Molecular and Cellular Probes (1999) 13, 291–302

Article No. mcpr.1999.0251, available online at http://www.idealibrary.com on

Semi-automated fluorogenic PCR assays (TaqMan) for

rapid detection of Escherichia coli O157:H7 and other
Shiga toxigenic E. coli

V. K. Sharma,∗ E. A. Dean-Nystrom and T. A. Casey

Enteric Diseases and Food Safety Research Unit, National Animal Disease Center,
USDA, Agricultural Research Service, Ames, Iowa, 50010 USA

(Received 13 March 1999, Accepted 13 May 1999)

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-

O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain
reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence
genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used
for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene
of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were
included in one reaction to simultaneously amplify and detect any of the three targeted virulence
genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected
in independent reactions. The specificity of these assays was evaluated using suspensions of STEC
and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC
harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified
types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-
O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:
H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within
8–10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial
screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1
and stx2 of STEC and eaeA of EHEC O157:H7.

KEYWORDS: STEC, EHEC, haemorrhagic colitis, haemolytic uremic syndrome, intimin, food
safety.

INTRODUCTION the vast majority of outbreaks and sporadic cases of

Enterohaemorrhagic Escherichia coli (EHEC) sero-
types, a subset of Shiga toxin-producing E. coli (STEC),
are predominantly associated with haemorrhagic col-
itis, haemolytic-uremic syndrome and thrombotic
thrombocytopenic purpura in humans.1,2 Although
several EHEC serotypes have been associated with
human infections, EHEC O157:H7 is implicated in
bloody diarrhoea.3–6 The pathogenicity of EHEC O157:
H7 is associated with a number of virulence factors,
including Shiga toxins 1 and 2 (encoded by genes
stx1 and stx2),7–9 and intimin (encoded by the gene
eaeA).10,11 Shiga toxins are believed to play a major role
in the pathogenesis of haemorrhagic colitis and HUS
through cytopathic effect on vascular endothelial cells
of the kidneys, intestines, central nervous system and

Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information
and does not imply recommendation or endorsement by the US Department of Agriculture.
∗ Author to whom all correspondence should be addressed at: USDA, ARS, National Animal Disease Center, PO Box 70, Ames, IA
50010, USA. Tel: +1515 663 7406/7279; Fax: +1515 663 7458; E-mail: vsharma@nadc.ars.usda.gov

0890–8508/99/040291+12 $30.00/0  1999 Academic Press

292 V. K. Sharma et al.
other organs.3,12 Intimin facilitates the adherence to
intestinal villi by the process of attachment and ef-
facement.13 Enterohaemorrhagic E. coli strains which
produce one or more Shiga toxins and intimin are also
considered more virulent for calves than strains that
produce Shiga toxins only.14,15
Cattle are a major reservoir for EHEC O157:H7 and
many other non-O157:H7 STEC.16 Most disease out-
breaks have involved foods of bovine origin, such as
beef and raw milk, that become contaminated with
bovine faeces at slaughter houses or dairy farms.
Methods for automated and high-throughput screening
of foods and faeces of bovine origin to detect the pres-
ence of EHEC O157:H7 and STEC are needed for im-
proving food safety assurance and studying the ecology
and epidemiology of these pathogenic bacteria.
Several methods have been developed for the de-
tection of STEC and EHEC O157:H7. Detection of STEC
can be accomplished either by testing broth culture
supernatants of suspected foods or faeces in Vero
cell cytotoxicity assays7,17 or by enzyme-linked im-
munosorbent assays using anti-Shiga toxin anti-
bodies.18 However, immunological methods are
unable to detect all Shiga toxins with equal efficiency
and some Shiga toxins may not be detected at all be-
cause of the existence of antigenic variants of these
toxins.3,19,20 Moreover, methods that rely on Shiga toxin
detection alone cannot differentiate more virulent
STEC such as EHEC O157:H7 from other less virulent
STEC. Special biochemical media21,22 and diagnostic
kits containing latex reagents directed against O157
and H7 antigens23 have been developed for selective
isolation and specific detection, respectively, of EHEC
O157:H7. These methods, however, require 4–5 days
to complete and are difficult to automate. Genotypic
detection of STEC and EHEC may be accomplished
by DNA colony blot hybridization to identify genes
encoding Shiga toxins and intimin.24 The use of radio-
active isotopes and the time required make this method
unsuitable for many diagnostic laboratories.
Polymerase chain reaction has become a useful
diagnostic tool because it is quick, specific, sensitive
and relatively inexpensive. Several PCR assays have
been developed for the detection of genes stx1 and
stx2.25–27 These assays, however, do not distinguish
EHEC from other STEC. The detection of EHEC by PCR
has been achieved by amplification of the sequences
located in the 5′ two-thirds of the gene eaeA.28 This
region of eaeA gene is highly conserved among EHEC
serotypes. Polymerase chain reaction assays that allow
differentiation of EHEC O157:H7 from other EHEC use
primers that have homology in the 3′ one-third of the
eaeA gene.28,29 The 3′ end of the eaeA gene is less
conserved among EHEC serotypes. Although the use
of these primers in PCR facilitated the detection of all
EHEC O157:H7 strains, these primers also detected
enteropathogenic E. coli (EPEC) strains of serotype
O55:H7 and an EHEC strain of serotype O145:NM.
Most of these methods are based on the use of 18-
h cultures and lengthy DNA isolation protocols, and
require laborious, less sensitive and specific gel-based
analysis of PCR amplified products.
New and improved PCR methods incorporate fluo-
rogenic probes for automated and specific detection
of amplified products. One such system is the TaqMan
PCR detection system developed for detection of Sal-
monella30 spp. and Listeria monocytogenes.31 In Taq-
Man detection assays a fluorogenic oligonucleotide
probe, conjugated to a fluorescent reporter dye at the
5′ end and a fluorescent quencher dye at the 3′ end,
binds to its complement in the region of the target gene
selected for amplification by flanking primers. During
the amplification, the 5′ to 3′ nuclease activity of Taq
DNA polymerase displaces the probe from its com-
plementary sequence and cleaves off the reporter dye.
The fluorescence emission intensity of the free reporter
dye increases because it is no longer quenched by
the proximal quencher dye. The emission intensity is
measured by a fluorometer and the emission data is
analysed by the detection software to provide a ‘+’ or
‘−’ response, indicating amplification or no am-
plification of target genes.
The objectives of the present study were to develop
semi-automated TaqMan-based PCR that could be
used in a multiplex format for specific detection of all
STEC (except STEC that carry the gene stx2e and cause
edema disease in swine32) and in a non-multiplex for-
mat for deducing the Shiga toxin profile of these strains
and for distinguishing EHEC O157:H7 from non-
O157:H7 STEC. We described the effects of different
culture conditions, the duration of growth before ex-
tracting DNA and the presence of non-target bacterial
flora on the sensitivity of fluorogenic PCR assay in de-
tecting EHEC O157:H7 in foods and faeces of bovine
origin. We optimized these assays to obtain re-
producible and efficient amplification and detection of
targeted genes using crude DNA preparations thereby
eliminating the need to use high-purity DNA that could
take additional time to prepare. Our entire assay can
be completed in 8–13 h.
MATERIALS AND METHODS
Bacterial strains, culture media and growth
conditions
The bacterial strains used in this study, relevant char-
acteristics and sources of these strains are listed in
Table 2. Strains were propagated and maintained on
 Fluorogenic PCR assays for STEC and EHEC
Trypticase soy agar (TSA) plates. Liquid cultures were
obtained by growing bacteria in Trypticase soy broth
(TSB) for 18–20 h at 37°C with continuous agitation
(200 rpm). Trypticase soy agar and MacConkey agar
were used to enumerate bacteria. Trypticase soy agar,
TSB and MacConkey agar were purchased from BBL
(Becton Dickson Microbiology Systems, Cockeysville,
MD, USA). Novobiocin (Sigma Chemical Co., St
Louis, MO, USA) was used at 20 lg ml−1 in modified
TSB (mTSB).33
Culture of spiked beef and faecal samples
Ground beef (80% lean) was purchased on three
different occasions from a retail grocery store. The
faecal samples were obtained from cattle that were
housed at the National Animal Disease Center, Ames,
IA, USA. One gram of each sample type was added
to 9 ml of TSB, samples were vortexed for 30 s, and
then 10-fold dilutions of these samples were plated
on TSA and MacConkey plates and incubated for
18 h at 37°C to determine total aerobic and coliform
bacteria. The remainder of the beef- or faeces-broth
mixture was incubated for 18 h at 37°C with shaking.
These samples were centrifuged for 2 min at 1000×g
to remove large particles. Culture supernatant (0·2 ml)
was added to 0·8 ml of TSB and centrifuged at
12,000×g for 3 min. The DNA was isolated (see
below) from pelleted bacteria and it was used as a
template in the multiplex PCR assay containing
primers to amplify 80, 120, 150 bp fragments of
stx1, stx2 and eaeA, respectively. The amplification
products were analysed by electrophoresis through a
4% agarose gel (FMC BioProducts, Rockland, ME,
USA) followed by ethidium bromide staining. The
molecular size of visible bands was estimated from
a DNA-sizing ladder (50 bp ladder, Gibco-BRL). The
beef and faecal samples (1 g portions) determined
negative for the predicted size fragments of stx1, stx2
and eaeA genes by the multiplex PCR assay were
spiked with 0·1 ml of 10-fold dilutions of EHEC O157:
H7 strain 2409 followed by the addition of 8·9 ml of
TSB or mTSB. Some beef and faecal specimens were
also spiked with the non-target E. coli strain 63 to
obtain different ratios of target (EHEC O157:H7 strain
2409) to non-target strains. These spiked samples
were incubated for 0, 2, 4, 6 or 18 h at 37°C. Aliquots
(0·2 ml) from these cultures were diluted 1:5 in TSB,
bacteria were recovered by centrifugation and pro-
cessed for DNA isolation. All centrifugation steps
were performed at 4°C.
293
Isolation of DNA
Pure cultures of EHEC O157:H7 strain 2409, grown
for 18 h at 37°C, were diluted 10-fold and 0·2 ml of
each dilution was centrifuged to pellet bacteria. The
pellets were suspended in 0·2 ml of lysis buffer (10 m
Tris HCl, 1 m Na2 EDTA, pH 8·0 and 0·5% Triton
X-100).34 Bacteria were recovered from 0·2 ml super-
natants of spiked beef and faecal cultures that were
grown as described above. The pellets were sus-
pended in 0·2 ml of InstaMatrix (Bio-Rad Laboratories,
Richmond, CA, USA). The bacterial suspensions were
heated at 100°C for 10 min and then centrifuged at
12,000×g for 5 min. The supernatant containing
DNA was stored at −70°C.
Synthetic oligonucleotide primers and
fluorogenic reporter probes
The published nucleotide sequences of stx1,35 stx236
and eaeA37 were imported into the Primer Express
Software (Perkin-Elmer, Foster City, CA, USA) to de-
sign primer pairs to amplify 80 and 120 bp regions
of the stx1 and stx2 genes, respectively, of STEC and
150 bp fragment of the eaeA gene. This software was
also used in designing fluorogenic reporter probes to
specifically detect amplified sequences of stx1 and
stx2 of STEC and eaeA of EHEC O157:H7. The eaeA
probe was designed to distinguish EHEC O157:H7
from other EHEC serotypes and from bacterial species,
such as Citrobacter freundii and Hafnia alvei, that
may harbour an eaeA-like gene.28,29 However, this
probe was not capable of distinguishing the eaeA
gene of EHEC O157:H7 from the eaeA gene of EPEC
O55:H7 and other related EPEC serotypes because
eaeA genes of these serotypes share extensive nuc-
leotide sequence homology with the eaeA gene of
O157:H7.38 The nucleotide sequence and location of
these primers and probes in the genes of their origin
is shown in Table 1. Fluorogenic probes were gen-
erated by conjugating a reporter dye (FAM or 6-
carboxy-fluorescein) at the 5′ end and a quencher
dye (TAMRA or 6-carboxytetramethyl-rhodamine) at
the 3′ end (Integrated DNA Technologies, Coralville,
IA, USA).
PCR amplification
A PCR master mixture suitable for use in both mul-
tiplex and non-multiplex PCR assays was developed
by testing different concentrations of MgCl2, different
primer and probe ratios and different amounts of
DNA polymerase. Polymerase chain reactions were
294 V. K. Sharma et al.

Table 1. Nucleotide sequence of primers and fluorogenic probes

Primer Sequence (5′ → 3′) Gene Locationb GenBankc

or probea detected within the accession
gene number

VS1 (Forward) CAT AGT GGA ACC TCA CG ACG CAG T stx1 830–854 M16625
VS2 (Reverse) TTT GCC GAA AAC GTA AAG CTT CA stx1 917–886 M16625
VS4 (Forward) GGG CAG TTA TTT TGC TGT GGA stx2 442–462 X07865
VS5 (Reverse) TGT TGC CGT ATT AAC GAA CCC stx2 562–542 X07865
VS8 (Forward) GGC GGA TTA GAC TTC GGC TA eaeA 1896–1916 X60439
VS9 (Reverse) CGT TTT GGC ACT ATT TGC CC eaeA 2047–2028 X60439
VS3 (Probe) TGT GGC AAG AGC GAT GTT ACG GTT TG stx1 856–881 M16625
VS6 (Probe) CTA TCA GGC GCG TTT TGA CCA TCT TCG stx2 481–507 X07865
VS10 (Probe) AAC GCC GAT ACC ATT ACT TAT ACC GCG ACG eaeA 1925–1954 X60439
a
Probes were conjugated at 5′ and 3′ ends with fluorescent dye FAM and TAMRA, respectively.
b
The positions of the oligonucleotides are listed relative to the initiation codon (+1 adenine) of the respective gene.
c
The nucleotide sequences submitted with these accession numbers were used in designing primers and probes for targeted gene
amplification and detection.

performed by adding 5 ll of DNA to 45 ll of PCR
master mixture. The master mixture for multiplex PCR
assay contained 10 m Tris-HCl (pH 8·3), 50 m KCl,
10 m Na2 EDTA, 5 m MgCl2, 0·2 m deoxy-
nucleoside triphosphates, 100 n of stx1, 150 n of
stx2 and 200 n of eaeA primer pairs, 65 n of stx1-
and stx2-specific probes, 45 n of O157:H7-specific
eaeA probe, 60 n of reference dye ROX (carboxy-
X-rhodamine) and 2·5 units of AmpliTaq Gold DNA
polymerase (Perkin-Elmer, Foster City, CA, USA). The
pre-PCR fluorescence intensity of this master mixture
was in the range of 4000 to 4500 fluorescent units.
The non-multiplex PCR assay was performed in three
independent PCR reactions using the same master
mix as was used in the multiplex assay except that
each non-multiplex reaction contained only one
primer pair and the corresponding probe. The pre-
PCR fluorescence intensity of each non-multiplex
assay was in the range of 2000 to 2500 units. The
PCR reactions were dispensed into a 96-well Mi-
croAmp plate and amplified using a PTC-200 thermal
cycler (MJ Research Inc., Watertown, MA, USA).
Samples were heated to 95°C for 10 min to denature
the DNA and activate the AmpliTaq DNA polymerase.
This was followed by 40 cycles of denaturation at
94°C for 20 s, annealing at 60°C for 30 s and poly-
merization at 72°C for 30 s. The final extension was
carried out at 72°C for 5 min followed by cooling of
samples to 4°C.
Fluorogenic detection of amplified fragments
and data interpretation
The PCR-amplified products were detected by per-
forming fluorescence readings on LS-7200 Sequence
Detection System (Perkin-Elmer, Applied Biosystems).
The fluorescence of each sample was measured prior
to (pre-PCR read) and after PCR cycling (post-PCR
read). A series of six no-template controls were in-
cluded in each reaction plate to establish the baseline
emission intensity of the quenched reporter dye. This
baseline intensity measured at 600nm is subtracted
from pre- and post-PCR read for each sample to
yield the normalized pre- and post-PCR fluorescence
intensity. The differential of normalized post- and pre-
PCR values represented the net change in fluor-
escence (DRn). A threshold DRn was established based
on the standard deviation of fluorescence intensity
of six no-template controls at [99·5% confidence
interval. Samples with DRn higher than the threshold
were assigned a ‘+’ score indicating that targeted
gene(s) were amplified. Samples with DRn equal to
or less than the threshold were assigned a ‘−’ score
indicating no detectable amplification of targeted
gene(s) occurred in these samples.
DNA sequence analysis
The nucleotide sequences of 80, 120 and 150 bp
fragments of stx1, stx2 and eaeA, respectively, were
determined from the same E. coli O157:H7 (strain
2409) that was used in spiking beef and faecal
samples. The nucleotide sequence of these amplicons
was determined by Sanger dideoxynucleotide chain
termination method using a Dye Terminator Ther-
mosequenase Kit (Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ, USA). The sequenced samples were
analysed using ABI 377 DNA sequencer (Perkin-
Elmer, Applied Biosystems).
 Fluorogenic PCR assays for STEC and EHEC
1 2 3 4 5 M
200 bp
eae 150 bp
stx2
100 bp
stx1
50 bp
Fig. 1. Specificity of stx1-, stx2- and eaeA-specific
primers to amplify 80, 120 and 150 bp fragments,
respectively. The DNA from Escherichia coli strain 63
that lacks stx1, stx2 and eaeA genes and an EHEC O157:
H7 strain 2409 that has stx1, stx2 and eaeA genes were
used as a template in multiplex and non-multiplex
polymerase chain reaction (PCR) assays and amplified
products were analysed on 4% agarose gels. Lanes: M,
molecular size markers (50 bp ladder; Gibco-BRL); 1,
DNA from E. coli strain 63 amplified with all three
primer pairs in the multiplex PCR assay; 2, DNA from
EHEC O157:H7 strain 2409 amplified with stx1-specific
primer pair; 3, DNA from strain 2409 amplified with
stx2-specific primer pair; 4, DNA from strain 2409
amplified with eaeA-specific primer pair; 5, DNA from
2409 amplified with three primer pairs in multiplex PCR
assay. The relative position in the gel of three predicted
size PCR products is indicated by arrowheads on the left
side of the gel. Arrowheads on the right side of the gel
mark the position of 50, 100, 150 and 200 bp bands of
the molecular size markers.
RESULTS
Primer specificity
The DNA from EHEC O157:H7 strain 2409 containing
stx1, stx2 and eaeA genes or E. coli strain 63 lacking
these three genes was used as a template in multiplex
and non-multiplex PCR assays to determine the ability
of primers, listed in Table 1, to amplify 80, 120 and
150 bp conserved regions of stx1, stx2 and eaeA,
respectively. As shown in Fig. 1, the stx1-specific
primer set amplified an 80-bp fragment, the stx2-
specific primer set amplified a 120-bp fragment, and
295
the eaeA-specific primer set amplified a 150-bp frag-
ment in non-multiplex PCR assays. All three am-
plicons were easily detected even when three primer
sets were combined in the multiplex assay. The amp-
lified products were obtained only from EHEC O157:
H7 strain 2409 and not from E. coli strain 63. The
nucleotide sequence analysis revealed that each amp-
lified fragment matched the predicted sequence amp-
lified by each flanking primer pair (data not shown).
Specificity of fluorogenic multiplex PCR assay
for STEC
The specificity of the fluorogenic multiplex PCR assay
was evaluated by using 26 STEC and 21 non-STEC
strains. When DNA from STEC strains was subjected
to PCR amplification and detection in fluorogenic
multiplex PCR assay, DRn greater than the threshold
was generated for all STEC strains. These samples
were scored positive for amplification by the fluo-
rogenic detection system (Table 2). On the other hand,
all non-STEC isolates, except EPEC O55:NM and
O55:H6, resulted in DRn values less than or equal to
the threshold values and these samples were scored
negative for amplification by the detection system.
Specificity of fluorogenic non-multiplex PCR
assay for stx1 and stx2 of STEC and eaeA of
EHEC O157:H7
We evaluated the ability of the stx1-, stx2- and EHEC
O157:H7-specific eaeA non-multiplex fluorogenic
PCR assays to identify the virulence gene profile of
27 STEC strains. As shown in Table 3, the stx1-specific
PCR detected only those STEC possessing stx1, the
stx2-specific PCR detected STEC that carried the gene
stx2, and the EHEC O157:H7-specific eaeA PCR de-
tected EHEC O157:H7, EHEC O157:NM, and three
of the four EPEC isolates belonging to the serogroup
O55. The cross-reactivity of EHEC O157:H7-specific
eaeA probe with the eaeA gene of EHEC O157:NM
and EPEC O55:H7 has been observed in other PCR
assays.39,40 However, the EHEC O157:H7-specific
probe did not cross react with the eaeA gene of
several other serotypes of EHEC or Hafnia alvei.
Sensitivity limits of fluorogenic PCR assays
The detection limits of fluorogenic multiplex PCR
performed with DNA from 18 h broth cultures of
EHEC O157:H7 strain 2409 were 2×104 cfu ml−1
(Fig. 2). The detection limits of three non-multiplex
296 V. K. Sharma et al.

Table 2. Specificity of fluorogenic multiplex polymerase chain reaction assay for detecting STEC

Bacterial strains tested Strain Serotype Genotypea Origin/ Amplificationb

number Disease

Non-toxigenic Escherichia coli 63 078:H16 Dog −

912 OX3:H11 Pig/normal −
Enterotoxigenic Escherichia coli (ETEC) 431 O101 staP Pig/diarrhoea −
1477 O149:H10 elt estB staP Pig/diarrhoea −
Enteropathogenic Escherichia coli (EPEC) 1861 O119 eaeA Human/diarrhoea −
1987 O111:H2 eaeA Human −
1988 O26:NM eaeA Human −
1989 O55:NM eaeA Human +
5722 O55:H6 eaeA Human +
Shiga toxigenic Escherichia coli (STEC) 5702 O103:H2 stx1 Human/HUS +
3883 O111:NM stx1, eaeA Calf/normal +
5709 O111:NM stx1, stx2, eaeA Human/HUS +
3128 O113 stx1, stx2 Calf/normal +
3244 O119:H16 stx2 Calf/normal +
5705 O121:H19 stx1, stx2 Human/HUS +
5701 O126:H27 stx1 Human/HUS +
1524 O132 eaeA Rabbit/diarrhoea −
2409 O157:H7 stx1, eaeA Food +
2725 O157:H7 stx1, stx2, eaeA HUS +
2871 O157:H7 stx2, eaeA Calf/normal +
3081 O157:H7 stx1, stx2, eaeA Calf/normal +
4700 O157:H7 stx1, stx2, eaeA Calf +
4718 O157:H7 stx1, stx2, eaeA Human/HUS +
5354 O157:H7 stx1, eaeA Calf/normal +
5570 O157:H7 stx2, eaeA Human/diarrhoea +
2799 O157:NM stx2, eaeA Lab +
2873 O157:NM stx1, stx2, eaeA Calf/normal +
3048 O157:NM stx1, stx2, eaeA Calf/normal +
5264 O157:NM stx1, stx2, eaeA Calf/normal +
5981 O157:NM eaeA Calf +
1043 O22 stx1, stx2 Calf/diarrhoea +
5710 O26:H11 stx1, stx2, eaeA Human/HUS +
5708 O45:H2 stx2 Human/HUS +
5699 O91:H21 stx2 Human/HUS +
3861 OX3:H2 stx1, stx2 Calf/normal +
Hafnia alvei c eaeA Unknown −
Citrobacter freundii Unknown −
Enterobacter cloacae Unknown −
Klebsiella pneumoniae Unknown −
Proteus vulgaris Unknown −
Pseudomonas aeruginosa Unknown −
S. cholerae-suis Unknown −
S. dublin Unknown −
S. enteritidis Unknown −
Salmonella typhimurium Unknown −
Staphylococcus aureus Unknown −
Yersinia enterocolitica Unknown −
a
The presence of these genes was determined by colony blot hybridization using DNA probes specific for these genes (unpubl. data).
b
+or −indicates presence or absence, respectively, of amplified products that resulted in DRn greater than threshold in DRn.
c
Harley Moon, Veterinary Medical Research Institute, Iowa State University, Ames, IA, USA.

PCR assays were also 104 cfu ml−1 (data not shown).
Since DNA equivalent to 1/400 of the total sample
was used in each PCR reaction, the actual number
of cfu required to produce a positive signal were
50 for the multiplex PCR and 17–170 for the non-
multiplex PCR assays.
Sensitivity limits of fluorogenic PCR assays to
detect EHEC O157:H7 in beef
The infective dose of EHEC O157:H7 and probably
of other EHEC serotypes is believed to be in the range
of 10 to 100 cfu.41 However, as shown in Fig. 2, a
 Fluorogenic PCR assays for STEC and EHEC 297

Table 3. Specificity of fluorogenic non-multiplex polymerase chain reaction

assays to detect stx1 and stx2 of shiga toxin-producing Escherichia coli
(STEC) and eaeA of EHEC O157:H7

STEC Genotype Detection of

serotype tested

O157:H7-specific eaeA stx1 stx2

O22 stx1, stx2 − + +

O26:H11 stx1, stx2, eaeA − + +
O26:NM eaeA − − −
O45:H2 stx2 − − +
O55:H30 − − −
O55 eaeA + − −
O55:NM eaeA + − −
O55:H6 eaeA + − −
O91:H21 stx2 − − +
O103:H2 stx1 − + −
O111:H2 eaeA − − −
O111:NM stx1, eaeA − + −
O111:NM stx1, stx2, eaeA − + +
O113 stx1, stx2 − + +
O119:H16 staP, stx2 − − +
O121:H19 stx1, stx2 − + +
O126:H27 stx1 − + −
O157:H7 stx1, eaeA + + −
O157:H7 stx1, stx2, eaeA + + +
O157:H7 stx2, eaeA + − +
O157:H7 stx1, stx2, eaeA + + +
O157:H7 stx1, stx2, eaeA + + +
O157:H7 stx1, stx2, eaeA + + +
O157:H7 stx1, eaeA + + −
O157:H7 stx2, eaeA + − +
O157:NM stx2, eaeA + − +
O157:NM stx1, stx2, eaeA + + +
O157:NM stx1, stx2, eaeA + + +
O157:NM eaeA + − −
O157:NM stx1, stx2, eaeA + + +
OX3:H2 stx1, stx2 − + +

minimum of 104 cfu ml−1 of EHEC O157:H7 strain
2409 culture were required to generate a positive
amplification signal in our fluorogenic PCR assays.
In order to improve the sensitivity of the fluorogenic
PCR assay to detect very low numbers of EHEC O157:
H7 (less than 100 cfu) in a food sample, 1-g portions
of ground beef (containing 103 cfu of endogenous
bacteria per gram of beef; these bacterial counts were
determined as described in Materials and Methods)
were spiked with 3×10−3 to 104 cfu of EHEC O157:
H7 strain 2409 and was cultured for 0, 2, 4, 6 or
18 h before isolation of DNA. The isolated DNA was
tested in fluorogenic PCR assays to detect EHEC
O157:H7. Table 4 shows the type of amplification
signal generated for each sample when tested in
the multiplex fluorogenic PCR assay designed for
detection of any STEC or in the non-multiplex fluo-
rogenic PCR assay designed for detection of EHEC
O157:H7-specific eaeA gene. The sensitivity limits of
these assays were 3 cfu g−1 of beef that was cultured
in TSB or mTSB for 6–18 h.
Effect of non-target bacteria on the detection
of EHEC O157:H7 in beef
The fluorogenic PCR assays were able to detect 3 cfu
of EHEC O157:H7 in ground beef, containing very
low numbers of non-target bacteria, that was cultured
in broth for 6 h (Table 4). Next, we determined the
sensitivity of the fluorogenic PCR assays to detect
very low numbers of EHEC O157:H7 in food samples
containing very high numbers of non-target bacteria.
Bacterial DNA was isolated from ground beef, spiked
with different ratios of target (O157:H7) to non-target
(E. coli strain 63) strains, after 6 h of growth in TSB
and was tested in the non-multiplex fluorogenic PCR
298 V. K. Sharma et al.

80

70

60

50
∆Rn

40

30

20

10

0
0 1 2 3 4 5 6 7
0 2 × 10 2 × 10 2 × 10 2 × 10 2 × 10 2 × 10 2 × 10 2 × 10
–1
cfu ml

Fig. 2. Sensitivity limits of the multiplex fluorogenic polymerase chain reaction (PCR) assay. The DNA from 10-fold
dilutions of an overnight culture of EHEC O157:H7 strain 2409 was used in the multiplex fluorogenic PCR assay. The
fluorescence measurement and data analysis were performed as described under Materials and Methods. The
fluorescent values (DRn) were plotted against cfu per ml. The threshold DRn is indicated by the dashed line. The
dilutions with DRn values above the threshold value were scored positive indicating the amplification of targeted genes.
Errors bar indicate the standard deviation of the mean (n=3).

assay designed for detection of EHEC O157:H7-spe-
cific eaeA gene. The sensitivity limits of this assay
were 5·8, 58 and 580 cfu of EHEC O157:H7 in beef
that contained 106, 107 and 108 cfu of the non-target
strain, respectively.
Sensitivity limits of fluorogenic PCR assay to
detect EHEC O157:H7 in bovine faeces
Detection of low numbers of pathogens in faeces is
a challenge because of the presence of large number
of faecal coliforms and other bacteria. We evaluated
the ability of fluorogenic PCR assays to detect very
low numbers of EHEC O157:H7 in bovine faeces
containing different ratios of target to non-target bac-
teria. Faeces, spiked with different ratios of target
(EHEC O157:H7 strain 2409) to non-target (E. coli
strain 63) strains, were cultured for 6 h and DNA
from these samples was tested in the non-multiplex
fluorogenic PCR assay designed for detection of EHEC
O157:H7-specific eaeA gene. The sensitivity limits of
this assay were 1·2, 12 and 1200 cfu of EHEC O157:
H7 per gram of faeces that contained 0, 108 and

Table 4. Sensitivity limits of fluorogenic polymerase chain reaction assays to detect EHEC O157:H7 in beefa

Detection of O157:H7 in beef-broth mixtures that were cultured for

O157:H7 cfu ml−1 in 0h 2h 4h 6h 18 h

beef-broth mixture

TSB m-TSB TSB m-TSB TSB m-TSB TSB m-TSB TSB m-TSB

0 − − − − − − − − − −
3×10−3 − − − − − − − − − −
3×10−2 − − − − − − − − − −
3×10−1 − − − − − − − − − −
3×100 − − − − − − + + + +
3×101 − − − − − − + + + +
3×102 − − − − + + + + + +
3×103 − − + + + + + + + +
3×104 − + + + + + + + + +
a
Endogenous bacterial count in beef before inoculation with O157:H7 was 103 cfu ml−1.
 Fluorogenic PCR assays for STEC and EHEC 299

80
6
1 × 10 E. coli strain 63
70 1 × 107 E.coli strain 63
1 × 108 E.coli strain 63
60

50
∆Rn

40

30

20

10

0
–1 0 1 2 3 4 5
0 5.8 × 10 5.8 × 10 5.8 × 10 5.8 × 10 5.8 × 10 5.8 × 10 5.8 × 10
EHEC O157:H7 cfu g–1 beef

Fig. 3. Sensitivity limits of the fluorogenic polymerase chain reaction (PCR) assay for detecting EHEC O157:H7 in beef
containing different levels of non-target bacteria. One-gram portions of beef were spiked with 106, 107, or 108 cfu of
Escherichia coli strain 63 (non-target strain) and that was followed by the addition of 5·8×10−1 to 5·8×105 cfu of EHEC
O157:H7 strain 2409 (target strain). The DNA isolated from these samples was used in the non-multiplex fluorogenic
PCR assay designed for detecting enterohaemorrhagic E. coli (EHEC) O157:H7-specific eaeA. The fluorescence
measurement and data analysis were performed as described under Materials and Methods. The threshold DRn is
indicated by the dashed line. The dilutions with DRn values above the threshold value were scored positive indicating
the amplification of targeted genes. Errors bar indicate the standard deviation of the mean (n=3).

109 cfu, respectively, of the non-target strain plus
105 cfu of faecal bacteria (Fig. 4).
DISCUSSION
The fluorogenic PCR assays reported in this study
were optimized to provide excellent specificity for
semi-automated detection of EHEC O157:H7 and
other STEC and for determining virulence gene pro-
files of these strains. Although the specificity of these
assays is comparable to numerous other gel-based
PCR assays facilitating the detection of STEC25–27,42
and EHEC O157:H7,28,29,34,43,44 the assays described
in this report offer two unique advantages as com-
pared to other PCR-based assays. First, the fluorogenic
multiplex PCR assay can be used as a screen to test
large number of samples as it allows rapid detection
of STEC carrying any individual or any combination
of stx1, stx2 and eaeA genes. Although this method
does not identify the individual gene or gene com-
bination harboured by the detected STEC (unless
samples are analysed on an agarose gel), this method
provides immediate warning as to the number of
samples that may contain STEC. Second, the three
individual non-multiplex PCR reactions can be used
when identity of Shiga toxin genes harboured by a
STEC needs to be determined, and when it is essential
to characterize STEC as EHEC O157:H7 and non-
O157:H7 STEC.
Several parameters were optimized to enable the
fluorogenic PCR assays to detect EHEC O157:H7 and
other STEC with specificity approaching 100%. First,
we designed novel sets of primers that generated short
PCR products (less than 200 bp as compared to primer
pairs reported and evaluated previously for PCR
amplification of stx1, stx2 and eaeA to detect
STEC25,26,42 and EHEC O157:H728,29,34,43,44 in gel-based
PCR assays) so that PCR cycling could be completed in
a short time, enzyme and other reaction components
would not become limiting factors and poor template
integrity in crude DNA preparations would not com-
promise the yield of amplified products. The ability
of these primers to amplify predicted size fragments
of targeted genes was demonstrated both in multiplex
and in non-multiplex PCR assays. Second, we de-
signed fluorogenic probes that allowed specific de-
tection of EHEC O157:H7 and other STEC. The
fluorogenic probes specific for stx1 and stx2 detected
all STEC (except STEC that harbour stx2e32 were
not detected by the stx2 probe; data not shown)
harbouring these genes and the fluorogenic probe
specific for EHEC O157:H7 eaeA gene detected EHEC
O157:H7 and EHEC O157:NM but not other STEC.
The cross-reactivity of the EHEC O157:H7-specific
eaeA probe with the eaeA gene of EHEC O157:NM
300 V. K. Sharma et al.

80
1 × 105 faecal bacteria (fb)
8
70 fb + 1 × 10 E.coli strain 63
9
fb + 1 × 10 E.coli strain 63
60

50
∆Rn

40

30

20

10

0
0 1 2 3 4 5 6 7
0 1.2 × 10 1.2 × 10 1.2 × 10 1.2 × 10 1.2 × 10 1.2 × 10 1.2 × 10 1.2 × 10
–1
EHEC O157:H7 cfu g faeces

Fig. 4. Sensitivity limits of the fluorogenic polymerase chain reaction (PCR) assay for detecting EHEC O157:H7 in
faeces containing different levels of non-target bacteria. One-gram portions of faeces were first spiked with 0, 108 and
109 cfu of Escherichia coli strain 63 (non-target strain) followed by the addition of 1·2×100 to 1·2×107 cfu of EHEC
O157:H7 strain 2409 (target strain). The DNA isolated from these samples was used in the non-multiplex fluorogenic
PCR assay designed to detect enterohaemorrhagic E. coli (EHEC) O157:H7-specific eaeA. The threshold DRn is indicated
by the dashed line. The dilutions with DRn values above the threshold value were scored positive indicating the
amplification of targeted genes. Errors bar indicate the standard deviation of the mean (n=3).

has been observed in other eaeA-based PCR
assays39,40 and it is considered advantageous because
EHEC O157:NM has been increasingly isolated from
patients with HUS.41 The identification of O157:NM
by O157:H7-specific eaeA probe is plausible in light
of the recent studies showing the presence of the
gene fliC encoding the H7 flagellar antigen in O157:
NM isolates, and suggesting that many O157:NM
isolates are the non-motile variants of O157:H7.45
The EHEC O157:H7-specific eaeA probe also cross-
reacted with EPEC O55, O55:H6 and O55:NM. Sim-
ilar cross-reactivity of the EHEC O157:H7-specific
eaeA probe with the eaeA gene of EPEC O55:H7 has
been observed in other eaeA-based PCR assays.34,39
This cross-reactivity is due to the presence of virtually
identical eaeA genes in these strains,38 and that pre-
cludes the design of primers and probes to suc-
cessfully differentiate O157:H7 from EPEC, especially
O55:H7 and other motile and non-motile members
of the serogroup O55. However, EPEC are not de-
tected by stx1- and stx2-based PCR assays because
these strains lack genes encoding Shiga toxins.46
Moreover, O55:H7 lacks O157:H7-specific rfbE, re-
sponsible for O157-specific O-antigen biosynthesis.47
The gene rfbE has been used as an additional target
gene in PCR assays to distinguish O157:H7 from
O55:H7.45 Thus, new primers and fluorogenic probes
can easily be incorporated into our fluorogenic PCR
assays to distinguish Shiga toxin-negative EPEC be-
longing to the serogroup O55 from EHEC O157:H7.
The fluorogenic PCR assays described in this study
were highly sensitive in detecting EHEC O157:H7 in
beef and faecal cultures containing very high numbers
of non-target bacteria. The detection limit of less
than 10 cfu for EHEC O157:H7 in beef cultures is
equivalent to the detection limits of a few recently
reported fluorogenic PCR assays for detecting STEC
in beef.26,48 Unlike our assays, previously described
assays were unable to distinguish EHEC O157:H7
from other STEC and did not provide specific iden-
tification of Shiga toxin genes. A 5′ nuclease assay
employing a fluorogenic EHEC O157:H7-specific
eaeA probe for the detection of EHEC O157:H7 and
EHEC O157:NM was able to detect 100 cfu after
secondary culture of target cells that were captured
using EHEC O157:H7-specific immunomagnetic
beads from primary beef cultures.39
Another advantage of the fluorogenic PCR assays
described in this study is that they provide automated
and specific detection of amplified products thereby
eliminating the need for agarose gel analysis of amp-
lified samples. Coupling of this automated PCR
amplification detection system with automated pro-
cedures for DNA extraction and liquid transfer will
make these methods suitable for complete automation
and high-throughput screening of food and faecal
samples for rapid detection of EHEC O157:H7 and
other STEC. These methods will be useful in helping
the meat industry satisfy federal regulations, such
as Hazard Analysis and Critical Control Points
 Fluorogenic PCR assays for STEC and EHEC
(HACCP),49 concerning the presence of harmful bac-
teria in foods. These methods will help veterinary
and clinical microbiologists to detect EHEC O157:
H7 and STEC in samples of interest and to monitor
the shedding of these pathogenic strains in cattle.
ACKNOWLEDGEMENTS
The authors thank Drs Steve Carlson and Jay Ellingson for
critical review of this manuscript, Robert Morgan for his
technical assistance and Sandy Johnson for her help in
preparation of this manuscript.
1999 US Government
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