Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR

Abstract

Escherichia coli, which causes diarrhea in humans, can be classified into the following heterogenous groups: enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC). These diarrheagenic E. coli categories are differentiated on the basis of their infection and pathogenic mechanisms. Serotyping, phenotypic assays, and molecular detection assays are very useful in identifying these diarrheagenic E. coli categories (2, 12).Since several virulence factors and DNA sequences of diarrheagenic E. coli have been identified, their molecular analyses have been performed using genetic detection assays, including PCR and DNA hybridization (5, 21, 26). The PCR method, which is a rapid gene detection assay, is particularly effective in identifying these diarrheagenic E. coli categories. Several PCR methods for detecting the various virulence factors have been reported elsewhere (1,6,22).However, conducting the separate PCR reactions that are required for the detection of the virulence factors in order to assign an isolated E. coli strain to one of the five categories is very laborious and time comsuming. Therefore, various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction (15,18,20,23). Using the multiplex PCR method, we will be able to save the time and effort involved in analyzing various virulence factors. Some multiplex PCR systems have been reported for the rapid detection of specific virulence factors that distinguish EHEC O157 from other serotypes of EHEC (10,11,14,16,17). Cebula et al. (3) reported the multiplex PCR systems for the simultaneous detection of stx1, stx2, and uidA genes, which are specific to EHEC O157:H7. Wang et al. (27) have also reported the combination of certain multiplex PCR systems for the detection of stx1, stx2, stx2 variants, eaeA, EHEC hlyA, and rfbE O157 , which are specific to the O157 serotype, and fliC H7 , which is specific to the flagellum H7 serotype. Pass et al. (15) and Toma et al. (25) reported the use of multiplex PCR systems for the detection of 11 and 6 virulence genes, respectively. However, the systems described by them for simultaneous categorization of E. coli have not been completely effective for the simultaneous categorization of all diarrheagenic E. coli. The multiplex PCR system reported Abstract: A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR ...

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

“…However, these methods alone are not sufficient to identify all five pathotypes of DEC (Brandal et al 2007). The genes encoding virulence factors are extensively studied and characterized, and several PCR methods have been developed to identify the virulence genes of DEC (Kimata et al 2005). Numerous multiplex PCR assays have been developed to identify DEC, and assays were recently described that are capable of distinguishing the five pathotypes of DEC (Aranda et al 2004, Nguyen et al 2005.…”

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

“…Detection assays rely on the amplification of eltA alone, eltA plus either sta1 or sta2, or all three toxin genes. [6][7][8][9][10][11][12][13][14][15][16] The DNA hybridization assay is a culture-dependent method that requires transfer of cultured lactose-positive colonies from MacConkey agar to Whatman filter paper (Fisher Scientific, Waltham, MA) followed by cell lysis and probe hybridization. Probes targeting ST P (sta1), ST H (sta2), and LT (eltA) are then used for ETEC detection 17,18 ; remarkably, many clinical manuscripts reporting the prevalence of ETEC use this less-sensitive DNA hybridization method or use PCR assays that target eltA plus only a single allele of ST. As a result, these studies are likely to have underestimated the prevalence of ETEC infections.…”

Rapid Detection Method for Enteroaggregative <i>Escherichia coli</i> Using Simple Clump Formation and Aggregative Assay

“…Some researchers believe that a typical EAggEC possesses both aggR and pCVD432 genes that are present in the virulence plasmid pAA [17,18]. Kimata et al [19] identified AA was characterized by the prominent autoagglutination of bacterial cells as well as adherence to glass cover slips without HEp-2 cells. The sine qua non of AA, however, was the characteristic layering of the bacteria, best described as a stacked-brick configuration.…”