A Multiplex Droplet Digital PCR Assay for Quantification of HTLV-1c DNA Proviral Load and T-Cells from Blood and Respiratory Exudates Sampled in a Remote Setting

Abstract

19During human T-cell leukemia virus type-1 (HTLV-1) infection the frequency of cells 20 harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk

Impact of Hepatitis B Virus Coinfection on Human T-Lymphotropic Virus Type 1 Clonality in an Indigenous Population of Central Australia

“…These 15 samples were therefore not analyzed further. c To compare the 2 groups, the HTLV-1 proviral load of T cells was remeasured by droplet digital PCR ([9]). Sufficient DNA was not available for 7 samples from the previous study [4].…”

“…Genomic DNA was extracted from peripheral blood buffy coats for the initial study in asymptomatic HTLV-1–monoinfected subjects [4] and from peripheral blood mononuclear cells in the coinfection study. To compare the proviral load of HTLV-1 between these 2 different sets of samples, we quantified the proviral load in T cells by a ddPCR assay [9]. The proviral load in HTLV-1–infected subjects coinfected with HBV (median, 1.7 × 10 4 proviral copies per 10 6 T cells) was not significantly different from that of HTLV-1–monoinfected asymptomatic subjects (median, 4.3 × 10 4 proviral copies per 10 6 T cells; Figure 2A; Wilcoxon rank-sum test, P = .10).…”

“…A , HTLV-1 proviral load was measured in duplicate by droplet digital polymerase chain reaction, using a tax probe as a viral specific target and RPP30 as an internal reference gene. The T-cell count was then determined by estimating the frequency of non–T cells by using a set of primers targeting the unrearranged T-cell receptor Dβ1-Jβ1.1 intergenic sequences [9]. The limit of detection of 98 copies per million T cells is indicated by the dashed lines.…”

“…The HTLV-1 proviral load was measured as previously described [9] by droplet digital polymerase chain reaction (ddPCR) using 100 ng of gDNA mixed with forward (5ʹ-TCCAGGCCTTATTTGGACAT-3ʹ) and reverse (5ʹ-CGTGTGAGAGTAGGACTGAG-3ʹ) primers (each 900 nM) targeting HTLV-1 tax, and TaqMan MGB probe (5ʹ-FAM-CATGATTTCCGGGCCTTGC-MGBNFQ-3ʹ) (250 nM) (Thermo Scientific), and (×20) PrimePCR ddPCR copy number assay: ribonuclease P/MRP 30 kDa subunit (RPP30-HEX) human primer/probe mix (Bio-Rad), and (×2) Bio-Rad Supermix for Probes (no deoxyuridine triphosphate).…”

“…All samples were run in duplicate, and the reported HTLV-1 subtype c (HTLV-1c) proviral load is the mean of the 2 measurements. The T-cell population was determined by measuring the loss of the Dβ1-Jβ1.1 intergenic sequences, as previously described [9].…”