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United States Department of Agriculture

Agricultural Research Service

Title: Semi-Automated Fluorescent (Taqman) Pcr Assays for Rapid Detection of Escherichia Coli O157:h7 and Other Shiga Toxigenic E. Coli (Stec)

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

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: June 3, 1999
Publication Date: N/A

Technical Abstract: Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) have emerged in recent years as important foodborne pathogens. Methods for high-throughput screening of foods contaminated with these bacteria are constantly being sought. In this report, we describe the development and evaluation of a TaqMan-based PCR assays to detect STEC and O157:H7. Three primer pairs were designed to amplify 80, 120, and 150 bp regions of stx1, stx2, and eaeA genes, respectively. To detect amplified products, we designed fluorogenic oligonucleotide probes capable of binding to the amplified regions of stx1 and stx2 genes of STEC and the eaeA gene of O157:H7. These primer pairs and probes were either combined for use in a multiplex assay or a single primer pair and the corresponding probe were used in a nonmultiplex assay. These assays were tested with suspensions of various serotypes of STEC and other bacterial species lacking stx1, stx2, eaeA. All serotypes of STEC we tested were positive, whereas other bacterial species tested were negative. The nonmultiplex assay also facilitated the determination of types of Shiga toxin genes carried by a STEC and characterization of STEC as O157:H7 or non-O157:H7. When ground beef or feces were spiked with an O157:H7 strain and cultured overnight, both assays could detect inoculated strain at 3 to 30 CFU per g of beef and 1 to 10 CFU per g of feces. Since 96 samples can be screened in 15 minutes to detect amplified products, these assays offer the potential for high-throughput screening of large number of samples.

Last Modified: 4/22/2015
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