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United States Department of Agriculture

Agricultural Research Service

Title: Development and Application of a Real-Time Pcr to Detect and Quantify Enterohemorrhagic Escherichia Coli O157:h7 and Other Shiga Toxin-Producing E. Coli

Author
item Sharma, Vijay

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 24, 2001
Publication Date: N/A

Technical Abstract: Enterohemorrhagic E. coli (EHEC) O157:H7 causes a broad spectrum of diseases in humans, ranging from diarrhea to hemolytic-uremic syndrome. Contaminated undercooked ground beef, fruit juices, vegetables, and water have been implicated in the transmission of this pathogen to humans, resulting in sporadic and large-scale disease outbreaks. Real-time PCR is a rapid, sensitive, and reliable technique to detect and quantify bacterial pathogens. We have developed a real-time PCR assay to detect and quantify EHEC O157:H7 and other Shiga toxin-producing E. coli (STEC) in meats and bovine feces. In this assay, a segment of the gene eae specific to EHEC O157:H7 was amplified along with the DNA segments specific to stx1 and stx2 genes found in EHEC and STEC. The amplified products were detected by incorporating fluorescent dye-labeled gene-specific probes in the PCR reaction. This method correctly detected and distinguished EHEC O157:H7 from STEC carrying various combinations of eae and stx genes. Detection of all three genes was linear for DNA isolated from samples containing 10**2 to 10**7 CFU per ml of a pure culture of EHEC O157:H7. When tested with DNA prepared from beef and feces, inoculated with EHEC O157:H7 and cultured overnight, samples containing low levels (1 to 10**3 CFU per g of beef or feces) of EHEC O157:H7 could be distinguished from samples with higher (>/= 10**4 CFU per g beef or feces) levels. The sensitivity of this assay ranged from 1 to 10 CFU per g of beef or feces. The real-time PCR assay proved to be a highly sensitive test for detection and quantification of low levels of EHEC O157:H7 in biological matrices.

Last Modified: 4/18/2014