Affinity-based collection of amplified viral DNA: application to the detection of human immunodeficiency virus type 1, human cytomegalovirus and human papillomavirus type 16.
We have devised a sensitive and convenient hybridization technique by combining the polymerase chain reaction (PCR) with affinity-based hybrid collection. In this method 5'-biotinylated primers are used to introduce biotin residues into the DNA fragments during the amplification. The amplified DNA fragments are detected by liquid hybridization using a 32P- or 35S-labelled oligonucleotide as probe. For measurement the hybrids are collected on polystyrene microparticles or onto microtitre wells taking advantage of the biotinavidin interaction. The method is highly sensitive allowing the detection of 30 molecules of DNA. It involves few and simple operations, and is thus suitable for routine diagnostics. The applicability of the method to the detection of HIV-1 DNA from blood, HCMV DNA from urine and HPV-16 DNA from cervical scrapes was evaluated.
Harju
L
L
Orion Pharmaceutica, Biotechnology, Helsinki, Finland.
Jänne
P
P
Kallio
A
A
Laukkanen
M L
ML
Lautenschlager
I
I
Mattinen
S
S
Ranki
A
A
Ranki
M
M
Soares
V R
VR
Söderlund
H
H
D001483:Base Sequence; D001710:Biotin; D003587:Cytomegalovirus / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000175:diagnosis; D015342:DNA Probes / Q000138:chemical synthesis; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005784:Gene Amplification; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008828:Microbiological Techniques; D008863:Microspheres; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D014412:Tumor Virus Infections / Q000175:diagnosis; D014777:Virus Diseases / Q000175:diagnosis*
D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral; D001710:Biotin