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Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae)
OBJECTIVES: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shig...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992677/ https://www.ncbi.nlm.nih.gov/pubmed/29880035 http://dx.doi.org/10.1186/s13104-018-3457-8 |
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author | Anglès d’Auriac, Marc B. Sirevåg, Reidun |
author_facet | Anglès d’Auriac, Marc B. Sirevåg, Reidun |
author_sort | Anglès d’Auriac, Marc B. |
collection | PubMed |
description | OBJECTIVES: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. RESULTS: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-018-3457-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5992677 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-59926772018-06-21 Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) Anglès d’Auriac, Marc B. Sirevåg, Reidun BMC Res Notes Research Note OBJECTIVES: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. RESULTS: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-018-3457-8) contains supplementary material, which is available to authorized users. BioMed Central 2018-06-07 /pmc/articles/PMC5992677/ /pubmed/29880035 http://dx.doi.org/10.1186/s13104-018-3457-8 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Note Anglès d’Auriac, Marc B. Sirevåg, Reidun Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title | Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title_full | Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title_fullStr | Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title_full_unstemmed | Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title_short | Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx(1) and stx(2)) and the Intimin gene (eae) |
title_sort | multiplex pcr for the simultaneous detection of the enterobacterial gene weca, the shiga toxin genes (stx(1) and stx(2)) and the intimin gene (eae) |
topic | Research Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992677/ https://www.ncbi.nlm.nih.gov/pubmed/29880035 http://dx.doi.org/10.1186/s13104-018-3457-8 |
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