Abstract
AbtractEstablishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides ‘absolute’ quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.
Highlights
A routine method for quantification of HIV RNA viral load, real-time quantitative PCR, is increasingly being used for measuring HIV DNA associated with the viral reservoir9. qPCR requires calibration and for this to be reproducible it is essential that the calibrator must be stable when shared between laboratories
It has been used to value assign a variety of qPCR calibrators, including those for BCR-ABL21 and Mycobacterium tuberculosis22. Digital PCR (dPCR) has been used in the direct quantification of HIV DNA from patients in a number of studies[23,24,25,26,27] and unlike qPCR has the advantage of not requiring an external calibration standard
Analysis of clinical samples by qPCR and dPCR. 18 peripheral blood mononuclear cell samples (PBMC) DNA samples from HIV-positive patients were analysed by dPCR and qPCR using the PDH/HIV LTR-gag duplex assays
Summary
A routine method for quantification of HIV RNA viral load, real-time quantitative PCR (qPCR), is increasingly being used for measuring HIV DNA associated with the viral reservoir9. qPCR requires calibration and for this to be reproducible it is essential that the calibrator must be stable when shared between laboratories. A routine method for quantification of HIV RNA viral load, real-time quantitative PCR (qPCR), is increasingly being used for measuring HIV DNA associated with the viral reservoir. DPCR has been used in the direct quantification of HIV DNA from patients in a number of studies[23,24,25,26,27] and unlike qPCR has the advantage of not requiring an external calibration standard. False positives and issues surrounding threshold determination have been reported to limit the usefulness of dPCR when employed for the most sensitive measurements of HIV DNA28. In this study we investigated the application of dPCR instruments in the context of HIV DNA measurement, both for comparison with qPCR analysis of patient samples and as a method for value assigning 8E5 calibration standards from three different sources
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