20.500.12556/DiRROS-19309 An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1 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. imunski sistem digitalni PCR virusi true false true Angleški jezik Slovenski jezik Neznano 2024-07-16 03:40:38 2024-07-16 04:37:54 2024-07-17 03:59:10 0000-00-00 00:00:00 2022 0 0 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; str. [34]-40 Vol. 201 May 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2022-05-01 577 1046-2023 10.1016/j.ymeth.2021.03.006 83727875 https://doi.org/10.1016/j.ymeth.2021.03.006 1 69637456-4314-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26633 RAZ_Falak_Samreen_2022.pdf RAZ_Falak_Samreen_2022.pdf 1 59CF6A5B70A39431A8C01A99430F6BA8 b8a1c32c8daf2a49d484c031ac1dedea1af09fba5a64d4311ccd60648402dc6d b0d599f3-431c-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26675 Nacionalni inštitut za biologijo 0 0 0