¹Division of Clinical Virology, Department of Laboratory Medicine and Pathology, ²Section of Infectious Diseases, Department of Medicine, Veterans General Hospital, Taipei, ³Meditech Biotechnology Co., Ltd. ⁴National Yang-Ming University Medical College, ⁵Institute of Biotechnology in Medicine National Yang-Ming University, Taipei, Taiwan

A sensitive, specific and rapid in house-nested reverse transcriptase polymerase chain reaction (IHN RT-PCR) method for direct detection of human immunodeficiency virus type-1 (HIV-1) is established. It was suitable for measuring plasma specimens collected within 6 hours. This method described here used AMV-reverse transcriptase coupled with Tth DNA polymerase in a single-tube for both reverse transcription and PCR. A sequence homologous to that of a high conserved region of the virus pol gene was selected as primer for this one-tube system. In this system, the reverse transcription was carried out shortly at an elevated temperature of 53°C for 10 minutes and an extra addition of 10% DMSO to overcome the possible RNA secondary structure that might inhibit the reaction. This system could also improve the cDNA yield as well. The first one-tube RT-PCR product was then directly used to process nested PCR for semi-quantitation of the viral RNA. Fifty HIV-1 antibody positive plasma samples were directly detected by this IHN RT-PCR method, and it was compared with AMPLICOR MONITOR HIV-1 RT-PCR assay (Roche). The sensitivity between two methods is 100 and 98.0% respectively. A good linear correlation of the viral load between two assays was obtained (p<0.001). The variability existed in the HIV-1 pol gene was revealed by single-strand conformation polymorphism (SSCP) analysis. This standardized SSCP analysis system (GenePhor, Pharmacia Biotech) seemed to be well suited for the detection of HIV-1 variant for referring the disease progression of the infected patients.