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Applied and Environmental Microbiology, June 2000, p. 2513-2519, Vol. 66, No. 6
Infectious Diseases and Immunity Program,
School of Public Health, University of California, Berkeley,
California 947201; U.S. Department of
Agriculture, Agricultural Research Service, Albany, California
947102; and Food Animal Health and
Management Center, College of Veterinary Medicine, Kansas State
University, Manhattan, Kansas 665063
Received 22 December 1999/Accepted 17 March 2000
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an
important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using
a method based on restriction site polymorphisms.
Restriction-site-specific PCR (RSS-PCR) involves the amplification of
DNA fragments using primers based on specific restriction enzyme
recognition sequences, without the use of endonucleases, to generate a
set of amplicons that yield "fingerprint" patterns when resolved
electrophoretically on an agarose gel. The method was evaluated in a
blinded study of E. coli isolates obtained from
environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal
antibody test as belonging to O157:H7 serotype. They were retested by
anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent
assay (ELISA). The RSS-PCR method identified all 28 isolates that were
shown to be E. coli O157:H7 by the monoclonal antibody
ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains
as non-O157:H7. The specificity of the RSS-PCR results correlated
better with the monoclonal antibody ELISA than with the polyclonal
antibody latex agglutination tests. The RSS-PCR method may be a useful
test to distinguish E. coli O157:H7 from a large number of
E. coli isolates from environmental samples.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Restriction-Site-Specific PCR as a Rapid Test To
Detect Enterohemorrhagic Escherichia coli O157:H7 Strains
in Environmental Samples
*
Corresponding author. Mailing address: Infectious
Diseases and Immunity Program, School of Public Health, University of
California, 140 Warren Hall, Berkeley, CA 94720. Phone: (510) 642-9200. Fax: (510) 642-6350. E-mail:
lwriley{at}uclink4.berkeley.edu.
Applied and Environmental Microbiology, June 2000, p. 2513-2519, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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