STANDARDIZED NESTED POLYMERASE CHAIN REACTION-BASED ASSAY FOR DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 DNA IN WHOLE-BLOOD LYSATES

The routine detection of human immunodeficiency virus type 1 (HIV-1) p roviral DNA in clinical samples requires a standardized, simple, and s ensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AM PLICIS test). In order to simplify the general process, whole-blood ly sates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the fi nal amplified products allowed us to define precise interpretive crite ria to determine the positivity level of the test. Three new primer se ts located in the gag and pol structural genes and in the tat regulato ry gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPL ICIS test to be used for HIV-1 provirus detection: 53 of 54 of the ser opositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial di lutions of blood lysates from 26 HIV-1-seropositive patients.