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:	Monia Ardhaoui, Emna Ennaifer, Anna Christina De Matos Salim, Flávio Marcom Gomez, Thalja Laasili, Med Samir Boubaker, Ikram Guizani
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/70418b75eda44b4d860346046fe87b32
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spelling	oai:doaj.org-article:70418b75eda44b4d860346046fe87b322021-12-02T20:18:22ZNested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.1932-620310.1371/journal.pone.0255914https://doaj.org/article/70418b75eda44b4d860346046fe87b322021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0255914https://doaj.org/toc/1932-6203The 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.Monia ArdhaouiEmna EnnaiferAnna Christina De Matos SalimFlávio Marcom GomezThalja LaasiliMed Samir BoubakerIkram GuizaniPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 8, p e0255914 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Monia Ardhaoui Emna Ennaifer Anna Christina De Matos Salim Flávio Marcom Gomez Thalja Laasili Med Samir Boubaker Ikram Guizani Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.
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.
format	article
author	Monia Ardhaoui Emna Ennaifer Anna Christina De Matos Salim Flávio Marcom Gomez Thalja Laasili Med Samir Boubaker Ikram Guizani
author_facet	Monia Ardhaoui Emna Ennaifer Anna Christina De Matos Salim Flávio Marcom Gomez Thalja Laasili Med Samir Boubaker Ikram Guizani
author_sort	Monia Ardhaoui
title	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_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_sort	nested pcr followed by ngs: validation and application for hpv genotyping of tunisian cervical samples.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/70418b75eda44b4d860346046fe87b32
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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.

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