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United States Department of Agriculture

Agricultural Research Service

Title: Development of Fluorogenic Pcr Assays for Rapid Detection of Virulence Genes of Escherichia Coli O157:h7 and Other Shiga Toxigenic E. Coli (Virulence Mechanisms of Bacterial Pathogens, 9/12-15/99, Ames, Ia)

item Sharma, Vijay
item Nystrom, Evelyn
item Casey, Thomas

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 15, 1999
Publication Date: N/A

Technical Abstract: The genes stx1 and stx2 are important virulence attributes of Shiga toxin-producing E. coli (STEC). Enterohemorrhagic E. coli (EHEC), such as serotype O157:H7, possess additional genes, such as eaeA, mediating attachment to enterocytes. In this report, we describe the development and evaluation of fluorogenic PCR assays to detect these genes in beef and fecal cultures. We amplified 80, 120, and 150 bp regions of stx1, stx2, and eaeA genes using specific primers. The amplified products were detected by fluorogenic probes specific for stx1 and stx2 of STEC and the eaeA of O157:H7. These primers and probes were either used in a multiplex assay or used singly in a nonmultiplex assay. These assays were tested with various serotypes of STEC and other bacterial species lacking stx1, stx2, and eaeA. All serotypes of STEC were tested positive and other bacterial species tested were negative. The nonmultiplex assay identified Shiga toxin genes carried by a STEC and characterized STEC as O157:H7 or non-O157:H7 based on the presence or absence of the O157:H7-specific eaeA gene. These assays were able to detect these genes in bacterial cultures of beef or feces seeded with less than 50 cfu of O157:H7 and grown for 6 h to overnight.

Last Modified: 4/21/2015