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. 2022 Dec 16;3(4):101681.
doi: 10.1016/j.xpro.2022.101681. Epub 2022 Sep 28.

Protocol for high-throughput reservoir quantification across global HIV subtypes using a cross-subtype intact proviral DNA assay

Carolyn S Fish  1 , Noah A J Cassidy  1 , Claire N Levy  2 , Sean M Hughes  2 , Keith R Jerome  3 , Julie Overbaugh  1 , Florian Hladik  4 , Dara A Lehman  5
Affiliations

Protocol for high-throughput reservoir quantification across global HIV subtypes using a cross-subtype intact proviral DNA assay

Carolyn S Fish et al. STAR Protoc. .

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).

Keywords: Genomics; High throughput screening; Microbiology; Molecular biology.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Example plate layout for performing CS-IPDA and RPP30/deltaD reference assay in a single 96-well experiment RPP30/deltaD reference assay reactions are completed in singlet (blue). CS-IPDA reactions are completed in triplicate (white). Experimental controls include all-target plasmid mix gating control (yellow), triple positive plasmid gating control (orange), HIV-positive control, such as J-lat 5A8 or 8.4 gDNA (green), HIV-negative gDNA control (gray), and RPP30 gDNA gating control, such as HIV-negative PBMCs or J-lat 5A8 or 8.4 gDNA (red).
Figure 2
Figure 2
Example of ddPCR data analysis using QuantaSoft Analysis Pro software (A) RPP30/deltaD reference assay wells are gated using extracted gDNA from provirus-containing cells mixed with HIV-negative PBMCs as a reference. Assay probe targets for each cluster are indicated by text. (B) RPP30/deltaD gating thresholds determined by the control are applied to a clinical sample. Assay probe targets for each cluster are indicated by text. (C) CS-IPDA gating thresholds are determined using a mixture of plasmids containing all targets. (D) CS-IPDA gating thresholds applied to gDNA extracted from provirus containing cells (approximately 200 HIV DNA copies/reaction). Assay probe targets for each cluster are indicated by text. See also Methods video S1.
See this image and copyright information in PMC

References

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