Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.

<h4>Background</h4>Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.<h4>Methodology/principal finding...

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Autores principales:	Sueli M Nakatani, Carlos A Santos, Irina N Riediger, Marco A Krieger, Cesar A B Duarte, Marco A Lacerda, Alexander W Biondo, Flair J Carrilho, Suzane K Ono-Nita
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/35fe83d4422742a59f45c986123ff866
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spelling	oai:doaj.org-article:35fe83d4422742a59f45c986123ff8662021-11-25T06:24:35ZDevelopment of hepatitis C virus genotyping by real-time PCR based on the NS5B region.1932-620310.1371/journal.pone.0010150https://doaj.org/article/35fe83d4422742a59f45c986123ff8662010-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20405017/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.<h4>Methodology/principal findings</h4>Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a.<h4>Conclusions/significance</h4>The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.Sueli M NakataniCarlos A SantosIrina N RiedigerMarco A KriegerCesar A B DuarteMarco A LacerdaAlexander W BiondoFlair J CarrilhoSuzane K Ono-NitaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 4, p e10150 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sueli M Nakatani Carlos A Santos Irina N Riediger Marco A Krieger Cesar A B Duarte Marco A Lacerda Alexander W Biondo Flair J Carrilho Suzane K Ono-Nita Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
description	<h4>Background</h4>Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.<h4>Methodology/principal findings</h4>Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a.<h4>Conclusions/significance</h4>The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.
format	article
author	Sueli M Nakatani Carlos A Santos Irina N Riediger Marco A Krieger Cesar A B Duarte Marco A Lacerda Alexander W Biondo Flair J Carrilho Suzane K Ono-Nita
author_facet	Sueli M Nakatani Carlos A Santos Irina N Riediger Marco A Krieger Cesar A B Duarte Marco A Lacerda Alexander W Biondo Flair J Carrilho Suzane K Ono-Nita
author_sort	Sueli M Nakatani
title	Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
title_short	Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
title_full	Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
title_fullStr	Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
title_full_unstemmed	Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.
title_sort	development of hepatitis c virus genotyping by real-time pcr based on the ns5b region.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/35fe83d4422742a59f45c986123ff866
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Development of hepatitis C virus genotyping by real-time PCR based on the NS5B region.

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