Protocol for high-throughput reservoir quantification across global HIV subtypes using a cross-subtype intact proviral DNA assay
Carolyn S. Fish, Noah A.J. Cassidy, Claire Levy, Sean M. Hughes, Keith R. Jerome, Julie Overbaugh, Florian Hladik, Dara A. Lehman
Abstract
The cross-subtype intact proviral DNA assay (CS-IPDA) is a high-throughput method to quantify HIV reservoir size in populations infected with any of the dominant global HIV-1 subtypes. Our protocol includes genomic DNA isolation optimized to minimize DNA shearing, a reference droplet digital PCR (ddPCR) assay to quantify T cells and assess DNA shearing, and a multiplex ddPCR targeting three distinct regions across the HIV genome to quantify intact proviruses as an estimate of replication-competent proviruses in the reservoir. For complete details on the use and execution of this protocol, please refer to Cassidy et al. (2022).