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J Clin Microbiol. Nov 2004; 42(11): 5125–5132.
PMCID: PMC525216

Detection of Campylobacter spp. in Chicken Fecal Samples by Real-Time PCR

Abstract

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at −18°C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.

Thermophilic Campylobacter spp. are the major cause of bacterial enteric infection in humans in Denmark, and the incidence of infection has been increasing in all industrialized countries, particularly since 1990 (1). The reason for this dramatic rise in the number of human cases of campylobacteriosis is not known, but poultry is often implicated as a main source of human infections, due to the high prevalence of Campylobacter in broilers. Approximately 42% of all Danish broiler flocks harbored Campylobacter at the point of slaughter in 2002 (2).

As food safety has become an increasing concern for consumers, there is a growing need for fast and sensitive methods for specific detection and identification of zoonotic microorganisms. The PCR technique has several advantages over classical bacteriology with respect to detection limit, speed, and the potential for automation and has succesfully been applied to the detection of Campylobacter spp. (27, 28, 32, 37, 52).

However, the conventional PCR technique has certain drawbacks. In most analyses, the PCR products are separated on an agarose gel and vizualised on a UV board, with the size of the vizualised bands as the sole confirmation of specificity (12, 28). Further specificity can be added by performing hybridization using blotting of PCR products (13, 51) or a PCR enzyme-linked immunosorbent assay (ELISA) procedure (9, 15), but both are laborious and time-consuming processes.

A disadvantage in the diagnostic application of conventional PCR is the production of false-positive results. Theese are attributable to contamination by nucleic acids, particularly from previously amplified material (carryover) (25). Any contaminant, even the smallest airborne remnant carried over from the previous PCR procedure, may be multiplied and produce a false-positive result. The real-time mode of amplification has abolished the need to open the PCR tubes following amplification and thereby has drastically reduced the risk of carryover contamination. In addition, the liquid hybridization assay (e.g., TaqMan probes) adds further specificity to the system, comparable to hybridization techniques using blotted PCR products (16, 22). The elimination of postamplification steps increases the reliability and reproducibility of the assay and decreases the time needed to perform the procedure.

A common problem of both conventional and real-time PCR is failure of DNA amplification due to the presence of inhibitory substances in the samples. Inhibition influences the outcome of the PCR by lowering or completely preventing the amplification, producing false-negative results (54). The European Standardization Committee, in collaboration with the International Standards Organization, has proposed a general guideline for PCR testing that requires the presence of an internal control in the PCR (3). Thus, if a PCR assay is to be validated through a multicenter collaborative trial, it must contain an internal control (19). An internal control can be included either by including primers for a ubiquitous cellular gene sequence, which is expected to be present in all specimens (24, 40) or by adding synthetic DNA with primer regions identical to the target DNA (18, 45). Alternatively, DNA (7, 14) or a control organism can be added before DNA extraction. Whatever method is used, it is important to ensure that the internal control has a comparable sensitivity to inhibition as has the target DNA.

Conventional PCR assays for detection of Campylobacter species, which include an internal control, have been reported (33) and several real-time PCR assays for detecting Campylobacter jejuni have been reported (39, 44, 55), but these assays did not include an internal control. To our knowledge, only two real-time PCR assays detecting more species than C. jejuni in natural samples have been reported (21, 31), and both included two rounds of PCR.

Here we describe a fast and reliable real-time PCR analysis for rapid and sensitive detection of thermophilic Campylobacter spp. in chicken feces, including an internal control for both DNA isolation and PCR.

MATERIALS AND METHODS

Bacterial strains.

The bacterial strains used in this study and their sources are listed in Table Table1.1. Strains were stored at −80°C in brain heart infusion broth (Difco, Detroit, Mich.) containing 20% glycerol. For testing the specificity of the primers used in the assay, DNA was isolated directly from the storage medium by centrifugation of 0.1 ml of medium at 15,870 × g for 7 min and then the pellet was subjected to DNA extraction as described below. Approximately 1 ng of DNA was used per PCR.

TABLE 1.
List of strains used for validation of specificity of the Campylobacter real-time assay

Preparation of media.

Bolton broth was prepared as recommended by manufacturer. Briefly, 13.8 g of Bolton broth (CM0983; Oxoid, Basingstoke, United Kingdom) was dissolved in 500 ml of distilled water and autoclaved for 15 min at 121°C. After it had cooled to 50°C, a mixture of 25 ml of laked horse blood (SR0048; Oxoid) was added aseptically, as well as one vial of Bolton broth selective supplement (SR0183E; Oxoid).

Modified charcoal cefoperazone deoxycholate agar (mCCDA) was prepared as recommended by the manufacturer. Briefly, 45.5 g of Campylobacter blood-free selective agar (CM0739; Oxoid) was suspended in 1,000 ml of distilled water and brought to boiling to dissolve completely. The agar was autoclaved at 121°C for 15 min; after it had cooled to 50°C, two vials of CCDA selective supplement SR0155 (Oxoid) reconstituted with 2 ml each of sterile distilled water were added aseptically.

Blood agar plates were prepared as recommended by the manufacturer. Briefly, 40 g of blood agar base no. 2, (CM271; Oxoid) was dissolved in 1,000 ml of distilled water and autoclaved for 15 minutes at 121°C; after it had cooled to 45°C 50 ml, calf blood (Danish Institute for Food and Veterinary Research) was added aseptically.

Fecal samples.

Two types of fecal samples were used. A total of 111 pooled cloacal swab samples from the Danish surveillance program for Campylobacter in broilers were collected. Each batch of broilers was sampled using cloacal swabs from 10 individual broilers at slaughter. The swabs were placed in a tube containing a medium suitable for bacterial transport (Transwab; Medical Wire & Equipment Co. Ltd., Corsham, England) and transported to the laboratory by ordinary mail. On arrival, the 10 swabs were pooled in a tube containing 3 ml of sterile water. The swabs were whirl-mixed in the tube and left for approximately 5 min at room temperature to release the bacteria.

Sock samples were collected as described by Skov et al. (48). Briefly, a pair of sock samples consisted of two elastic cotton tubes (Tubigrip D no. 1451; Seton Healthcare Group plc) approximately 20 cm long. The sock samples were prewetted in water and put on by being pulled over the footwear (the ball of the foot) and turning the sock when walking around in the chicken house until all parts had been in contact with the floor. The socks were transported to the laboratory by ordinary mail, diluted 1:10 by weight in buffered peptone water, and stomached at medium level for 1 min. The socks were then left for approximately 5 min at room temperature to release the bacteria.

Detection of Campylobacter spp. by bacterial culture methods.

Thermophilic Campylobacter spp. were isolated both by direct inoculation and by selective enrichment. For the direct inoculation, 10 μl of fecal suspension was streaked onto mCCDA and incubated microaerobically (6% O2, 7% CO2, 7% H2, 80% N2) at 42°C for 18 to 24 h. For the selective enrichment, 1 ml of each sample was added to 9 ml of Bolton broth and incubated microaerobically as above at 42°C for 24 h, after which 100 μl of the broth was streaked onto mCCDA plates and incubated microaerobically, as above, at 42°C for 2 to 3 days.

Campylobacter spp. were identified by observation of Campylobacter-like growth on mCCDA plates followed by microscopy of Campylobacter-like colonies and conventional PCR of colony material. This was done by resuspending a few colonies from the plate in 100 μl of 0.9% NaCl and performing Campylobacter-specific PCR on 3 μl of the suspension as previously described (34). Briefly, PCR amplification was performed with 50-μl volumes containing 3 μl of the DNA samples, 25 μl of a PCR master mix (Promega, Madison, Wis.), 2 μl of a 25 mM MgCl2 solution, 0.5 μl of a 10-mg/ml bovine serum albumin solution, and 20 pmol of each primer. The PCR amplification was performed in a Peltier PTC-200 thermal cycler (MJ Research Inc., Waltham, Mass.). Cycling conditions were 1 cycle of 95°C for 2 min, 58°C for 1 min, and 72°C for 1 min, followed by 34 cycles of 95°C for 15 s, 58°C for 40 s, with a final cycle of 72°C for 40 s. The last elongation step lasted 5 min. An 18-μl volume of the PCR product was loaded onto a 2% agarose gel (BioWhittaker, Inc.) containing 0.1 μg of ethidium bromide per ml. The gel was visualised on an UV board. The primers used for detection of Campylobacter spp. in this PCR amplification were C412F and C1288R (28).

Internal control for DNA isolation and PCR amplification.

An internal control was included in the assay by adding a small amount (2 × 104 CFU) of the bacterium Yersinia ruckeri (DVI-Å83), which is the causative agent of enteric redmouth disease in salmonid fish species (11). The bacterium, which is not found naturally in chicken feces, was added to the sample before DNA extraction in order to have a control of the DNA extration as well as the PCR amplification. The bacterium was grown on blood agar plates at 20°C for 2 days. The bacteria on the plate were scraped together and resuspended in 10 ml of sterile 0.9% NaCl. The CFU was determined, and a dilution of 2 × 107 CFU/ml was prepared in 0.9% NaCl. A 1-μl volume of this dilution was added to each sample before DNA extraction. Aliqots of the 2 × 107-CFU/ml dilution was kept at −80°C and thawed on ice for use.

DNA isolation.

A 1-ml volume of the fecal suspension from the pooled sample was transferred to a microcentrifuge tube and centrifuged at 15,870 × g for 7 min. DNA was isolated from the pellet on a KingFisher machine (Thermo Labsystems, Helsinki, Finland) using a DNA isolation kit for blood, cells and tissue (Thermo Labsystems) as specified by the supplier. Briefly, lysis buffer from the kit was added to the pellet in the microcentrifuge tube together with 2 × 104 CFU of Y. ruckeri (internal control), and the mixture was vortexed and left at room temperature for 5 min. Lysed samples, magnetic beads, washing solution, and resuspension solution were added to an ELISA format plate (Thermo Labsystems). The plate was placed in the KingFisher machine. C. jejuni reference strain CCUG 11284 (105 CFU) and Y. ruckeri (2 × 104 CFU) were used as positive controls, whereas sterile water served as the negative control. DNA preparations were used immediately for PCR amplification or stored at −20°C.

Real-time PCR.

The PCR master mix was prepared in a large volume, and 22-μl volumes were prealiquoted into PCR tubes (Bio-Rad Laboratories, Hercules, Calif.) and kept at −20°C until used. The 22 μl of master mix consisted of 12.5 μl of PCR Master Mix (ABgene House), 10 pmol each of campF2 and campR2, 3 pmol each of yersF1 and yersR1, 10 pmol of the campP2 probe, and 10 pmol of the yersP1 probe. A 3-μl volume of sample DNA was added to the master mix.

Primers and probes for both Campylobacter spp. and Y. ruckeri were located in the 16S rRNA gene sequence. Sequences were chosen by alignment of 16S rRNA gene sequences from Campylobacter spp., Arcobacter spp., and Yersinia spp. using CLUSTALW Multiple Alignment. Recommended guidelines for designing fluorogenic probes for 5′ nuclease assays were followed (29). Campylobacter primers were designed to capture the four Campylobacter species C. jejuni, C. coli, C. lari, and C. upsaliensis, but not necessarily only these species. Y. ruckeri primers were designed to capture exclusively Y. ruckeri. Candidate primers and probes were tested theoretically by comparison to sequence databases (BLAST Sequence Comparisons [National Institutes of Health]). The primers selected for detection of Campylobacter spp. were campF2 (5′-CACGTGCTACAATGGCATAT-3′) and campR2 (5′-GGCTTCATGCTCTCGAGTT-3′), and the TaqMan probe was campP2 (5′-FAM-CAGAGAACAATCCGAACTGGGACA-BHQ1-3) (MWG Biotech AG, Ebersberg, Germany). The primers selected for detection of the internal control were yersF1 (5′-GGAGGAAGGGTTAAGTGTTA-3′) and yersR1 (5′-GAGTTAGCCGGTGCTTCTT-3′), and the TaqMan probe was yersP1 (5′-Hex-GCGAGTAACGTCAATGTTCAGTGC-BHQ1-3′) (MWG Biotech AG).

The PCR amplification was performed on an Icycler (Bio-Rad) as follows: 1 cycle at 95°C for 10 min, followed by 40 or 50 cycles of 15 s at 95°C, 30 s at 58°C, and 30 s at 72°C, with a final cycle of 5 min at 72°C. After real-time data acquisition, the baseline cycles for the FAM signal were set from cycle 2 to one cycle below the cycle at which the first signal appeared and the threshold value at the point at which the fluorescence exceeded 10 times the standard deviation of the mean baseline emission. For the HEX signal, the baseline cycles were set from 5 to 25 and the threshold value at the point where fluorescence exceeded 10 times the standard deviation of the mean baseline emission. All samples were run in duplicate. Samples in which both duplicates had a threshold cycle (Ct) value below 40 were regarded as positive.

Fecal inhibitor.

Fecal suspension from samples negative for Campylobacter tested by both culture and real-time PCR was centrifuged in a microcentrifuge at 15,870 × g for 7 min, and the supernatant was used as a fecal inhibitor in PCR amplifications. Various amounts of inhibitor were added to the tubes containing master mix and a positive DNA sample, and real-time PCR was performed.

Sensitivity.

To determine the detection limit of the assay in both swab and sock samples, we collected the fecal suspensions from 20 pooled swab samples and from socks that were negative both by selective enrichment culture and by PCR. Serial dilutions from a broth of the C. jejuni reference strain CCUG 11284 or the C. coli reference strain CCUG 11283 were added to the pellet from 1 ml of the Campylobacter-negative fecal samples, and the material was used for DNA isolation and PCR.

For data management and calculations, Microsoft Excel 97 SR 2 and SAS Systems version 8 (SAS, Cary, N.C.) were used. The level of agreement according to precision was expressed as the kappa statistic, defined as the proportion of potential agreement beyond chance exhibited by two tests. The diagnostic specificity was calculated as d/(b + d), where d is the number of samples negative by both PCR and culture and b is the number of samples positive by PCR but negative by culture. The level of agreement between two tests was calculated as (a + d)/n, where a is the number of samples positive by both PCR and culture, d is the number of samples negative by both methods, and n is the total number of samples under examination (36, 49). The performance of the real-time PCR was compared to that of culture with direct plating and that of culture by selective enrichment by using McNemar's test (43).

RESULTS

Specificity and sensitivity of the PCR assay.

The specificity of the PCR assay was tested against a panel of Campylobacter and non-Campylobacter DNA templates. The assay detected C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus, and C. hyointestinalis but none of the other Campylobacter species tested. No signal was observed for any of the Arcobater, Helicobacter, or other non-Campylobacter species tested. The sensitivity of the assay was evaluated using Campylobacter-negative fecal samples spiked with various amounts of Campylobacter. Figure Figure11 shows a plot of the Ct values against the concentrations of C. jejuni (Fig. (Fig.1A)1A) and C. coli (Fig. (Fig.1B)1B) in spiked negative fecal swab samples and negative fecal sock samples, respectively.

FIG. 1.
Log-linear plot of the amplification profile for fecal samples spiked with serial 10-fold dilutions of C. jejuni in eight replicates (A) and C. coli in six replicates (B). The quantity of Campylobacter in the samples from which the DNA is isolated is ...

At 7 CFU of C. jejuni/ml, three of the eight PCR amplifications had a Ct value below 40, and at 12 CFU of C. coli/ml, four of the six PCR amplifications had a Ct value below 40. At 70 CFU of C. jejuni/ml and 120 CFU of C. coli/ml, all PCR amplifications had a Ct value below 40; hence, all reactions were positive. Several different spiking series were performed using both negative swab samples and negative sock samples. For both types of samples, the detection limit for the PCR assay in spiked fecal samples was 50 to 100 CFU/ml of fecal suspension for both species.

Diagnostic specificity.

A total of 111 of pooled samples from the Danish poultry surveillance program of Campylobacter in broilers were tested for the occurrence of Campylobacter spp. both by culture and by real-time PCR directly from the feces. The results for the 111 samples are shown in Table Table2.2. A total of 41 samples were positive by real-time PCR and 43 samples were positive by culture with selective enrichment. Six samples were positive by culture with selective enrichment but negative by real-time PCR. Four samples were positive by real-time PCR but negative by culture with selective enrichment.

TABLE 2.
Diagnostic specificity of Campylobacter real-time PCR detection

A total of 32 samples were positive by direct plating, with no culture-positive/real-time PCR-negative samples.

McNemar's test showed no difference in performance between real-time PCR and culture by selective enrichment (P < 0.5), whereas the difference between real-time PCR and direct plating was statistically significant (P < 0.01). The diagnostic specificity for the comparison to culture by selective enrichment was 0.96, with an agreement of 0.92.

For the direct plating, all positive samples were also positive by real-time PCR and selective enrichment but 15 samples were negative by direct plating and positive by either real-time PCR or selective enrichment. A comparison of the results for these 15 samples is shown in Table Table3.3. Five of the samples were negative by direct plating, six were negative by both direct plating and real time PCR, and the other four were negative by both culturing methods.

TABLE 3.
Results from 15 divergent samples

Variabilities.

Fecal samples were spiked with C. jejuni in the range of approximately 40 to 40,000 CFU/ml of fecal suspension, and DNA was isolated. To obtain values for the intra- and interassay variation of the real-time PCR assay, DNA from the spiking series was subjected to PCR in quadruplicate, with four different mixes performed on different days. The results are presented in Table Table4.4. The coefficient of variation (Cv) for the four different intra-assay experiments ranged from 0.99 to 6.17%, while the interassay variation ranged from 0.93 to 6.3%. The interassay variation for the whole assay, including the DNA isolation procedure, was also estimated by performing PCR on DNA isolated from eight spiking series. The results are presented in Table Table5.5. The Cv values in this test varied from 3.04 to 4.65%. Corresponding results for the internal control were an intra-assay coefficient of variation of 0.61 to 7.6%, an interassay variation of 0.41 to 7.6%, and and interassay variation including DNA isolation of 3.24 to 9.0%, with the highest variation being found in samples with smallest amount of Campylobacter. When intra-assay and interassay variabilities were measured for dilutions of pure DNA in the range of approximately 102 to 106 genomic copies, the Cv values for intra-assay and interassay variabilities were consistently lower than 2%, except for one series at the lowest copy numbers, where the Cv was 3.81%.

TABLE 4.
Intra- and interassay variabilities of Campylobacter real-time PCR
TABLE 5.
Interassay variabilities including the DNA extraction procedure

Inhibition.

To detect the effect of fecal inhibitors on the PCR of both Campylobacter and internal control, DNA from fecal samples spiked with 40 to 34,000 CFU of C. jejuni per ml was subjected to real-time PCR in triplicate with increasing amount of fecal inhibitor added. An example of this kind of experiment is given in Table Table6.6. The table shows that in samples that were spiked with 40 CFU of C. jejuni per ml, the Campylobacter signal disappeared with the addition of 0.75 μl of fecal inhibitor to the reaction mixture. The signal for the internal control did not disappear, but the Ct value rose 0.9 cycle from 29.6 to 30.5. In samples spiked with 160 CFU of C. jejuni per ml, the Campylobacter signal disappeared when 1.5 μl of fecal inhibitor was added to the reaction mixture. The signal for the internal control did not disappear, but the Ct value rose 1.4 cycles from 28.9 to 30.3. When the sample contained 3,400 CFU of C. jejuni per ml, the Campylobacter signal disappeared when 3 μl of fecal inhibitor was added to the reaction mixture and the Ct value for the internal control rose 3.5 cycles. Finally, in the sample that contained 34,000 CFU of C. jejuni, the Campylobacter signal persisted even when 3 μl of fecal inhibitor was added to the reaction mixture; in contrast, the signal for the internal control disappeared. In eight experiments, the rise in the Ct value for the internal control at the point of disappearance of Campylobacter was from 3.3 to more than 13 at a Campylobacter concentration of 800 to 1,200 CFU/ml.

TABLE 6.
Effect of faecal inhibitor on Campylobacter and internal control PCR

To find the mean and variation of the Ct value for the internal control in samples containing no inhibitor, real-time PCR was performed on DNA from 50 negative fecal swab samples. The Ct values for the internal control in negative fecal samples containing no inhibitor were between 26.6 and 32.5. The average was 28.6 with a standard deviation of 1.1. That none of the samples contained any inhibitor was shown by performing real-time PCR on a weak positive control with or without the addition of 5 μl of the DNA isolated from the negative sample. When the Ct values for the pairwise reactions were compared, no significant difference was found for any of the negative samples.

DISCUSSION

The method presented in this paper was developed for routine detection of a large number of samples on a daily basis. This was done by using a semiautomated DNA isolation procedure, and all handling of samples after the centrifugation step was performed using multichannel pipettes. Furthermore, the real-time PCR mixture was prepared in a large volume, aliquoted into PCR tubes, and frozen until used. In this way, the performance of the mixture could be tested before use with routine samples.

To our knowledge, no real-time PCR assay detecting thermophilic campylobacters has been described in the literature. The primers and the probe of this assay were constructed to detect at least the four thermophilic campylobacters C. jejuni, C. coli, C. lari, and C. upsaliensis, which are the Campylobacter species mainly found in poultry (4, 53). The primers and probe also detected C. helveticus and C. hyointestinalis, which are found mostly in cats and dogs (50) and pigs (41), respectively, and are thus of neglible importance in poultry.

The sequences of the Campylobacter primers and probe are identical to sequences from C. lanienae, which should be detected by the assay, but this was not tested empirically. C. lanienae was isolated first from abattoir workers (30) and later in bovine feces, but it has not been isolated from broilers (47).

The detection limit was 50 to 100 CFU/ml of fecal suspension, corresponding to 4 to 8 CFU per PCR when using spiked control material from swab or sock samples. A 1-ml sample of fecal suspension from the fecal swab samples contained 0.2 g of fecal material, and the detection limit thus corresponds to 250 to 500 CFU/g of feces. In comparison to other Campylobacter real-time assays, Yang et al. (55) found a sensitivity of 6 to 15 CFU per PCR in a real-time assay for detecting C. jejuni in poultry, milk, and environmental water, while a sensitivity of 103 CFU/g of stool sample was found in a duplex real-time SYBR Green assay (10).

A sensitivity of approximately 12 genome equivalents per PCR was found in a real-time assay for detecting C. jejuni in enrichment cultures from foods (46), and less than 10 CFU C. jejuni/ml of chicken rinse water was detected in a SYBR Green real-time PCR assay on enrichment cultures (6). With conventional PCR, Campylobacter assay sensitivities of 35 to 120 CFU/ml of chicken dung (42), 36 CFU/ml of chicken fecal suspension (34), and 104 CFU/g of bovine feces (20) in direct PCR have been obtained. In a PCR ELISA, a sensitivity of 40 CFU/ml of chicken carcass rinse water was obtained for C. jejuni and C. coli (17). Thus, the sensitivity of the real-time PCR assay was comparable to both other real-time assays and conventional PCR assays.

The assay was performed with 111 pooled swab samples and compared to culture by direct plating and by selective enrichment. The selective enrichment was the more sensitive of the two culture assays. A higher sensitivity of selective enrichment compared to direct plating has also been found by others (5, 35). Six samples were positive by selective enrichment but negative by both the PCR assay and direct inoculation. Thus, the six samples contained very little Campylobacter since the sensitivity of direct plating was 36 CFU/ml of fecal material (34). Nine samples were negative by direct plating but positive by real-time PCR. Ct values for these samples were all higher than 29, reflecting the fact that the amount of Campylobacter was not very large. Four of the samples were also negative by selective enrichment. It is possible that these samples contained viable but nonculturable or dead Campylobacter organisms.

McNemar's test showed no difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity for the comparison to the enrichment culture was 0.96, with an agreement of 0.92. A discrepancy was found in 8.1% of the samples; thus, there was a good agreement between the two methods.

Previous comparisons of both real-time PCR assays and conventional PCR assays with culture have yielded different results. Similar sensitivities between direct culture and conventional PCR assays have been reported (27, 34), as well as between selective enrichment and a real-time PCR assay detecting C. jejuni, but in another study the real-time PCR assay was performed on enrichment broth and not directly from samples (46). Others have reported the same sensitivity of PCR and selective enrichment for the species C. jejuni and C. coli but a higher sensitivity of the conventional PCR assay (26) or the real-time PCR assay (31) for other Campylobacter species due to the bias of selective enrichment toward C. jejuni and C. coli. A significantly higher sensitivity for the real-time PCR assay detecting C. jejuni in poultry, milk, and environmental water was found compared to selective enrichment culture; this was explained by the occurrence of mainly dead or nonculturable bacteria in samples collected under suboptimal conditions (55). Thus, the sensitivity of PCR assays and culture assays are dependent on factors such as the Campylobacter species detected, method of culture, and sample treatment, which should be taken into consideration when comparing PCR assays with culture methods.

The Cv values for the intra-assay and interassay variabilities did not differ significantly. This may indicate that the main reason for variation is not due to pipetting errors in setting up the PCR assay but may be caused by inhibitors and contaminants from the fecal samples. These inhibitors and contaminants may interfere with uniform and consistent dilution as well as the amplification of target DNA. This is supported by the low Cv values of intra-assay and interassay variations with purified Campylobacter DNA. When interassay variation was measured with the DNA isolation included, the Cv values was on average higher than the values for interassay variation without the DNA isolation included, but overall, the variabilities of the whole assay were similar to those found in other studies (8, 23, 38).

The function of the internal control as an indicator of the presence of inhibitor was evaluated. This is an important isssue because some positive samples might be detected as false negative if the PCR amplification of the internal control is more resistant to the inhibitors than is the PCR amplification of the target. On the other hand, if the PCR amplification of the internal control is more sensitive to inhibition than is the PCR amplification of the target, true-negative samples might have to be retested, because the internal control has disappeared. Therefore, it is important to ensure that the PCR amplification of the internal control has the same sensitivity to inhibition as has the PCR amplification of the target at the desired sensitivity.

Apparently the DNA isolation method performed on swab samples yielded very pure DNA since 50 negative samples investigated did not contain any inhibitor of the PCR assay. However, this does not exclude the possibility that in samples containing larger amounts of feces, blood samples, or, e.g., cecal material, some inhibitors might be retained in the DNA. We tested the effect of fecal inhibitors on the Ct values for both the Campylobacter signal and the internal control signal. The experiments were repeated several times with different concentrations of C. jejuni and C. coli, and although great variations were seen, it was a general pattern that the rise in the Ct value for the internal control at the point of disappearance of Campylobacter increased with increasing concentrations of Campylobacter in the sample. We found that in fecal samples containing 800 to 1,200 CFU of Campylobacter per ml, the Ct value of the internal control rose at a minimum 3.3 cycles at the point where the Campylobacter signal disappeared due to heavy loading of fecal inhibitor.

If a rise in the Ct value for the internal controls should be used for detection of inhibition, it would be necessary to know the variation of the Ct value for the internal control in samples containing no inhibitor. We found that the Ct values for the internal control in 50 negative fecal samples containing no inhibitor spanned 5.9 cycles between 26.6 and 32.5, with an average of 28.6. We have found that a Ct value of the internal control between 26 and 32.5 cycles is acceptable in Campylobacter-negative samples, but Campylobacter-negative samples with a higher Ct value of the internal control should be reinvestigated.

In some samples containing less than 800 to 1,200 CFU/ml, the amount of inhibitor could be large enough to abolish the weak Campylobacter signal but still too small to cause a significant rise in the internal control; therefore, these samples would appear as false negative. However, in most of the samples, the presence of inhibitors of the PCR assay is detected.

In summary, we have described a real-time PCR assay that directly detects thermophilic campylobacters in chicken feces in less than 4 h. The assay is easy and reliable and contains an internal control in the PCR, which makes the assay suitable for testing in a multicenter collaborative trial, according to recommended guidelines from The European Standardization Committee.

Acknowledgments

We thank Elsebeth Boulund Sørensen and the technical staff of the Danish National Campylobacter Surveillance Laboratory for excellent technical assistance.

This work was partly financed by the Danish Agricultural and Veterinary Research Council (SUE project 2027-00-0008), by the Danish Poultrymeat Association, and by the Danish Ministry of Food, Agriculture and Fisheries (DFFE project 3401-66-03-5).

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