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Development of a multiplex PCR targeting eae, stx and cdt genes in genus Escherichia and detection of a novel cdtB gene in Providencia rustigianii.

This study was aimed to develop a multiplex PCR (m-PCR) for the detection of Escherichia coli attaching and effacing (eae), Shiga toxin (stx) and cytolethal distending toxin (cdt) genes encoding important virulence factors of diarrheagenic E. coli such as EPEC, STEC, and Escherichia albertii. For this purpose, the m-PCR was designed to detect eae, all the subtypes of stx (stx1, stx2a-g except stx2f) and cdt (I-V) genes. The m-PCR was validated with 58 and 55 target gene-positive and negative strains of different sources, respectively. Sensitivity and specificity of the m-PCR were 100%. The m-PCR could also detect the eae, stx and cdt genes in bacteria spiked into stool specimens with or without enrichment culture. Clinical specimens collected from children with diarrhea were tested by the m-PCR, and 27 eae and 32 cdt genes were detected. Among them, three cdt-II and one untypable cdt gene-positive bacteria were isolated and identified as E. albertii and Providencia rustigianii, respectively. This is the first report demonstrating the presence of cdtB gene in P. rustigianii. These results indicate that the m-PCR is useful for surveillance of eae, stx and cdt gene-positive bacteria, not only EPEC, STEC and E. albertii but also P. rustigianii.