Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform
Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted ‘intact proviral DNA assay’ (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was ori...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-06-01
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| Series: | Journal of Virus Eradication |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2055664023000213 |
| _version_ | 1797790070624747520 |
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| author | Carolin Tumpach Catherine R. Cochrane Youry Kim Jesslyn Ong Ajantha Rhodes Thomas A. Angelovich Melissa J. Churchill Sharon R. Lewin Sushama Telwatte Michael Roche |
| author_facet | Carolin Tumpach Catherine R. Cochrane Youry Kim Jesslyn Ong Ajantha Rhodes Thomas A. Angelovich Melissa J. Churchill Sharon R. Lewin Sushama Telwatte Michael Roche |
| author_sort | Carolin Tumpach |
| collection | DOAJ |
| description | Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted ‘intact proviral DNA assay’ (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5’ defective and 3’ defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3’ defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5’ defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses. |
| first_indexed | 2024-03-13T01:59:31Z |
| format | Article |
| id | doaj.art-9667ec6b75ac4d299079c26788a17b22 |
| institution | Directory Open Access Journal |
| issn | 2055-6640 |
| language | English |
| last_indexed | 2024-03-13T01:59:31Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Virus Eradication |
| spelling | doaj.art-9667ec6b75ac4d299079c26788a17b222023-07-02T04:16:31ZengElsevierJournal of Virus Eradication2055-66402023-06-0192100335Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platformCarolin Tumpach0Catherine R. Cochrane1Youry Kim2Jesslyn Ong3Ajantha Rhodes4Thomas A. Angelovich5Melissa J. Churchill6Sharon R. Lewin7Sushama Telwatte8Michael Roche9Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, AustraliaInfectious and Inflammatory Diseases Theme, School of Health and Biomedical Sciences, RMIT University, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Infectious and Inflammatory Diseases Theme, School of Health and Biomedical Sciences, RMIT University, Melbourne, Australia; Life Science, Burnet Institute, Melbourne, AustraliaInfectious and Inflammatory Diseases Theme, School of Health and Biomedical Sciences, RMIT University, Melbourne, Australia; Life Science, Burnet Institute, Melbourne, Australia; Departments of Microbiology and Medicine, Monash University, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Australia; Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Corresponding author. Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, AustraliaDepartment of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; Infectious and Inflammatory Diseases Theme, School of Health and Biomedical Sciences, RMIT University, Melbourne, AustraliaQuantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted ‘intact proviral DNA assay’ (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5’ defective and 3’ defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3’ defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5’ defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses.http://www.sciencedirect.com/science/article/pii/S2055664023000213 |
| spellingShingle | Carolin Tumpach Catherine R. Cochrane Youry Kim Jesslyn Ong Ajantha Rhodes Thomas A. Angelovich Melissa J. Churchill Sharon R. Lewin Sushama Telwatte Michael Roche Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform Journal of Virus Eradication |
| title | Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform |
| title_full | Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform |
| title_fullStr | Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform |
| title_full_unstemmed | Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform |
| title_short | Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform |
| title_sort | adaptation of the intact proviral dna assay to a nanowell based digital pcr platform |
| url | http://www.sciencedirect.com/science/article/pii/S2055664023000213 |
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