Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling

S. Crovella, D. Pirulli, D. De Santo, F. De Seta, M. Boniotto, L. Braida, F. Boaretto, S. Guaschino, A. Amoroso

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

In this study we developed an in situ protocol for quantitative detection of high-risk human papillomavirus (HPV), based on direct in situ polymerase chain reaction (PCR) with SYBR Green I labeling and GeneAmp 5700 Sequence Detection System technology. This protocol was applied on cytological specimens of patients with cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). We performed direct in situ quantitative PCR on cell smears, uninfected human skin fibroblasts, Hela and Caski cells. After in situ amplification, slides were counterstained with propidium iodide and analyzed under a fluorescent microscope in order to localize high-risk HPV and verify preservation of morphology. After PCR optimization, we obtained the following results. The Hela cells showed values ranging from 15 to 33 copies of high-risk HPV per cell, the Caski cell line from 220 to 300 high-risk HPV copies per cell and the cell smear (both CIN and SCC) around 20-35 copies of high-risk HPV per cell. No high-risk HPV amplification was detected in uninfected human fibroblasts, healthy controls, non-amplification control, and non-specific primer control. A positive intranuclear high-risk HPV amplification was detected in cell smears from 20 patients with CIN and 10 with SCC. In conclusion, our in situ quantitative protocol for high-risk HPV detection on cell smears combines both quantitative data and in situ localization of the target, with preservation of morphology. For this reason it could be used as a rapid screening tool when both morphological and quantitative results are requested on the same slide.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalClinical and Experimental Medicine
Volume2
Issue number1
Publication statusPublished - 2002

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Labeling
Polymerase chain reaction
Cervical Intraepithelial Neoplasia
Amplification
Fibroblasts
Squamous Cell Carcinoma
HeLa Cells
Polymerase Chain Reaction
SYBR Green I
Propidium
Skin
Screening
Microscopes
Cells
Technology
Cell Line
Epithelial Cells

Keywords

  • Cell smears
  • Fluorescent detection
  • High-risk human papillomavirus
  • In situ quantitative PCR

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Clinical Biochemistry

Cite this

Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling. / Crovella, S.; Pirulli, D.; De Santo, D.; De Seta, F.; Boniotto, M.; Braida, L.; Boaretto, F.; Guaschino, S.; Amoroso, A.

In: Clinical and Experimental Medicine, Vol. 2, No. 1, 2002, p. 1-6.

Research output: Contribution to journalArticle

Crovella, S, Pirulli, D, De Santo, D, De Seta, F, Boniotto, M, Braida, L, Boaretto, F, Guaschino, S & Amoroso, A 2002, 'Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling', Clinical and Experimental Medicine, vol. 2, no. 1, pp. 1-6.
Crovella, S. ; Pirulli, D. ; De Santo, D. ; De Seta, F. ; Boniotto, M. ; Braida, L. ; Boaretto, F. ; Guaschino, S. ; Amoroso, A. / Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling. In: Clinical and Experimental Medicine. 2002 ; Vol. 2, No. 1. pp. 1-6.
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AU - Boniotto, M.

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