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1 December 2002

Colony Multiplex PCR Assay for Identification and Differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus


A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.
Pathogens belonging to the genus Campylobacter have fastidious growth requirements, making conventional detection and identification procedures problematic. As a consequence, rapid and reliable detection procedures are required. Methods based on DNA probe technology have been developed for these organisms; however, they are generally of low sensitivity in food products (9, 10, 15, 18). A number of genetically based detection and typing methods have been developed for detecting Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus in clinical, environmental, and food samples. Such assays have used 23S rRNA PCR-restriction fragment length polymorphism (8, 20), sequence analysis of the GTPase gene (22), multiplex PCR for C. jejuni and C. coli (7), DNA-based PCR (ceuE) detection (11), variable region analysis of 16S rRNA (17), dot blot hybridization using a digoxigenin-labeled C. fetus-specific oligonucleotide probe (3), and DNA hybridization. A combination of PCR and hybridization methods has also been used (4, 5).
A colony multiplex PCR was developed and optimized to simultaneous identify the 23S rRNA from Campylobacter spp.; the hipO gene (hippuricase) from C. jejuni subsp. jejuni; the glyA gene (serine hydroxymethyltransferase) from C. coli, C. lari, and C. upsaliensis; and the sapB2 gene (surface layer protein) from C. fetus subsp. fetus. The multiplex PCR protocol was capable of detecting the type strains and clinical isolates from all five species with a high degree of specificity.
A total of 137 strains of various species of enterobacteria were evaluated, and of these 124 were campylobacters: 70 from C. jejuni subsp. jejuni; 21 from C. coli; 7 from C. lari; 6 each from C. upsaliensis, C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. hyointestinalis; and 1 each from C. sputorum biovar bubulus and C. sputorum biovar fecalis. In addition, three Arcobacter butzleri, three A. butzleri-like, three Helicobacter pylori, two Escherichia coli, and two Aeromonas hydrophila isolates were also examined. All isolates were obtained from the culture collection of the National Laboratory for Enteric Pathogens, and these included both clinical isolates and well-established laboratory isolates. The Campylobacter isolates were grown on Mueller-Hinton agar (Oxoid, Nepean, Ontario, Canada) supplemented with 10% sheep blood and incubated at 37°C in a microaerobic atmosphere containing 5% O2, 10% CO2, and 85% N2.

DNA template preparation.

DNA was prepared by the whole-cell procedure. Each PCR template was prepared by using approximately half a loopful of culture transferred to 1 ml of brain heart infusion broth (Oxoid). The optical density was adjusted to give a reading of 0.3 at 600 nm. The optimized whole-cell DNA preparations from all Campylobacter species were further diluted 1:500 in distilled water and were heated at 100°C for 10 min in a 0.5-ml Eppendorf tube. Templates were used immediately for PCRs or were kept at 4°C for up to 1 month.

Primers and PCR sensitivity.

Oligonucleotides, ranging from 18- to 24-mers, were selected from the published DNA sequences of the various Campylobacter species (Table 1) using Oligo software (version 3.4). Synthesis of oligonucleotides was carried out at the DNA Core Facility in the National Microbiology Laboratory, Winnipeg, Canada. The six pairs of primers were designed to identify the genes hipO from C. jejuni; glyA from C. coli, C. lari, and C. upsaliensis; sapB2 from C. fetus subsp. fetus; and the internal control 23S rRNA. The primer sequences used in the multiplex PCR are outlined in Table 1. The colony PCR sensitivity was determined by both PCR and plate count methods; in brief, 10-fold serial dilutions of up to 10−14 were made in triplicate in brain heart infusion broth from 1- ml cultures with starting optical density readings of 0.3 at 600 nm. From these diluted samples, 100-μl aliquots from C. jejuni, C. coli, C. lari, C. fetus subsp. fetus, and C. upsaliensis isolates were plated onto Mueller-Hinton agar. Duplicate samples of each were used to perform standard colony counts and to evaluate the sensitivity of the PCR assay using agarose gel electrophoresis relative to actual plate counts within the range of 102 to 1013 CFU/ml. It appears that a range of 108 to 1013 CFU/ml was most effective for all the Campylobacter colony multiplex PCR assays.

Multiplex PCR conditions.

Each multiplex PCR tube contained 200 μM deoxynucleoside triphosphate; 2.5 μl of 10× reaction buffer (500 mM Tris-HCl [pH 8.3], 100 mM KCl, and 50 mM [NH4]2SO4); 20 mM MgCl2; 0.5 μM C. jejuni and C. lari primers; 1 μM C. coli and C. fetus primers, 2 μM C. upsaliensis primers; 0.2 μM 23S rRNA primer (Table 1); 1.25 U of FastStart Taq DNA polymerase (Roche Diagnostics, GmbH, Mannheim, Germany), and 2.5 μl of whole-cell template DNA. The volume was adjusted with sterile distilled water to give 25 μl. DNA amplification was carried out in a Perkin-Elmer thermocycler using an initial denaturation step at 95°C for 6 min followed by 30 cycles of amplification (denaturation at 95°C for 0.5 min, annealing at 59°C for 0.5 min, and extension at 72°C for 0.5 min), ending with a final extension at 72°C for 7 min. The six primer sets were evaluated individually for primer specificity using the reference strains C. jejuni NCTC 11168, C. coli NCTC 11353, C. lari NCTC 11352, C. upsaliensis ATCC 43954, C. fetus subsp. fetus ATCC 27374, C. fetus subsp. venerealis ATCC 19438, C. hyointestinalis ATCC 35217, C. sputorum biovar fecalis ATCC 33711, A. butzleri ATCC 49616, and A. butzleri-like CDCD2887. Only the corresponding strains showed the expected PCR amplification products. Reproducibility of the multiplex PCR assay was evaluated using 137 clinical and environmental samples.
Figure 1 illustrates the PCR-amplified products with the Campylobacter reference strains as templates following 1.5% agarose gel electrophoresis. In the assay, six bands were detected from a mixture of DNA containing each of the five Campylobacter spp. (Fig. 1, lane 8). The amplicons from the control strains were subjected to further confirmation and characterization by digestion using restriction endonucleases with cleavage sites within the amplicon. The restriction enzymes used and the predicted product sizes are shown in Table 2. Enzyme fragments with the anticipated sizes were obtained in each case (data not shown).
All 124 Campylobacter samples were identified by using biochemical assays and 16S rRNA-PCR (14). Complete agreement was obtained with the species-specific primers used in the present assay for all isolates examined. The amplicon for the Campylobacter 23S rRNA primers was present in all tested Campylobacter, Arcobacter, and Helicobacter isolates but failed to amplify E. coli and A. hydrophila isolates (Table 3). The sensitivity range of the colony multiplex PCR in number of CFU per milliliter was 108 to 1013 for C. jejuni, 106 to 1013 for C. coli and C. upsaliensis, 107 to 1013 for C. lari, and 102 to 1013 for C. fetus subsp. fetus.
Case control studies demonstrate that human campylobacteriosis is a food-borne disease with infection most frequently resulting from handling and consuming contaminated poultry meat (21). Indeed, in one study it was reported that 73.2% of 489 meat samples were contaminated with the pathogen (12). Clinically the most important campylobacters are the members of the thermophilic group that includes C. jejuni, C. coli, C. lari, and C. upsaliensis, with C. jejuni responsible for the majority of human cases (1). C. fetus is also recognized as a human and animal pathogen and has been identified in 12.5% of ox liver samples (12). Accurate identification of these organisms is required in order to decide upon appropriate therapeutic measures, to understand the pathology of disease, and to provide clinical and epidemiological data for disease control. A number of protocols (3-8, 11, 17, 20, 22) have been described in the literature for use in the differentiation of the closely related thermophilic C. jejuni, C. coli, C. lari, and C. upsaliensis species as well as of C. fetus. Most of these methods are based on DNA probe technology or rRNA PCR-restriction fragment length polymorphism. Due to a requirement for restriction enzymes or hybridization steps with species-specific probes, both are relatively complex to perform. An assay combining PCR and DNA hybridization was developed for the rapid detection of C. fetus (5), but it is not capable of differentiating C. fetus subsp. fetus from C. fetus subsp. venerealis. In a recent study multiplex PCR was used for simultaneously differentiating C. jejuni, C. coli, and C. lari (6). This assay was based on the sequence information of the gene encoding the oxidoreductase subunit of C. jejuni (23; GenBank accession no. AL139075 ), the aspartokinase gene for C. coli (13), and 16S rRNA for C. lari (16).
Detection by PCR of the hipO gene, shown to be highly conserved in C. jejuni, provided an effective identification marker for C. jejuni (19, 20). In addition, PCR hybridization confirmed that the Campylobacter glyA gene can be used as the target to identify and differentiate C. jejuni, C. coli, C. lari, and C. upsaliensis at the species level (2). Furthermore, the sapB2 gene of C. fetus subsp. fetus was recognized as a suitable target for identifying C. fetus (4).
In this study, a colony multiplex PCR-based diagnostic protocol was developed to simultaneously detect five genes specific to each of the five pathogenic Campylobacter species, while the 23S rRNA probe was included to serve as an internal validation control to monitor PCR conditions and reagents. The 23S rRNA was found in all of the Campylobacter, Arcobacter, and H. pylori species investigated.
The present colony multiplex PCR assay proved to be accurate and simple to perform and could be completed within 3 h. It had the added advantage of detecting the hipO gene in C. jejuni strains that were hippuricase negative by phenotypic methods and therefore difficult to differentiate from C. coli (5, 20). In addition to clinical use, the method has potential as a diagnostic kit for detecting thermophilic campylobacters in complex samples, such as foods in which low pathogen numbers (<103 CFU/ml) are frequently present. The present PCR assay offers an effective alternative to traditional biochemical typing methods for the identification and differentiation of C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus isolated from humans and poultry.
FIG. 1.
FIG. 1. Amplification fragments of multiplex PCR detection and identification. Lanes 1 and 9: 123-bp ladder (Bethesda Research Laboratories Inc., Gaithersburg, Md.); lane 2: C. coli NCTC 11353, 126-bp fragment; lane 3: C. upsaliensis ATCC 43954, 204-bp fragment; lane 4: C. lari NCTC 11352, 251-bp fragment; lane 5: C. jejuni NCTC 11168, 323-bp fragment; lane 6: C. fetus subsp. fetus ATCC 27374, 435-bp fragment; lane 7: C. sputorum biovar fecalis ATCC 33711, 650-bp fragment of 23S rRNA (which occurred in all Campylobacter spp., Arcobacter and Helicobacter isolates tested); and lane 8: PCR-positive control with DNA mixture.
TABLE 1. Primer sequences used in the multiplex PCR assay and the expected sizes of the products
PrimerSize (in bp)Sequence (5′-3′)GenBank accession no.Target geneGene location (bp)
CJF323ACTTCTTTATTGCTTGCTGCZ36940C. jejuni hipO1662-1681
CUF204AATTGAAACTCTTGCTATCCAF136496C. upsaliensis glyA63-82
23SF650TATACCGGTAAGGAGTGCTGGAGZ29326C. jejuni 23S rRNA3807-3829
TABLE 2. Predicted sizes of restriction fragments and enzymes used for restriction fragment length polymorphism analysis of amplified products of multiplex PCR
GenePCR amplicon size (in bp)EnzymeExpected size of restriction fragments (bp)
hipO323BsrDI109, 214
C. coli glyA126AluI11, 36, 79
C. lari glyA251ApoI79, 172
C. upsaliensis glyA204DdeI31, 173
C. fetus subsp. fetus-sapB2435BclI130, 305
C. jejuni 23S rRNA650HhaI212, 438
TABLE 3. PCR results by multiplex PCR analysis of Campylobacter strainsa
StrainNo. of strains testedNo. of strains that were PCR positive for:     
  C. jejuniC. coliC. lariC. upsaliensisC. fetus subsp. fetus23S rRNA
C. jejuni707070
C. coli212121
C. lari777
C. upsaliensis666
C. fetus subsp. fetus666
C. hyointestinalis66
C. fetus subsp. venerealis66
C. sputorum biovar bubulus11
C. sputorum biovar fecalis11
A. butzleri33
A. butzleri-like33
H. pylori33
E. coli2
A. hydrophila2
—, tested negative.


We thank Roman Benes for technical assistance.


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Information & Contributors


Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 40Number 12December 2002
Pages: 4744 - 4747
PubMed: 12454184


Received: 26 April 2002
Revision received: 30 July 2002
Accepted: 13 September 2002
Published online: 1 December 2002


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National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Clifford G. Clark
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Tracy M. Taylor
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Chad Pucknell
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Connie Barton
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Lawrence Price
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
David L. Woodward
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Frank G. Rodgers
National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Canada
Present address: Department of Microbiology, Rudman Hall, University of New Hampshire, Durham, NH 03824.

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