APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1992, p. 3804-3808

Vol. 58, No. 12

0099-2240/92/123804-05$02.00/0

Rapid and Sensitive Detection of Campylobacter spp. in Chicken Products by Using the Polymerase Chain Reaction B. A. J. GIESENDORF, 12* W. G. V. QUINT,1'3 M. H. C. HENKENS,2 H. STEGEMAN,2 F. A. HUF,2 AND H. G. M. NIESTERS" 3 Department of Molecular Biology, Diagnostic Center SSDZ, PaO. Box 5010, 2600 GA Delft, 1 Department of Microbiology, State Institute for Quality Control of Agricultural Products, 6700 AE Wageningen, 2 and University Hospital Dijkzigt, 3015 GD Rotterdam, 3 The Netherlands Received 13 May 1992/Accepted 29 September 1992

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacterjejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.

fragment by enzymatic amplification of the target DNA in vitro. Although the PCR is by now widely used in clinical settings, only a few reports of its use in food microbiology have been published (4, 6, 23, 25, 26). One important reason for this is that the complex composition of food matrices can hinder the PCR and lower its sensitivity. In addition, the level of contamination of food products is lower than that in clinical samples. In this paper we describe an improved method for the rapid detection and identification of Campylobacter spp. in chicken products; this method is a combination of a short enrichment culture and the PCR technique. A specific PCR and probe hybridization assay based on the 16S rRNA gene sequence was developed.

Campylobacter spp. are recognized as one of the most important causes of acute diarrheal disease in humans throughout the world (1, 11, 17, 24). The three species most commonly regarded as being involved in campylobacter enteritis are Campylobacterjejuni, Campylobacter coli, and Campylobacter lari. Because the thermotolerant Campylobacter spp. are closely related, differentiating C. jejuni and C. coli is difficult and solely based on the hippurate hydrolysis test (11). This distinction, however, is only accurate in approximately 90% of all cases (13). C jejuni, the most predominant species, has been demonstrated in stools of up to 14% of all patients with diarrhea and is responsible for as many as two million cases of infection in the United States yearly (1, 9). Campylobacter spp. are usually transmitted by raw milk and animal meat, particularly chicken. Infection can be caused by a few hundred bacteria (17). Detection of Campylobacter spp. depends upon isolation of the bacteria with selective media followed by identification and determination of antimicrobial susceptability patterns (11, 18); a presumptive identification, which cannot discriminate between species, requires 4 days with aselective agar. The comparison of 16S rRNA sequences not only is a powerful method for the systematic classification of microbial organisms but also provides an opportunity to develop specific DNA-RNA probes (8, 15, 22, 27). The rRNA, an essential part of prokaryotic and eukaryotic ribosomes, is genetically stable and consists of conservative and variable regions. The latter may vary considerably among different bacterial species and can therefore be targets for specific oligonucleotide probes. In this study the 16S rRNA gene of C. jejuni and parts of the 16S rRNA genes of C. coli and C. lan were sequenced. Sequence comparison of the variable regions indicated several specific oligonucleotides that could be used as primers and probes for the polymerase chain reaction (PCR). The recently developed PCR technique can detect a specific gene *

MATERIALS AND METHODS Bacterial strains. The strains used in this study (Table 1) were obtained from the American Type Culture Collection (Rockville, Md.), Rikilt-DLO (Wageningen, the Netherlands), and the Diagnostic Center SSDZ (Delft, the Netherlands). Culture conditions and enumeration techniques. Chicken products, mainly wings, were obtained from local supermarkets and poultry shops. A total of 10 g of the skin was suspended 1:5 in Brucella broth (Difco) in a stomacher. Samples of 10 ml were enriched in 40 ml of Preston broth (Oxoid) and incubated microaerobically at 42.5°C for 18 h. Then 100 ,ul was taken for nucleic acid isolation and subsequent PCR, and another 100 ,ul was plated on Skirrow agar and incubated under microaerophilic conditions at 42.5°C for 48 h. Typical colonies were examined under a phase-contrast microscope, and typing was confirmed by Gram staining and biochemical tests (11). For the preparation of inoculated chicken products, samples were sterilized by irradiation with 60Co. Samples were checked for sterility by enrichment culture only. Volumes of 1 ml of an overnight broth culture of C. jejuni 85Y242 were mixed with 10-g samples in 40 ml of Brucella broth to achieve inoculum levels of 5 to 5,000 cells per g. After 18 h

Corresponding author. 3804

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USE OF PCR TO DETECT CAMPYLOBACTER SPP. IN CHICKEN TABLE 1. Strains used in this study Species and strain

A

Remarks

Campylobacterjejuni ATCC 29428 85Y242 ............... 85Y500 ............... 85Y401 ...............

Clinical Clinical Clinical Clinical Clinical

86Y092 ...............

Y354/85 LAI ...............

isolate isolate isolate isolate isolate

Campylobacter lari ATCC 35221 EF 10370 ...............................

Rikilt, Wageningen

Campylobacter coli ATCC 33559

6247/87

...............................

Rikilt, Wageningen Clinical isolate

...............................

ES 4658/88 ...............................

Clinical isolate

Campylobacter fetus .............................. Clinical isolate Helicobacter pylori ATCC 43504 Salmonella typhimurium .............................. Clinical isolate Salmonella enteritidis .............................. Clinical isolate Yersinia enterocolitica 03

Shigella flexneri

............................

..............................

agglomerans

Enterobacter cloacae Klebsiella

pneumoniae

Proteus mirabilis

............................

..............................

..............................

..............................

.................

Rikilt, Wageningen Rikilt, Wageningen Rikilt, Wageningen

Rikilt, Wageningen

Rikilt, Wageningen

Streptococcus agalactiae ............................. Rikilt, Wageningen Streptococcus faecalis

8043

..........................

396 CGTGGAGGATGACACTTTTCGGAGCGTAA 424

H. pylori

374.

E. col i S. enteritidis S. typhimurium Y. enterocoli tica

402 399 399 397

C. jejuni C. coli C. laridis

790

H. pyl ori E. S. S. Y.

... .... ... ....

AGG.... A.. TT .

T.T .A. .AGGCC...GTT.... T.T .A. .AGGCC . GTT .... T.T .A. .AGGCC...GTT.... TGT. .. AGGCC...GTT....

..T ........... CT ...... .

.....

402 430 427 427 425

GG .........

827

..TG 778

coli enceritidis

821

ryphimurium enterocolitica

818 815

818

G.TG

AG............

TA

TCTC

816

CG... T.GA.G.T.T.CC..T-.AGGCG.GGC.TCC.G 858 .CT .T.GA.G.T.T. CC. .T-.AGGCG.GGC.TCC.G 855 .CT... .T.GA.G.T.T.CC..T-.AGGCG.GGC.TCC.G 855 .CG... T.GA.G.T.T.CC..T-.AGGCG.GGC.TCC.G 852

FIG. 1. Alignment of 16S rRNA sequences of Campylobacter and several related microorganisms in the region before V3 (A) and in V5 (B) (positions 402 through 430 and 821 through 858, respectively). Only nucleotides different from those of C. jejuni are indicated. Dashes indicate deletions. The underlined sequences in panels A and B are primer C442 and the complementary strand of primer C490, respectively. spp.

Clinical isolate

Pseudomonas aeruginosa ............................. Rikilt, Wageningen Staphylococcus aureus ATCC 6538 P Staphylococcus epidermidis Ec/36

C. jejuni C. coli C. laridis

Clinical isolate

Escherichia coli ATCC 11775 Listeria monocytogenes 4b NCTC 10527 Bacillus cereus subsp. mycoides ATCC 11778 Bacillus subtilis ATCC 6633 Clostridium perfringens ATCC 12916 Enterobacter

3805

Rikilt, Wageningen

of incubation, 100-,I samples were taken for the PCR. The number of viable thermotolerant Campylobacter spp. cells in the broth was determined by direct plating of 100 ,l of bacterial suspension onto Skirrow agar. Extraction of nucleic acids. Nucleic acids were extracted by standard procedures (7). Briefly, 100 pl of bacterial pellet was suspended in 150 of 50 mM glucose-10 mM EDTA-25 mM Tris-HCl (pH 8.0) and incubated for 5 min at room temperature with 10 ,1u of lysozyme (125 mg/ml). Then 12.5 ptl of proteinase K (1 mg/ml) and 12.5 of 10% sodium dodecyl sulfate (SDS) were added, and incubation was continued for 30 min at 37°C. Nucleic acids were extracted with phenol, phenol-chloroform-isoamyl alcohol (25:24:1), and then chloroform-isoamyl alcohol (24:1). Nucleic acids were precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 96% ethanol, washed, dried, and resuspended in double-distilled water. Nucleic acid concentrations were estimated on a 1% agarose gels with lambda DNA as a reference. DNA cloning and sequencing. The 16S rRNA genes of C. jejuni, C. coli, and C. lan were amplified in the PCR by using primers in the conservative regions within the 16S rRNA genes (3' primer, aaaggatcctgcagACCTTGITACGACTTCA CCCCA; 5' primer, atattggatccGAGAGTlTGATCCTGG CTCAG). Linkers (lowerca1seletters) containing restriction sites for BamHI and PstI (underlined) were attached to the

primers. After digestion, a 1.6-kb fragment was cloned into phage M13mpl8 and M13mpl9 and transfected into Escherichia coli PC2495 (7). The sequences of the 16S rRNA genes of C. jejuni, C. coli, and C. lan were determined by using the Pharmacia T7 polymerase sequencing system (Pharmacia, Uppsala, Sweden) and [a-32P]dATP (Amersham). Sequencing was performed according to the instructions of the manufacturer. Gels were wrapped in plastic wrap and exposed to X-Omat S film (Eastman Kodak Co., Rochester, N.Y.) at -80°C for 24 h. Sequence comparisons were carried out with Microgenie software (Beckman). Primers and probes were synthesized on an Applied Biosystems 381A DNA synthesizer by the ,B-cyanoethyl phosphoamadite method. Pretreatment of samples for amplification. Nucleic acids were extracted from the enriched culture and culture dilutions essentially as described by Boom et al. (2). Briefly, a 100-,u aliquot was added to 1 ml of buffer (120 g of guanidinium isothiocyanate, 100 ml of 0.1 M Tris [pH 6.4], 22 ml of 0.2 M EDTA [pH 8.0], 2.6 g of Triton X-100) and 20 p,l of Celite (10 g in 50 ml of H20 and 500 pul of 32% [wtlvol] HCl). The samples were vortexed and incubated at 65°C for 10 min. After centrifugation at 12,000 x g (Eppendorf centrifuge, fixed angle), the pellets were washed twice with 120 g of guanidinium isothiocyanate-100 ml of 0.1 M Tris (pH 6.4), twice with 70% ethanol, and once with acetone. The pellets were dried and suspended in 100 ,ul of 10 mM Tris (pH 8.0). After incubation at 56°C for 10 min, the samples were centrifuged at 12,000 x g; 25 pI of the supematant was used for the PCR. PCR. Two synthetic oligonucleotides, one homologous to the region before V3 and one homologous to V5, were selected and used to generate a 426-bp DNA fragment from the C. jejuni 16S rRNA gene in the PCR (Fig. 1). The isolated Campylobacter spp. target DNA was amplified essentially as described by Saiki et al. (16). A spatial separation of the different steps of the technique was routinely used to prevent contamination of the samples (5). Distilled water and noninfected samples were used as negative controls and C. jejuni DNA was used as a positive control in each experiment. All results were confirmed by repeat testing. A standard PCR

3806

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GIESENDORF ET AL.

C. jejuni C. coli C. lari

437 AGGG----AAG--AAT--------TC--T-GAC-GGTACCTAAG 462

H. pyl ori

427 ..--------... AT.---------------.--..-------T.... C 449

E.

coli

451

GAGTA

S.

enreritidis

448

TGTTGTG.TT

AACCGCAGCAAT.-

448 typhimurium Y. enterocolitica 445

TGTTGTG.TT

AACCGCAGCAAT.-...-.T

S.

TT..

.CATAA.G.TT.

ACCTTTGC.

AT.-.. .-.T

AACCTTTG.GAT.-.

CGCA

...-.T.... .CGCA

.-.T.

CGCA

492

489 489

TCGCA 486

FIG. 2. Alignment of the Campylobacter 16S rRNA sequences in the V3 region (E. coli 16S rRNA positions 451 through 492). The 16S rRNA sequences of related microorganisms are also shown. Only nucleotides different from those of C. jejuni are indicated. Dashes indicate deletions, and the underlined sequence is probe C631.

pl) contained 25 ,ul of the test sample, 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 2.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 200 p,M each deoxynucleoside triphosphate, 50 pmol of each primer, and 1 U of Taq DNA polymerase (Promega). This solution was covered with 3 drops of mineral oil (no. M3516; Sigma) to prevent evaporation and subjected to 40 cycles of amplification in a PCR processor (Biomed). Each cycle consisted of 1 min at 94°C, 1 min at 52°C, and 1 min at 74°C. Analysis of the amplified samples. Amplified samples were analyzed by electrophoresis on 2% agarose gels with ethidium bromide staining (7). For Southern blotting, the gel was denatured in 0.4 N NaOH and transferred to a nylon membrane (Hybond N+; Amersham). For dot spots, 45 RI of each PCR sample was denatured with 165 RI of 0.5 N NaOH at 65°C. Each sample was neutralized by adding 30 pl of 10 N ammonium acetate on ice. The samples were spotted with a Bio-Dot apparatus (Bio-Rad Laboratories, Richmond, Calif.). The oligonucleotide probes were labelled by standard procedures with [-y-32P]ATP (Amersham) and T4 polynucleotide kinase (Pharmacia). Standard hybridization techniques (7) were used. Briefly, the membranes were prehybridized in 5x SSC (lx SSC is 15 mM sodium citrate-150 mM NaCl), 5x Denhardt's solution (lx Denhardt's solution is 0.02% polyvinylpyrrolidone-0.02% Ficoll-0.02% bovine serum albumin), 0.5% SDS, and 100 ,ug of denatured, sonicated herring sperm DNA per ml at 42°C for 2 h. Samples were hybridized for 16 h at 42°C in S x SSC, 5 x Denhardt's solution, 0.5% SDS, 100 ,g of denaturated, sonicated herring sperm DNA per ml, and 2 x 106 cpm of a 32P-5'-endlabelled oligonucleotide probe per ml. After hybridization, the blots were washed twice for 15 min at 420C in 2x SSC-0.1% SDS and once for 15 min at 55°C in 0.5x SSC-0.1% SDS. The blots were autoradiographed for 3.5 h with Kodak Royal X-Omat films and Dupont intensifying screens at -80°C. reaction mixture (100

RESULTS Sequencing of the 16S rRNA Gene. The sequences of the 16S rRNA genes from a number of enterobacteria were determined after amplification with conserved primers and subsequent cloning into M13mpl8 and M13mpl9. The C. jejuni 16S rRNA gene sequence and the 16S rRNA sequences of Salmonella typhimurium, Salmonella enteritidis, Yersinia enterocolitica, Helicobacterpylori, C. coli (partial sequence), and C. lan (partial sequence) were determined. The 16S rRNA sequence of E. coli was obtained from GenBank, and partial 16S rRNA sequences of C. coli, C. lan, and Campylobacterfetus were obtained from Romaniuk et al. (15) and Thompson et al. (20).

FIG. 3. Specificity of the PCR with different Campylobacter strains. The amplification products were hybridized with probe C631. Lanes: 1, C. jejuni ATCC 29428; 2, C. jejuni 85Y242; 3, C lan ATCC 35221; 4, C. lan EF 10370; 5, C. coli ATCC 33559; 6, C. coli 6247/87; 7, H. pylon ATCC 43504; 8, C. fetus; 9, E. coli ATCC 11775; N, negative control. The molecular weight marker used was pBR322 digested with Hinfl, giving Fragments of 1,632, 517, 506, 396, 344, 298, 221, 220, 154, and 75 bp.

Figures 1 and 2 show the sequence alignments in the region before V3, V3, and V5 from the 16S rRNA sequences of Campylobacter spp. and related microorganisms. In general, very few differences between the thermotolerant Campylobacter spp. (C. jejuni, C. coli, and C. lan) were found. However, there was some interspecies variability in the region before V3 and in V5, corresponding to positions 402 through 430 and 821 through 858, respectively, according to the International Union of Biochemistry numbering of E. coli 16S rRNA (3). Development of the PCR assay. Primers were selected from the region before V3 (C442, nucleotides 399 through 420; Fig. 1A) and V5 (C490, nucleotides 803 through 825; Fig. 1B). A probe was selected from V3 (C631, nucleotides 439 through 459; Fig. 2). To investigate the specificity of the selected primers and probe, phenol-chloroform-extracted DNA from a panel of different Campylobacter species and nonrelated microorganisms (Table 1) was tested in the PCR and by Southern blot analysis. The use of primers C442 and C490 at an annealing temperature of 52°C resulted in amplified products of 426 bp for C. jejuni, C. coli, and C. lan only. The other microorganisms tested (all strains from Table 1) showed no amplification products. The results were confirmed by hybridization with oligonucleotide probe C631 (Fig. 3). To determine the sensitivity of the PCR assay as used, serial dilutions of C. jejuni 85Y242 in Preston broth were made. DNA was isolated with guanidinium isothiocyanate and analyzed by the PCR. Dilutions containing 12.5 CFU per reaction yielded a positive result (data not shown). This corresponds to 500 CFU/ml. Enrichment procedure. To estimate the minimal period of time needed for culturing, 10 naturally infected samples were cultured. Equal aliquots from this culture were taken at different time intervals both for the PCR and viable plate counts. Figure 4 shows that the PCR positivity increased after 14 h of enrichment. PCR-positive samples contained at least 500 CFU/ml. Samples containing fewer than 500 CFU/ml were PCR negative. Samples taken before the enrichment were always PCR negative, and samples taken

FIG. 4. PCR after different culture times with naturally infected chicken samples. The amplification products were hybridized with probe C631. Lanes 1 through 4 and 5 through 8 contain PCR products from two different samples after 12, 14, 16, and 18 h of enrichment, respectively.

USE OF PCR TO DETECT CAMPYLOBACTER SPP. IN CHICKEN

VOL. 58, 1992 1

428

bp-

2

3

4

5

e

_gpauI4.

FIG. 5. Detection limit with Southern blot hybridization of C. jejuni in 10-g chicken samples inoculated with strain 85Y242. Lanes 1 to 6 contain samples inoculated with 500, 250, 100, 25, 10, and 0 CFU/g, respectively. The molecular weight marker used was pBR322 digested with Hinfl, giving fragments of 1,632, 517, 506, 396, 344, 298, 221, 220, 154, and 75 bp.

after 16 to 18 h of culturing were PCR positive. Therefore, a standard enrichment time of 18 h was chosen. Detection of Campylobacter spp. in inoculated samples. The detection limit of the PCR-culture assay was investigated by inoculating irradiated 10-g samples with 0 to 500 CFU of C. jejuni 85Y242. Figure 5 shows the results of the PCR after Southern blot hybridisation. Positive results were obtained from samples infected with 25 CFU/g (Fig. 5, lane 4). Negative control samples from noninfected chicken skin remained negative after amplification. Plate counts after 18 h of incubation of the samples infected with 25 CFU/g (data not shown) indicated that the detection limit of the PCRculture assay was 500 CFU/ml of Preston broth. Comparison of the PCR and conventional enrichment in naturally contaminated samples. Forty-five chicken samples from local poultry shops and supermarkets were screened for the presence of Campylobacter spp. in 2-g skin samples by using the PCR-culture assay and conventional enrichment. In this survey, 36 (80%) of 45 samples were positive with both the PCR and conventional enrichment, and 9 (20%) samples were negative with both methods. High levels (106 to 108 CFU/g) of other contaminating bacteria did not effect the PCR-culture assay specificity. DISCUSSION

The PCR technique based on the 16S rRNA gene was used to screen poultry products for Campylobacter spp. in this study. Since little sequence information was available on the 16S rRNA gene of C jejuni, the complete sequence was determined. The entire 16S rRNA gene was amplified by using primers homologous to the conserved regions flanking the gene and cloned into M13mpl8 and M13mpl9. With this procedure, the sequences of the Campylobacter 16S rRNA genes could be determined, and specific oligonucleotides could be selected. The primers selected from the region before V3 and from VS enabled discrimination of thermotolerant Campylobacter spp. from other microorganisms. Although some sequence variation was present in V5, the thermotolerant Campylobacter spp. could not be discriminated because the three species are closely related; previous reports described difficulties in discriminating among these species with biochemical tests (11) or DNA probes (9, 14, 19). An efficient assay with a sensitivity of 12.5 CFU/PCR reaction, determined by using dilutions of a pure C. jejuni culture treated with guanidinium isothiocyanate, was developed. This sensitivity is in agreement with findings from Wernars et al. (25). To develop the PCR assay for the detection of Campylobacter spp. in chicken products, the PCR was combined with a short enrichment culture. This combination had two important implications. First, the combined method provided a

3807

high sensitivity. Chicken products inoculated with at least 25 CFU/g generated a positive PCR result. Further, the minimal detectable number of CFU present after 18 h of culturing was 500 CFU/ml of culture broth (12.5 CFU per PCR reaction). Compared with tests that use DNA probes for the detection of Campylobacter spp. in infected stool samples and on crude cell lysates (3sensitivity, 105 to 107 CFU/g), the PCR assay is at least 10 -fold more sensitive (9, 20, 21). Regarding the low infectious dose of Campylobacter spp. (a few hundred bacteria [17]), one possible way to increase the sensitivity of the PCR-culture assay is to use the rRNA, instead of DNA, as a target. Each cell contains at least 103 to 104 copies of rRNA. This could increase the sensitivity of the PCR or reduce the length of the enrichment culture period. Second, the combination of the PCR with a short culture increased the level of viable cells, whereas the nonculturable or dead cells were diluted. Samples taken before the 18-h culture did not generate a positive PCR signal. Thus, even when dead or nonculturable cells were present, the PCR assay results were not positive. The comparison of the PCR-culture assay with the conventional method showed that both methods detect the same number of positive and negative samples. One possible way to make sure that even viable but nonculturable Campylobacter spp. cells are detected is to perform direct viable counts (acridine orange direct counts) on PCR- and culture-negative samples (12). Although the transition of the viable but nonculturable stage has been described for cells after a prolongued stay in an aqueous environment, the absence of Campylobacter spp. from chicken meat estimated by conventional methods should be considered with some caution (10). When the results of the naturally infected samples are compared with the results of the inoculated samples, it might be concluded that the initial infection level of all the naturally infected samples is more than 25 CFU/g. Total counts of the naturally infected samples however, showed infection levels below 25 CFU/g. This suggests that Campylobacter spp. grow better on naturally infected samples and that the sensitivity of the PCR-culture assay, estimated with inoculated samples, is in practice even higher. A more direct way to discriminate between dead and viable cells is to use RNA instead of DNA as a template for the PCR. This method however, strongly depends on the stability of RNA in dead cells, which requires further investigation. It is also possible to use mRNA from a unique gene as a target for amplification. The 16S rRNA gene not only offers the possibility for the development of a genus- or species-specific PCR but also can be used to simultaneously detect different pathogens in one sample. This would require a set of primers homologous to conserved regions in combination with several speciesspecific probes or two or more sets of species-specific primers in one PCR. To achieve this, primer sets that function under the same reaction and thermocycling conditions have to be used. Also, they must produce products of sufficiently different sizes so as to be clearly distinguishable. In the experiments described in this paper, PCR results were confirmed by hybridization with an oligonucleotide probe labelled with 32P. Results of the complete PCR assay were available within 48 h. Compared with conventional culturing and biochemical identification (requiring at least 96 h), the PCR-culture assay is both sensitive and rapid. The use of nonradioactive probes would further reduce the time needed for the assay. We are investigating the use of the 23S rRNA gene and 16S-23S spacer region to discriminate among thermotolerant

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Campylobacter spp. This could increase both specificity and sensitivity of the PCR-culture assay. ACKNOWLEDGMENTS We thank Alex van Belkum for critical reading of the manuscript, Martha Canning for correcting language use and spelling, and Ankie Koeken for part of the sequence work. REFERENCES 1. Blase, M. J., and L. B. Beller. 1981. Campylobacter enteritis. N. Engl. J. Med. 305:1444-1452. 2. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503. 3. Brosius, J., J. L. Palmer, J. P. Kennedy, and H. F. Noller. 1978. Complete nucleotide sequence of 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801-4805. 4. Hill, W. E., S. P. Keasler, W. W. Trucksess, P. Feng, C. A. Kayser, and K. A. Lampel. 1991. Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl. Environ. Microbiol. 57:707-711. 5. Kwok, S. 1990. Procedures to minimize PCR-product carryover, p. 142-145. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, Calif. 6. Lampel, K. A., J. A. Jagow, M. Trucksess, and W. E. Hill. 1990. Polymerase chain reaction for detection of invasive Shigella fle-xneri in food. Appl. Environ. Microbiol. 56:1536-1540. 7. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Neefs, J. M., Y. van de Peer, L. Hendriks, and R. de Wachter. 1990. Compilation of small ribosomal subunit RNA-sequence. NAR Sequence Suppl. 18:2237-2317. 9. Olive, D. M., M. Johny, and S. K. Sethi. 1990. Use of an alkaline phosphatase labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli. J. Clin. Microbiol. 28:1565-1569. 10. Park, R. W. A., P. L. Griffiths, and G. S. Moreno. 1991. Sources and survival of Campylobacters: relevance to enteritis and the food industry. J. Appl. Bacteriol. 70:97S-106S. 11. Penner, J. L. 1988. The genus Campylobacter. Clin. Microbiol. Rev. 1:157-172. 12. Rollins, D. M., and R. R. Colwell. 1986. Viable but nonculturable stage of Campylobacterjejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microbiol. 52:531538. 13. Romaniuk, P. J., and T. J. Trust. 1987. Identification of Campylobacter species by Southern hybridisation of genomic DNA using an oligonucleotide probe for 16S rRNA genes. FEMS Microbiol. Lett. 43:331-335. 14. Romaniuk, P. J., and T. J. Trust. 1989. Rapid identification of Campylobacter species using oligonucleotide probes to 16S

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Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gen...
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