Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA (original) (raw)
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Quantitative Competitive RNA PCR for Quantitation of Virion-Associated HIV-1 RNA
Methods, 1997
Quantitative competitive PCR is a highly sensitive technique technique is, however, labor intensive and is limited in that allows accurate quantitation of small amounts of RNA. We its ability to distinguish small fluctuations in titers of have modified the original method to include the use of an interinfectious virus in body fluids. Measurement of virionnal standard at all stages of sample analysis. In this way, the associated genomic viral RNA by PCR-based apmethod can accommodate for variations in the recovery of viral proaches represents an extremely valuable tool for exparticles and in the isolation of genomic RNA as well as provide amining virus load in body fluids and for monitoring a suitable competitive substrate during quantitative RNA PCR. fluctuations in virus load at distinct stages of disease We have used this method to characterize changes in virus load (2-8) and monitoring efficacy of drug combinations (9, in plasma of HIV-1-seropositive individuals following their vacci-10). Commercial assays for measurement of virion-asnation against opportunistic infections. ᭧ 1997 Academic Press sociated RNA have been developed. These commercial assays are exquisitely sensitive and linear over a wide target copy number range. However, these assays represent a considerable cost outlay for those investigators who may need quantitative measurements of virus load Analysis of virus load in peripheral blood of HIVon smaller sample numbers. For this reason, we have 1-infected individuals indicates an inverse correlation developed a protocol for quantitation of virus load in between virus load and circulating CD4 / T lymphobody fluids that, through the use of an internal stancytes and a positive correlation between high virus load dard, allows the investigator to accommodate losses and severity of disease (1-8). Examination of virus load due to sample preparation and the presence of inhibihas proven a valuable approach for evaluating the effitors that affect the efficiency and kinetics of RT-PCR. cacy of various antiviral strategies targeted to either In using RNA PCR to examine viral particle numviral or immune functions (9-15). Thus, quantitative ber in a biological sample, the investigator must enmeasurements of HIV-1 replication represent an imsure that the amount of final amplification product portant tool for gauging the efficacy of a drug regimen is directly proportional to the target copy number and evaluating clinical status of HIV-1-infected indipresent in the original specimen (21). However, there viduals.
2000
The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log 10 estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log 10 in most comparisons. Interlaboratory differences across runs were <0.10 log 10 at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log 10 HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had >50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml; P ؍ 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.
Journal of Clinical Virology, 2015
Background: Quantitative measurement of HIV-1 RNA levels in plasma ('viral load') plays a central role in clinical management. The choice of assay platform can influence results and treatment decisions. Objective: To compare the analytical performance of the new TMA-based Hologic Aptima ® HIV-1 Quant Dx assay with that of three PCR-based assays: Abbott RealTime HIV-1, Qiagen Artus ® HI Virus-1 QS-RGQ, and Roche CAP/CTM HIV-1 Test v2. Study design: Assay performance was evaluated using Acrometrix HIV-1 RNA Standard panels; the 3rd WHO HIV-1 RNA International Standard (12-500 copies/ml; 6 dilutions; 9 replicates); and plasma samples from 191 HIV-positive patients. Results: Aptima showed high (>0.99) precision, accuracy and concordance with the Acrometrix Standards across a wide dynamic range (2.0-6.7 log 10 copies/ml). Variance caused up to 2.1 (Aptima), 1.7 (RealTime), 7.5 (Artus), and 1.9 (CAP/CTM) fold changes in the International Standard quantifications at 50-500 copies/ml. HIV-1 RNA detection rates in plasma samples were 141/191 (74%), 119/191 (62%), 108/191 (57%), and 145/191 (76%) for Aptima, RealTime, Artus and CAP/CTM, respectively. For categorising samples either side of 50 copies/ml, Aptima had excellent agreement with RealTime (kappa 0.92; 95% CI 0.87-0.98); lowest agreement was with Artus (kappa 0.79; 95%CI 0.70-0.88). Aptima quantifications were mean 0.12 and 0.06 log 10 copies/ml higher compared with RealTime and CAP/CTM, respectively, and 0.05 log 10 copies/ml lower compared with Artus. Limits of agreement were narrowest when comparing Aptima to RealTime. Conclusions: The new Aptima HIV assay is sensitive, precise, and accurate. HIV assays exhibit discordance at low HIV-1 RNA copy numbers.
A Novel PCR Assay for Quantification of HIV-1 RNA
Current assays for quantification of HIV-1 virions rely on real-time reverse transcriptase (RT)-PCR detection of conserved regions of HIV-1 RNA and can be limited by detection of contaminating viral or plasmid DNA. We developed a novel RT-PCR assay using a reverse primer that hybridizes with the poly(A) tail of HIV-1 mRNAs, anchored by conserved viral nucleotides at the most distal region of the transcript. This assay can detect and quantify HIV-1 RNA with high specificity and sensitivity.
Journal of Visualized Experiments, 2011
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate. The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15 ,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.