oru.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Digital droplet PCR (ddPCR) for the detection and quantification of HPV 16, 18, 33 and 45: a short report
Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Laboratory Medicine.ORCID iD: 0000-0001-9992-266X
Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Laboratory Medicine.ORCID iD: 0000-0003-2317-5738
2017 (English)In: Cellular Oncology, ISSN 2211-3428, E-ISSN 2211-3436, Vol. 40, no 5, p. 521-527Article in journal (Refereed) Published
Abstract [en]

Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR).

Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR. Droplets positive for VIC and FAM fluorescence were read in a QX200 Droplet reader (TM) (BIO-RAD) after which the viral load was calculated using Quantasoft software.

We found that DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples yielded lower amplification signals compared to those obtained from liquid based cytology (LBC) samples, but they were clearly distinguishable from negative background signals. The viral limit of detection was 1.6 copies of HPV 16, 2.8 copies of HPV 18, 4.6 copies of HPV 33 and 1.6 copies of HPV 45. The mean inter-assay coefficients of variability (CV) for the assays ranged from 3.4 to 7.0%, and the mean intra-assay CV from 2.6 to 8.2%. The viral load in the different cohorts of tumor samples ranged from 154 to 340,200 copies for HPV 16, 244 to 31,300 copies for HPV 18 and 738 to 69,100 copies for HPV 33. One sample positive for HPV 45 contained 1331 viral copies. When comparing qPCR data with ddPCR copy number data, the qPCR values were found to be 1 to 31 times higher.

Separation of fragments in nanodroplets may facilitate the amplification of fragmented human and viral DNA. The method of digital droplet PCR may, thus, provide a new and promising tool for evaluating the HPV viral load in clinical samples.

Place, publisher, year, edition, pages
Springer, 2017. Vol. 40, no 5, p. 521-527
Keywords [en]
Human papilloma virus (HPV), Quantitation, Viral load, PCR
National Category
Cancer and Oncology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:oru:diva-61424DOI: 10.1007/s13402-017-0331-yISI: 000411570200008PubMedID: 28748500Scopus ID: 2-s2.0-85026909802OAI: oai:DiVA.org:oru-61424DiVA, id: diva2:1148722
Note

Funding Agencies:

Region Örebro County through ALF  

Örebro County Council Research Committee  

Available from: 2017-10-12 Created: 2017-10-12 Last updated: 2018-02-01Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMedScopus

Authority records BETA

Lillsunde-Larsson, GabriellaHelenius, Gisela

Search in DiVA

By author/editor
Lillsunde-Larsson, GabriellaHelenius, Gisela
By organisation
School of Medical SciencesÖrebro University Hospital
In the same journal
Cellular Oncology
Cancer and OncologyMedical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 67 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf