title: A genotypic assay for the amplification and sequencing of gag and protease from diverse human immunodeficiency virus type 1 group M subtypes.
abstract: In human immunodeficiency virus type 1 (HIV-1), an interaction exists between the in vivo evolution of Gag protein and protease to escape from antiretroviral drug selective pressure. Therefore, it was decided to develop a genotypic assay for the amplification and sequencing of HIV-1 gag and protease. As the HIV-1 pandemic is characterised by a large genetic diversity, the assay developed was evaluated on a panel of 28 genetically divergent samples belonging to the following subtypes A1, B, C, D, F1, F2, G, H, J, CRF01-AE, CRF02-AG and CRF13-cpx. The assay displayed a detection limit ranging between 500 RNA copies/ml and 5000 RNA copies/ml plasma. Full-length sequences could be obtained for 25 samples. The population sequences of the three other samples lacked a part of the sequence because of heterogeneous signal, probably due to the presence of quasi-species with insertions/deletions of a different length.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. kristel.vanlaethem@uz.kuleuven.ac.be
authors.forename: Kristel
authors.initials: K
authors.lastname: Van Laethem
authors.forename: Yoeri
authors.initials: Y
authors.lastname: Schrooten
authors.forename: Sarah
authors.initials: S
authors.lastname: Dedecker
authors.forename: Lies
authors.initials: L
authors.lastname: Van Heeswijck
authors.forename: Koen
authors.initials: K
authors.lastname: Deforche
authors.forename: Eric
authors.initials: E
authors.lastname: Van Wijngaerden
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
authors.forename: Anne-Mieke
authors.initials: AM
authors.lastname: Vandamme
mesh_terms: D017931:DNA Primers; D018076:DNA, Complementary; D024882:Drug Resistance, Viral / Q000235:genetics; D015750:Genes, gag; D015751:Genes, pol; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010949:Plasma / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D016333:HIV Protease