Food Control 25 (2012) 144e149

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Food Control
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Real-time PCR with internal amplication control for the detection of Cronobacter
spp. (Enterobacter sakazakii) in food samples
Xiang Wang a, Changqing Zhu a, b, Xinglian Xu a, *, Guanghong Zhou a
a
b

Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, PR China
Jiangsu Entry Exit Inspection and Quarantine Bureau, Nanjing 210001, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 1 June 2011
Received in revised form
9 October 2011
Accepted 15 October 2011

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening
infections in neonates. The organism has been isolated from a wide variety of foods and environments. In
this study, a Taqman real-time PCR assay incorporating an internal amplication control (IAC) was
developed and evaluated for specic detection of Cronobacter spp. in foods. Previously reported
macromolecular synthesis (MMS) operon sequence was selected for specicity, and 67 bacterial strains,
including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully
identied, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the
assay in pure culture and formula infant without enrichment was 1.2
103 CFU/ml (1.2
101 CFU/assay).
After 24 h enrichment in broth, as few as 100 CFU/ml or g of Cronobacter could be detected in articially
contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the
real-time PCR assay however remained unaffected in the presence of 108 CFU/ml Salmonella typhimurium
in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of
which two pork samples were found as positive by real-time PCR, whereas only one was detected by the
ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp.
in food samples with high specicity and sensitivity, and can prevent false negative results by using the
IAC.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Cronobacter spp. (Enterobacter sakazakii)
Real-time PCR
Internal amplication control
Detection
Food

1. Introduction
Cronobacter spp. is a group of emerging opportunistic foodborne pathogens. The genus was originally described as yellow
pigmented Enterobacter cloacae until it was dened as Enterobacter
sakazakii in 1980 (Farmer, Asbury, Hickman, & Brenner, 1980).
Recently, it was reclassied as a new genus consisting of six
genomospecies (Iversen et al., 2008), all considered pathogenic due
to retrospectively linking each to clinical cases of infection in either
infants or adults (FAO/WHO, 2008). Clinical symptoms of Cronobacter infection include necrotizing enterocolitis, bacteremia and
meningitis with high fatality rates in neonates and infants (Healy
et al., 2010). Although the primary source of Cronobacter spp. has
yet to be determined, the organism has been isolated from a wide
variety of foods including infant formula, milk powders, cheese,
cereals, fruits, vegetables, herbs and meat (Chap et al., 2009;

* Corresponding author. Tel.: 86 025 84395939; fax: 86 025 84395730.

E-mail addresses: wangxiangnjau@126.com (X. Wang), xlxu@njau.edu.cn
(X. Xu).
0956-7135/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.10.037

Friedemann, 2007; Jaradat, Ababneh, Saadoun, Samara, &

Rashdan, 2009; Jung & Park, 2006; Kandhai et al., 2010; Leclercq,
Wanegue, & Baylac, 2002), as well as in environmental samples
such as soil, food factory environments and consumer homes
(Edrington et al., 2009; Jaradat et al., 2009; Kandhai, Reij, Gorris,
Guillaume-Gentil, & van Schothorst, 2004; Shaker, Osaili, AlOmary, Jaradat, & Al-Zuby, 2007).
Conventional methods available for identication of Cronobacter
spp. (FDA, 2002; ISO, 2006) are time consuming and often lead to
inaccurate results (Fox & Jordan, 2008; Iversen, Druggan, &
Forsythe, 2004; Iversen, Lehner, Mullane, Bidlas et al., 2007; Seo
& Brackett, 2005). Thus rapid and more accurate methods have
been developed and one of the most promising is real-time PCR. In
addition to being rapid and simple, real-time PCR is sensitive and
specic, as well as being able to avoid the possibility of cross
contamination (Ginzinger, 2002). Several real-time PCR methods
have been developed to detect Cronobacter spp., and target
sequences have been utilized including the macromolecular
synthesis (MMS) operon sequence (Seo & Brackett, 2005), the 16S
rRNA gene (Kang, Nam, & Hong, 2007; Malorny & Wagner, 2005),
the 16Se23S rRNA internal transcribed spacer (ITS) (Liu et al.,

X. Wang et al. / Food Control 25 (2012) 144e149

Table 1
Bacterial strains used for specicity tests and results.
Bacterial strains

Sourcea/Strain ID

Number

Resultb
MMS

IAC

Cronobacter sakazakii
Cronobacter sakazakii
Cronobacter spp.
Corynebacterium variabilis
Citrobacter breakii
Enterobacter aerogene
Enterobacter aerogenes
Enterobacter cloacae
Enterobacter cloacae
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
Enterococcus faecium
Escherichia coli
Escherichia coli O157
Klebsiella oxytoca
Lactobacillus plantarum
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Pseudomonas sp.
Salmonella enteritidis
Salmonella typhimurium
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus saprophyticus
Staphylococcus sciuri
Staphylococcus xylosus
Tetragenococcus halophilus
Yersinia enterocolitica
Yersinia enterocolitica

ATCC 29544
ATCC 12868
JSCIQ
NMC
NMC
ATCC 13048
NMC
ATCC 13047
NMC
ATCC 13433
NMC
ATCC 19434
NMC
NMC
CICC 21530
NMC
NMC
ATCC 15313
ATCC 19115
ATCC 19112
ATCC 19117
CICC 21583
NMC
JSCIQ
JSCDC 50013
NMC
NMC
NMC
NMC
NMC
NMC
CICC 21565
JSCDC

1
1
2
2
1
1
3
1
5
1
5
1
4
7
1
1
7
1
1
1
1
1
4
1
1
2
1
2
1
2
2
1
1
































a
JSCIQ, Jiangsu Entry-Exit Inspection and Quarantine Bureau; CICC, China Center
of Industrial Culture Collection; JSCDC, Jiangsu Provincial Center for Disease
Prevention and Control; NMC, National Meat Center of China.
b
, Positive; , Negative.

2006), the DNA region located between t-RNA-glu and 23S RNA
(Derzelle & Dilasser, 2006), and the outer membrane protein A
(OmpA) gene (Kandhai et al., 2010). However, not all of these
identication systems have been thoroughly tested for specicity.
The assay developed by Seo and Brackett (2005) has been validated
in a number of studies to have a high specicity for Cronobacter spp.
(Chen, Song, Brown, & Lampel, 2010; Iversen, Lehner, Mullane,
Marugg et al., 2007; Lampel & Chen, 2009; Molloy et al., 2009),
and recently, FDA has included it as its revised ofcial method for
Cronobacter detection and isolation in infant formula as a promising
screening and conrmation tool (Chen, Hammack, Song, & Lampel,
2009). However its lack of an internal amplication control (IAC)
required for elimination of false negative results can be a critical
shortcoming.
In this study, a Taqman real-time PCR assay based on the MMS
sequences for Cronobacter detection was improved by incorporating a competitive IAC. The effectiveness of the method was
determined with both articially and naturally contaminated food
samples.

145

cultured overnight (12e18 h) at 37  C in nutrient broth (LuQiao

company, Beijing, China). Bacterial genomic DNA was extracted
from 1 ml of overnight cultures using lysis by boiling in 200 ml TE
buffer (pH 7.6) supplemented with 1% Triton X-100
(Krascsenicsova, Trncikova, & Kaclikova, 2008).

2.2. PCR primers and probes

The primers and TaqMan probe described by Seo and Brackett
(2005) were used to detect Cronobacter spp. The primers and
probe (Table 2) of the IAC were designed with the help of Beacon
designer software (version 7.0). They were synthesized by GenRay
(Shanghai, China). The Cronobacter spp. target probe was labeled at
the 50 -end with the reporter dye 6-carboxyuorescein (FAM) and at
the 30 end with the BHQ-2 Dark Quencher. The IAC probe was
labeled at the 50 end with the reporter dye HEX and at the 30 end
with the BHQ-2 Dark Quencher.

2.3. IAC control

An IAC was constructed as previously described (Mohan Nair &
Venkitanarayanan, 2006). The control DNA (156-bp PCR product)
comprised IAC probe sequence anked by the target Cronobacter
primer sequences MMS-F and MMS-R (Table 2). The IAC target DNA
was amplied from pTG19-T vector (GeneRay, Shanghai, China)
with primers IAC-F and IAC-R (Table 2). The target DNA was then
cloned into the pTG19-T vector and transformed into DH5a cells.
Recombinant plasmid carrying the IAC template sequence was then
amplied with M13-47 and RV-M primers resulting in a 353-bp
fragment. The PCR product was puried with the Wizard DNA
purication kit (Promega, Madison, USA), its concentration was
estimated spectrophotometrically using a NanoDrop 2000 spectrophotometer (Thermo Scientic, MA, USA) and the number of
copies was calculated and adjusted. The optimal copy number of
IAC for inclusion into real-time PCR reaction was determined by
serial dilutions of IAC (101e106 copies/reaction).
2.4. Real-time PCR amplication
Real-time PCR was carried out in an ABI 7500 real-time PCR
system (Applied Biosystems, USA). A PCR mixture (25 ml) contained
12.5 ml Premix Ex Taq (2
) buffer (Takara, Dalian, China), 0.5 ml
50
ROX Reference Dye , 600 nM of MMS-F and MMS-R primers,
150 nM of MMS probe, 200 nM of IAC probe, 2 ml of sample DNA and
1 ml of 1000 copies of IAC template (puried 353 bp product). All
runs included a negative control without target DNA. Thermal
cycling conditions were as follows: 95  C for 30 s, followed by 40
cycles of 95  C for 5 s and 60  C for 34 s. The cycle threshold (Ct) was
calculated using ABI 7500 Sequence Detection System Software.
Negative values or lack of amplication were considered for Ct
values of >40.

Table 2
Primers and probes used in this study.

2. Material and methods

2.1. Bacterial strains, growth media and DNA extraction
The bacterial strains used in this study and their sources are
listed in Table 1. A total of 67 strains including 4 strains of Cronobacter spp. were used for specicity tests. All the strains were

Designation

Sencence(50 e30 )

Positions

MMS-F
MMS-R
MMS-Probe
IAC-F
IAC-R
IAC-Probe

ggg ata ttg tcc cct gaa aca g

cga gaa taa gcc gcg cat t
aga gta gta gtt gta gag gcc gtg ctt ccg aaa g
ggg ata ttg tcc cct gaa aca gcg ctg cgc ctt atc
cga gaa taa gcc gcg cat tca ccg cct aca tac ctc
ccg cct aca tac ctc gct ctg cta atc ct

201e222
278e260
225e258

146

X. Wang et al. / Food Control 25 (2012) 144e149

2.5. Sensitivity in pure culture and infant formula

3.2. Sensitivity and detection limit in pure bacterial culture

The sensitivity of the real-time PCR assay was determined using

the standard curves prepared with genomic DNA from Cronobacter
sakazakii (ATCC 29544) pure culture in broth as well as in infant
formula.
A 1 ml aliquot of overnight bacterial culture was prepared for
DNA extraction, and 10-fold dilutions (to 109) of the DNA samples
were used as template in the real-time PCR assay. The population of
the overnight Cronobacter pure culture was estimated by plate
counting on nutrient agar (LuQiao Company, Beijing, China).
The 1 ml of Cronobacter overnight cultures were serially diluted
(10-fold) with 8 ml of sterile 0.85% NaCl and 1 g infant formula
which was determined to be free of Cronobacter by standard
method of ISO. The articially inoculated infant formula samples
were analyzed using real-time PCR assay after DNA extraction.

Two hundred microliters of genomic DNA was extracted from

1 ml Cronobacter overnight culture which contained 1.2
109 CFU
of the cells. 10-fold dilutions of DNA were amplied in triplicate. For
reconstituted milk, overnight culture of Cronobacter was serially
diluted to achieve a nal concentration of 1.2
101e1.2
108 CFU/
ml. Standard curves were constructed by plotting the Ct versus the
log of the CFU (Fig. 1a and b), both of them showed good linearity.
Ct values of IAC were relatively stable. The detection limit of the
real-time PCR was 1.2
103 CFU/ml (1.2
101 CFU/assay) (Table 3)
in pure cultures or infant formula, however due to the competition
for the primers, no IAC signals were detected when the concentration of Cronobacter cells was 1.2
109 CFU/ml.

2.6. Detection of articial contamination food samples after

enrichment
To test the effect of the sample matrix and background ora on
the real-time PCR assay, food samples (milk powder, sterilized milk
and chicken meat) used to test the method were obtained from
local market. 25 g or ml of samples were homogenized in 225 ml
nutrient broth, and 9 ml from each samples was inoculated with
a 1 ml 10-fold serial dilutions of C. sakazakii (ATCC 29544) culture to
obtain a level of contamination from 1
100 to 1
104 CFU/ml or g.
After gentle mixing, the samples were incubated at 37  C for 24 h
prior to testing. The viable cell counts of natural background ora of
chicken meat were determined by following the methods
described by Malorny et al. (2003).

3.3. Detection of Cronobacter spp. in articially-contaminated food

samples
Cronobacter-negative infant formula, sterilized milk and chicken
meat were inoculated with C. sakazakii (ATCC 29544) at different
levels (from 100 to 104 CFU/ml). After 24 h of enrichment, the realtime PCR assay detected as few as 100 CFU/ml of Cronobacter in the
various food samples (Table 4). Samples which were not inoculated

2.7. Detection of Cronobacter spp. in the presence of Salmonella

typhimurium
To investigate the capability of the assay detecting Cronobacter
in the presence of other bacteria, infant formula having different
levels of Cronobacter spp. and S. typhimurium were taken as model
sample. The infant formula samples having C. sakazakii (ATCC
29544) 101e107 CFU/ml were inoculated with 108 CFU/ml
S. typhimurium, while those containing 103 CFU/ml Cronobacter spp.
were inoculated with 101e107 CFU/ml S. typhimurium. After
enrichment for 24 h, 1 ml was taken from each sample and DNA was
extracted as template for real-time PCR.
2.8. Detection of Cronobacter in food samples
A total of 92 food samples, including milk (n 10), milk powder
(n 6), chicken meat (n 18), pork (n 37), sausages (n 9) and
vegetables (n 12) were collected from local supermarkets and
retailers (Nanjing, China). All the samples were analyzed in parallel
by ISO standard method and real-time PCR after enrichment.
3. Results
3.1. Specicity of real-time PCR assay
A total of 67 strains including four Cronobacter spp. strains were
subjected to test specicity. Most were food-borne organisms and
pathogens (Table 1). All the Cronobacter spp. strains gave positive
signals (FAM channel) in the real-time assay, whereas the other 63
strains generated only IAC (HEX channel) signals (Table 1). No falsepositive or false-negative results were obtained, thus the addition
of IAC did not affect the specicity, the real-time PCR detection
system is specic for the detection of Cronobacter spp.

Fig. 1. Standard curves showing the linear relationship between Ct and log CFU for 10fold series dilution of Cronobacter and the IAC in pure culture (a) and in infant formula
(b). The coefcient of determination and the slope of the regression curve are indicated
respectively.

X. Wang et al. / Food Control 25 (2012) 144e149

Table 3
Sensitivity of real-time PCR for Cronobacter detection in pure culture and infant
formula.
Cronobacter
concentration
(CFU/ml)

Cronobacter
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
a

109
108
107
106
105
104
103
102

Table 5
Detection of Cronobacter in the presence of S. typhimurium.

14.36
18.35
21.84
25.67
29.32
33.21
36.59
ND









0.03
0.02
0.11
0.05
0.10
1.06
0.55

Infant formula
IAC
NDa
29.01
27.87
28.40
28.76
28.88
28.94
28.51









0.05
0.02
0.01
0.18
0.03
0.09
0.55

Cronobacter

IAC

N/A
18.23
22.07
25.61
29.95
32.75
36.48
ND

N/A
29.65
28.80
29.03
29.32
29.28
29.08
28.89








0.17
0.48
0.52
0.09
0.16
0.26









0.10
0.23
0.08
0.01
0.07
0.07
0.38

Not detected.

with Cronobacter did not yield a uorescence signal. With the same
amount of Cronobacter inoculated into the various food samples,
the Ct values of chicken meat were higher than that of infant
formula or sterilized milk. A high level of competition with background ora might have been responsible for this nding. The total
viable colony counts of aerobically grown bacteria of chicken meat
were about 3.1
105 CFU/g.
3.4. Detection of Cronobacter spp. in the presence of
S. typhimurium
The presence of S. typhimurium did not affect the detection limit
of the real-time PCR. Even when inoculated with 108 CFU/ml of
S. typhimurium, the method was capable of detecting as few as
101 CFU/ml Cronobacter after enrichment (Table 5). When inoculated with 103 CFU/ml of Cronobacter, all the inoculated samples
were found positive after enrichment with Ct value of 18.15e23.71
(data not shown).
3.5. Detection of Cronobacter spp. in food samples
Of all the 92 food samples tested, only one pork sample was
found positive by the ISO-IDF method and two pork samples were
positive for Cronobacter by real-time PCR, giving more than 98%
concordance between both methodologies.
4. Discussion
Traditional methods for the detection of Cronobacter, based on
biochemical and serological approaches, are labor-intensive, timeconsuming and the accuracy has been subjected to question. Realtime PCR assays using TaqMan probe have been shown to be useful
for detection of pathogenic bacteria in food samples (Kang et al.,
2007). In the present study, the sensitive and accurate Taqman
real-time PCR assay developed by Seo and Brackett (2005) was
improved by introduction of an IAC. The addition of IAC was to
control false-negative results, which may be caused by the

Ct  SD (n 3)

Pathogen (CFU/ml)

Ct  SD (n 3)
Pure culture

147

S. typhimurium

Cronobacter

Cronobacter

108

107
106
105
104
103
102
101

22.43
21.67
21.32
22.08
26.21
27.43
30.76









0.17
0.79
0.19
0.12
0.12
0.08
0.23

IAC
31.34
30.28
30.01
30.54
31.01
30.47
30.01









0.11
0.07
0.35
0.54
0.37
0.21
0.12

malfunction of the thermal cycler, incorrect PCR mixture, inhibitory

substances etc (Rossen, Norskov, Holmstrom, & Rasmussen, 1992).
Inclusion of IAC has now become mandatory for the detection of
food-borne pathogens (Hoorfar et al., 2003, 2004; ISO, 2005; Moore
& Feist, 2007). A competitive IAC was designed in this study, where
both the target DNA and IAC were amplied by the same primer set.
However, the target Ct values remained unaffected within a range
of IAC concentrations from zero to 106 copy/reaction (data not
shown). The target amplication signal decreased when the IAC
concentration was 105 copy/reaction or higher; thus 1000 copies of
IAC template were added in the PCR mixture. At this concentration,
competition with high concentrations of target DNA is expected to
be the main cause of IAC amplication failure; however IAC
amplication is unnecessary when the target is amplied (Hoorfar
et al., 2004).
The standard curves showed a strict inverse correlation between
Ct values and concentration of Cronobacter in broth as well as in
infant formula. The detection limit was about 1.2
103 CFU/ml
(1.2
101 CFU/assay), somewhat lower than that previously reported; 1.0
102 CFU/ml (Seo & Brackett, 2005). This may have
resulted from self-degradation and release of uorescent signals of
probes after the 40 cycles used in this study as compared to the 50
cycles used by Seo and Brackett (2005). Positive signals of the
negative control were also observed after 40 cycles.
The real-time PCR was able to detect 100 CFU/ml or g of Cronobacter in articially contaminated food samples after 24 h of
enrichment in broth. The results agreed with previous reports
where the detection limits of real-time PCR assay were improved
after enrichment (Liu et al., 2006; Seo & Brackett, 2005). Due to the
fact that the present level of Cronobacter is very low in food
(Kandhai et al., 2010), an enrichment step is required to improve
the detection limit by multiplying the number of viable cells.
Another advantage of enrichment is to effectively dilute the
inhibitory components of the food samples. Moreover, after the
enrichment step, the number of dead cells became negligible, thus
overcoming the problem of the inability of PCR to distinguish
between living and dead organisms, which would cause potential
false-positive results (Mafu, Pitre, & Sirois, 2009). It is noted that the
infectious dose associated with Cronobacter was estimated to be 103
(Iversen & Forsythe, 2004) or 104 CFU (FAO/WHO, 2004). Thus, such
detection limit of the real-time PCR assay after enrichment would
be very useful to detect Cronobacter in foods. The time required for

Table 4
Detection of Cronobacter in articially-contaminated food samples.
Sample

Ct values of Cronobacter by contamination level (CFU)a

100

101

102

103

104

Infant formula
Sterilized milk
Chicken meat

24.45  1.52
20.15  0.93
33.20  1.07

25.17  1.62
20.09  1.51
33.33  0.79

22.37  1.02
21.01  1.30
30.98  0.99

22.27  1.67b
19.59  1.24
25.02  0.76

21.90  1.71
20.14  1.05
27.21  0.71

a
b

The Ct value are means  standard deviation of three replicate experiments.

A failure in the sample amplication but not IAC.

148

X. Wang et al. / Food Control 25 (2012) 144e149

enrichment has been mentioned as an important factor in isolation

efciency (Spira & Ahmed, 1981). The time required for the detection of Cronobacter is usually 24  2 h (Chen et al., 2010). Overgrowth by competitors, even at 24 h in this study, did not seem to
be a problem.
So far only infant formulas have been denitely attributed to
cases of Cronobacter spp. infection, and most of the investigative
attention has been focused on these products (Kandhai et al., 2010).
Other foods may also be potential sources of Cronobacter spp.
contamination in the food chain (Healy et al., 2010). In this study,
besides infant formula and milk, chicken meat was also tested and
the results showed that the detection limit was not inuenced by
background ora or matrix. Moreover, our results showed that even
in the presence of high level of S. typhimurium (108 CFU/ml), the
detection limit was not reduced.
Among the 92 food samples tested in this study, only one pork
sample was detected positive by both the methods. It is apparent
that the prevalence rates of Cronobacter in food are equally low as
found in previous reports (Jung & Park, 2006; Kandhai et al., 2010),
and as far as we know, this is the rst report that Cronobacter were
detected in meat in China.
In conclusion, the real-time PCR assay described in this paper is
rapid, specic, and sensitive for the detection of Cronobacter in food
samples of various origins. The application of IAC can effectively
eliminate false negative results. The assay can detect very low
numbers of Cronobacter cells in food samples contaminated with
high levels of background ora and common PCR inhibitors of food
samples after 24 h of enrichment.

Acknowledgments
This research was joint supported by the Ministry of Science and
Technology of China (Grant No: 2009DFA31770) and the National
Science and Technology Major Projects-Breeding of New Variety for
Transgenic Biology (Grant No: 2008ZX08011-004).

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