Highly Precise Measurement of HIV DNA by Droplet Digital PCR

Matthew C Strain,Sara Gianella,Valeri H Terry,Douglas D Richman,Steven M Lada,Christopher H Woelk,Celsa A Spina,Steffney E Rought,Tiffany Luong,Yuntao Wu

doi:10.1371/journal.pone.0055943

Matthew C Strain, Sara Gianella + Show 8 more

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https://doi.org/10.1371/journal.pone.0055943

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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.

Highlights

Exponential amplification of nucleic acids by the polymerase chain reaction (PCR) is the cornerstone of modern molecular biology
This paper focuses on the application of droplet digital PCR [8], in which micro-partitioning is achieved by emulsification of the aqueous PCR reaction mixture in a thermostable oil
To determine the maximum amount of cellular deoxyribonucleic acid (DNA) that could be loaded into a single well, 10, 100 or 1000 copies of pNL43 or 2-LTR plasmid were spiked into a background of salmon sperm DNA prior to droplet formation (Fig. 1)

Summary

Introduction

Exponential amplification of nucleic acids by the polymerase chain reaction (PCR) is the cornerstone of modern molecular biology. Numerous methods have been developed to obtain quantitative information from PCR about the concentration of deoxyribonucleic acid (DNA) before amplification. The most widely used form of quantitative PCR (qPCR) is ‘‘real-time’’ PCR (qPCR), in which initial concentrations are extrapolated from sequential measurements during the cycling reaction [1]. The quantification and dynamics of HIV burden in infected patient were originally elucidated using quantitative PCR to measure viral ribonucleic acid (RNA) in blood plasma [9,10],[9,10] and viral load testing remains the standard clinical tool to assess the rate of disease progression. Proviral HIV DNA is the most widely used measure of cellular reservoir size, and other forms, including 2-LTR (long terminal repeat) circles and cell-associated RNA and DNA, provide additional information about viral dynamics

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