journals

1. Molecular and Cellular Probes 21 (2007) 177–181
Real-time PCR assay for rapid detection and quantification of
Campylobacter jejuni on chicken rinses from poultry processing plant
Aradom Debretsiona
, Tsegaye Habtemariamb
, Saul Wilsonc
,
David Nganwab
, Teshome Yehualaesheta,Ã
a
Department of Pathobiology, College of Veterinary Medicine, Nursing & Allied Health, Tuskegee University, AL 36088, USA
b
Center for Computational Epidemiology, Bioinformatics & Risk Analysis, College of Veterinary Medicine, Nursing & Allied Health,
Tuskegee University, AL 36088, USA
c
International Center for Tropical Animal Health, College of Veterinary Medicine, Nursing & Allied Health, Tuskegee University, AL 36088, USA
Received 19 January 2006; accepted 30 October 2006
Available online 14 November 2006
Abstract
Campylobacter jejuni (C. jejuni) is the leading cause of food-borne gastroenteritis in the United States. Detection of Campylobacter in
food samples by conventional culture is cumbersome; therefore, there is a need to develop rapid and cost-effective detection and
quantification methods. Eighty-four whole chicken rinses were collected at different stages of processing at three poultry processing
plants. After chicken wash collection and DNA extraction, the samples were directly subjected to real-time PCR (rtPCR) without
enrichment and also culture. The assay specificity was determined with a range of Campylobacter species, related, and unrelated
organisms. Of the 84 samples collected 65 (77%) of the samples were positive by the rtPCR assay and 27 (32%) of the samples tested
positive by direct plating to selective agar media. The results were positively concordant for 27 (32%) of the samples. The whole rtPCR
assay can be completed within 90 min with a detection limit of 1 CFU, compared to 5–7 days for enrichment and sub culturing in selective
agar. This assay is the first report of rtPCR method capable of detecting and quantifying C. jejuni from chicken rinses without an
enrichment step and could be an important, rapid and quantification model for other food-borne pathogens.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Campylobacter jejuni; Chicken; Real-time PCR; Without enrichment
1. Introduction
In many industrialized countries, the incidence of
campylobacteriosis exceeds that of salmonellosis. Campy-
lobacter species are transmitted to humans mainly in food,
especially in poultry meat products [1]. They are zoonotic
bacteria, with many wild and domesticated animals serving
as potential reservoirs. Sources of sporadic campylobacter-
iosis are seldom identified, but contaminated water, pets,
and especially poultry products are known to be associated
with the illness [2]. Epidemiological evidence suggests that
animals, particularly poultry, cattle, wild birds, pigs and
domestic pets are the reservoirs for the strains that infect
humans [3,4].
Traditional diagnostic methods are commonly based on
selective enrichment of the target pathogens [5]. Even
though these methods are standardized and efficient, they
suffer from serious disadvantages, such as that they are
time-consuming and expensive, correct analysis can be
difficult due to lack of expression of phenotypic properties,
and detection of viable but-non-culturable cells (VBNCs) is
almost impossible at present. In an effort to overcome
these limitations, DNA-based detection methods have been
developed [6]. One of the most promising methods is real-
time PCR (rtPCR) due to its speed, cost effectiveness and
sensitivity, specificity, selectivity, high degree of automa-
tion and the possibility of target quantification [7,8].
A number of conventional PCR assays have been
described for the identification and characterization of
Campylobacter species from a spectrum of sample types,
including stools [9–11], food products [12,13], water [14]
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doi:10.1016/j.mcp.2006.10.006
ÃCorresponding author. Tel.: +334 727 8107; fax: +334 724 4110.
E-mail address: teyehual@tuskegee.edu (T. Yehualaeshet).

2. and cultures [15]; using a variety of gene targets such as,
hipO, glyA, 23S rRNA, ceuE and mapA. The rtPCR
method that identifies species-specific detection of Campy-
lobacter species in naturally infected chicken fecal samples
after an enrichment step has been developed [8]. To our
knowledge, no studies have been done on naturally infected
chicken skin, using rtPCR without enrichment to detect
and quantify C. jejuni.
The objective of this study was to develop an rtPCR
assay capable of optimizing and detecting thermophilic
Campylobacter species as well as distinguishing C. jejuni
from other Campylobacter species in a single rtPCR
reaction without enrichment. This protocol was also
capable of quantifying C. jejuni with high degrees of
analytical sensitivity and specificity of rtPCR from poultry
carcass rinses without enrichment.
2. Materials and methods
2.1. Media, bacterial isolates, and culture conditions
Campylobacter species and related reference strains were
used to determine the specificity of the assay (Table 1).
Arcobacter butzleri and Escherichia coli were grown
aerobically at 35 1C on modified Campy-Cefex media.
Helicobacter species were grown microaerobically without
Campylobacter selective supplement at 37 1C.
The reference bacterial isolates were recovered from
À80 1C storage and grown on selective agar or broth,
modified Campy-Cefex agar (MCC), charcoal cefopera-
zone deoxycholate agar (CCDA) and Brucella broth.
Triplicate plates were each spread with 0.1 ml of the
carcass rinses, then incubated at 42 1C for 48 h in Glad
Fresh Protect bags (The Glad Products Company, Oak-
land, CA) and flushed with a microaerophilic gas mixture
(BOC Gases, Hixson, TN) containing 85% N2, 10% CO2
and 5% O2. Plates without Campylobacter species, growth
after 48 h, were incubated for an additional 24 h to ensure
maximum recovery.
2.2. Poultry samples and DNA extraction prior to
enrichment
A total of 84 chickens were collected from poultry
processing plants and processed for the detection and
quantification of C. jejuni. The whole chicken was placed
into a sterile chicken rinse plastic bag (Biotrace Interna-
tional Bioproducts, Muncie, IN), and then, 400 ml of
buffered peptone water (Biotrace International Biopro-
ducts, Muncie, IN) was added, the bag was twisted to seal,
and the contents were swirled for a minimum of 2 min.
After immediate transfer of the chicken carcass rinses,
the DNA template was extracted directly from the rinse
without enrichment as follows. Briefly, 1 ml of the rinse was
transferred into a micro-centrifuge tube and then centri-
fuged at 16,000 rpm for 15 min at 4 1C. The supernatant
was discarded and the pellet was re-suspended in 2 ml of
buffered peptone water. One milliliter of the pellet
suspension mixture was used for DNA extraction with
200 ml of PrepMan Ultra diluents as recommended by the
manufacturer (Applied Biosystems, Foster City, CA).
Simultaneously, the chicken rinses were subjected to the
appropriate selective media.
2.3. Real-time PCR primers and probe
The primers and probe used for the rtPCR to detect
C. jejuni were 50
-GAATGAAATTTTAGAATGGGG-30
(forward primer) and 50
-GATATGTATGATT-TTATCC-
TGC-30
(reverse primer) and TTTAACTTGGCTAA-
AGGCTAAGGCT (probe). The amplification product
detected was 358-bp. The PCR primers, VS1 sequence
(GB AN X71603), were selected to target the C. jejuni-
specific region [16]. The probe was labeled with a
fluorescent dye in the 50
-nuclease PCR; in this case
6-carboxyflourescein (FAM), and 6-carboxytetramethylrho-
damine (TAMRA) to contribute to the fluorescent spectra,
generating an overlapping composite spectrum. Campylo-
bacter jejuni species-specific primers and probe were
purchased from Integrated DNA Technologies (IDT,
Coralville, IA) and TaqMan probe from Applied Biosys-
tems (AB, Foster City, CA).
2.4. Real-time PCR assay
The PCR assay in the detection and quantification of C.
jejuni involves several critical steps, such as DNA extrac-
tion from specimens, PCR amplification, and detection of
amplicons. In particular, when specific clinical specimens,
such as chicken rinse, with only a few bacteria present are
tested by PCR, each procedure must be carefully designed
and performed. The rtPCR assay was carried out in a total
volume of 25 ml using the Cepheid Smart Cycler II
(Cepheid, Sunnyvale, CA). Each of the amplification
reactions (25 ml) contained 1 ml of the target DNA sample
solution ($100 pg), 0.4 mM of each forward and reverse
C. jejuni-specific primers (IDT, CoralVille, IA), 0.1 mM
ARTICLE IN PRESS
Table 1
Reference strains of Campylobacter, and related bacterial strains used in
rtPCR assay
Species Source
Positive controls
Campylobacter jejuni strain ATCC 35918
ATCC DNA genome 33560D
Campylobacter jejuni strain CDC strain collections
Negative controls
Campylobacter coli strain ATCC 43484
Campylobacter lari ATCC 35223
Campylobacter fetus ATCC 27374
Escherichia coli ATCC 33625
Helicobacter cinaedi ATCC BAA-847
Helicobacte hepaticus ATCC 51448
Arcobacter butzleri ATTC 49616
A. Debretsion et al. / Molecular and Cellular Probes 21 (2007) 177–181178

3. C. jejuni-specific probe (AB, Foster City, CA). The 25 ml
reconstituted (OmniMix Hs, Sunnyvale, CA) PCR reagent
contains 1.5 U TaKaRa hot start TaqTM Polymarase,
200 mM dNTP, 4 mM MgCl2 and 25 mM HEPES buffer at
pH 8.070.1. C. jejuni strain (ATCC 35918) was used for
preliminary optimization of the PCR and cycling para-
meters. A part of the amplified sample was analyzed using
1.5% agarose gel electrophoresis. DNA bands were stained
with ethidium bromide, visualized with an ultraviolet
transilluminator, and photographed.
2.5. Determination of sensitivity and specificity of the
rtPCR assay
The analytical sensitivity of the rtPCR assay was
determined using a 0.5 McFarland turbidity suspension.
The suspension was prepared in PBS from colonies grown
in Brucella broth containing FBP, cefoperazone and
amphotericin B at 10-fold dilutions of DNA isolated from
known C. jejuni strains. The specificity of the assay was
determined using 106
genome copies per PCR (1 ml) of
DNA template from bacterial species found in skin
washing rinse of the chicken.
The non-Campylobacter species specificity of the assay
was further characterized using chicken-rinse screening
strategy: the DNA was isolated from five Campylobacter-
negative samples and examined with the rtPCR assay to
ensure the specificity of the primers and probe for C. jejuni
only.
2.6. Statistical analysis
Pearson correlation coefficient was used to measure the
linear relationship between the two quantitative variables:
the amount of amplicon as logarithmic colony forming unit
(CFU) counts and the proportion of fluorescence as a cycle
threshold (CT). The correlation coefficient assumes a value
between À1 and +1. If one variable increases as the other
decreases, then the correlation coefficient is negative.
Conversely, if the two variables increase together, then
the correlation coefficient is positive. The correlation
coefficient was calculated using the following formula for
the two variables x and y: r ¼ Sðx À mxÞðy À myÞ=
ðn À 1ÞSxSy, where mx and Sx were the sample mean and
the standard deviation for the first variable x and my and
Sy were the sample mean and standard deviation for the
second variable y, respectively.
3. Results
3.1. Optimization and specificity of rtPCR for C. jejuni
In the current study, we sought to address detection,
specificity as well as the sensitivity associated with the
rtPCR detection of C. jejuni from chicken rinses without
enrichment. The overall positive and negative results were
recorded and expressed as percentages. The assay’s
specificity was further demonstrated by its ability to
exclude all non-C. jejuni bacterial species. The specificity
of the primers and probe were optimized and tested against
three strains of other Campylobacter species as well as a set
of four species belonging to genetically related or common
food-borne microorganisms, all of which were found to be
negative (Table 1).
The rtPCR results were compared to culture, selective
agar medium and phenotypic identification. A total of 84
chicken rinses were collected from three poultry processing
plants and processed by the routine culture protocol and
the rtPCR assay. Thirty-four samples were collected from
poultry processing plant I, out of which 22 samples were
positive by the rtPCR and 9 samples grew on selective
media. From Plant II, 30 samples were subjected to rtPCR
and culture out of which 26 and 11 were found positive,
respectively. Seventeen of 20 chicken rinses were rtPCR
positive from processing plant III and seven chicken rinses
were positive by selective culture media. Overall 65 samples
(77%) out of the 84 chicken rinses were positive by the
rtPCR assay samples for C. jejuni and 27 (32%) samples
were positive by direct plating on selective media (Table 2).
Twenty-seven of the samples were positively concordant by
both rtPCR and culture. Agarose gel analysis indicated a
clearly distinguishable band at 358-bp which is specific to
C. jejuni and no amplification bands were present in all of
the other bacterial isolates (Fig. 1). The total time required
for the detection of C. jejuni using the rtPCR was about 2 h
(30 min for the DNA extraction and 90 min for the rtPCR
assay) with the results being available immediately at the
end of PCR cycling.
ARTICLE IN PRESS
Table 2
Comparison of rtPCR assay and DPSAa
results to detect C. jejuni from chicken rinses
Poultry processing
plant
Total samples No. of rtPCR samples No. of DPSA samples No. of positive samples for
both (rtPCR and DPSA)
+ À + À
Plant I 34 22 12 9 25 9
Plant II 30 26 4 11 19 11
Plant III 20 17 3 7 13 7
Total 84 65 (77%) 19 (23%) 27 (32%) 57 (68%) 27 (32%)
a
DPSA—direct plating on selective agar.
A. Debretsion et al. / Molecular and Cellular Probes 21 (2007) 177–181 179

4. rtPCR-positive/culture-negative samples had CT values
of 39.02 or greater, with the mean value being 43.2,
whereas the 27 PCR-positive/culture-positive samples had
CT values of between 20.8 and 37.5, with the mean value
being 26.2.
3.2. Detection limits and determination of linear range
quantification of rtPCR
The detection and quantification limits of the rtPCR
assays were determined by using genomic DNA isolated
from C. jejuni. Ten-fold serial dilutions of DNA were
prepared and a CFU standard curve was constructed.
Amplification reactions were performed with a range of
DNA concentrations. As shown in Fig. 2, DNA extracted
from approximately 1 Â 106
C. jejuni cells was serially
diluted (106
–100
CFU; panel B–H) and subjected to rtPCR.
The standard curve based on the dilutions of DNA showed
a linear relationship between log CFU and threshold cycles
(CT). This was compared to the standard curve used as a
reference for the study (Fig. 3).
DNA standard curves showed that the detection limit of
rtPCR assay was 1 CFU from pure cultures. The number of
C. jejuni cells was described by the following equations:
CT ¼ À3.417logCFU+34.63 and CFU ¼ 10½ðCTÀ34:63Þ=À3:417Š
.
4. Discussion
Detection of bacterial DNA has become more sensitive
and laboratories are becoming proficient, it is likely that
the PCR methodology will usurp other microbiological
diagnostic methods. The use of rtPCR/PCR with DNA
extracts has several advantages, including a more rapid
time to the end point compared to that for culturing the
bacteria [13]. If rtPCR method is intended for quantitative
measurements, it is a prerequisite to obtain information on
both linear range and amplification efficiency in order to
ensure correct quantification. The sensitivity and detection
limit of rtPCR can be considerably affected by several
factors, including presence of inhibitory substances and
quality of the DNA. The detection limit of any PCR and
rtPCR should always be assessed in association with the
detection probability to illustrate the reliability of the assay
[17].
The rtPCR assay in this study demonstrated a linear
range of the quantification over six orders of magnitude
and a quantitative limit of detection of approximately 10
genome equivalents, with detection below these levels being
inconsistent. Previously reported sensitivities of detection
by TaqMan assays varies, approximately 50 CFU per
reaction for the Listeria monocytogenes assay [18] to
1075 CFU per reaction for the E. coli 0157 assay [19].
Nogva and Lillehaug reported TaqMan Salmonella PCR
detection kit in a real-time format and demonstrated
ARTICLE IN PRESS
1 2 3 6 7 8
200
800
400
4 5
Fig. 1. Representative results of agarose gel analysis to demonstrate the
amplification of a 358-bp product isolated from bacterial cultures and
chicken rinses. Lane 1, Low DNA Mass Ladder; lane 2, Campylobacter
jejuni (ATCC, 35918); lane 3, Campylobacter jejuni (ATCC, 33560D);
lanes 4. Campylobacter jejuni (retail chicken): lanes 5, 6 and 7,
Campylobacter jejuni (from chicken rinses); and lane 8, Campylobacter
coli (ATCC 43484).
A B C D E F G
H
Fig. 2. Standard curve for real-time PCR on the SmartCycler instrument
using 10-fold dilution of Campylobacter jejuni DNA. Line A correspond to
reference sample (Campylobacter jejuni, ATCC, 35918); B to H represents
10-fold diluted DNA (106
–100
), respectively.
Fig. 3. Application curve for serial 10-fold diluted DNA curve of C. jejuni.
A. Debretsion et al. / Molecular and Cellular Probes 21 (2007) 177–181180

5. quantification of Salmonella cells over six orders of
magnitude in pure culture [20]. It has been also reported
that real-time quantitative assays for L. monocytogenes and
C. jejuni, both of which had a linear range of quantification
of at least six orders of magnitude [20,21]. However, none
of these assays were applied to the quantitative detection of
these species in naturally contaminated foods.
C. jejuni was detected by both methods from samples
artificially contaminated with 1 or 10 CFU of C. jejuni per
10 g, after 24 h of enrichment [22]. Basically this finding is
similar to our study; however, our rtPCR detection level
was higher for the chicken rinses from poultry processing
plants and no enrichment was applied. Many factors such
as the program of the PCR cycler, combination of primers,
annealing temperature, concentration of primers and DNA
extraction protocol may affect the results of rtPCR.
In terms of sensitivity and specificity, the rtPCR assay
described here was comparable to conventional PCR and
rtPCR-based assays previously reported for the identifica-
tion of Campylobacter species from clinical specimens [10].
The rtPCR assay reported here demonstrated to be more
sensitive than the conventional culture methods (Table 2)
and significantly reduced the time taken for detection. In
conclusion, we report highly specific, sensitive, and reliable
rtPCR assays for the quantitative detection of C. jejuni
from chicken rinses without enrichment. The assays show
excellent quantification characteristics in terms of both
linear dynamic range and relative accuracy with respect to
the standard plate count technique, thus offering
a promising alternative to traditional microbiological
methods.
To the best of our knowledge, this assay is the first report
of the application of a real-time quantitative PCR assay for
the detection of C. jejuni in naturally contaminated chicken
rinses without enrichment, and this protocol can poten-
tially be adopted as a model for other food-borne
pathogens.
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