We are currently setting up a technique that will detect HIV infected cells in a population of uninfected ones in the absence of detergent or phenol treatment using In Situ Polymerase Chain Reaction. The rationale of the technique is to set up a sensitive and reproducible test that will screen out infected cells non- isotopically by visual examination under an optical microscope. The technique will fix the cells onto the wells on a teflon coated slide. The cells will then be washed and made permeable with a quick proteinase K digestion. They are then reacted with PCR reaction mixture containing SK38/39 primers for 35 cycles at 91, 55, and 65 degrees C after covering with coverslips. The amplified products within the cells can be hybridized with biotinylated SK 19 probe and reacted with a streptavidin- peroxidase system for color development. The positive cells stain brownish-red and can be scored visually under optical microscope. At the present time, several methods of fixation and several fluorophores for labelling of primers and probes are being explored. Methods for detection of amplified material include analysis by flow cytometry. We will use the technique in detection of HIV virus in different physiologic materials, in assessing the effectiveness of anti-viral drugs against HIV and in understanding the role of host and viral factors in pathogenesis of the disease.
