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Sci Rep. 2015 Sep 9;5:13811. doi: 10.1038/srep13811.

Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir.

Author information

1
Department of Medical Microbiology, Virology, University Medical Centre Utrecht, The Netherlands.
2
Department of Internal Medicine, HIV Translational Research Unit, University Ghent &University Hospital Ghent, Belgium.

Abstract

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01±0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA.

PMID:
26350506
PMCID:
PMC4563360
DOI:
10.1038/srep13811
[Indexed for MEDLINE]
Free PMC Article

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