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Eloise Busby 1, Alexandra Whale1, R. Bridget Ferns 2, Paul R. Grant3, Gary Morley1, Jonathan Campbell1, Carole Foy1, Eleni Nastouli3,4, Jim Huggett1,5, Jeremy A. Garson 2,6 1 Molecular and Cell Biology Team, LGC, Teddington, UK 2 Department of Infection, Division of Infection and Immunity, University College London, London, UK 3 Department of Clinical Virology, University College London Hospital NHS Foundation Trust, and the UCL/UCLH NIHR Biomedical Research Centre, London, UK 4 Department of Population Policy and Practice, UCL GOS Institute of Child Health, London, UK 5 School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford GU2 7XH, UK 6 National Transfusion Microbiology Laboratories, NHS Blood and Transplant, Colindale, London, UK |
Abstract
Quantification of HIV DNA associated with the viral reservoir is increasingly used in research as a tool to study latent disease. Such a target could potentially be used clinically to assist moni- toring of disease progression or decline as it has been reported to correlate with viral outgrowth, and could serve as a biomarker for monitoring chronic infection. In addition to measurement of RNA viral load, qPCR is used as the method of choice for quantification of HIV DNA. Results are normalised to a human genomic target and reported as HIV DNA copies per 1,000,000 cells. When per- forming qPCR this is commonly achieved by calibrating against the 8E5 cell line reported to contain one stably integrated HIV genome per cell. In this study we used dPCR to investigate the stability of the HIV genome in the 8E5 cell line and demonstrated how accurate value assignment of this calibrator impacts upon qPCR measurement of HIV DNA. DNA from three separate sources of 8E5 cells, which included serially passaged fresh cells, was analysed using two droplet-based dPCR platforms. We identified genetic instability in the 8E5 cell line, which supports very recent find- ings of other researchers, and resulted in HIV DNA quantification in patient samples varying by a factor of ∼50. dPCR value assign- ment of the calibrators removed this error. Appropriately calibrated quantitative methods afford greater accuracy and measurement harmonisation when using qPCR to measure a specific sequence. We show here that dPCR is a method that can effectively be used to quantify calibration materials for this purpose.
http://dx.doi.org/10.1016/j.bdq.2017.02.039
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