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Abstract
American Society for Microbiology The 103rd General Meeting(2003.5.18-22)
Rapid Detection of Shiga toxin-producing Escherichia coli by Multiplex LAMP, a New DNA Amplification Method
J. Nemoto, M. Yoshino, Y. Momoda, A. Sunada, T. Annaka, H. Miyajima, Y. Ohta, S. Kohjiya, S. Sadamoto, T. Kojima, M. Ikedo;
Eiken Chemical Co., Ltd., Tochigi, JAPAN.
Background:
Shiga toxin-producing Escherichia coli (STEC) is a major cause of serious outbreaks and sporadic causes of hemorrhagic colitis and hemolytic uremic syndrome. Considering the clinical significance of STEC, rapid, sensitive, and specific detection methods are required. To detect STEC, a new DNA amplification method was developed and evaluated. Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity under isothermal conditions at 65ーC. This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA (1).

Methods:
STEC may produce either Shiga toxin (Stx)1 or Stx2 toxin or both. The two primer sets were designed for Stx1 and Stx2, respectively, and these primer sets were combined for detection of STEC. LAMP was carried out in a reaction mixture containing Bst DNA polymerase large fragment and its appended buffer, primers, dNTPs. The reaction was monitored in realtime on the ABI PRISM 7700 system in the presence of fluorescent intercalating dye. The sensitivity of the assay was examined with series of 10-fold dilutions of Stx1 and Stx2 producing E. coli, respectively. The specificity of the LAMP method was evaluated with 71 STEC and 143 non-STEC strains.

Results:
The detection limit of this method was 60 colony-forming units per reaction. Significant amplification was observed within 40 minutes in all 71 STEC strains, but there was no amplification in 143 of non-STEC strains after 120 minutes incubation.

Conclusion:
The Multiplex-LAMP method is able to detect Shiga toxin-producing E. coli with high specificity, rapidity and simple procedure. This method is very useful to detect STEC from food and clinical specimens.

(1) T. Notomi et al. 2000. Nucleic Acids Research. 28 (12) : E63.

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