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- Title
Multiplex PCR for the simultaneous detection of the Enterobacterial gene <italic>wecA</italic>, the Shiga Toxin genes (<italic>stx</italic><sub>1</sub> and <italic>stx</italic><sub>2</sub>) and the Intimin gene (<italic>eae</italic>).
- Authors
Anglès d'Auriac, Marc B.; Sirevåg, Reidun
- Abstract
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 <italic>E. coli</italic> outer membrane protein (Eae) or intimin, encoded by the <italic>stx</italic> and <italic>eae</italic> 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 <italic>stx</italic> and <italic>eae</italic> variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. Results: The <italic>wecA</italic> gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of <italic>stx</italic> and <italic>eae</italic> were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the <italic>stx</italic> 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.
- Subjects
POLYMERASE chain reaction; VEROCYTOTOXINS; INTIMIN; ENTEROBACTERIACEAE; VIRULENCE of Escherichia coli; MICROBIAL virulence genetics
- Publication
BMC Research Notes, 2018, Vol 11, Issue 1, pN.PAG
- ISSN
1756-0500
- Publication type
Article
- DOI
10.1186/s13104-018-3457-8