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Updated: Sep 18, 2025
Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing
Published on: October 16, 2018
Considerations and limitations for establishing an Intact Proviral DNA Assay (IPDA) on a chip-based digital PCR
Jasmin Tschumi1, Kathrin Neumann2, Dominique L Braun1
1Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
None:
The intact proviral DNA assay (IPDA) has become a gold standard for HIV-1 reservoir quantification in HIV-1 latency research, as well as in the evaluation of HIV-1 cure strategies. In this work, we adjusted the IPDA assay to a chip-based digital PCR (dPCR) system, established the use of CCR5 as a different reference gene, and evaluated the performance on cell lines, clinical samples from people with HIV (PWH) off and on antiretroviral therapy (ART), and different HIV-1 subtypes. Our adapted IPDA performs well on the chip-based dPCR system with no false positive intact HIV-1 in negative controls and with less hands-on time compared to droplet-based dPCR systems. Undetectable intact HIV-1 DNA is common in clinical samples on ART and correlated with total HIV-1 reservoir size and sample input concentration for HIV-1 subtype B samples. Performance on non-B subtypes varies depending on the subtype and should be improved with subtype specific primer/probe combinations.
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