Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology


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pISSN 2288-0585 eISSN 2288-6850

Detection of Shiga Toxin-Producing Escherichia coli by In Situ Hybridization and Sequence Analysis of Stx2

Original article

Annals of Clinical Microbiology (Ann Clin Microbiol) 2000 December Volume 3, Issue 2, pages 94-98.

Detection of Shiga Toxin-Producing Escherichia coli by In Situ Hybridization and Sequence Analysis of Stx2

Eui Chong Kim, M.D.¹,², Dong Young Lee, M.D.¹, Hae Shim Choi, M.S.¹, Se Ik Joo, M.S.¹, Jung Hee Lee, M.S.³, Sang Hyun Kim, M.D.² and Sung Hwan Ban, M.D.³

¹Department of Clinical Pathology, Seoul National University College of Medicine; ²Clinical Research Institute, Seoul National University Hospital, Seoul; ³Department of Pediatrics, Kumi Hospital, Soonchunhyang University College of Medicine, Kumi, Korea


Background: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E. coli including O157 serotype. Sorbitol-MacConkey agar may be useful for the detection of E. coli O157, but is not helpful for the detection of sorbitol-fermenting STEC other than O157. Moreover, some strains of E. coli O157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and sequence analysis of a part of shiga toxin gene was performed.

Methods: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stx1 and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotyping and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser.

Results: An stx1-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stx1 and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the O antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T→C) of 95th nucleotide and amino acid sequence was identical each other.

Conclusions: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stx1– and stx2-specific primers, and in situ hybridization should be performed on PCR-positive specimens for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak. (Korean J Clin Microbiol 2000;3:94-98)


Shiga toxin, Escherichia coli, PCR, in situ hybridization, digoxigenin DNA probe