Detection of HIV-1 DNA by polymerase chain reaction incorporation of digoxigenin-11-dUTP and hybridization to immobilized probes.
We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
Faculdade de Farmácia da Universidade de Lisboa, Departamento de Microbiologia, Portugal.
N C
NC
Taveira
P C
PC
Gomes
M O
MO
Ferreira
J M
JM
Pereira
D001483:Base Sequence; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004076:Digoxigenin / Q000031:analogs & derivatives* / Q000378:metabolism; D015894:Genome, Human; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*
D015342:DNA Probes; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin