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

需要逆转录病毒感染细胞的直接和灵敏的测定

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.,