title: Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling.
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.
authors.affiliation: Dipartimento di Scienze della Riproduzione e dello Sviluppo, Università di Trieste, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Crovella
authors.forename: D
authors.initials: D
authors.lastname: Pirulli
authors.forename: D
authors.initials: D
authors.lastname: De Santo
authors.forename: F
authors.initials: F
authors.lastname: De Seta
authors.forename: M
authors.initials: M
authors.lastname: Boniotto
authors.forename: L
authors.initials: L
authors.lastname: Braida
authors.forename: F
authors.initials: F
authors.lastname: Boaretto
authors.forename: S
authors.initials: S
authors.lastname: Guaschino
authors.forename: A
authors.initials: A
authors.lastname: Amoroso
mesh_terms: D052160:Benzothiazoles; D002294:Carcinoma, Squamous Cell / Q000821:virology*; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009930:Organic Chemicals; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000821:virology*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I