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Jpn. J. Infect. Dis.

, 62, 476-480, 2009

Method

Rapid Diagnostic Method for the Detection of Diarrheagenic


Escherichia coli by Multiplex PCR
Miyuki Fujioka*, Kosuke Kasai, Tomisato Miura, Tatsusuke Sato, and Yoshimitsu Otomo
Division of Medical Life Sciences, Hirosaki University Graduate School of
Health Sciences, Aomori 036-8564, Japan
(Received May 11, 2009. Accepted October 6, 2009)
SUMMARY: Two novel multiplex polymerase chain reaction (mPCR) assays were originally developed for
detecting nine targeted virulence-associated genes of five categorized diarrheagenic Escherichia coli (DEC).
The mPCR assay 1 included five primer sets (stx1, eaeA, invE, STp gene, and astA), and assay 2 included four
primer sets (stx2, aggR, STh gene, and LT gene). The two mPCRs showed 100% specificity in identifying the
reference strains without nonspecific bands, and 51 DEC and 38 astA gene-positive E. coli strains from 683 E.
coli-like isolates. Our mPCR methods showed high sensitivity and specificity for detecting nine virulence genes
of DEC strains. We proved that these methods will contribute to reducing the cost for the reagents of mPCR
reported elsewhere and could, therefore, contribute to the diagnosis of DEC in clinical laboratories.
(16) was used for the detection of 12 virulence genes (stx1,
stx2, eaeA, invE, STh gene, STp gene, astA, esth, bfpA, aggR,
EAF, and CVD432), although the sequences of four primer
sets mentioned in the study were not shown in the paper.
Therefore, this system may not be cost effective at routine
diagnostic laboratories. Toma et al. (14) and Aranda et al.
(17) reported one-shot mPCR, whose primers did not contain
stx1, stx2, STh gene, or STp gene, which were essential for
epidemiological and pathogenical research for DEC infection.
In this study, we selected some primer sets whose sequences
were described previously (18-21) and whose amplicons could
be identified clearly in the appropriate size. However, the ST
gene for ETEC was not detected by developed mPCR combined various primers. Proceeding empirically, we selected the
STp (also referred to as STIa) gene and STh (also referred to
as STIb) gene instead of the ST gene, which harbors both the
STh and STp genes. We ultimately prepared nine target genes
(stx1, stx2, eaeA, invE, aggR, STh gene, STp gene, LT gene,
and astA) that could distinguish five categories of DEC as
follows: stx1, stx2, and eaeA for STEC, eaeA for EPEC, invE
for EIEC, STh gene, STp gene, LT gene, and astA for ETEC,
and aggR and astA for EAggEC, respectively. We have attempted to develop a one-shot mPCR system to identify nine
target genes, but to date no one has succeeded in detecting all
of the target genes by one-shot mPCR. Because of this, we
first tried the develop two mPCR assay systems at one time.
The mPCR assay 1 used five primer sets (stx1, eaeA, invE,
STp gene, and astA), and assay 2 used four primer sets (stx2,
aggR, STh gene, and LT gene). These two assays were performed at a same time in each of two reaction tubes.

INTRODUCTION
Escherichia coli that causes diarrhea in humans is referred
to as the diarrheagenic E. coli (DEC) and has been categorized
into the following six groups: Shiga toxin-producing E. coli
(STEC), enterotoxigenic E. coli (ETEC), enteropathogenic E.
coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative
E. coli (EAggEC), and diffusely adhering E. coli (DAEC)
(1). These groups of DEC are differentiated on the basis of
their virulence mechanisms, such as their specific toxin productions, ability to attach to or invade target cells, etc., and
thus different immunological, infectious, and molecular assays have been developed for the identification of different
groups of DEC (2). However, identification of the six groups
of DEC is very laborious, time consuming, and costly, and
therefore many clinical laboratories routinely perform only
serotyping assays for the identification, which is sometimes
very useful for the detection of STEC of the serotype O157,
O26, and so on (3).
In recent years, the genes related to the pathogenicity of
the DEC, except for DAEC, have been identified (1), which
helped in the development of different molecular detection
assays like DNA hybridization (4), real-time polymerase chain
reaction (PCR) (5,6), loop-mediated isothermal amplification
(LAMP) (7), the DNA microarray method (8), and multiplex
PCR (mPCR) (9). Among these techniques, the mPCR assay
is one of the most useful routine diagnostic methods, and
some mPCR systems have already been reported for the identification of several categories of DEC (10-13), or for all the
five categories for which it was intended (14-17). Aranda et
al. (15) reported two mPCR assays to identify eight target
genes (eae, bfpA, CVD432, LT gene, ST gene, ipaH, stx1,
and stx2) except aggR and astA, coding for the transcriptional
activator of adherence fimbriae and the enterotoxin, respectively. The one-shot mPCR assay reported by Kimata et al.

MATERIALS AND METHODS


Bacterial strains and template DNA preparation: A total of seven control strains were used in this study (Table 1).
The control strains were grown on Kajiton agar (Eiken Chemical Co., Tokyo, Japan) at 37C for 20 2 h and stored at 4C
until use. For the extraction of DNA, each control strain was
grown in 3 ml of Luria-Bertani (LB)-broth with shaking at
37C, overnight. A 100-l portion of bacterial culture was

*Corresponding author: Mailing address: Division of Medical Life


Sciences, Hirosaki University Graduate School of Health Sciences,
66-1 Honcho, Hirosaki, Aomori 036-8564, Japan. Tel: +81-17239-5971, Fax: +81-172-39-5972, E-mail: mfujioka@cc.hirosakiu.ac.jp
476

suspended in 900 l of Tris-EDTA (TE) buffer, boiled for 5


min, and centrifuged at 10,000 g for 5 min. The supernatant was then used as the pathogenic gene-positive template
for PCR.
Primers and PCR amplification: PCR primers are listed
in Table 2. The oligonucleotides were synthesized at Sigma
Genosys Japan (Ishikari, Japan). Pathogenic genes of the control strains described above were confirmed with single primer
sets of PCR. To develop the mPCR, we tested the progressive incorporation of primers corresponding to the different
genes and several combinations of melting temperatures (Tm)
and primer concentrations. Several variants of stx1, stx2, and
eae genes have been reported elsewhere (22-32). In this
study, our primer sets of stx1, stx2, and eaeA can theoretically
detect each variant of the following: stx1OX3 and stx1CB by
stx1 (but stx1c and stx1d are undetectable) (22-26); stx2dO111, stx2d-oxa, stx2d-ount, stx2d-NV206, activatable stx2d,
stx2c (vt2c), stx2e, and stx2g by stx2 (but stx2f and stx2va are
undetectable) (24,27-29); eae-, eae-, eae-, eae-, eae, eae-, and eae- by eaeA (but eae-, eae-, and eae are undetectable) (30-32).
The mPCR assays were performed as follows: each 25 l
of reaction mixture contained 20 mM Tris-HCl (pH 8.0), 100
mM KCl, 1.5 - 2.0 mM MgCl2, 2.5 mM of deoxynucleoside
triphosphates, 1.25 U of Taq DNA polymerase (TaKaRa Bio

Inc., Shiga, Japan and Nippon Gene Co., Tokyo, Japan), 25 50 M of each primer, and 2.5 l of the DNA template. These
samples, which had been preheated at 94C for 1 min, were
amplified for 25 cycles using a thermal cycler (TaKaRa PCR
Thermal cycler MP [TaKaRa], and BioRAD i-cycler [BioRad, Hercules, Calif., USA]). Each cycle consisted of denaturation at 94C for 1 min, annealing at 55C for 1 min, and
extension at 72C for 1 min. A final extension step was performed at 72C for 10 min. The PCR products were separated
by electrophoresis (100 V, 40 min; Mupid-21; Cosmo Bio
Ltd., Tokyo, Japan) in 3% agarose gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and stained with ethidium
bromide.
Applying mPCR assays to clinical isolates of E. coli:
To demonstrate the diagnostic usefulness of the two mPCR
assay systems, we examined stool specimens collected from
outpatients attending different private hospitals around
Hirosaki, Aomori, and Sendai cities in Japan, between February 2005 and November 2006. These stool specimens were
examined for the presence of Salmonella enterica, Shigella,
Yersinia, Vibrio, Campylobacter, and E. coli following standard identification methods. A total of 683 E. coli-like isolates
were pure cultured on 5% sheep-blood agar plates (Becton
Dickinson Co., Sparks, Md., USA) and serotyped with O antisera purchased from Denka Seiken. They were then identified
by the Enterotube II (Becton Dickinson). All these strains
were grown on Kajiton agars that were preserved at 4C
before the mPCR assays were performed, as described above.

Table 1. Control strains of DEC


Strain

Description

Target gene

E76
E32
E31
E-O169
E1231
E33
E1407

STEC
ETEC
ETEC
ETEC
EPEC
EIEC
EAggEC

stx1, stx2, eaeA


STh gene, astA
STh gene, LT gene, astA
STp gene, astA
eaeA
invE
aggR, astA

RESULTS
The control strains were analyzed by each single primer
set of PCR to detect the virulence genes (stx1, stx2, eaeA,
invE, aggR, LT gene, ST gene, and astA). However, the ST
gene was detected as a weak band (data not shown). At first,
we tried simultaneously the eight primer sets of PCR, but
only a few genes were detected. Next we devided these primer
sets into a group comprising primers for stx1, eaeA, LT gene,
and astA and a group comprising primers for stx2, invE, aggR,

DEC, diarrheagenic Escherichia coli; STEC, shiga toxinproducing E. coli; ETEC, enterotoxigenic E. coli; EPEC,
enteropathogenic E. coli; EIEC, enteroinvasive E. coli;
EAggEC, enteroaggregative E. coli.

Table 2. PCR primers used in this study


Target
gene
stx1
stx2
eaeA
invE
aggR
ST gene
STp gene
STh gene
LT gene
astA

Sequence (5 to 3)
AGTTAATGTGGTGGCGAA
GACTCTTCCATCTGCCG
TTCGGTATCCTATTCCCG
TCTCTGGTCATTGTATTA
AAACAGGTGAAACTGTTGCC
CTCTGCAGATTAACCTCTGC
ATATCTCTATTTCCAATCGCGT
GATGGCGAGAAATTATATCCCG
GTATACACAAAAGAAGGAAGC
ACAGAATCGTCAGCATCAGC
TTTATTTCTGTATTGTCTTT
ATTACAACACAGTTCACAG
TCTGTATTATCTTTCCCCTC
ATAACATCCAGCACAGGC
CCCTCAGGATGCTAAACCAG
TTAATAGCACCCGGTACAAGC
AGCAGGTTTCCCACCGGATCACCA
GTGCTCAGATTCTGGGTCTC
GCCATCAACACAGTATATCC
GAGTGACGGCTTTGTAGTCC

477

Amplicon
size (bp)

Concentration of
primers (M)

Reference

817

25

18

474

25

18

454

25

19

382

25

10

254

25

20

171

50

10

186

50

21

166

25

21

130

25

10

106

25

19

and the ST gene. As a result, the target genes (stx1, stx2, eaeA,
invE, aggR, LT gene, and astA) were detected as clear bands.
However, the ST gene was either not detected or was detected only with the single PCR assay, but not with mPCR.
Therefore, the ST gene primer was divided into the STp gene
and STh gene. The completed mPCR was comprised of two
combinations: one group with stx1, eaeA, invE, STp gene,
and astA, and the other group with stx2, aggR, STh gene, and
LT gene (Fig. 1).
All the 683 clinical E. coli-like isolates were examined by
mPCR, and 51 DEC and 38 astA gene-positive E. coli strains
(Table 3) were detected (EPEC, 21; EAggEC, 15; ETEC, 9;
STEC, 6; and astA positive strains, 38). Five (eaeA, three;
aggR, four) of these DEC strains were O antigen untypable
(OUT) strains. In addition, we analyzed all these 683 E. coli
strains for confirmation tests with individual nine-kind PCR
with each primer set, and the results were identical to those

obtained by mPCR assays.


Specificity and sensitivity: Nine target genes of seven control strains were detected by two developed mPCRs. The
specificity of our two mPCR assays was 100%, and the
sensitivity was 104 CFU/mL for all strains.
DISCUSSION
Suspected DEC strains from diarrheal patients are usually
assayed by a single PCR. However, conducting a single PCR
required for the detection of the virulence factors to isolate
E. coli strains in one of five categories is very laborious and
time consuming. In this study, the nine kinds of primer sets
were divided into a group comprised of the primers for stx1,
eaeA, invE, STp gene, and astA, and a group comprised of the
primers for stx2, aggR, STh gene, and LT gene. We found that
using these two primer sets saved time and effort in analyzing various virulence factors of E. coli isolates.
Typical EPECs may possess adherence factors of bundleforming pilus (bfpA) and EPEC adherence factor (EAF) in
addition to the eaeA (E. coli attaching and effacing lesion).
However, neither bfpA nor EAF are steady, and in this study
our target for only eaeA detection was stable. As a result, the
strains containing the eaeA gene were confirmed by seven
isolates and one STEC serovar O157 conserving both stx1
and stx2.
Some researchers believe that a typical EAggEC possesses
both CVD432 and aggR that are present in the virulence plasmid pAA. However, Kimata et al. (16) showed a few
CVD432-positive and aggR-negative strains, and vice versa.
These findings eliminate the difficulty for the strict evaluation of virulence for EAggEC by PCR only. Therefore, we

Fig. 1. The results of control DEC by mPCR. Lane 1, E76 + E33 + EO169; 2, E76; 3, E33; 4, E-O169; 5, DNA molecular size markers
(100-bp ladder); 6, E76 + E1407 + E31; 7, E76; 8, E1407; 9, E31.

Table 3. Pathogenic factors of DEC isolated in this study


Serotype
O1
O8
O15
O16
O18
O20
O25
O26
O44
O55
O78
O103
O111
O114
O119
O125
O126
O128
O142
O145
O153
O157
O158
O159
O164
O166
O167
O169
OUT
Total

stx1 + stx2
+ eaeA

stx1 + eaeA

No. of the strains harboring DEC-related genes


STh gene
eaeA + astA eaeA aggR + astA aggR
+ astA

STp gene
+ astA

LT gene

astA
5
3
1

1
1
1
4

3
2
5

3
1
4
1

1
1
1
3
1

1
2
2

4
2

1
3
1
1

1
3
2
2

1
1
1

1
2
1
4
3
2

20

OUT, O antigen untypable.

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11

38

and Childrens Center, for supplying the reference DEC strains.


This work was partly supported by Grant-in-Aid for Scientific Research
from the Japanese Association of Medical Technologists, Japan.

used only aggR for detecting pAA to avoid the complication


of PCR methods. Consequently, two clinical isolates harboring aggR were also isolated and confirmed as possessing
CVD432 (data not shown).
On the other hand, three of the reference ETEC strains and
all of the STh gene-positive isolated strains possessed the
astA gene. Savarino et al. (33) established that astA encodes
the EAggEC heat-stable enterotoxin (EAST-1), but astA is
widely distributed in DEC and the pathogenicity of astA is
non-obvious. However, Yatsuyanagi et al. (34) reported that
astA was associated with a waterborne outbreak of diarrhea
in Japan. In this study, nine clinical strains contained astA,
and three of them contained the STh gene. Further study
should be conducted to evaluate the significance of EAST1
as a virulence factor of DEC.
No relationship was found between the serogroups and the
inclusion of pathogenic genes in the detected clinical DEC
strains. Therefore, detecting DEC only by serotyping in many
clinical laboratories may be incomplete. Recently, many
serotypes of STEC, other than O157 and O26, have been
reported. However, Eklund et al. (35) reported that serotyping
is effective only for particular serotypes. Therefore, further
examination is necessary to confirm the relationships between
the serotype and the pathogens of DEC.
Toma et al. (14) developed a one-shot mPCR for detecting
eight genes of five categories of DEC. However, the STp
genes band as the ST gene was not detected, and the gene
showed many extra bands when we used Tomas method (14).
Their method required twice as much Taq DNA polymerase
compared with our mPCR method, which used 1.25 U. The
STp gene has been detected in strains originating from both
pigs and humans, but the STh gene was detected in strains
isolated from humans only. It seems very important for the
epidemiological assay of outbreaks to distinguish the STh
gene from the STp gene. The specificity of our method was
100%, and the sensitivity of our assay was as high as that of
Tomas assay, except for sensitivity to the STp gene. Kimata
et al. (16) also developed a one-shot mPCR for detecting 12
virulence genes of five categories of DEC and showed the
usefulness of the assay to identify the 208 strains isolated
clinically. However, five of nine primer pairs in their method
were not publicly available, and that method showed some
extra bands in the 400- to 700-bp range for mPCR profiles.
Furthermore, their method required the use of three times as
much Taq DNA polymerase, 3.75 U, as our method. In our
two developed mPCR assays, all of the nine primer pairs
were synthesized cheaply because those had been previously
described elsewhere. Also, some amplicons derived from
mPCR were observed faintly, but they could be detected correctly because there were no extra bands.
The two novel mPCR assays we developed proved useful
because with them one could easily detect nine virulence
genes of five categorized DEC strains. The use of this method
will contribute to reducing the cost for the reagents of mPCR
reported elsewhere so far. Therefore, accumulating data by
these developed mPCR assays would assist clinical laboratories in the diagnosis of DEC.
Recently, several stx1, stx2, and eae variants have been
reported (22-32), so we must develop ways to detect these
variants.

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ACKNOWLEDGMENTS
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