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United States Department of Agriculture

Agricultural Research Service

Title: Simultaneous Identification of Salmonella and Enterohemorrhagic Escherichiacoli (Ehec) O157:h7 in Feces and Foods Using An Enrichment Broth Culture Fluorogenic Multiplex Pcr Assay

item Sharma, Vijay
item Carlson, Steven

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 25, 2000
Publication Date: N/A

Technical Abstract: Enteric infections due to foodborne bacterial pathogens account for annual losses of $3.6 billion in the U.S. Since many of these pathogens are present in the intestines of livestock, fecal contamination of meats is the most important source for their transmission to humans. Rapid and accurate diagnostic tests for simultaneous detection of multiple foodborne pathogens, therefore, are needed in industrial, clinical, and veterinary diagnostic laboratories. Herein we describe the development and evaluation of a method that rapidly detects EHEC O157:H7 and all pathogenic serotypes of Salmonella. Foods and feces of cattle were inoculated with these two pathogens and were cultured in a modified-GN broth (6 h or overnight). The DNA was extracted and used in a multiplex fluorogenic PCR assay to amplify 250 bp and 150 bp fragments of sipB/C virulence gene segment of pathogenic Salmonella and the virulence gene eaeA of EHEC O157:H7, respectively. Specific detection of the two amplified fragments was achieved by including fluorogenic reporter oligonucleotides in PCR assays. This assay was found to be 100% specific for both Salmonella and O157:H7 detection. With this method, we detected less than 10 cfu of each pathogen after 6 h or overnight growth in a single enrichment broth.

Last Modified: 4/22/2015
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