Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2

Hedberg, Sara Thulin; Eriksson, Lorraine; Demontis, Maria Piera; Mölling, Paula; Sundqvist, Martin; Taylor, Graham P.; Malm, Kerstin; Andersson, Sören

doi:10.1016/j.jviromet.2018.07.003

Cited by 11 publications

(6 citation statements)

References 19 publications

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“…Apart from a few exceptions, the ddPCR showed relatively low intra-/inter-assay CV, as the variance among replicates approached the mean over replicates (Brunetto et al, 2014;Liu et al, 2019). Results indicate that our ddPCR can provide a repeatable and reproducible quantification of HPV DNA, whereas it can be potentially used for patient longitudinal monitoring, given that HPV clearance is recorded over time (Thulin Hedberg et al, 2018). Following ddPCR set up, as first approach on biological/clinical samples, we extended the assay on restriction enzyme-undigested DNA isolated from 45 CIN tissues and 4 human cells lines, including 3 HPV-positive cervical cancer cell lines.…”

Section: Discussionmentioning

confidence: 86%

Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

et al. 2020

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Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts. There has been progressive demand for more analytical assays for HPV DNA quantification. A novel droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV DNA from different HPV types. DdPCR was initially tested for assay sensitivity, accuracy, specificity as well as intra-and inter-run assay variation employing four recombinant plasmids containing HPV16, HPV18, HPV11, and HPV45 DNAs. The assay was extended to investigate/quantify HPV DNA in Cervical Intraepithelial Neoplasia (CIN, n = 45) specimens and human cell lines (n = 4). DdPCR and qPCR data from clinical samples were compared. The assay showed high accuracy, sensitivity and specificity, with low intra-/inter-run variations, in detecting/quantifying HPV16/18/11/45 DNAs. HPV DNA was detected in 51.1% (23/45) CIN DNA samples by ddPCR, whereas 40% (18/45) CIN tested HPV-positive by qPCR. Five CIN, tested positive by ddPCR, were found to be negative by qPCR. In CIN specimens, the mean HPV DNA loads determined by ddPCR were 3.81 copy/cell (range 0.002-51.02 copy/cell), whereas 8.04 copy/cell (range 0.003-78.73 copy/cell) by qPCR. DdPCR and qPCR concordantly detected HPV DNA in SiHa, CaSki and Hela cells, whereas HaCaT tested HPV-negative. The correlation between HPV DNA loads simultaneously detected by ddPCR/qPCR in CINs/cell lines was good (R 2 = 0.9706, p < 0.0001). Our data indicate that ddPCR is a valuable technique in quantifying HPV DNA load in CIN specimens and human cell lines, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.

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Section: Discussionmentioning

confidence: 86%

Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

et al. 2020

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“…Quantification of PVL using ddPCR has been established for human retroviruses, wherein the PVL in HTLV1 infections was used as a marker of disease progression. 45 Based on the similarities in pathogenesis between HTLV and BLV, we optimized ddPCR for BLV in peripheral blood of naturally infected cattle and experimentally infected sheep. The resulting ddPCR was highly sensitive and specific for BLV from various infected animals, making it suitable for use in early detection, to clarify inconclusive samples analyzed by qPCR, as well as for PVL quantification.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to nucleic acid detection, ddPCR is a platform designed to provide high precision for quantification of target molecules because it carries out direct quantification without requiring a calibration curve. 17,45 In contrast, the rtPCR assays used widely in diagnostic laboratories around the world show significant variability in the measurement of the BLV proviral DNA copy number among laboratories, even when qPCR is performed by experienced staff. 28,32,40 We found that PVLs were not concordant between qPCR and ddPCR.…”

Section: Discussionmentioning

confidence: 99%

Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus

et al. 2022

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Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants—a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

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“…The coefficient of variation for the C q values found was low for intra‐ and inter‐assay. Lower provirus concentrations in these samples may be one of the possible factors for variability found (Thulin Hedberg et al 2018). HTLV‐1 DNA was detected in all of the 12 samples.…”

Section: Discussionmentioning

confidence: 99%

Use of synthetic oligonucleotides for determination of HTLV‐1 proviral load by real‐time PCR: a helpful alternative approach in the clinical management

et al. 2020

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Aims: To evaluate the potential use of synthetic oligonucleotides as a standard curve for proviral load (PVL) of human T-cell leukaemia virus type 1 (HTLV-1) quantification in peripheral blood mononuclear cells (PBMC) of HTLV-1infected individuals by quantitative real-time polymerase chain reaction (qPCR) analysis. Methods and results: Synthetic oligonucleotides based on HTLV-1 genome were customized to use as a standard curve. Twelve anti-HTLV-1-positive samples with known HTLV-1 PVL, previously quantified by qPCR assay using TARL-2 cells as a conventional standard curve, were submitted to the new protocol. The proviral quantification levels had a high concordance with qPCR results using a conventional standard curve. The results demonstrate that the conventional standard curve can be replaced by a synthetic standard curve due to its ability to quantification based on the linearity and qPCR efficiency and similar results with a validated qPCR assay using a conventional standard curve. Conclusions: Synthetic oligonucleotides standard curves could be a very useful tool on HTLV-1 diagnosis and absolute HTLV-1 PVL quantification. Significance and Impact of the Study: HTLV-1 PVL determination using synthetic oligonucleotides standard curve by qPCR could be a helpful alternative for the laboratories that monitor infected patients as an important prognostic factor in HTLV-1-associated diseases progression. Also, it can decrease costs and overcome the biological limitations of the plasmid curve.

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Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2

Abstract: The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.

Cited by 11 publications

References 19 publications

Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus

Use of synthetic oligonucleotides for determination of HTLV‐1 proviral load by real‐time PCR: a helpful alternative approach in the clinical management

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