A colorimetric IsoPCR for the rapid and sensitive visual detection of high-risk HPV16 in clinical samples with hydroxynaphthol blue

M Daskou; D Tsakogiannis; D S Alexopoulou; T G Dimitriou; D Mossialos; G D Amoutzias; C Kottaridi; P Markoulatos

doi:10.1016/j.jviromet.2021.114072

A colorimetric IsoPCR for the rapid and sensitive visual detection of high-risk HPV16 in clinical samples with hydroxynaphthol blue

J Virol Methods. 2021 Apr:290:114072. doi: 10.1016/j.jviromet.2021.114072. Epub 2021 Jan 21.

Authors

M Daskou¹, D Tsakogiannis², D S Alexopoulou¹, T G Dimitriou¹, D Mossialos¹, G D Amoutzias³, C Kottaridi⁴, P Markoulatos¹

Affiliations

¹ University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Biopolis, 41500, Larissa, Greece.
² University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Biopolis, 41500, Larissa, Greece. Electronic address: ditsakog@uth.gr.
³ Bioinformatics Laboratory, University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Biopolis, Larissa, Greece.
⁴ Second Department of Pathology, Attikon University Hospital, National and Kapodistrian University of Athens, Chaidari, Athens, 12462, Greece; Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, GR, 54124, Greece.

PMID: 33485938
DOI: 10.1016/j.jviromet.2021.114072

Abstract

HPV16 infection is found in more than 50 % of cervical cancer cases worldwide, triggering the development of numerous molecular techniques for viral diagnosis. The present study focuses on the development of a colorimetric IsoPCR for HPV16 DNA detection. The methodology combines the advantages of PCR and LAMP, while the most significant aspect of the new established methodology is the visual detection of amplification products through hydroxynapthol blue dye, thus minimizing the time and labor needed. An experimental cut-off value was tested through reconstitution experiments, while the specificity was evaluated by assessing clinical samples. The analytical sensitivity of the new colorimetric IsoPCR was found to be 0.1 viral DNA copy per reaction, while the specificity was 100 % for the detection of HPV16 DNA. The assay enabled the amplification of viral DNA in cases with viral load lower than 1 copy. In conclusion, the new established colorimetric IsoPCR can be regarded as an attractive molecular tool that facilitates the specific, rapid and highly sensitive visual detection of HPV16 DNA even at the very early stages of viral infection.

Keywords: Cervical cancer; Colorimetric assay; HPV16; Hydroxynapthol blue; IsoPCR.

MeSH terms

Colorimetry*
Human papillomavirus 16* / genetics
Humans
Naphthalenesulfonates
Nucleic Acid Amplification Techniques*
Sensitivity and Specificity

Substances

Naphthalenesulfonates
trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate