QUANTIFICATION OF HUMAN IMMUNO-DEFICIENCY VIRUS TYPE-1 BY AN IN HOUSE NESTED RT-PCR AND IDENTIFICATION OF VARIANTS WITH SSCP ANALYSIS
WU Y.C.1,3, WONG W.W.2, KUO B.I.T.2,4, LIU C.Y.2, CHEN C.T.3, LIU W.T.1,5
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.
Copyright 1997 Hong Kong Medical Technology Association .
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