A novel internally controlled real-time reverse transcription-PCR assay for HIV-1 RNA targeting the pol integrase genomic region.
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.
Müller
Jens
J
Institute of Experimental Haematology and Transfusion Medicine, Rheinische Friedrich-Wilhelms-University, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. jens.mueller@ukb.uni-bonn.de
Eis-Hübinger
Anna Maria
AM
Däumer
Martin
M
Kaiser
Rolf
R
Rox
Jutta Maria
JM
Gürtler
Lutz
L
Hanfland
Peter
P
Pötzsch
Bernd
B
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
D015684:Gene Products, pol; D012367:RNA, Viral; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D019427:HIV Integrase