title: Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes.
abstract: Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput. The detection limit was found to be 100 copies/ml, the dynamic range up to 500,000,000 copies/ml. Intra and inter assay imprecision were 1.6% and 2.0% (n=10), respectively. The assay was calibrated against WHO Standard 97/656. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HIV-1 Monitor (r = 0.954; p < 0.001). This rapid (3 h) and cost effective assay is suitable for both HIV-1 detection and disease monitoring with the ability to detect and quantify all HIV-1 subtypes including O1 and O2.
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany.
authors.forename: Manabu
authors.initials: M
authors.lastname: Abe
authors.forename: Corinne
authors.initials: C
authors.lastname: Klett
authors.forename: Eberhard
authors.initials: E
authors.lastname: Wieland
authors.forename: Sascha
authors.initials: S
authors.lastname: Gille
authors.forename: Olfert
authors.initials: O
authors.lastname: Landt
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral