Biocontrol Science, 2013, Vol. 18, No.

4, 227−232

Note

Detection of Emetic Bacillus cereus by Real-Time PCR in Foods

SHIGEKO UEDA＊, MANAMI YAMAGUCHI,
MIKI IWASE, AND YOSHIHIRO KUWABARA

Laboratory of Hygiene, Kagawa Nutrition University, 3-9-1, Chiyoda, Sakado-shi, Saitama 350-0214, Japan

Received 27 March, 2013/Accepted 8 May, 2013

 The simplex real-time PCR assays based on the TaqMan probe and SYBR green I，which
target cereulide synthetase genes（ces genes）were used for rapidly, reliably and sensitively
identifying the emetic strains from among Bacillus cereus strains isolated from different
sources. Only the emetic strains showed positive reactions to the real-time PCR assays, but all
examined strains of diarrheal B. cereus, other Bacillus species and other gram positive and
negative bacteria gave negative results. The final identification of emetic B. cereus was possible
within 1 to 1.5 h in both simplex real-time PCR procedures. The detection limit of emetic strains
in food was suggested to be 104 – 105 cfu/g. Both simplex real-time PCR assays were found to
be rapid, sensitive and reliable diagnostic tools that complement the different cellular, immuno-
logical and chemical detection methods for cereulide- producing B. cereus, even if a relatively
expensive device is required for the assays.

Key words：Bacillus cereus / Emetic toxin（cereulide）/ Real-time PCR / TaqMan-probe / SYBR green I.

 While Bacillus cereus is ubiquitous in the soil and systems have been developed for the identification of
outdoor air, the bacterium is well-known as the emetic strains and the detection of the toxin gene
etiological agent of foodborne illnesses, characterized （ Ehling-Schultz et al., 2004, 2005; Altayar and
by abdominal cramps and diarrhea 8 – 16 h（the Surtherland, 2006）. However, the conventional PCR
diarrheal type）, and vomiting 1 – 5 h after ingestion of requires post-PCR analysis by gel electrophoresis and
the contaminated food（the emetic type） （Gilbert and visualization steps, which take valuable time and may
Kramer, 1984; Schoeni and Wong, 2005）. The diarrheal bear the risk of giving false-positive results due to
type of disease is caused by either a single protein, laboratory contamination. In contrast to conventional
cytotoxin K1（Lund, et al., 2000）or two enterotoxin PCR, real-time PCR is highly specific and sensitive,
complexes, hemolysin BL（Beecher et al., 1995）and reduces significantly the risk of cross-contamination,
the non-hemolytic enterotoxin（Lund, et al., 1995）. On and makes possible high-throughput performance and
the other hand, the emetic syndrome is caused by a easy automation（Mackay, 2004）. Different real-time
cyclic peptide, cereulide（Agata et al., 1995）which is PCR procedures utilizing fluorescent probes and dyes
encoded by nonribosomal peptide synthetase genes intercalating with double stranded DNA have been
（ces genes） （Ehling-Schulz et al., 2005）. In addition to developed（Arya et al., 2005）. Furthermore, Fricker et
cellular and immunological tests（Ueda et al., 2011）for al.（2007）used the biosynthetic code for the ribosomal
the detection of emetic B. cereus, the identification and synthesis of cereulide（Ehling-Schulz et al., 2005;
quantification of cereulide has been attempted by using Magarvey et al., 2006）to set the TaqMan primers and
the chemical methods such as HPLC-MS（Biesta- probes as well as the SYBR green I primers for the real-
Peters et al., 2010; Ueda et al., 2012）. Furthermore, time PCR assays. The present study was performed to
the molecular assays based on conventional PCR evaluate the efficiency of a TaqMan real-time PCR assay
and a SYBR green I real-time PCR assay for the rapid
＊
Corresponding author. Tel:+81-49-282-7343, Fax:+81-49- and reliable detection of emetic strains from among
282-3717, E-mail:ueda（a）eiyo.ac.jp different types of B. cereus isolated from food materials.
 228 S. UEDA ET AL.

 All strains tested were grown on Brain Heart TaqMan real-time PCR assay of DNAs from B. cereus
Infusion （BHI） agar （BBL） over night at 35℃, are shown in Fig.2. The representative emetic strain
followed by incubation of one colony in 5 ml of BHI type（B. cereus strain 13）illustrated an amplified
broth（BBL）at 35℃ for 24 h. After a 10 μl aliquot of pattern, but the diarrheal strain（B. cereus strain 48）
the BHI culture was inoculated into 90μl TE buffer did not.
（Tris-HCl buffer containing 1 mM EDTA）and heated  For the SYBR green I real-time PCR assay, a 20-μl
at 90℃ for 20 min for sporeforming bacteria and for 5 PCR mixture, consisting of 10 μl Fast SYBRR Green
min for non-sporeformers, the supernatant obtained Master Mix（2×）, 0.4 μl ces-SYBR-forward primer
by centrifugation at 10,000 rpm for 3 min was used （10 pM）, 0.4μl ces-SYBR-reverse primer（10 pM）,
as the template DNA. 7.2 μl distilled water and 2μl of the template DNA,
 Primers and probes（Table 1）designed by Fricker was amplified as follows. Typical cycling conditions
et al.（2007）, targeting a region of the ces genes, using a StepOne TM real-time PCR system （Life
were synthesized by Life Technologies Japan TM and Technologies）were 95℃ for 20 s for AmpliTaq
used for conventional PCR and real-time PCR assays. activation and followed by 40 cycles at 95℃ for 15 s
 For the conventional PCR, a 10 μl aliquot of the and 60℃ for 60 s. In order to allow subsequent melting
template DNA was added to 90μl of a reaction solution point analysis, PCR products were heated to 95℃ for
consisting of 10 μl of a 10-fold reaction buffer, 2.5 mM 15 s, cooled to 60℃ for 60 s and slowly reheated to
dNTP mixture, mixture of the ces-forward primer and 95℃ at a rate of 0.3℃/s. The melting curves of SYBR
the ces-reverse primer, 2.5 U of Taq DNA polymerase green I real-time PCR products of an emetic（strain 13）
（Takara）and double-distilled water. The PCR was run
for 35 cycles with denaturation at 94℃ for 60 s,
annealing at 55℃ for 60 s, and extension at 72℃ for 60
s. A 10 μl aliquot of the PCR product was subjected to
electrophoresis in a 2 %（W/V）agarose gel（Sigma）
with Tris-actate EDTA buffer, and then the gel was
stained with ethidium bromide and the DNA bands were
visualized with UV light. The DNA band of emetic B.
cereus strains（strain 13 and strain 4312）, amplified
with the PCR primer set, was in the site of 176
bp-amplicon length, as shown in Fig.1.
 For the TaqMan-based PCR assay, a 20-μl PCR
mixture, consisting of 10 μl TaqManR Gene Expression
Master Mix（2×）, 1.8μl ces-forward primer（10 pM/
μl）, 1.8μl ces-reverse primer（10 pM/μl）, 1 μl
TaqManR probe（5 pM/μl）, 3.4μl distilled water and 2
μl of the template DNA, was amplified as follows. The
typical cycling conditions under a StepOneTM real-time
PCR system（Life Technologies）were 50℃ for 2 min
FIG. 1. Amplification products (176 bp) of emetic B. cereus
for uracil N-glycosylase（UNG）activation and 95℃ for obtained with the ces-primer set for PCR assay.
10 min for AmpliTaq Gold R activation and UNG Lane 1: emetic strain 13; 3: emetic strain 4312;
deactivation, followed by 35 cycles at 95℃ for 15 s and Lanes 2, 4 and 5: diarrheal strain.
60℃ for 60 s. The amplification curves based on Lane M: pHY size marker.

TABLE 1. Primer sets and probes used

PCR assay Primer set Sequence from 5' to 3' end Amplicon
ces-for CACGCCGAAAGTGATTATACCAA 176 bp
Conventional PCR
ces-rev CACGATAAAACCACTGAGATAGTG
SYBR-green 1 ces_SYBR_F CACGCCGAAAGTGATTATACCAA 176 bp
assay ces_SYBR_R CACGATAAAACCACTGAGATAGTG
Real-Time PCR ces_TaqMan-for CGCCGAAAGTGATTATACCAA 103 bp
TaqMan assay ces_TaqMan_rev TATGCCCCGTTCTCAAACTG
ces_TaqMan_probe１） FAM-GGGAAAATAACGAGAAATGCA-MGB２）
1）FAM: 6-carboxyfluorescein; 2）MGB: Minor groove binder.
 DETECTION OF EMETIC B. CEREUS BY RTPCR 229

FIG. 2. Amplification curves of TaqMan real-time PCR products from B. cereus emetic strain
13 and diarrheal strain 48.

＊The PCR product of the emetic strain 13 began to amplify in 20 cycles or more, but that
of the diarrheal strain did not.

and diarrheal strain（strain 48）are illustrated in Fig. 3.

The emetic strain（strain 13）revealed a distinct melting
point of 75℃, but the diarrheal strain（strain 48）was
not found to be amplified. The final identification of
emetic B. cereus was possible within 1 to 1.5 h in either
simplex real-time PCR assay.
 The evaluation of the specificities of a conventional
PCR assay and two simplex real-time PCR assays for
the detection of emetic B. cereus were examined by
using 168 B. cereus strains including 116 emetic type
strains, 51 diarrheal strains and a type culture of
IFO13484, and 38 B. thuringiensis subspp. and other 9
Bacillus strains such as B. subtilis, B. licheniformis, B.
pumilus, B. sphericus, B. coagulans, B. circulans and
B.firmus. In addition, other gram positive and gram
negative species tested for exclusivity were as follows:
Clostridium perfringens, C. botulinum, Staphylococcus
aureus, Listeria monocytogenes, Escherichia coli,
K l e b s i e l l a a e ro g e n e s , C i t r o b a c t e r f r e u n d i i ,
Salmonella Enteritidis, S. Typhimurium, V ibrio
parahaemolyticus, V. alginolyticus, Campylobacter
jejuni, Yesinia enterocolitica, Aeromonas hydrophila. The
test strains had been isolated from the type cultures,
clinical specimens, food materials, air and water, as
FIG. 3. Melting curves of SYBR green I real-time PCR prod-
shown in Table 2. In consequence, all 116 emetic B. ucts used for the detection of emetic strain 13 and diarrheal
cereus strains were found to give positive results in the strain 48 of B. cereus.
3 assays, but other Bacillus strains such as diarrheal B.
cereus, and B. thuringiensis showed negative results ＊The melting point of emetic strain 13 in the singleplex reac-
（Table 2）. In addition, other gram-positive and negative tion was shown to be 75℃, while diarrheal strain 48 did
bacterial species cultures showed negative results. In not show melting at 75℃.
230 S. UEDA ET AL.

this respect, the PCR assays could selectively and hamburger, boiled rice and boiled spaghetti. A 10 %
specifically detect the emetic strains from among B. suspension of each sample was prepared with sterile
cereus strains from various sources. saline solution except for apple juice and milk. After the
 To further evaluate the sensitivity of the real-time PCR suspensions were inoculated with B. cereus strain 13 at
assays, B. cereus strain 13 was used as a the level of 101, 102, 103, 104 or 105 cfu/ml, mixed with
representative of the emetic B. cereus strains in the a vortex and made to stand for 1 h at room temperature
following way. The strain was inoculated into BHI broth, （25℃）, real-time PCR assays were carried out on the
TSB（BBL）or 1% glucose-casein hydrolysate-yeast food suspensions. The assays were shown to be
extract broth（CHYEB）at levels of 102 cfu/ml and capable of detecting emetic B. cereus at the level of 104
incubated at 35℃, and the enumerations of the B. cfu/ml from food samples, similar to those in the
cereus counts and the PCR assays of the cultures were enrichment broths（Table 4）. Consequently, the
carried out on the aliquots sampled from cultures at 2, detection limits of both assays were found to be 104 to
6, 8, and 24 h of incubation. The bacterial counts in all 105 cfu/g or ml of food sample. Fricker et al.（2007）
cultures increased up to 10 4 cfu/ml during the 4 described that the detection limit of the TaqMan-based
h-incubation, and the results of both real-time PCR assay was 105 cfu/g in artificially contaminated rice, and
assays gave distinct DNA amplification patterns（Table that of the SYBR green I assay was 10 3 cfu/g. Also,
3） . Wehrle et al.（2010）revealed the detection limit of 105
 Food samples contaminated artificially with the strain cfu/g for the emetic strain in the SYBR green I assay.
13 were also used to evaluate the sensitivity of the Therefore, the sensitivities of both real-time PCR assays
assays. Food samples used were lettuce, vegetable were generally in line with these previously reported
salad, apple juice, liquid whole egg, cow milk, results in the present research. Furthermore, a reliable

TABLE 2. Results of the SYBR green I and TaqMan real time PCR assays for different bacterial species.
（No. of Real Time PCR
Bacterial species Origin PCR
strains） SYBR-green TaqMan
Bacillus sp.
 Emetic B. cereus food poisoning （116） ＋ ＋ ＋
 Diarrheal B. cereus food poisoning （ 51） − − −
 B. cereus IFO13484 （ 1） − − −
 B. thuriengiensis１） type cultures （ 38） − − −
 B. subtilis PCI218 （ 1） − − −
 B. licheniformis ATCC14580, air （ 2） − − −
 B. pumilus air （ 2） − − −
 B. sphericus air （ 1） − − −
 B. coagulans food （ 1） − − −
 B. circulans food （ 1） − − −
 B. firmus air （ 1） − − −
Clostridium sp.
 C. botulinum type A food （ 1） − − −
 C. perfringens Hobbs type 13 food （ 1） − − −
Staphylococcus aureus２） food poisoning （ 8） − − −
Listeria monocytogenes 4b food （ 1） − − −
Escherichia coli３） foods, type cultures （ 12） − − −
Klebsiella aerogenes food （ 1） − − −
Citrobacter freundii food （ 1） − − −
Salmonella Enteritidis egg （ 1） − − −
Salmonella Typhimurium human faeces （ 1） − − −
V. parahaemolyticus fish （ 1） − − −
Vibrio alginolyticus fish （ 1） − − −
Campylobacter jejuni patient （ 1） − − −
Yersinia enterocolitica patient （ 1） − − −
Aeromonas hydrophila water （ 1） − − −
1）38 subspecies of B. thuringiensis were tested.
2）including enterotoxin A, B, C, D, E and H producers, and isolates from foods.
3）including EPEC, ETEC, EIEC, VTEC and type culture.
 DETECTION OF EMETIC B. CEREUS BY RTPCR 231

TABLE 3. Sensitivities of SYBR green I and TaqMan real-time PCR assays for detection of emetic B. cereus cells.

Incubation BHI cultures TSB cultures 1% glucose-CHYEB cultures

time B. cereus Real-time PCR B. cereus Real-time PCR B. cereus Real-time PCR
(h) counts1） SYBR TaqMan counts SYBR TaqMan counts SYBR TaqMan
0 2.0 − − 2.1 − − 2.2 − −
4 3.8 ＋ ＋ 3.9 ＋ ＋ 4.0 ＋ ＋
6 5.8 ＋ ＋ 5.4 ＋ ＋ 5.4 ＋ ＋
8 7.2 ＋ ＋ 6.6 ＋ ＋ 6.8 ＋ ＋
24 8.1 ＋ ＋ 8.1 ＋ ＋ 8.6 ＋ ＋
1）The bacterial count was expresed as log of cfu/ml.

TABLE 4. Detection limits of real-time PCR assays for emetic B. cereus strain 13 in different food samples.
Level of cfu/ml spiked into food samples
1）
Food sample Blank 102 103 104 105
SYBR2） Taq3） SYBR Taq SYBR Taq SYBR Taq SYBR Taq
Lettuce − − − − − − ＋ ＋ ＋ ＋
Vegetable salad − − − − − − ＋ ＋ ＋ ＋
Apple juice − − − − − − ＋ ＋ ＋ ＋
Liquid whole egg − − − − − − ＋ ＋ ＋ ＋
Cow milk − − − − − − ＋ ＋ ＋ ＋
Hamburger − − − − − − ＋ ＋ ＋ ＋
Boiled rice − − − − − − ＋ ＋ ＋ ＋
Boiled spaghetti − − − − − − ＋ ＋ ＋ ＋
Control:TE-buffer4） − − − − − − ＋ ＋ ＋ ＋
1）All food samples, except apple juice and cow milk, were used as 10% suspension.
2）SYBR: SYBR green I real-time PCR assay.
3）Taq: TaqMan real-time PCR assay.
4）TE-buffer: Tris-HCl buffer containing 1 mM EDTA.

detection of less than 102 cfu/g of emetic B. cereus in

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