A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.

Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineag...

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Main Authors: Raul O Cosentino, Fernán Agüero
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS Neglected Tropical Diseases
Online Access:http://europepmc.org/articles/PMC3409129?pdf=render
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spelling doaj-de07c1fa95f1442a91bd45a5850d03042020-11-25T01:43:05ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352012-01-0167e177710.1371/journal.pntd.0001777A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.Raul O CosentinoFernán AgüeroTrypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI-TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.http://europepmc.org/articles/PMC3409129?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Raul O Cosentino
Fernán Agüero
spellingShingle Raul O Cosentino
Fernán Agüero
A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
PLoS Neglected Tropical Diseases
author_facet Raul O Cosentino
Fernán Agüero
author_sort Raul O Cosentino
title A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
title_short A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
title_full A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
title_fullStr A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
title_full_unstemmed A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
title_sort simple strain typing assay for trypanosoma cruzi: discrimination of major evolutionary lineages from a single amplification product.
publisher Public Library of Science (PLoS)
series PLoS Neglected Tropical Diseases
issn 1935-2727
1935-2735
publishDate 2012-01-01
description Trypanosoma cruzi is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the T. cruzi species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of loci.We present a simple typing assay for T. cruzi, based on the amplification of a single polymorphic locus: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI-TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.Based on these findings we propose a simple typing assay for T. cruzi that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- locus sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.
url http://europepmc.org/articles/PMC3409129?pdf=render
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