Volume 11 Supplement 1

16th Interntional Conference on Human Retroviruses: HTLV and Related Viruses

Open Access

Digital droplet PCR for precise quantification of human T-lymphotropic virus 1 proviral loads

  • Giovanna S Brunetto1,
  • Raya Massoud1,
  • Joan Ohayon1,
  • Kaylan Fenton1,
  • Irene Cortese1 and
  • Steven Jacobson1
Retrovirology201411(Suppl 1):P13

https://doi.org/10.1186/1742-4690-11-S1-P13

Published: 7 January 2014

Elevated HTLV-1 proviral load (PVL) is thought to be the major risk factor for developing HAM/TSP in HTLV-1 infected subjects, and a high cerebrospinal fluid (CSF) to peripheral blood mononuclear cells (PBMCs) PVL ratio might be diagnostic of the condition. However, the standard method for quantification of HTLV-1 PVL, Real time PCR, has multiple limitations: the inter-assay variability increases at low PVL and low cell numbers in CSF often precludes accurate quantification. Thus, we are evaluating a novel technique, Digital Droplet PCR (ddPCR), as a potentially more reliable tool. For ddPCR, DNA samples are partitioned into thousands of nanoliter -sized droplets, amplified on a thermocycler, queried for fluorescent signal and normalized to a housekeeping gene. Due to the high number of DNA molecules and number of “independent” events (droplets), Poisson algorithms are used to determine absolute copy numbers and are independent of a standard curve. Our results suggest that ddPCR is very accurate: Intraassay variability evaluated by calculating the coefficient of variation of ten replicates of three samples of DNA in three different ranges of PVL (low < 5%, medium 5-10%, and high > 10%) was 13.0%, 7.1% and 9.5%, respectively. Interassay variability, was evaluated by calculating the CV of duplicates of PVL from three independent runs and three independent extractions was 4.5% with a standard deviation of 0.008. Additionally, ddPCR is reliable in quantifying PVL in the CSF where we have confirmed and extended previous observations of increased HTLV-I PVL in CSF of HAM/TSP compared to the periphery.

Authors’ Affiliations

(1)
Viral Immunology Section, Neuroimmunology Branch, National Institutes of Neurological Disorders and Stroke

Copyright

© Brunetto et al; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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