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 infected individuals using the polymerase chain reaction (PCR). This is useful if only serum, or other specimens which would not be expected to harbour proviral DNA, is available for diagnostic testing. Viral RNA present in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any proteins present, specifically ribonucleases. After washing the RNA/silica pellet, the RNA is eluted in water and reverse transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is amplified using nested PCR and the products of amplification are detected by gel electrophoresis and ethidium bromide staining. The identity of bands on the gel is confirmed using a digoxigenin-labelled oligomer probe. The method is a general one applicable to amplification of any RNA species.
Gibson
K M
KM
Virus Reference Division, Central Public Health Laboratory, London, UK.
Mori
J
J
Clewley
J P
JP
D001483:Base Sequence; D002460:Cell Line; D005188:False Negative Reactions; D006146:Guanidines; D015658:HIV Infections / Q000097:blood / Q000382:microbiology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D013861:Thiocyanates; D014766:Viremia / Q000382:microbiology*
D006146:Guanidines; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012822:Silicon Dioxide; D012194:RNA-Directed DNA Polymerase