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Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR

https://doi.org/10.1016/j.jviromet.2012.08.019Get rights and content

Abstract

Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets.

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.

References (11)

  • M.K. Liszewski et al.

    Detecting HIV-1 integration by repetitive-sampling (Alu) under-bar-gag PCR

    Methods

    (2009)
  • E.M. Anderson et al.

    Single-copy detection of plasma HIV-1 RNA using droplet digital PCR technology (Abstract V-1002)

  • A. Brussel et al.

    Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA

    Methods in Molecular Biology

    (2005)
  • S.L. Butler et al.

    Human immunodeficiency virus cDNA metabolism: notable stability of two-long terminal repeat circles

    Journal of Virology

    (2002)
  • B.J. Hindson et al.

    High-throughput droplet digital PCR system for absolute quantitation of DNA copy number

    Analytical Chemistry

    (2011)
There are more references available in the full text version of this article.

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