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

需要对逆转录病毒感染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， 治疗 我们建议首先开发一种检测感染 用聚合酶链反应（PRC）扩增HTLV-I病毒， DNA和非放射性夹心杂交测定（在ELISA中 微孔形式）以检测扩增产物。这种分析应 能够在10 '6个未感染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 以及开发HTLV-I和β-珠蛋白内部对照测定。 随后将测试结合HTLV-I的可行性 检测与我们现有的HIV-1检测在PCR水平以及 检测分析水平。一个组合测试将大大降低成本， 确认逆转录病毒暴露，使用DNA探针，通过节省时间，劳动 和DNA样本当选择最终测定形式时，临床样品 将进行检测，HTLV-I/HIV-1 DNA结果与患者相关 抗体、临床和病毒分离数据。后来在这个过程中， 根据该提案，我们将开发针对HIV-2和HTLV-II的类似检测方法。后 开发了单独的测定法，将两种测定法的组合 测试和最终选择的格式。临床样本将使用 联合试验形式，以确定灵敏度和特异性 的测定，

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.,