Short communicationLow-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR
Highlights
► ddPCR provides quantitation of sequences without the use of standard curves. ► The ability of ddPCR to detect HIV-1 DNA from patient samples was evaluated. ► The ability of ddPCR to detect and quantitate HIV-1 DNA was similar to RT-PCR. ► ddPCR is a promising novel technology for the study of HIV-1 reservoirs. ► Further optimization would enhance the detection of low-level viral genetic targets.
Introduction
Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitation of target sequences without relying on the use of standard curves. To perform ddCPR, the DNA target, fluorescently-labeled probe and the ingredients for a PCR reaction are partitioned into an emulsion of approximately 20,000 droplets, each of which ideally contains 1 or less copies of the target DNA. Using 96-well plates, 2 million PCR reactions can be performed simultaneously (Hindson et al., 2011). Following PCR amplification, enumeration of both fluorescing and non-fluorescing droplets is performed, allowing the absolute quantitation of target molecules present in the original sample. To date, ddPCR has been validated primarily for measurement of germline copy number variation, detection and quantitation of rare alleles, and absolute quantitation of circulating fetal and maternal DNA (Hindson et al., 2011, Pinheiro et al., 2012).
The large-scale partitioning involved with ddPCR leads to greater precision and sensitivity for these applications when compared with real-time PCR (RT-PCR) (Hindson et al., 2011, Pinheiro et al., 2012). Partitioning reactions in picoliter droplets may allow ddPCR to handle relatively large amounts of input DNA with little interference from PCR inhibitors. As result, ddPCR is a promising platform for investigation of very low-levels of viral genetic material, and there has been considerable interest in this technology for the study of HIV-1 reservoirs. Data are emerging that support ddPCR for the quantitation of low-levels of plasma HIV-1 RNA (Anderson et al., 2012), but the performance and applicability to the study of cell-associated HIV-1 DNA have yet to be fully described. Natural history studies of HIV-1 infection in the setting of antiretroviral therapy show that HIV-1 can persist indefinitely (Siliciano et al., 2003) because the viral genome integrates into host cell chromosomes. HIV-1 cDNA can also enter the cell nucleus, but fail to integrate, resulting in circularized molecules containing one or two copies of the long terminal repeat (LTR) region of HIV (Brussel et al., 2005, Butler et al., 2002, Morlese et al., 2003). The development of novel methods to detect and quantitate low-levels of HIV-1 DNA is important to advance the study of viral persistence and eradication strategies. We evaluated the dynamic range of ddPCR and tested the ability of ddPCR to detect and quantitate HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy compared with traditional RT-PCR methods.
Section snippets
Patient samples, DNA extraction and preparation
Approval for the use of human material by the Brigham and Women's Hospital/Partners Healthcare Institutional Review Board was obtained prior to study initiation. In order to compare the performance of ddPCR and traditional RT-PCR for HIV-1 DNA, 5 million peripheral blood mononuclear cells (PBMCs) were obtained from 3 HIV-1-infected viremic patients and 4 patients on suppressive antiretroviral therapy. The PBMCs were washed in Hanks balanced salt solution (HBSS) and pelleted. Following
Real-time PCR and ddPCR quantitation of HIV-1 DNA
Copy numbers determined by ddPCR of serial dilutions of HIV-1 or CCR5 DNA standards are shown in Table 1. Linear regression correlation coefficients (R2) for the log-transformed copy number between ddPCR results and expected copy numbers were all >0.98 and slopes were all >0.92. Expected copy numbers measured by ddPCR were comparable to input standard values with the exception of the HIV-1 DNA amplicon standard; ddPCR detected approximately 60% fewer HIV-1 DNA copies than expected by the input
Discussion
Digital droplet PCR is a promising novel technology for the detection and quantitation of viral genetic material. In this study, we demonstrate that the platform is generally comparable to traditional RT-PCR methods for both total HIV-DNA and episomal 2-LTR circles. Despite good correlations of both linear and episomal DNA standards between the two methods, differences in the number of genomic HIV-1 DNA copies and episomal 2-LTR circles were observed. Whereas ddPCR is a direct measure of target
Acknowledgments
We would like to thank Bio-Rad Laboratories, Inc. for providing trial reagents and equipment. Funding: NIH/NIAID K23AI098480 to T.J.H.
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