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ARS Home » Plains Area » Bushland, Texas » Conservation and Production Research Laboratory » Livestock Nutrient Management Research » Research » Publications at this Location » Publication #144150

Title: Design and evaluation of PCR primers which differentiate Escherichia coli 0157:H7 and related serotypes

item Rice, William

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2008
Publication Date: 1/1/2009
Citation: Rice, W.C. 2009. Design and evaluation of PCR primers which differentiate Escherichia coli 0157:H7 and related serotypes. Journal of Applied Microbiology. 106:149-160.

Interpretive Summary: A detection method based solely upon DNA analysis using the polymerase chain reaction was developed which allowed for the rapid detection of E. coli O157:H7 serotypes. This method herein referred to as amplicon length polymorphism utilized a number of DNA primers to develop DNA marker patterns characteristic of various E. coli serotypes. Application of these DNA markers to collections of E. coli O157:H7 will allow one to rapidly characterize and assign various unknown environmental isolates of E. coli O157:H7 into distinct groups based upon the DNA backbone contained within that particular strain. This will facilitate the subsequent analysis and characterization of these isolates based on group properties.

Technical Abstract: Methods to detect and differentiate E. coli O157:H7 and related serotypes by amplicon length polymorphism (ALP) analysis were developed based on identifying DNA sequence deletions within highly homologous regions of all three sequenced E coli strains. Potential primer locations along the ancestral genomic backbone were identified and evaluated against these three sequenced genomes and then applied to a reference set of pathogenic E coli strains. All sixteen primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes. Information regarding the genetic composition of the ancestral backbone was obtained using the ALP analysis method developed herein. Thus, this study defines a useful collection of primers distributed along the E coli genome that can be applied to ALP analysis methods to detect E coli O157:H7 and successfully differentiate various E coli serotypes. The principles underlying this method should be able to be applied to other microbial organisms.