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Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay

High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments withou...

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Autores principales: Reslová, Nikol, Škorpíková, Lucie, Slaný, Michal, Pozio, Edoardo, Kašný, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701189/
https://www.ncbi.nlm.nih.gov/pubmed/29176674
http://dx.doi.org/10.1038/s41598-017-16329-x
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author Reslová, Nikol
Škorpíková, Lucie
Slaný, Michal
Pozio, Edoardo
Kašný, Martin
author_facet Reslová, Nikol
Škorpíková, Lucie
Slaný, Michal
Pozio, Edoardo
Kašný, Martin
author_sort Reslová, Nikol
collection PubMed
description High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites–nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments–cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa.
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spelling pubmed-57011892017-11-30 Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay Reslová, Nikol Škorpíková, Lucie Slaný, Michal Pozio, Edoardo Kašný, Martin Sci Rep Article High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites–nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments–cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa. Nature Publishing Group UK 2017-11-24 /pmc/articles/PMC5701189/ /pubmed/29176674 http://dx.doi.org/10.1038/s41598-017-16329-x Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Reslová, Nikol
Škorpíková, Lucie
Slaný, Michal
Pozio, Edoardo
Kašný, Martin
Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title_full Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title_fullStr Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title_full_unstemmed Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title_short Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay
title_sort fast and reliable differentiation of eight trichinella species using a high resolution melting assay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701189/
https://www.ncbi.nlm.nih.gov/pubmed/29176674
http://dx.doi.org/10.1038/s41598-017-16329-x
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