title: Development and application of a high-throughput HIV type 1 genotyping assay to identify CRF02_AG in West/West Central Africa.
abstract: In West/West Central Africa, CRF02_AG is the most prevalent HIV-1 strain and circulates in the milieu of rare subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs). The molecular complexity of HIV-1 epidemics in this region and the need to extensively sample large populations, such as in the case of vaccine trials, pose seemingly conflicting requirements between full-genome sequencing and high-throughput low-resolution assays. Here we describe the development and evaluation of a multiregion hybridization assay (MHAcrf02) for the efficient genotyping of CRF02_AG in West/West Central Africa. Subtype A, G, and CRF02_AG-specific fluorescent probes were designed flanking five recombination breakpoints in CRF02_AG and were used in real-time PCRs. A panel representing West/West Central African HIV-1 genetic diversity was evaluated by MHAcrf02. The sample set, previously characterized by full-genome sequencing, included CRF02_AG and CRF02_AG-containing recombinants (n = 28), other subtypes, CRFs, and URFs (n = 34). DNA from peripheral blood mononuclear cells, cocultures, and plasmids was used as template. When the patterns of probe reactivity were evaluated. CRF02_AG was identified with a 100% specificity and sensitivity. In conclusion, MHAcrf02 will permit more efficient characterization of HIV-1 in West/West Central Africa, where CRF02_AG is an important strain. Together with other regional genotyping assays MHAcrf02 will contribute to the development of a global picture of HIV-1 diversity and geographic distribution, providing a strong foundation for intervention, including vaccine development.
authors.affiliation: The Henry M. Jackson Foundation, Rockville, Maryland 20850, USA. gkijak@hivresearch.org
authors.forename: Gustavo H
authors.initials: GH
authors.lastname: Kijak
authors.forename: Eric
authors.initials: E
authors.lastname: Sanders-Buell
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Wolfe
authors.forename: Eitel
authors.initials: E
authors.lastname: Mpoudi-Ngole
authors.forename: Bohye
authors.initials: B
authors.lastname: Kim
authors.forename: Bruce
authors.initials: B
authors.lastname: Brown
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Robb
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Birx
authors.forename: Donald S
authors.initials: DS
authors.lastname: Burke
authors.forename: Jean K
authors.initials: JK
authors.lastname: Carr
authors.forename: Francine E
authors.initials: FE
authors.lastname: McCutchan
mesh_terms: D000350:Africa, Central; D000354:Africa, Western; D001483:Base Sequence; D017931:DNA Primers; D005456:Fluorescent Dyes; D005838:Genotype; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes