Nonradioactive, Colorimetric Microplate Hybridization Assay for Detecting Amplified Human Immunodeficiency Virus DNA

procedure was used to assay human immunodeficiency virus (H IV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150 000 cells were detected by ampli-fying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative indi-viduals yielded negative results (104 of 104), whereas samples from seropositive individualsyielded>99 % pos-itive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acndinium ester-la-beled probe and in a solution hybridization assay in which a 32P-labeled probe was used. Indexing Terms: polymerase chain reaction mononuclear blood cells The polymerase chain reaction (PCR) may be used to axnpliQy very small quantities of nucleic acid up to several million times (1)3 Nucleic acids of infectious agents, such as human immunodeficiency virus (HIV), that are fre-quently not detectable by conventional molecular biology methods can be amplified to detectable amounts by PCR. The amplified H1V DNA is identified by means of DNA probes labeled by various isotopic (2, 3) and nonisotopic methods (4-7). Tests with 32P-labeled DNA probes usu-ally require 3-18 h and are complicated by the isotope’s short half-life and the potential biohazards of the radio-labeled detector probes. Here we report a colorimetric, nonradioactive microplate hybridization assay for detect-ing amplified HIV DNA. MaterIals and Method