title: Highly precise measurement of HIV DNA by droplet digital PCR.
abstract: Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it.
authors.affiliation: University of California San Diego, La Jolla, California, United States of America. mstrain@ucsd.edu
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Strain
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lada
authors.forename: Tiffany
authors.initials: T
authors.lastname: Luong
authors.forename: Steffney E
authors.initials: SE
authors.lastname: Rought
authors.forename: Sara
authors.initials: S
authors.lastname: Gianella
authors.forename: Valeri H
authors.initials: VH
authors.lastname: Terry
authors.forename: Celsa A
authors.initials: CA
authors.lastname: Spina
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Woelk
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Richman
mesh_terms: D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D015658:HIV Infections / Q000097:blood* / Q000188:drug therapy / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D016012:Poisson Distribution; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D059629:Signal-To-Noise Ratio; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D004279:DNA, Viral; D054302:pol Gene Products, Human Immunodeficiency Virus