Detection of Diarrheagenic Escherichia coli Using a Two‐System Multiplex‐PCR Protocol

Abstract

The protocol is specific for DEC Shigella and is suitable for clinical laboratories.

Impact of Aeromonas and diarrheagenic Escherichia coli screening in patients with diarrhea in Paraná, southern Brazil

“…In addition, serotyping is rarely sufficient to reliably identify a strain as diarrheagenic. In this study, we used multiplex PCR for detection of virulence genes that characterize the DEC pathotypes [11]. The DEC strains ranked third among the enteropathogens recovered, with a frequency of 5.2%, with atypical EPEC being the predominant pathotype among them (Table 1).…”

“…These strains are increasingly being implicated as important causes of gastroenteritis around the world [3]. In Switzerland, a frequency of 8% of DEC was found among children with diarrhea [20], while among Caribbean-Colombian children, a rate of 14.4% was reported [26], and in Brazil, rates from 7.6% to 48% [11,[27][28][29][30] have been reported. In India, a frequency of 52% of DEC was found among children under five years of age with diarrhea [31], and in Ghana, these bacteria were recovered from 77% of children and 38% of adults with diarrhea [32].…”

“…The procedure described in [11] was followed. Briefly, for screening of diarrheagenic E. coli (DEC), the stool samples were inoculated in MacConkey agar.…”

“…After overnight incubation at 36°C, a loopful of the area of confluent growth was resuspended in 500 μL of sterile water and used for DNA extraction by the boiling method. The extracts were centrifuged at 14,000 rpm for two minutes, and 3 μL of the supernatants were used in the two-system multiplex-PCR method developed by Fialho et al [11]. The multiplex-PCR system 1 contains primers for detection of Shiga-toxin producing E. coli (STEC; stx1, stx2),…”

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.…”