Studies on Detection of Human Immunodeficiency Virus Using Double Polmerase Chain Reaction

BACKGROUND: Serological methods for screening blood and blood products for the presence of antibodies to human immunodeficiency virus( HIV) are efficient and sensitive. In repeatedly reactive cases confirmational tests such as Western blot are available. However, direct viral detection may be needed for a patient in seronegative window period and a newborn from a infected mother. In addition, a direct assay for the virus would provide a means to monitor both latent and actively replicating virus in patients on therapeutic drugs. However, direct detection of HIV in patient samples is difficult and disappointing even with co-cultivation and the successful recovery rate varies from 10 to 75%. Polymerase chain reaction (PCR) may provide the answer because it can do in vitro amplification of viral genome integrated into human genome (provirus). However, actual results of clinical application of conventional PCR do not show favorable sensitivity especially in samples containing very small amounts of HIV molecule copies. PURPOSE: We comparatively analyzed the sensitivity of single ( primary)PCR and double ( secondary) PCR in the detection of HIV to define whether double PCR can overcome the limited sensitivity of single( primary) PCR and if it can be a clinically promising method for detecting HIV. MATERIALS AND METHODS: Ten peripheral blood samples from individuals who had antibodies to human immunodeficiency virus were prepared and centrifuged in Ficoll-Hypaque to isolate lymphocytes and monocytes. After DNA extraction from the cell, 35 cycles of primary PCR was performed and a part of the PCR product of individual specimen was electrophoresed to elucidate the results of primary PCR. Secondary PCR with the other part of the individual primary PCR product was followed to compare the efficacies of single and double PCR. RESULTS: With primary PCR, only one specimen among 10 showed a suspicious corresponding band on polyacrylamide gel electrophoresis using ethidium bromide. The results of double PCR presented a striking contrast to those of primary PCR, elucidating 100O% sensitivity without using radioisotope. CONCLUSIONS: This study suggeststhat double PCR is a very potent method in detection of human immunodeficiency virus genome incorporated in human white blood cells.ope