A solid phase plate assay for HIV-1 genotyping.
A prototype solid phase plate assay (SPPA) for the detection and genotyping of HIV-1 subtypes was developed using a PCR-based capture hybridization format. Well characterized HIV-1 reference controls of known subtypes and subtype specific capture oligonucleotide probes targeting several regions of the envelope (env) gene of HIV-1 were selected to develop the assay. The subtype specific oligonucleotide probes were covalently bound to microtubes in an ordered pattern and biotin labelled primers were used to amplify the target sequences. The PCR products were hybridized to the corresponding oligonucleotide probes, and colorimetrically detected by a chromogenic reaction using a standard microplate reader. All the HIV-1 subtype reference controls specifically hybridized to the corresponding capture probes and negligible cross-hybridization between subtypes was observed. To demonstrate the performance and reproducibility of the SPPA system and its validation with clinical samples, several human plasma samples of unknown and known HIV-1 subtypes were tested. The SPPA is highly specific and unambiguously identify the major subtypes of the HIV-1 M and O groups. This assay could be a useful method for subtyping samples of HIV-1 infected individuals and for disease management.
González-Villaseñor
L I
LI
BBI-Biotech Research Laboratories, Gaithersburg, MD 20877, USA.
Wu
K
K
Yang
L
L
D019921:Biotinylation; D003116:Color; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D015686:Gene Products, env / Q000032:analysis / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D005838:Genotype; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility
D017931:DNA Primers; D004279:DNA, Viral; D015686:Gene Products, env; D015345:Oligonucleotide Probes