Typing of Verotoxins by DNA Colony Hybridization with Poly‐ and Oligonucleotide Probes, a Bead‐Enzyme‐Linked Immunosorbent Assay, and Polymerase Chain Reaction

Abstract

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo-and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Distribution of Virulence Genes Related to Adhesions and Toxins in Shiga Toxin-Producing Escherichia coli Strains Isolated from Healthy Cattle and Diarrheal Patients in Japan

“…The overnight cultures, which were positive for stx genes, were serially diluted with 10 mM phosphate-buffered saline (pH 7.0) (PBS) and 100 l of each dilution was spread on MacConkey agar (Becton Dickinson) plates. The colonies were transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by a replica blotting method and a colony hybridization assay was carried out under highstringent condition (50% formamide in hybridization buffer and 65C incubation for washing) by using 32 P-labeled stx1 or stx2 gene as a probe as described previously [41]. For stx1-and stx2-specific gene probes, an internal 0.9-kb HincII-HpaI fragment of the stx1 gene [41] and an internal 0.86-kb PstI-SmaI fragment of the A subunit of the stx2 gene were used, respectively.…”

“…The colonies were transferred to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by a replica blotting method and a colony hybridization assay was carried out under highstringent condition (50% formamide in hybridization buffer and 65C incubation for washing) by using 32 P-labeled stx1 or stx2 gene as a probe as described previously [41]. For stx1-and stx2-specific gene probes, an internal 0.9-kb HincII-HpaI fragment of the stx1 gene [41] and an internal 0.86-kb PstI-SmaI fragment of the A subunit of the stx2 gene were used, respectively. These fragments were labeled by the random priming method by using Multiprime DNA Labeling System (Amersham Biosciences Corp. Piscataway, NJ, U.S.A.) and [- Total 61 58 2 59 58 11 60 2 1 a) Nonmotile.…”

Genetic Characteristics and Antimicrobial Resistance of Escherichia Coli From Japanese Macaques (Macaca Fuscata) in Rural Japan

“…DNA extracted with a DNeasy TM Tissue Kit (QIAGEN, Hilden, Germany), or boiled enrichment culture was used as templates. Oligonucleotide primers (Table 2) for PCR were as follows: SK1, SK2 for eae (Oswald et al, 2000), Vtcom-u, Vtcomd for stx (Yamasaki et al, 1996), LT L , LT R for elt (Toma et al, 2003), and AL65, AL125c for est (Toma et al, 2003). PCR was performed under the following conditions: initial denaturation for 5 min at 94 C, 30 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C, and a final extension step of 7 min at 72 C. PCR amplicons were electrophoresed on 2% SeaKem GTG agarose gel (Takara, Otsu, Shiga, Japan) in 13 TAE buffer.…”

Molecular characterization and evaluation of antimicrobial susceptibility of enteropathogenic E. coli (EPEC) isolated from minas soft cheese

“…The presence of pathogenic E. coli strains in Minas soft cheese has been reported by several studies (NASCIMENTO; SABIONI; PIMENTA, 1988;PANETO et al, 2007).…”