1. Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirusAlexandra Bogožalec Košir, Tašja Cvelbar, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Mojca Milavec, 2020, izvirni znanstveni članek Povzetek: Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir.
Ključne besede: digital PCR, antimicrobial-drug resistance, HCMV, polymerase chain reaction, viruses
Objavljeno v DiRROS: 22.07.2024; Ogledov: 125; Prenosov: 102
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2. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Samreen Falak, Rainer Macdonald, Eloise J. Busby, Denise M. O'Sullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett, 2022, izvirni znanstveni članek Povzetek: 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.
Objavljeno v DiRROS: 16.07.2024; Ogledov: 119; Prenosov: 63
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3. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four yearsMojca Milavec, Jernej Pavšič, Alexandra Bogožalec Košir, Gerwyn M. Jones, Denise M. O'Sullivan, Alison S. Devonshire, Fran Van Heuverswyn, Maria Karczmarczyk, Jannika Neeb, Annabell Plauth, Philippe Corbisier, Heinz Schimmel, Andreas Kummrow, Jörg Neukammer, Carole A. Foy, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Jim F. Huggett, 2022, izvirni znanstveni članek Objavljeno v DiRROS: 15.07.2024; Ogledov: 213; Prenosov: 62
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