Highly Precise Measurement of HIV DNA by Droplet Digital PCR

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.

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

“…The false positive rate for wells using the CD3Z assay is extremely low and consistent with a background of 0.1 to 0.4 false positive events per non template control as reported using non-bisulfite treated DNA templates. [30][31][32] By contrast, T cell lymphopenia (<200 CD3/uL) is associated with approximately 400-700 positive droplets using our protocol; thus, the ddPCR provides ample sensitivity for applications in clinically relevant settings. The results of T cell proportions were highly correlated between the 2 assays ( Fig.…”

Asymptomatic CMV Replication During Early Human Immunodeficiency Virus (HIV) Infection Is Associated With Lower CD4/CD8 Ratio During HIV Treatment

“…Total CMV and EBV DNA was quantified by droplet digital polymerase chain reaction (PCR) as previously described [20,21]. Copy numbers were calculated as the mean of replicate PCR measurements and normalized to 1 million cells, determined by RPP30 [22].…”

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

“…cfDNA is comprised of fragments of extracellular DNA found mainly in the blood circulation, but it can also be detected in other bodily fluids such as saliva and urine. Of the range of DNA detection methods developed to date, the droplet digital polymerase chain reaction (ddPCR) provides high-precision, absolute quantification of nucleic acid target sequences, and has been shown to be even more sensitive than qPCR particularly in pathogen detection, cancer and prenatal testing (Hudecova, 2015;Miotke et al, 2014;Strain et al, 2013;Sze et al, 2014). …”

“…Therefore the concentrations of schistosome cfDNA in other body fluids such as urine (with the exception of S. haematobium which parasitises the bladder) and saliva, where direct contact with the parasite stages does not occur and the exact mechanisms of their release is yet unclear, cfDNA will be present in low amounts (Weerakoon and McManus, 2016). Hence, particularly in low intensity infections, cfDNA detection requires a highly sensitive diagnostic approach, using more technically advanced, novel DNA based methods, such as ddPCR (Hudecova, 2015;Miotke et al, 2014;Strain et al, 2013;Sze et al, 2014). When compared with cPCR and qPCR assays, ddPCR has higher diagnostic accuracy, and is able to provide absolute quantification, both features being beneficial in the support of disease control and elimination programs.…”

“…We noted that with higher amounts of template DNA, both droplet generation and amplification were impaired. The effect on droplet production may be due to chemical reactions at the oil water interface which are known to occur with higher DNA concentrations (Strain et al, 2013). Impaired droplet generation in turn affects the ddPCR reaction.…”