title: Performance characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System.
abstract: The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a "gold standard" reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of >or=1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples.
authors.affiliation: Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, Colorado, USA. dkuritzkes@partners.org
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Kuritzkes
authors.forename: Robert M
authors.initials: RM
authors.lastname: Grant
authors.forename: Paul
authors.initials: P
authors.lastname: Feorino
authors.forename: Marshal
authors.initials: M
authors.lastname: Griswold
authors.forename: Marie
authors.initials: M
authors.lastname: Hoover
authors.forename: Russell
authors.initials: R
authors.lastname: Young
authors.forename: Stephen
authors.initials: S
authors.lastname: Day
authors.forename: Robert M
authors.initials: RM
authors.lastname: Lloyd
authors.forename: Caroline
authors.initials: C
authors.lastname: Reid
authors.forename: Gillian F
authors.initials: GF
authors.lastname: Morgan
authors.forename: Dean L
authors.initials: DL
authors.lastname: Winslow
mesh_terms: D000925:Anticoagulants; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000097:blood* / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods
chemical_list: D000925:Anticoagulants; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D054303:HIV Reverse Transcriptase; D016333:HIV Protease