Samreen Falak (Author), Rainer Macdonald (Author), Eloise J. Busby (Author), Denise M. O'Sullivan (Author), Mojca Milavec (Author), Annabell Plauth (Author), Martin Kammel (Author), Heinz Zeichhardt (Author), Hans-Peter Grunert (Author), Andreas Kummrow (Author), Jim F. Huggett (Author)

Abstract

Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.

Keywords

imunski sistem;digitalni PCR;virusi;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: NIB - National Institute of Biology
UDC: 577
COBISS: 83727875 Link will open in a new window
ISSN: 1046-2023
Views: 43
Downloads: 10
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Other data

Secondary language: Slovenian
Secondary keywords: imunski sistem;digitalni PCR;virusi;
Source comment: Soavtorji: Rainer MacDonald, Eloise J. Busby, Denise M. OʼSullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett;
Pages: str. [34]-40
Issue: ǂVol. ǂ201
Chronology: May 2022
DOI: 10.1016/j.ymeth.2021.03.006
ID: 24536857