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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Escherichia Coli O157:h7 by Multiplex Polymerase Chain Reaction

Authors
item Fratamico, Pina
item Sackitey, Solomon
item Wiedmann Martin, - CORNELL U - FOOD SCI DEPT
item Deng, Ming

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 5, 1995
Publication Date: N/A

Interpretive Summary: Escherichia coli O157:H7 has emerged as a foodborne pathogen of major public health importance. Many current techniques for this pathogen in foods are nonspecific, nonreliable and time-consuming. A method involving simultaneous amplification of three DNA segments (toxin gene, attachment gene and plasmid) possessed by E. coli O157:H7 has been developed. A positive test result for all three DNA segments is obtained only with toxin-producing E. coli O157. This method is very specific, rapid, and approximately 1000-fold more sensitive than most other rapid methods. It can be used for identification and detection of E. coli O157:H7 in food, milk, water and other samples. The new DNA amplification method permits the food industry and regulatory agencies to more readily evaluate food products for the presence of this pathogen and will thus enhance the safety of the food supply.

Technical Abstract: In order to develop a specific PCR assay for Escherichia coli O157:H7, a portion of the 60 MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then developed employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxin I and II and for the 60 MDa plasmid. PCR products of 1087 bp (eaeA), 227/224 bp (SLTI/SLTII) and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC serotype O157.

Last Modified: 8/21/2014