title: Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma.
abstract: An ultrasensitive version of an 'in-house' reverse transcription-competitive polymerase chain reaction assay described previously for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma was developed. The increase in sensitivity from 400 to 50 HIV-1 RNA copies/ml was achieved by pelleting virus particles from 1.8 ml plasma by centrifugation prior to RNA extraction, modifying competitor DNA structure and amounts, and redesigning primers. Quantitation of HIV-1 RNA in 130 samples tested previously by the standard assay showed that the two procedures yield comparable results (mean absolute difference, 0.26+/-0.20 log) and that the ultrasensitive version detects HIV-1 RNA below the threshold of sensitivity of the standard method. The ultrasensitive 'in-house assay' and the reference QUANTIPLEX HIV-1 RNA 3.0 had the same sensitivity and gave equivalent results (mean absolute difference, 0.19+/-0.11 log), as shown by parallel blinded testing of 47 plasma samples. Titration experiments with reconstructed plasma samples allowed the determination of a dynamic range of 50-500000 HIV-1 RNA copies/ml for the 'in-house' system. The interassay coefficient of variation for samples nominally containing 200, 4000 and 80000 HIV-1 RNA copies/ml were 33.4, 22.9 and 38.2%, respectively. The performance, turnaround time, and cost-effectiveness of this system make it suitable for medium-scale clinical application.
authors.affiliation: Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Università di Siena, Siena, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Venturi
authors.forename: R
authors.initials: R
authors.lastname: Ferruzzi
authors.forename: L
authors.initials: L
authors.lastname: Romano
authors.forename: M
authors.initials: M
authors.lastname: Catucci
authors.forename: P E
authors.initials: PE
authors.lastname: Valensin
authors.forename: M
authors.initials: M
authors.lastname: Zazzi
mesh_terms: D002498:Centrifugation; D017931:DNA Primers / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D017931:DNA Primers; D012367:RNA, Viral