Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma
| Autor: | Rebecca Ferruzzi, Giulietta Venturi, Pier Egisto Valensin, M. Catucci, Maurizio Zazzi, Laura Romano |
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| Rok vydání: | 2000 |
| Předmět: |
Reverse Transcriptase Polymerase Chain Reaction
Coefficient of variation RNA Centrifugation HIV Infections Viral Load Biology Sensitivity and Specificity Molecular biology Reverse transcriptase Virus chemistry.chemical_compound chemistry Virology HIV-1 Humans RNA Viral RNA extraction Viral load Quantitative analysis (chemistry) DNA DNA Primers |
| Zdroj: | Journal of Virological Methods. 87:91-97 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/s0166-0934(00)00151-8 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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