Development of a One-Step PCR Assay with Nine Primer Pairs for the Detection of Five Diarrheagenic Escherichia coli Types

Abstract

Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 10(1) CFU/ml for EHEC, 5 × 10(3) CFU/ml for ETEC expressing lt and sth, 5 × 10(4) CFU/ml for ETEC expressing stp, 5 × 102 CFU/ml for EPEC, 5 × 10(4) CFU/ml for EAEC, and 5 × 10(2) CFU/ml for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection

“…These genes are: (1) bfpA (bundle-forming pilus) that codifies for pili type IV involved in bacterial clustering and formation of tight microcolonies on tissue culture cells as an adherence pattern 41 and (2) eaeA that codifies for the bacterial adherent in the translocation mechanism of intracellular signals. 42 , 43 In Figure 6 , a positive control (lanes: 2, 6 and 10) of gene amplification directly from E. coli EPEC genomic DNA was included. Lanes 4, 5, 8, 9, 12, and 13 show PCR amplification from biofunctionalized films using standardized conditions after antigen–antibody interactions (detection).…”

Comparative Evaluation of Seegene Allplex Gastrointestinal, Luminex xTAG Gastrointestinal Pathogen Panel, and BD MAX Enteric Assays for Detection of Gastrointestinal Pathogens in Clinical Stool Specimens

“…The DNA templates were subjected to multiplex PCR with specific primers for the detection of the following virulence markers: aggR and sepA for enteroaggregative E coli (EAEC), stx1/2 for STEC, ipaH for enteroinvasive E coli (EIEC)/Shigella spp, eaeA and escV for enteropathogenic E coli (EPEC), elt and estib for enterotoxigenic E coli (ETEC), and InvA for Salmonella spp, along with uidA as internal control as previously described elsewhere. [9][10][11] Xpert C difficile Assay The Xpert C difficile assay (Cepheid) was performed on the Cepheid GeneXpert Dx System. First, a swab was dipped into the stool specimen and it was placed in the sample reagent and capped.…”

First Isolation of a Hybrid Shigatoxigenic and Enterotoxigenic <i>Escherichia coli</i> Strain Harboring the <i>stx2</i> and <i>elt</i> Genes in Korea

“…In addition, the O-antigen of E. coli was examined by an agglutination method, using the O antisera (O1-O181; Universidad de Santiago de Compostela, Lugo, Spain), and the presence of H antigen was confirmed using Denka E. coli Antisera Set 2 (Denka Seiken, Tokyo, Japan). The EC14-3526 strain was characterized by testing for the presence of toxin-encoding genes (stx1, stx2, elt, and st), virulence genes: attachment-effacement gene (eae), hemolysin genes (hly, ehx, and clyA), adhesion-related genes (iha, saa, efa1, tir, bfpA, aggregative adherence regulator gene [aggR], and toxB) and type III secretion genes (espA and ipaH), and colonization factors (CFs) by PCR (3,4). Furthermore, antimicrobial susceptibility was evaluated, and a multilocus sequence typing (MLST) assay was performed.…”