Highly precise measurement of HIV DNA by droplet digital PCR
Highly precise measurement of HIV DNA by droplet digital PCR
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.
e55943
Strain, M.C.
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Lada, S.
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Luong, T.
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Rought, S.E.
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Gianella, S.
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Terry, V.
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Spina, C.A.
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Woelk, C.H.
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Richman, D.D.
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3 April 2013
Strain, M.C.
c42d99eb-f03f-4cdf-9116-8e4b09d1c0da
Lada, S.
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Luong, T.
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Rought, S.E.
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Gianella, S.
dd0d17ac-3711-4d97-932e-f64dec268d7c
Terry, V.
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Spina, C.A.
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Woelk, C.H.
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Richman, D.D.
1d5f38e4-c778-4b0b-b542-9d1160e63041
Strain, M.C., Lada, S., Luong, T., Rought, S.E., Gianella, S., Terry, V., Spina, C.A., Woelk, C.H. and Richman, D.D.
(2013)
Highly precise measurement of HIV DNA by droplet digital PCR.
PLoS ONE, 8 (4), .
(doi:10.1371/journal.pone.0055943).
(PMID:23573183)
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.
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Published date: 3 April 2013
Organisations:
Clinical & Experimental Sciences
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Local EPrints ID: 352907
URI: http://eprints.soton.ac.uk/id/eprint/352907
ISSN: 1932-6203
PURE UUID: a73e92cc-80a6-43cf-a21f-27fbdfe870ee
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Date deposited: 23 May 2013 14:42
Last modified: 14 Mar 2024 13:57
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Contributors
Author:
M.C. Strain
Author:
S. Lada
Author:
T. Luong
Author:
S.E. Rought
Author:
S. Gianella
Author:
V. Terry
Author:
C.A. Spina
Author:
C.H. Woelk
Author:
D.D. Richman
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