Detection Of Echerichia Coli O157:H7 By Multiplex Pcr

Detection of Escherichia coli O157:H7 by
multiplex PCR.
P M Fratamico, S K Sackitey, M Wiedmann and M Y Deng
J. Clin. Microbiol. 1995, 33(8):2188.

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1995, p. 2188–2191
0095-1137/95/$04.00 0
Copyright 1995, American Society for Microbiology

Vol. 33, No. 8

Detection ofEscherichia coli O157:H7 by Multiplex PCR
PINA M. FRATAMICO,1* SOLOMON K. SACKITEY,1 MARTIN WIEDMANN,2

AND

MING YI DENG1

Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia,
Pennsylvania 19118,1 and Department of Food Science, Cornell University, Ithaca, New York 148532
Received 10 March 1995/Returned for modification 11 April1995/Accepted 3 May 1995

mid of E. coli O157:H7 strain (933) was kindly provided by
James Nataro (Center for Vaccine Development, University of
Maryland School of Medicine, Baltimore) (14). pCVD419 was
digested with HindIII, and the isolated 3.4-kb fragment was
then digested with EcoRI, yielding fragments of approximately
1.4 and 1.9 kb in size. The 1.4-kb fragment was subcloned into
M13mp19 with an M13 cloning kit (Boehringer Mannheim
Corporation, Indianapolis, Ind.) according to the manufacturer’s instructions. A portion of the 1.4-kb fragment was then
sequenced with the Sequenase version 2.0 kit (United States
Biochemical Corporation, Cleveland, Ohio). Two PCR primers (Table 1) were designed to specifically amplify a 166-bp
amplicon within the 60-MDa plasmid sequence. Agene bank
search using FASTA (18) revealed a 63.4% homology between
the sequenced fragment and a 435-bp fragment of the chromosomal E. coli hlyA gene (4). Our results are in agreement
with Schmidt et al. (20) who recently reported that the large
plasmid (ca. 60 MDa) of E. coli O157:H7 EDL933 is associated
with hemolytic activity and that the gene encoding this hemolysin shared ca. 70% homologywith the hlyA gene present in
other pathogenic E. coli serotypes.
For PCR amplification of the plasmid fragment, a bacterial
colony was transferred from nutrient agar (Difco Laboratories,
Detroit, Mich.) to 200 l of a solution consisting of 0.5%
Triton X-100, 20 mM Tris (pH 8.0), and 2 mM EDTA, and the
bacterial suspensions were heated at 100 C for 10 min. The
PCR reaction (total volume of100 l) consisted of 5 to 10 l
of the crude cell lysates, 1.5 mM MgCl2, 20 mM Tris (pH 8.0),
50 mM KCl, 0.001% gelatin, 200 M each of the four deoxynucleoside triphosphates, 2.5 U of Taq DNA polymerase
(Gibco/BRL, Gaithersburg, Md.), and 50 pmol of each primer.
PCRs were performed in a thermal cycler (MJ Research, Inc.,
Watertown, Mass.) using the following cycling conditions: an
initialdenaturation at 94 C for 5 min and 35 cycles, with 1 cycle
consisting of 30 s at 94 C, 30 s at 55 C, and 90 s at 72 C. The
amplified product was visualized following ethidium bromide
staining of agarose (1.6%) gels. To confirm the identity of the
166-bp PCR product, amplified DNA was analyzed following
gel electrophoresis by Southern blotting (19) using a 3 -end-labeled (digoxigenin-11-ddUTP)internal oligonucleotide probe
(PS28) (Genius 5 kit, Boehringer Mannheim location), 5 -CCGT
ATCTTATAATAAGACGGATGTTGG-3 . A 166-bp hybridization signal was visible with all of the strains in which the amplification product was detectable on agarose gels (data not
shown).
To determine the sensitivity of the PCR, serial 10-fold dilutions in 1% peptone were prepared from a 4-h culture of E. coli...