title: A novel internally controlled real-time reverse transcription-PCR assay for HIV-1 RNA targeting the pol integrase genomic region.
abstract: Given the worldwide increasing spread of HIV-1 genetic variants, it is mandatory that assays used for nucleic acid testing for HIV-1 detect all existing groups and subtypes of HIV-1. In this report the development and evaluation of a quantitative real-time HIV-1 RT-PCR assay that targets a conserved region within the pol integrase domain is described. As an internal control reaction, endogenous glyceraldehyde-3-phosphate-dehydrogenase transcripts were detected in a multiplex configuration. The detection limit (95% cut-off value) was determined by probit analysis and calculated as 281 IU/ml of HIV-1 RNA. Within-run and between-run coefficients of variation were below 15 and 27%, respectively, indicating high reproducibility. The described assay detected all tested HIV-1 isolates representing groups M, O and N. Within group M, quantitative test results correlated well with viral loads as determined by the automated Abbott RealTime HIV-1 assay. Based on the testing of 1206 confirmed HIV-1 RNA negative blood donor samples, assay specificity was found to be 100%. The rate of inhibition was 0.37%. The described HIV-1 real-time RT-PCR was validated according to regulatory guidelines and is applicable to the screening of blood donors as well as the determination of HIV-1 viral load.
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, Rheinische Friedrich-Wilhelms-University, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. jens.mueller@ukb.uni-bonn.de
authors.forename: Jens
authors.initials: J
authors.lastname: Müller
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Eis-Hübinger
authors.forename: Martin
authors.initials: M
authors.lastname: Däumer
authors.forename: Rolf
authors.initials: R
authors.lastname: Kaiser
authors.forename: Jutta Maria
authors.initials: JM
authors.lastname: Rox
authors.forename: Lutz
authors.initials: L
authors.lastname: Gürtler
authors.forename: Peter
authors.initials: P
authors.lastname: Hanfland
authors.forename: Bernd
authors.initials: B
authors.lastname: Pötzsch
mesh_terms: D001782:Blood Donors; D015684:Gene Products, pol / Q000235:genetics*; D014644:Genetic Variation; D016679:Genome, Viral; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism*; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D019427:HIV Integrase / Q000235:genetics*; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015684:Gene Products, pol; D012367:RNA, Viral; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D019427:HIV Integrase