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Journal of Clinical Microbiology, December 2005, p. 6086-6090, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6086-6090.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Ramification Amplification Assay for Detection of Escherichia coli O157:H7 and Other E. coli Shiga Toxin-Producing Strains

Fan Li,¹ Chunyan Zhao,¹ Wandi Zhang,² Shenghui Cui,³ Jianghong Meng,³ Josephine Wu,² and David Y. Zhang^2*

Department of Pathogenobiology, School of Basic Medical Sciences, Jilin University, Changchun, China,¹ Department of Pathology, Mount Sinai School of Medicine, New York, New York,² Department of Nutrition and Food Science, University of Maryland, College Park, Maryland³

Received 10 February 2005/ Returned for modification 28 April 2005/ Accepted 17 August 2005

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx₂). Our results showed that as few as 10 copies of stx₂ could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx₂ gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.

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* Corresponding author. Mailing address: Molecular Pathology Laboratory, Mount Sinai School of Medicine, Box 1122, One Gustave Levy Place, Box 1122, New York, NY 10029. Phone: (212) 659-8173. Fax: (212) 427-2082. E-mail: David.Zhang{at}mssm.edu.

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Journal of Clinical Microbiology, December 2005, p. 6086-6090, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6086-6090.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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