A simple and rapid method for detecting human immunodeficiency virus by PCR.
A simple, sensitive and specific method using the polymerase chain reaction (PCR) for amplification of human immunodeficiency virus type 1 (HIV-1) is described. The method involves minimal manipulations. Peripheral blood mononuclear cells (PBMC) were prepared by a rapid Ficoll-Paque gradient method. Lymphocytes were lysed in PCR buffer containing Proteinase K and detergents, and subjected to amplification under stringent conditions, using two primer pairs. Amplified DNA sequences were hybridized with a 3'-end labelled probe, electrophoresed on agarose gels and visualised by ethidium bromide staining. Identification of amplified HIV-1 proviral DNA sequences was confirmed by autoradiography. HIV-1 sequences were amplified in all samples from 103 HIV-1 seropositive individuals, but not in 40 HIV-1 seronegative controls. The absence of contamination may be attributable in part to minimisation of manipulations before amplification.
Gibson
K M
KM
Virus Reference Laboratory, Central Public Health Laboratory, London, U.K.
McLean
K A
KA
Clewley
J P
JP
D001483:Base Sequence; D004279:DNA, Viral / Q000097:blood*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification
D004279:DNA, Viral