title: Novel multiregion hybridization assay for the identification of the most prevalent genetic forms of the human immunodeficiency virus type 1 circulating in Portugal.
abstract: The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.
authors.affiliation: Unidade de Microbiologia Médica-Grupo de Virologia/Unidade de Parasitologia e Microbiologia Médicas, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, P-1349-008 Lisboa, Portugal. ferdinandofreitas@gmail.com
authors.forename: Ferdinando B
authors.initials: FB
authors.lastname: Freitas
authors.forename: Aida
authors.initials: A
authors.lastname: Esteves
authors.forename: João
authors.initials: J
authors.lastname: Piedade
authors.forename: Ricardo
authors.initials: R
authors.lastname: Parreira
mesh_terms: D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D015658:HIV Infections / Q000453:epidemiology* / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011174:Portugal / Q000453:epidemiology; D011804:Quinolines; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D014764:Viral Proteins; C098022:SYBR Green I