DETECTION OF HIV-1 IN SERUM, USING REVERSE TRANSCRIPTION AND THE POLYMERASE CHAIN-REACTION (RT-PCR)

We have used a simple method for detecting HIV-1 in the serum of infec ted individuals using the polymerase chain reaction (PCR). This is use ful if only serum, or other specimens which would not be expected to h arbour proviral DNA, is available for diagnostic testing. Viral RNA pr esent in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any p roteins present, specifically ribonucleases. After washing the RNA/sil ica pellet, the RNA is eluted in water and reverse transcribed using r andom primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is ampli fied using nested PCR and the products of amplification are detected b y gel electrophoresis and ethidium bromide staining. The identity of b ands on the gel is confirmed using a digoxigenin-labelled oligomer pro be. The method is a general one applicable to amplification of any RNA species.