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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV geno...

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Autores principales: Ardhaoui, Monia, Ennaifer, Emna, De Matos Salim, Anna Christina, Gomez, Flávio Marcom, Laasili, Thalja, Boubaker, Med Samir, Guizani, Ikram
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357094/
https://www.ncbi.nlm.nih.gov/pubmed/34379683
http://dx.doi.org/10.1371/journal.pone.0255914
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author Ardhaoui, Monia
Ennaifer, Emna
De Matos Salim, Anna Christina
Gomez, Flávio Marcom
Laasili, Thalja
Boubaker, Med Samir
Guizani, Ikram
author_facet Ardhaoui, Monia
Ennaifer, Emna
De Matos Salim, Anna Christina
Gomez, Flávio Marcom
Laasili, Thalja
Boubaker, Med Samir
Guizani, Ikram
author_sort Ardhaoui, Monia
collection PubMed
description The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.
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spelling pubmed-83570942021-08-12 Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples Ardhaoui, Monia Ennaifer, Emna De Matos Salim, Anna Christina Gomez, Flávio Marcom Laasili, Thalja Boubaker, Med Samir Guizani, Ikram PLoS One Research Article The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies. Public Library of Science 2021-08-11 /pmc/articles/PMC8357094/ /pubmed/34379683 http://dx.doi.org/10.1371/journal.pone.0255914 Text en © 2021 Ardhaoui et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Ardhaoui, Monia
Ennaifer, Emna
De Matos Salim, Anna Christina
Gomez, Flávio Marcom
Laasili, Thalja
Boubaker, Med Samir
Guizani, Ikram
Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title_full Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title_fullStr Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title_full_unstemmed Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title_short Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples
title_sort nested pcr followed by ngs: validation and application for hpv genotyping of tunisian cervical samples
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357094/
https://www.ncbi.nlm.nih.gov/pubmed/34379683
http://dx.doi.org/10.1371/journal.pone.0255914
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