Use of Multiple Competitors for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma

Abstract

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative 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% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection 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 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Cooperative Oligonucleotides Mediating Direct Capture of Hepatitis C Virus RNA from Serum

“…However, repeated analysis of this diluted rRNA target occasionally showed up to a 25-fold improvement in capture with the prehybridization probe. We believe that this observed variation is not a result of differences in capture efficiency but rather reflects stochastic variation within end-diluted rRNA specimens (24). To further dissect the differences shown in Fig.…”

“…After incubation, the beads were washed and transferred to PCR tubes containing reagents and primers for outer PCR amplification of the HCV target region. A seminested inner PCR was carried out to allow for comparison at the PCR plateau level, at which all dilutions have reached saturation irrespective of the initial copy number (20,24). This is illustrated by the roughly equal intensity of the PCR fragments after gel electrophoresis.…”

“…To allow for generation of a 649-nt transcript by in vitro transcription, purified plasmids containing the 5Ј NTR of HCV (genotypes 1a, 2b, and 3a) were digested with NarI (258 bp downstream of the insert) and the resulting linearized DNA was precipitated and dissolved in 50 l of diethylpyrocarbonate (DEPC) (Sigma Chemical Co.)-treated H 2 O. Transcription from the T7 promoter was performed on 1.5 g of this linearized DNA, as previously described (24). After transcription, template DNA was fragmented by restriction digestion (with AvaI) and treatment with 8 U of RNase-free DNase I (Boehringer) at 37°C for 45 min.…”

Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples

“…Pooling of samples has a relatively small impact on the ability of a NAT to detect early infection, as HIV and HCV nucleic acids reach high levels rapidly after infection is established [7,8]. The RNA levels can be quantified by means of target or signal amplification techniques but, until recently, the quantitative units used in the various commercial assays did not represent the same amount of virus RNA in clinical samples.…”

RNA Internal Standard Synthesis by Nucleic Acid Sequence-Based Amplification for Competitive Quantitative Amplification Reactions

“…The development of quantitative competitive molecular amplification reactions such as the nucleic acid sequence-based amplification (NASBA) reaction, the PCR and reverse transcriptase PCR (RT-PCR) have been demonstrated. Quantitative competitive PCR or RT-PCR have been used for the detection of viral or bacterial pathogens as well as cellular RNA or DNA − since it was first described. − NASBA is an ideal alternative to RT-PCR since it does not react with contaminating DNA. Thus, in recent years, quantitative NASBA assays have been developed for the detection of various viral and bacterial RNA − ever since NASBA was first introduced …”