A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens

  • Negin Nikouyan
  • Ali Farhadi
  • Ali Akbar Gorzin
  • Bita Geramizadeh
  • Mohammad Ali Okhovat
  • Noorossadat Seyyedi
  • Gholamreza Rafiei Dehbidi
  • Reza Ranjbaran
  • Abbas Behzad-BehbahaniEmail author
Clinical Microbiology - Research Paper


Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin–labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.


High-risk human papillomavirus (HR-HPV) FFPE cervical samples Cervical cancer Microplate hybridization Fluorometric assay 



microplate colorimetric hybridization assay


microplate fluorometric hybridization assay


type-specific nested PCR



The authors wish to thank Mr. H. Argasi at the Research Consolation Center (RCC) at Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.

This work is based on a thesis in Virology by Negin Nikouyan (Project No. 93-7242), supported by Shiraz University of Medical Sciences, Shiraz, Iran.

Compliance with ethical standards

All the clinical specimens were obtained from the pathology department archive with local Ethics Committee approval at the Namazi Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. A statement of ethical approval is required to appear before the references for studies involving human or animal subjects.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Negin Nikouyan
    • 1
    • 2
  • Ali Farhadi
    • 2
  • Ali Akbar Gorzin
    • 1
  • Bita Geramizadeh
    • 3
  • Mohammad Ali Okhovat
    • 2
  • Noorossadat Seyyedi
    • 2
  • Gholamreza Rafiei Dehbidi
    • 2
  • Reza Ranjbaran
    • 2
  • Abbas Behzad-Behbahani
    • 2
    Email author
  1. 1.Department of Bacteriology and Virology, School of MedicineShiraz University of Medical SciencesShirazIran
  2. 2.Diagnostic Laboratory Sciences and Technology Research Centre, School of Paramedical SciencesShiraz University of Medical SciencesShirazIran
  3. 3.Organ Transplant Research CentreShiraz University of Medical SciencesShirazIran

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