical samples will be tested, using the combined assay formal, to determine the sensitivity and specificity of the assay.,