USE OF MULTIPLE COMPETITORS FOR QUANTIFICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA IN PLASMA

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in p lasma has rapidly become an important tool in basic HIV research and i n the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple com petitors was developed. Pour RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructe d. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by frag ment analysis with conventional automated sequencers. The coamplificat ion of known amounts of the RNA competitors provided the means to esta blish internal calibration curves for the individual reactions resulti ng in exclusion of tube-to-tube variations. Calibration curves were cr eated from the peak areas, which were proportional to the starting amo unt of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitativ e assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between -run coefficients of variation were <24% (range, 10 to 24) and <36% (r ange, 27 to 36), respectively. The high reproducibility (standard devi ation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detec tion variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 dis ease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.