1. Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR
- Author
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Lada, Steven M, Huang, Karissa, VanBelzen, D Jake, Montaner, Luis J, O'Doherty, Una, and Richman, Douglas D
- Subjects
Medical Microbiology ,Biomedical and Clinical Sciences ,Immunology ,HIV/AIDS ,Sexually Transmitted Infections ,Infectious Diseases ,Genetics ,Infection ,Good Health and Well Being ,DNA ,Viral ,Electrophoresis ,Gel ,Pulsed-Field ,Gene Products ,gag ,HIV Infections ,HIV Long Terminal Repeat ,HIV-1 ,Humans ,Leukocytes ,Mononuclear ,Proviruses ,Real-Time Polymerase Chain Reaction ,Reproducibility of Results ,Virus Integration ,HIV ,latent reservoir ,pulsed-field gel electrophoresis ,integrated DNA ,Biological Sciences ,Agricultural and Veterinary Sciences ,Medical and Health Sciences ,Microbiology ,Clinical sciences ,Medical microbiology - Abstract
We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.
- Published
- 2018