Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform

• DOI: 10.1016/j.jve.2023.100335
• Publication Date: 2023-06-28T06:51:46Z
• Authors: Carolin Tumpach; Catherine R. Coch...; Youry Kim; Jesslyn Ong; Ajantha Rhodes; Thomas A. Angelovich; Melissa J. Churchill; Sharon R. Lewin; Sushama Telwatte; Michael Roche
• Keywords: Public aspects of medicine; RA1-1270; Microbiology; QR1-502; 3. Good health; Original Research

Quantification of intact proviruses is a critical measurement in HIV cure studies both vitro and vivo. The widely adopted 'intact proviral DNA assay' (IPDA), designed to discriminate quantify genetically based on detection two sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR limited multiplexing capability (two-channel) labor- time intensive. To overcome some these limitations, we utilized nanowell-based platform (dPCR, QIAcuity from Qiagen) which fully automated system that partitions samples into nanowells rather than droplets. In this study adapted the IPDA assay assessed performance relative ddPCR. dPCR could differentiate between intact, 5' defective 3' sensitive single copy input. We found intra-assay inter-assay variability within acceptable ranges (with coefficient variation at or below 10%). When comparing ex vivo CD4+ T cells people with antiretroviral therapy, there strong correlation quantification (rs = 0.93; p < 0.001) 0.96; significant but less for 0.7; 0.04). demonstrate enables accurate similar greater speed efficiency. This flexible can be further optimized future, detect up 5 enabling more precise potentially replication-competent proviruses.