期刊
METHODS
卷 201, 期 -, 页码 34-40出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2021.03.006
关键词
Human immunodeficiency virus type 1; Ribonucleic acid; Digital PCR; Reverse transcriptase digital PCR; External quality assessment
资金
- European Metrology Programme for Innovation and Research (EMPIR) from the EMPIR programme [HLT07]
- European Union's Horizon 2020 research and innovation programme
- UK National Measurement System
This paper investigates the potential of reverse transcriptase dPCR (RT-dPCR) in quantifying HIV-1 genomic RNA without calibration. The study suggests that RT-dPCR could be used as a reference measurement procedure to assist in assigning values to HIV-1 reference materials.
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.
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