An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Falak, Samreen (Avtor) Macdonald, Rainer (Avtor) Busby, Eloise J. (Avtor) O'Sullivan, Denise M. (Avtor) Milavec, Mojca (Avtor) Plauth, Annabell (Avtor) Kammel, Martin (Avtor) Zeichhardt, Heinz (Avtor) Grunert, Hans-Peter (Avtor) Kummrow, Andreas (Avtor) Huggett, Jim F. (Avtor) 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.20222024-07-16 03:40:38Neznano19309UDK: 577ISSN pri članku: 1046-2023DOI: 10.1016/j.ymeth.2021.03.006COBISS_ID: 83727875sl