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Applied and Environmental Microbiology, September 2000, p. 4029-4036, Vol. 66, No. 9
MATFORSK, Norwegian Food Research Institute,
N-1430 Ås, Norway
Received 22 February 2000/Accepted 11 July 2000
Campylobacter jejuni is recognized as a leading human
food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the
possibility that this bacterium can enter a viable but nonculturable
state. Nucleic acid-based tests have emerged as a useful alternative to
traditional enrichment testing. In this article, we present a
5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions
381121 to 381206 of the published C. jejuni strain NCTC
11168 genome sequence was identified. When this probe was applied, the
assay was positive for all of the isolates of C. jejuni
tested (32 isolates, including the type strain) and negative for all
other Campylobacter spp. (11 species tested) and several
other bacteria (41 species tested). The total assay could be completed
in 3 h with a detection limit of approximately 1 CFU.
Quantification was linear over at least 6 log units. Quantitative
detection methods are important for both research purposes and further
development of C. jejuni detection methods. In this study,
we used the assay to investigate to what extent the PCR signals
generated by heat-killed bacteria interfere with the detection of
viable C. jejuni after exposure at elevated temperatures
for up to 5 days. An approach to the reduction of the PCR signal
generated by dead bacteria was also investigated by employing
externally added DNases to selectively inactivate free DNA and exposed
DNA in heat-killed bacteria. The results indicated relatively good
discrimination between exposed DNA from dead C. jejuni and
protected DNA in living bacteria.
Campylobacter jejuni has
come to be recognized worldwide as a leading cause of diarrheal disease
and food-borne gastroenteritis (37). C. jejuni is
zoonotic, with many animals serving as reservoirs for human disease
(21). Campylobacter cells may enter the
environment, including drinking water, through the feces of animals,
birds, or infected humans. C. jejuni is susceptible to a
variety of environmental conditions that make it unlikely to be
metabolically active for long periods of time outside the host
(21, 27). However, the organism may remain dormant in water
in a state that has been termed "viable but nonculturable" (VNC)
(32). These organisms are not able to grow but may survive
in the environment for several weeks (3, 4). Contaminated
water, raw milk, and poultry appear to be the most common vehicles of
transmission of C. jejuni in humans (37). It has
been estimated that as little as approximately 500 cells of C. jejuni can cause human illness (2, 31). However, little
is known about the virulence factors and the mechanisms of
pathogenicity (21).
There are several problems concerning Campylobacter
detection, including the low infective dosage numbers and the slow
growth rate of the organisms. The traditional methods currently used are time-consuming and laborious, requiring prolonged incubation and
selective enrichment to reduce the growth of the background flora to
enable biochemical identification. Campylobacter cells may
also enter the VNC state due to starvation and physical stress, which
may explain the failure of the culture techniques to isolate the
organisms from contaminated water samples implicated in outbreaks of
infection (14, 32). DNA-based methods such as the PCR have been increasingly used for rapid, sensitive, and specific
nonquantitative detection of C. jejuni (25, 40,
46). A quantitative assay, however, is still not available for
this organism. Quantitative assays are desirable for diagnostic,
legislative, and research purposes. Quantitation is especially
important for C. jejuni in relation to the VNC state in
elucidating otherwise elusive routes of infection. Among the various
quantitative PCR strategies available, those based on real-time
monitoring of the amplification reaction are the most accurate
(10, 12, 26).
There are, however, still some limitations to nucleic acid-based
diagnostics. A major obstacle encountered with the current DNA-based
tests is the separation of living and dead microorganisms (11,
18). In order to exploit the full potential of PCR for microbiological diagnosis, there is a great demand for sample preparation methods related to whether the organisms are living, VNC,
or dead. Most food decontamination and preservation techniques are
aimed on either inactivating or removing potential pathogens (5). The ability of the nucleic acids from the dead cells to generate PCR signals is affected by both the decontamination treatment and the organisms (11, 33). The intrinsic bacterial DNases (11, 36) may also affect the half-life of DNA. It is
important to know the stability of nucleic acids in order to interpret
whether a potential positive PCR signal is due to living pathogens.
An aspect that has not yet been widely exploited in PCR diagnostics is
the physical difference between living and dead cells. The nucleic
acids in living cells are protected because the cell walls and
membranes are intact. In dead cells, these barriers are compromised and
the nucleic acids are thus exposed to compounds added to the sample
(24, 30). The differential exposure of DNA in dead cells may
be utilized to destabilize or inactivate the exposed nucleic acids,
while the nucleic acids within living cells are protected from the
treatment by the cell membrane and wall.
In this paper, we describe the development and evaluation of a primer
and probe system directed toward a DNA fragment from C. jejuni which can be used in the quantification of C. jejuni using 5'-nuclease chemistry and the ABI Prism 7700 Sequence
Detection System from PE Biosystems (Foster City, Calif.). Using this
assay and DNA purification by magnetic beads, we investigated the
influence of nucleic acids from heat-killed bacteria on the detection
of viable C. jejuni. Finally, an approach to the reduction
of the background signal generated by DNA in heat-killed bacteria by using external DNases was evaluated. The results presented here provide
important background material for the development and interpretation of
diagnostic methods for C. jejuni in naturally contaminated
water and foods.
Bacterial strains, media, and cultures.
Thirty-two isolates
of C. jejuni, including the C. jejuni type
strain, DSMZ 4688T (Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Braunschweig, Germany), were used to test the
specificity of the primers and the probe. The isolates were collected
mostly from patients in Norway from 1996 to 1999, but the patients were
assumed to have been infected in different parts of the world.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Application of the 5'-Nuclease PCR Assay in
Evaluation and Development of Methods for Quantitative Detection of
Campylobacter jejuni
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
DNA isolation.
Samples (0.5 ml) of overnight cultures were
centrifuged at 6,000 × g for 7 min at 4°C, and the
supernatants were discarded. The pellets were stored at 
80°C. For
DNA isolation, pellets from the C. jejuni type strain were
resuspended in 1× TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0. Dynabeads
DNA Direct I (Dynal AS, Oslo, Norway), 200 µl, were then added to the
suspension of bacteria, and the bacterium-bead suspension was incubated
at 65°C for 20 min, followed by incubation at room temperature for
another 2 min. DNA bound to magnetic beads was then drawn to the wall
of the microcentrifuge tube by a magnet (MPC-E; Dynal AS) for 2 min. The supernatant containing salts, detergent, and cell debris was carefully removed without disrupting the Dynabead-DNA complex. The
beads were washed twice with a washing buffer (buffer 2 from the kit).
Finally, the DNA was removed from the beads by resuspension in 40 µl
of 10 mM Tris HCl, pH 8.0 (buffer 3 from the kit), and incubation at
65°C for 5 min. The beads, now released from the DNA, were collected
with the magnet, and the DNA-containing supernatant was transferred to
a fresh tube and used directly in the PCR.
TaqMan probe and primer design. The probe regions used were localized in the completed C. jejuni strain NCTC 11168 genome sequence (http://www.sanger.ac.uk/Projects/C_jejuni/). The Primer Express (version 1.0) ABI Prism (PE Biosystems) was used for the primer-probe design, together with guidelines from PE Biosystems (17). The GCG version of FastA (28) was used to search for similarities to other known sequences.
5'-nuclease-based PCR assay. Amplification reaction mixtures (50 µl) contained a DNA sample (1 µl); 1× TaqMan buffer A; 5 mM MgCl2; 200 µM each dATP, dCTP, and dGTP; 400 µM dUTP; 0.02 µM C. jejuni-specific probe; 0.3 µM each C. jejuni-specific primer; 1 U of AmpErase uracil N-glycosylase; and 2.5 U of AmpliTaq Gold DNA polymerase. PCR samples and controls were prepared in triplicate. Reaction tubes were MicroAmp Optical tubes, and tube caps were MicroAmp Optical caps. All consumables were supplied by PE Biosystems.
Before amplification, the PCR mixture was heated to 50°C in 5 min to let the uracil N-glycosylase destroy possibly contaminating PCR products and at 95°C for 10 min to denature the template DNA. The amplification profile was 40 cycles of 95°C for 20 s and 60°C for 1 min. Reactions were performed in the ABI Prism 7700 Sequence Detection System (PE Biosystems). Reaction conditions were programmed and data were analyzed on a power Macintosh 4400/20 (Apple Computer, Santa Clara, Calif.) linked directly to the ABI Prism 7700 Sequence Detection System using the SDS 1.6.3 application software (PE Biosystems) as described by the manufacturer. PCR products were detected directly by monitoring the increase in fluorescence from the dye-labeled C. jejuni-specific DNA probe. The TaqMan probe consisted of an oligonucleotide with a 5' reporter dye and a 3' quencher dye. The reporter dye carboxyfluorescein was covalently linked to the 5' end of the oligonucleotide. The fluorescence of the reporter was quenched by 6-carboxy-N,N,N',N'-tetramethylrhodamine, located at the 3' end. When the probe was intact, the proximity of the reporter dye to the quencher dye resulted in suppression of the reporter fluorescence. If the probe was cleaved, the reporter and quencher dyes were separated, causing the reporter dye fluorescence to increase. The amplification was plotted as
Rn, which was the normalized reporter signal
(reporter signal minus background), against the number of cycles. A
threshold signal was chosen, which intersected the amplification curves
in the linear region of the semilog plot. This gave the threshold cycle
(CT), which is defined as the PCR cycle where an
increase in fluorescence first occurred, for each amplification plot.
Different amplifications could then be compared by their respective
CTs. The CTs were plotted
against log input DNA or cells, which gave standard curves for
quantification of unknown samples and the ability to estimate the
amplification efficiency of the reaction (10, 29). The PCR
product was verified with ethidium bromide-stained 3% agarose gels
(SeaPlaque GTG Agarose; FMC BioProducts, Rockland, Maine). Agarose gel
electrophoresis was performed essentially as described by Sambrook et
al. (35).
Heat treatments. All experiments were performed in triplicate. Approximately 2.1 × 107 ± 0.4 × 107 cells of the C. jejuni type strain were used in each experiment. The C. jejuni cultures were pelleted at 6,000 × g for 7 min at 4°C, washed, resuspended in water, and transferred to microcentrifuge tubes. The tubes were then incubated at 25, 55, 72, and 100°C, and samples for PCR analysis and plating were investigated at intervals of 5 min, 1 h, 6 h, 24 h, and 5 days. The effect of heat treatment of the cultures and addition of DNase on DNA stability at room temperature was investigated. We used 1.7 × 107 ± 0.8 × 107 cells of the C. jejuni type strain with DNase and 5.0 × 107 ± 0.6 × 107 cells without DNase in these experiments. The cultures were incubated at 20, 55, 72, and 100°C for 5 min and 121°C for 15 min. One set of tubes was then incubated further at room temperature, and samples for PCR analysis were removed at intervals of 5 min, 15 min, and 30 min, 1 h, 6 h, 24 h, and 5 days. Ten units of RQ1 DNase (Promega, Madison, Wis.) and 1× DNase buffer were added to another set of tubes before the incubation at room temperature. Aliquots were analyzed after 5, 15, and 30 min and after 1, 6, and 24 h. For the PCR analysis, DNAs were purified from 10-µl aliquots taken at the respective time points and the subsequent 5'-nuclease PCR assay was performed as described above.
Stability of free DNA versus DNA in intact cells. Purified DNA from approximately 9.6 × 107 cells was added to a suspension containing 1.1 × 107 ± 0.8 × 107 living cells. The ability of DNase to selectively degrade the free DNA was investigated by addition of 10 U of RQ1 DNase (Promega) and 1× DNase buffer. After 1 h of incubation at room temperature, the cells were pelleted by centrifugation (for separate analysis of the free DNA only) and a 100-µl aliquot of the supernatant was immediately heated to 95°C for 5 min to inactivate the DNase. One microliter of the supernatant was then used in the 5'-nuclease PCR assay.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR fragment specificity.
Specific PCR primers and a probe
were designed for C. jejuni. The probe region was chosen to
optimize specificity and amplification efficiency. The putative primers
and probe were constructed using the primer express program, and then
these DNA sequences were subjected to a FastA search (28) in
the EMBL database (release 60). An 86-bp fragment including positions
381121 to 381206 of the published C. jejuni strain NCTC
11168 genome sequence (http://www.sanger.ac.uk/Projects/C_jejuni/) was
identified in these screenings. There were no known sequences in the
EMBL database with significant homology to this probe region. The most
closely related sequence had 57.7% identity and was located in the
putative gene yonO in the complete sequence of B. subtilis strain 168 (16). PCR primers were constructed
from the regions including positions 381121 to 381145 (forward) and
381206 to 381185 (reverse), while the probe includes positions 381147 to 381181 (Table 1).
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Quantitative aspects and detection limits.
Serial dilutions of
purified DNA or cells were made, and two types of standard curves were
constructed: DNA standard curves and cell standard curves. For DNA
standard curves, DNA isolated from approximately 4.2 × 107 C. jejuni cells was serially diluted 10-fold
in 1× TE buffer and subjected to PCR. The standard curve based on the
dilutions of DNA showed a linear relationship between log input DNA and threshold cycles (Fig. 2A). The slope of
the curve was 3.27, and the square regression coefficient after the
linear regression was 0.988. When a new serial 10-fold dilution from
the same DNA purification was used in a separate PCR experiment, the
slopes in the two runs were almost identical (3.20 and 3.27). When DNA from a separate isolation was used, the variation in the slopes of
the standard curves was no larger than that between different serial
dilutions from the same DNA isolation (3.28 and 3.27) and the
square regression coefficient (R2) remained
constant. For cell standard curves, approximately 4.2 × 107 cells were serially diluted 10-fold. DNA was isolated
from each dilution and subjected to PCR. The standard curve based on
six 10-fold dilutions of cells showed a linear relationship between log
input cells and the CTs (Fig. 2B). The slope of
the curve was 3.66, and the square regression coefficient after the
linear regression was 0.997. When the same serial 10-fold dilution of cells (slope, 3.68 versus 3.66) and a new serial 10-fold dilution of cells (slope, 3.46 versus 3.66) were used in separate PCR experiments, the slopes in the different runs were almost identical. When DNA from a separate isolation was used, the variation in the
slopes of the standard curves was still small (slope, 3.48 versus
3.66). The square regression coefficient (R2)
remained constant during all experiments. Both the DNA standard curves
and the cell standard curves showed a higher degree of variability
among the triplicates when the amount of template decreased. However,
the standard deviations were too small to be indicated in Fig. 2A and
B. When the detection limit in the cell standard curves was 1 CFU, the
slope of the cell standard curves were similar to that of the DNA
standard curves (e.g., 3.23 versus 3.27) and the square regression
coefficients were identical, i.e., R2 = 0.988. DNA standard curves showed that the detection limit of the PCR
assay was approximately 1 CFU per PCR (Fig. 2A). Cell standard curves
showed a detection limit of 10 CFU per PCR (Fig. 2B).
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Effect of heat treatment on C. jejuni DNA
stability.
The effect on DNA stability of prolonged exposure to
room temperature (approximately 25°C at the time of the experiments) or 55, 72, or 100°C for 5 min or 1, 6, 24, or 120 h was
investigated (Fig. 3). Both the
colony-forming ability of C. jejuni and the PCR signal
generated were determined.
|
Effect of externally added DNases on the stability of DNA in
heat-treated cells.
The effect of externally added DNases was
compared to control samples to which no DNase was added in cultures
incubated at 20, 50, 72, and 100°C for 5 min and at 121°C for 15 min. Results of incubation at room temperature (20°C) after the heat
treatments are shown in Fig. 4.
|
) and without
(
) DNase
treatment, are reported as copy numbers relative to those present
before heat treatment. The error bars show standard deviations.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Specific detection and quantification of C. jejuni. There is a requirement for rapid, quantitative, and accurate measurements of target organisms responsible for food poisoning. In the present study, a 5'-nuclease PCR system was constructed and applied to specifically detect and quantify C. jejuni. The preferred targets for pathogen detection are pathogen determinants. However, the mechanisms by which C. jejuni causes human disease is not completely understood (37). Colonization and/or infection appear to be dependent on intact motility and full-length flagella, and the flagellum gene flaA appears to be essential (22). However, the variation between the different strains was too extensive, so the flaA gene was unsuitable for design of the primers and probe necessary for a 5'-nuclease PCR assay. Empirical data suggest that a gene region in C. jejuni whose function is unknown is specific for this organism (41, 42, 45, 46). This region was compared with the most recently published sequences in the EMBL database (release 60) and the recently completed genome sequence of C. jejuni. The specificity of the constructed primers and probe was tested both by homology searches of nucleotide databases and by screening of a number of C. jejuni strains isolated from patients infected in several parts of the world. No false negatives were recorded among the 32 isolates tested, and no false positives were recorded among the other Campylobacter species or strains belonging to other genera. This demonstrates the high specificity of the designed primer-probe set. Furthermore, the amplification primers alone were also specific for C. jejuni, avoiding potential artifacts in a mixed population due to competition for the amplification primers through amplification of targets from other bacteria.
The square regression coefficients after the linear regressions indicated a good correlation between the amount of template (log input DNA or cells) and the amount of product (represented by the CTs) in the standard curves (R2 = 0.99). The linearity of the standard curves and the fact that the PCR operates with constant efficiency confirm that the assay is well suited for quantitative measurements. The detection limit of the PCR assay was estimated to be approximately 1 CFU/PCR. Our reported limit of detection is similar to those in other reports using a fluorogenic 5'-nuclease PCR assay for endpoint detection. Bassler et al. (1), obtained a detection level of approximately 50 CFU of L. monocytogenes/PCR, while Chen et al. (6) showed a detection limit as low as 2 CFU/PCR from a pure culture of S. enterica serovar Typhimurium. Quantitative DNA purification was carried out using the DNA Direct system because of both the reproducibility and the simplicity of the protocol (34). The detection limit of the DNA purification method was DNA from 10 cells per PCR. This is good recovery compared to, e.g., standard methods such as extractions with organic solvents (35). Furthermore, the 5'-nuclease assay is especially dependent on pure DNA because the amplification efficiency, and thus the quantification, can be affected by contaminants. No influence on PCR amplification efficiency or inhibition of the PCR was experienced when 12.5% of the purified material was used in the quantitative PCR.DNA as an indicator of viable C. jejuni. No real-time quantitative PCR studies have, until now, been performed on the degradation kinetics of DNA from dead bacteria. The few studies that have been done have been qualitative or semiquantitative, and endpoint analyses of PCR amplifications have been employed (11, 18). Generally, the assumption has been that the DNA molecule also persists after the bacteria are dead and thus is not a good marker for the separation of viable and dead bacteria. On the other hand, attempts have been made to use RNA as a living or dead cell marker in several studies (18, 19, 23, 36). However, the conclusions drawn from these experiments are that several assumptions have to be made in order to use RNA as a living or dead cell marker. The targeted gene has to be continuously expressed, the transcript has to be relatively unstable, and finally a specific region has to be identified in the targeted gene. Thus, if possible, it is preferable to use DNA as the target nucleic acid in relation to living versus dead cell studies.
Our data indicate a good correlation between CFU counts and the DNA-targeted 5'-nuclease assay for C. jejuni incubated in pure water at 25°C for up to 24 h (Fig. 3A). This may be due to degradation of DNA by internal DNases in the bacteria which die under these conditions. Furthermore, after 5 days, the 5'-nuclease assay indicated the presence of approximately 1% of the input DNA, while no culturable cells could be recovered. These results are in agreement with an earlier experiment in which C. jejuni strain CB258 was incubated in sterile water at 25°C and both direct viable counts and plate counts were determined. After 6 days, there was a 1- to 2-log reduction in the direct viable counts and a more-than-8-log reduction in the plate counts (20). There was an initial rapid loss of signal in all of the heat-treated samples (55°C, or above). The reason for this is still unknown, but it might be that some of the bacterial DNase activity was left. One can assume that this activity is lost over time, leading to more stable DNA. The generally rapid loss of the DNA signal has important implications for the use of DNA as a viability marker. If the heat treatment history of the sample is known, then it is possible to estimate the likelihood that DNA from bacteria present in the sample prior to the treatment will generate a positive signal. The ability of DNase to selectively degrade free DNA and DNA in heat-killed Campylobacter to further reduce the signal generated from dead cells was investigated. There were no significant differences between the DNase-treated and untreated samples at 20 or 55°C. The 20°C experiments show that DNA within intact cells is not degraded by externally added DNases and did not result in signal reduction. Although no viable cells could be recovered after the 55°C treatment, this temperature did not seem sufficient to expose the DNA to externally added DNases. For the samples heated to 72, 100, and 121°C, the addition of DNases nearly instantly reduced the amount of template by 1 log compared to the untreated samples. For these temperatures, the major fraction of DNA in the killed cells was not amplified in the assay. After 24 h, the signal from the autoclaved non-DNase-treated sample had increased. This increase may be due to reassociation of the single-stranded DNA. A reassociated double-stranded form may cover the whole amplification region, although each individual single-stranded fragment does not. Then, in the initial phase of the PCR amplification, the individual single-stranded DNA may be extended to cover the whole region. In conclusion, applying DNase treatment to reduce the noise signal generated by dead bacteria seems promising for samples that have been treated at temperatures above 72°C for 5 min or more. Work is also in progress to further increase the signal-to-noise ratio between living and dead Campylobacter cells through chemical inactivation of DNA in the dead cells (unpublished data).Comparison of culturing and 5'-nuclease PCR for detection of C. jejuni. It is evident that traditional culturing results in significant underreporting of potentially infectious C. jejuni. Pearson et al. (27) found the presence of VNC campylobacters in water as the only possibility of transmission to broiler chickens colonized by C. jejuni. Campylobacter in the VNC state may also be more resistant to food processing treatments than cells that can be cultured, especially at low temperatures (20, 32).
In contrast to traditional culturing, VNC C. jejuni may be detected through PCR amplification. However, samples may also test positive although C. jejuni has been inactivated. The direct detection of low cell numbers can also be a problem in food samples. Sensitivity, however, is apparently not a problem with water because bacteria in water samples can be concentrated in several ways (9). Quantitative detection systems are a requirement when estimating the risk of having infectious Campylobacter in food or water samples. Such risk assessments are also important for future legislative work. As demonstrated in this work, detailed studies of the degradation kinetics of DNA under different processing conditions reveal important information about what effect this DNA has on the detection of viable C. jejuni.Future developments. Adaptation of 5'-nuclease technology for quantification of C. jejuni in foods should presumably be feasible. When it comes to naturally contaminated foods, having access to proper protocols for the isolation of bacterial DNA or cells probably will be an important factor. A possible approach may be the use of magnetic beads for specific isolation of the bacteria, followed by isolation of DNA while the bacteria are still attached to the beads. Since paramagnetic beads are easy to manipulate in automated systems (A. Holmberg, A. Deggerdal, and F. Larsen, AMS '95, Third Int. Conf. Automation Mapping DNA Sequencing, abstr. A10, 1995), integrated cell concentration and DNA purification methods should be suited for high-throughput assays. Recently, there also have been efforts to miniaturize 5'-nuclease systems (13, 38) and integrate different processing steps (7, 44). Because of the microscopic size of the beads and the possibilities of performing 5'-nuclease PCR on a nanoliter scale (15), paramagnetic beads and real-time PCR may also be valuable tools in future miniaturized systems.
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ACKNOWLEDGMENTS |
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We are very grateful to Traute Vardund, BABG, National Institute of Public Health, Norway, who provided many of the Campylobacter isolates. We appreciate the advice of Lars Melin, PE Biosystems Sweden, on the design of the primers and probe for C. jejuni.
This work was financed by the Research Levy on certain agricultural products.
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FOOTNOTES |
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* Corresponding author. Mailing address: MATFORSK, Osloveien 1, N-1430 Ås, Norway. Phone: 47 64 97 01 00. Fax: 47 64 97 03 33. E-mail: knut.rudi{at}matforsk.no.
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REFERENCES |
|---|
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Bassler, H. A., S. J. A. Flood, K. J. Livak, J. Marmaro, R. Knorr, and C. A. Batt. 1995. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl. Environ. Microbiol. 61:3724-3728[Abstract]. |
| 2. | Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughs, and M. J. Blaser. 1988. Experimental Campylobacter jejuni infections in humans. J. Infect. Dis. 157:472-480[Medline]. |
| 3. |
Blaser, M. J.,
H. L. Hardesty,
B. Powers, and W. L. Wang.
1980.
Survival of Campylobacter fetus subsp. jejuni in biological milieus.
J. Clin. Microbiol.
11:309-313 |
| 4. | Blaser, M. J., D. N. Taylor, and R. A. Feldman. 1984. Survival of Campylobacter infections, p. 143-161. In J.-P. Butzler (ed.), Campylobacter infection in man and animals. CRC Press, Inc., Boca Raton, Fla. |
| 5. | Bolder, N. 1997. Decontamination of meat and poultry carcasses. Trends Food Sci. Technol. 5:384-389[CrossRef]. |
| 6. | Chen, S., A. Yee, M. Griffiths, C. Larkin, C. T. Yamashiro, R. Behari, C. Paszko-Kolva, K. Rahn, and S. A. De Grandis. 1997. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. J. Food Microbiol. 35:239-250[CrossRef][Medline]. |
| 7. | Cheng, J., E. L. Sheldon, L. Wu, A. Uribe, L. O. Gerrue, J. Carrino, M. J. Heller, and J. P. O'Connell. 1998. Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nat. Biotechnol. 16:541-546[CrossRef][Medline]. |
| 8. | Corry, J. E. L., P. E. Post, P. Colin, and M. J. Laisney. 1995. Culture media for the isolation of campylobacters, p. 129-162. In J. E. L. Corry, G. D. W. Curtis, and R. M. Baird (ed.), Culture media for food microbiology. Elsevier Science B.V., Amsterdam, The Netherlands. |
| 9. |
Giovannoni, S. J.,
E. F. DeLong,
T. M. Schmidt, and N. R. Pace.
1990.
Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton.
Appl. Environ. Microbiol.
56:2572-2575 |
| 10. |
Heid, C. A.,
J. Stevens,
K. J. Livak, and P. M. Williams.
1996.
Real time quantitative PCR.
Genome Res.
6:986-994 |
| 11. | Herman, L. 1997. Detection of viable and dead Listeria monocytogenes by PCR. Food Microbiol. 14:103-110. |
| 12. |
Holland, P. M.,
R. D. Abramson,
R. Watson, and D. H. Gelfand.
1991.
Detection of specific polymerase chain reaction product by utilizing the 5' 3' exonuclease activity of Thermus aquaticus DNA polymerase.
Proc. Natl. Acad. Sci. USA
88:7276-7280 |
| 13. | Ibrahim, M. S., R. S. Lofts, P. B. Jahrling, E. A. Henchal, V. W. Weedn, M. A. Northrup, and P. Belgrader. 1998. Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal. Chem. 70:2013-2017[Medline]. |
| 14. |
Jones, D. M.,
E. M. Sutcliffe, and A. Curry.
1991.
Recovery of viable but non-culturable Campylobacter jejuni.
J. Gen. Microbiol.
137:2477-2482 |
| 15. |
Kalinina, O.,
I. Lebedeva,
J. Brown, and J. Silver.
1997.
Nanoliter scale PCR with TaqMan detection.
Nucleic Acids Res.
25:1999-2004 |
| 16. | Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256[CrossRef][Medline]. |
| 17. | Livak, K. J., J. Marmaro, and S. J. A. Flood. 1995. Guidelines for designing TaqManTM fluorogenic probes for 5' nuclease assays. Perkin-Elmer Research News, ABI PRISMTM Sequence Detection System. The Perkin-Elmer Corp., Norwalk, Conn. |
| 18. | McKillip, J. L., L. A. Jaykus, and M. Drake. 1999. Nucleic acid persistence in heat-killed Escherichia coli O157:H7 from contaminated skim milk. J. Food Prot. 62:839-844[Medline]. |
| 19. |
McKillip, J. L.,
L. A. Jaykus, and M. Drake.
1998.
rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.
Appl. Environ. Microbiol.
64:4264-4268 |
| 20. | Medema, G. J., F. M. Schets, A. W. Van de Giessen, and A. H. Havelaar. 1992. Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni. J. Appl. Bacteriol. 72:512-516[Medline]. |
| 21. | Nachamkin, I. 1997. Campylobacter jejuni, p. 159-170. In M. P. Doyle, L. R. Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals and frontiers. ASM Press, Washington, D.C. |
| 22. |
Nachamkin, I.,
X. H. Yang, and N. J. Stern.
1993.
Role of Campylobacter jejuni flagella as colonization factors for three-day-old chicks: analysis with flagellar mutants.
Appl. Environ. Microbiol.
59:1269-1273 |
| 23. |
Norton, D. M., and C. A. Batt.
1999.
Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay.
Appl. Environ. Microbiol.
65:2122-2127 |
| 24. | O'Brien, M. C., and W. E. Bolton. 1995. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19:243-255[CrossRef][Medline]. |
| 25. | Olsen, J. E., S. Aabo, W. Hill, S. Notermans, K. Wernars, P. E. Granum, T. Popovic, H. N. Rasmussen, and O. Olsvik. 1995. Probes and polymerase chain reaction of food-borne bacterial pathogens. Int. J. Food Microbiol. 28:1-78[CrossRef][Medline]. |
| 26. | Orlando, C., P. Pinzani, and M. Pazzagli. 1998. Developments in quantitative PCR. Clin. Chem. Lab. Med. 36:255-269[CrossRef][Medline]. |
| 27. |
Pearson, A. D.,
M. Greenwood,
T. D. Healing,
D. Rollins,
M. Shahamat,
J. Donaldson, and R. R. Colwell.
1993.
Colonization of broiler chickens by waterborne Campylobacter jejuni.
Appl. Environ. Microbiol.
59:987-996 |
| 28. |
Pearson, W. R., and D. J. Lipman.
1988.
Improved tools for biological sequence comparison.
Proc. Natl. Acad. Sci. USA
85:2444-2448 |
| 29. | PE Biosystems. 1997. User bulletin 2: ABI PRISM 7700 Sequence Detection System. PE Biosystems, Foster City, Calif. |
| 30. | Riedy, M. C., K. A. Muirhead, C. P. Jensen, and C. C. Stewart. 1991. Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. Cytometry 12:133-139[CrossRef][Medline]. |
| 31. | Robinson, D. A. 1981. Infective dose of Campylobacter jejuni in milk. Br. Med. J. 282:1584. |
| 32. |
Rollins, D. M., and R. R. Colwell.
1986.
Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment.
Appl. Environ. Microbiol.
52:531-538 |
| 33. | Rossen, L., P. Nørskov, K. Holmstrøm, and O. F. Rasmussen. 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int. J. Food Microbiol. 17:37-45[CrossRef][Medline]. |
| 34. | Rudi, K., M. Kroken, O. J. Dahlberg, A. Deggerdal, K. S. Jakobsen, and F. Larsen. 1997. Rapid, universal method to isolate PCR-ready DNA using magnetic beads. BioTechniques 22:506-511[Medline]. |
| 35. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 36. |
Sheridan, G. E.,
C. I. Masters,
J. A. Shallcross, and B. M. MacKey.
1998.
Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells.
Appl. Environ. Microbiol.
64:1313-1318 |
| 37. | Solomon, E. B., and D. G. Hoover. 1999. Campylobacter jejuni: a bacterial paradox. J. Food Safety 19:121-136[CrossRef]. |
| 38. |
Taylor, T. B.,
E. S. Winn-Deen,
E. Picozza,
T. M. Woudenberg, and M. Albin.
1997.
Optimization of the performance in silicon-based microstructures.
Nucleic Acids Res.
25:3164-3168 |
| 39. |
Vanechoutte, M.,
P. Boerlin,
H. V. Tichy,
E. Bannerman,
B. Jager, and J. Bille.
1998.
Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates.
Int. J. Syst. Bacteriol.
48:127-139 |
| 40. |
Waage, A. S.,
T. Vardund,
V. Lund, and G. Kapperud.
1999.
Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay.
Appl. Environ. Microbiol.
65:1636-1643 |
| 41. | Wang, R.-F., M. F. Slavik, and W.-W. Cao. 1992. A rapid PCR method for direct detection of low numbers of Campylobacter jejuni. J. Rapid Methods Autom. Microbiol. 1:101-108. |
| 42. | Wang, R.-F., M. F. Slavik, W.-W. Cao, and P. J. Blore. 1992. Development of DNA probes specific for Campylobacter jejuni. J. Rapid Methods Autom. Microbiol. 1:83-92. |
| 43. |
Wang, W.-L.,
B. W. Powers,
N. W. Luechtefeld, and M. J. Blaser.
1983.
Effects of disinfectants on Campylobacter jejuni.
Appl. Environ. Microbiol.
45:1202-1205 |
| 44. | Waters, L. C., S. C. Jacobson, N. Kroutchinina, J. Khandurina, R. S. Foote, and J. M. Ramsey. 1998. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal. Chem. 70:158-162[Medline]. |
| 45. | Wesley, I. V., T. P. Sanderson, D. J. Larson, K. M. Harmon, J. J. Andrews, D. W. Miskimins, and D. H. Zeman. 1997. Application of multiplex polymerase chain reaction for rapid identification of Campylobacter jejuni and C. coli with reproductive failure. Am. J. Vet. Res. 58:1070-1075[Medline]. |
| 46. | Winters, D. K., A. E. O'Leary, and M. F. Slavik. 1997. Rapid PCR with nested primers for direct detection of Campylobacter jejuni in chicken washes. Mol. Cell. Probes 11:267-271[CrossRef][Medline]. |
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