Detection of HIV1 proviral DNA by PCR and hybridization with digoxigenin labelled probes.
One of the main obstacles for the introduction of PCR method to identify HIV1 proviral DNA in routine diagnostic laboratories is the use of radiolabelled oligodeoxynucleotide probes. Nonradioactive labelled probes have several advantages over radioactive labelling: they are stable for over 1 year, they can be produced easily in large amounts and they are safe. Polymerase chain reaction is an efficient and simple method to produce vector free inserts to use as probes. In this paper we describe a procedure for labelling DNA probes with digoxigenin-11-dUTP using the polymerase chain reaction. This non-radioactive labelling system was applied to detect HIV proviral sequences, amplified in vitro by PCR, from peripheral blood mononuclear cells DNA of infected subjects. We found identical sensitivities and specificities for probes synthesized with the non-radioactive and radioactive labelling procedures. The digoxigenin-11-dUTP can be efficiently incorporated during amplification of a DNA fragment using the polymerase chain reaction. This labelling and detection method proved to be specific, sensible and simple enough to be used in routine diagnostic laboratories for the detection of HIV1 infected individuals.
Taveira
N C
NC
Dep. Microbiologia, Faculdade de Farmácia, Universidade de Lisboa, Portugal.
Ferreira
M O
MO
Pereira
J M
JM
D001483:Base Sequence; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics*; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin