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Applicability of a rapid duplex real-time PCR assay for speciation of Campylobacter jejuni and Campylobacter coli directly from culture plates

Emma L. Best, Ella J. Powell, Craig Swift, Kathleen A. Grant, Jennifer A. Frost
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00845-0 237-241 First published online: 1 December 2003


A rapid duplex real-time polymerase chain reaction (PCR) assay for speciation of Campylobacter jejuni and Campylobacter coli using the ABI Prism 7700 sequence detection system (Applied Biosystems) was developed based on two of the genes used in a conventional multiplex PCR. A rapid turnaround time of 3 h was achieved with the use of boiled cell lysates. Applicability of the assay was tested with 6015 random campylobacter strains referred to the Campylobacter Reference Unit, with 97.6% being identified as either C. jejuni or C. coli by this technique. Rapidity, combined with specificity and sensitivity, makes this method for routine campylobacter speciation attractive to any laboratory with a Taqman system.

  • Real-time polymerase chain reaction
  • ABI Prism 7700
  • Campylobacter speciation

1 Introduction

Campylobacter jejuni and Campylobacter coli speciation is rarely undertaken in the majority of front-line laboratories, despite species determination being important for clinical and epidemiological purposes. Differences in antibiotic resistance exist between the two species especially in relation to erythromycin and ciprofloxacin, where resistance is more common in C. coli [1]. Furthermore, a population-based sentinel surveillance scheme from England and Wales has demonstrated the higher risk of obtaining an infection from a particular species by the exposure to certain food products [2].

A wide variety of polymerase chain reaction (PCR) assays for speciation of C. jejuni and C. coli have been designed based on genes, or parts of genes specific to either species, by the use of randomly generated DNA fragments, or PCR ELISA strategies [37].

Real-time PCR assays based on platforms such as the ABI Prism 7700 sequence detection system, more commonly referred to as the Taqman, have steadily become more established for the detection and identification of a variety of organisms including clinically important bacteria such as C. jejuni [89], Listeria monocytogenes [10] and Salmonella species [11]. The Taqman system exploits the 5′ nuclease activity of Taq DNA polymerase in conjunction with fluorogenic DNA probes. The probe is designed to hybridise to an internal region of the PCR product and is labelled at the 5′ end with a fluorescent reporter dye and at the 3′ end with a quencher dye. The intact probe is of sufficient length that, under normal conditions, the fluorescence of the reporter is suppressed due to its spatial proximity to the quencher. However, during PCR amplification the hybridised probe is hydrolysed by the 5′ nuclease activity of Taq polymerase, thus separating the reporter dye from the quencher. An increase in reporter dye fluorescence is a direct result of target amplification. Repeated PCR cycles result in an increase in fluorescence corresponding to amplification of the target PCR product, which is detected and recorded by the system.

The assay described herein is based on partial sequences of two genes employed in a multiplex PCR strategy described by Denis et al. [12]. For C. jejuni, the mapA gene was used, which encodes a species-specific 24-kDa membrane-associated protein identified by Stucki et al. [5] and for C. coli, the gene ceuE was used, encoding a periplasmic substrate binding protein (34.5 kDa) described by Richardson and Park [13]. A duplex Taqman assay for identification of the two species C. jejuni and C. coli was designed and evaluated prior to implementation for routine laboratory speciation.

2 Materials and methods

2.1 Bacterial strains and growth conditions

For the initial trial, a total of 60 isolates of C. jejuni and C. coli were selected randomly from those received into the reference laboratory, which underwent parallel characterisation by other methods. All isolates speciated thereafter were received over the next 6 months. Isolates were inoculated onto Columbia blood agar (Oxoid) and incubated for 48 h at 37°C in a variable atmosphere incubator (VAIN, Don Whitley Scientific) in a microaerobic atmosphere (H2 5%, CO2 3%, O2 5%, N2 87%). The following organisms were used as negative controls: Escherichia coli, Helicobacter pylori, Campylobacter lari, Campylobacter upsaliensis, Campylobacter curvus, Campylobacter helveticus and Campylobacter fetus. For specificity studies cultures of C. jejuni and C. coli were serially diluted 10-fold in sterile distilled water from a starting concentration of approximately 1×109 colony-forming units (CFUs) µl−1, enumerated by plate counts after incubation as above.

2.2 Validation of the assay by established speciation methods

The initial 60 samples were evaluated against conventional methods for speciation, including a phenotypic hippurate hydrolysis test [14], a conventional PCR for speciation based on different genes [6] and the serotyping scheme using the direct agglutination method for heat-stable antigens [15] where serotype is species-specific. Following the initial trial of 60 isolates, all subsequent isolates were serotyped, but only isolates with contradictory serotype (or non-serotypeable) and Taqman speciation were validated with the other methods above. Further validation was carried out where required, using a campylobacter 16S rRNA restriction fragment length polymorphism (RFLP) strategy based on amplification of a region of the 16S rRNA gene, followed by digestion with a combination of the restriction endonucleases DdeI, TaqI and BsrI to identify C. lari, C. upsaliensis or C. fetus [16]. Where required, sequencing of the mapA and ceuE genes was carried out by standard protocols using a Beckman CEQ8000 sequencer.

2.3 Preparation of boiled cell lysates

One colony was removed and emulsified in 100 µl of distilled sterile water in a 0.5-ml microcentrifuge tube and heated to 100°C for 10 min.

2.4 Taqman primer and probe design

Primers and probes were designed for C. jejuni gene mapA (X80135) and C. coli gene ceuE (X88849) using the criteria specified within Primer Express (Applied Biosystems version 1.5). All primers and probes were checked in BLASTN to ensure they were unlikely to amplify other genes from other species and organisms.


ceuE (amplicon size 102 bases): forward primer: 5′-AAGCTCTTATTGTTCTAACCAATTCTAACA-3′; reverse primer: 5′-TCATCCACAGCATTGATTCCTAA-3′; probe: 5′-VICTTGGACCTCAATCTCGCTTTGGAATCATTTAMRA-3′ (5′ fluorescent reporter dyes and 3′ quenchers underlined).

2.5 Nucleic acid amplification using the ABI Prism 7700 sequence detection system

Each 25 µl amplification reaction mix contained 1×Taqman Universal PCR Mastermix (containing AmpliTaq Gold™ DNA polymerase, AmpErase UNG, dNTPs with dUTP, Passive reference 1 (ROX) and optimised buffer components including 5 mM MgCl2), 300 nM primers, 100 nM of each probe and 5 µl of boiled cell lysate. Reactions were carried out in ABI Prism 96-well optical reaction plates with 96-well optical covers (Applied Biosystems, Warrington, Cheshire, UK). Amplification consisted of an initial hold for 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 s and annealing/extension at 60°C for 1 min. All reactions were carried out alongside a non-template control containing sterile water and a positive control containing 1 µg µl−1 DNA from the National Collection of Type Cultures (NCTC) reference strains NCTC 11168 (C. jejuni) and NCTC 12110 (C. coli).

PCR products were detected directly by the Taqman machine monitoring the increase in fluorescence where a numerical value, the CT value (threshold cycle), was assigned. This indicated the cycle number at which measured fluorescence increased above calculated background fluorescence, identifying amplification of the target sequence. For the non-template control the CT value was given as 40 (the total number of cycles) as there was no template present therefore no amplification and no signal after 40 cycles. For positive samples the CT was in the range 15–30, the cycle number at which amplification and hence fluorescence started to accumulate and be detected.

3 Results and discussion

3.1 Taqman assay specificity and sensitivity

This assay utilises distinct gene sequences for C. jejuni and C. coli in order to speciate strains referred for identification and typing. The assay showed positive results with CT values of between 15 and 30 for either the mapA probe and primers or ceuE probe and primers for isolates of C. jejuni and C. coli respectively. Optimisation of the assay was carried out to assess the best combination of probes and primers for maximum reaction efficiency, and the reaction volume was reduced by half to lower costs, without compromising specificity. There was no amplification with any of the unrelated microorganism isolates including E. coli, H. pylori, C. lari, C. upsaliensis, C. curvus, C. helveticus and C. fetus, confirming specificity of the assay (data not shown).

Cell standard curves were constructed in duplicate for both species (Fig. 1) where a linear relationship was obtained between CT and log CFU. The detection limit of the Taqman assay per PCR was approximately 20 CFU for the mapA and ceuE gene regions. The square regression coefficients showed a good correlation between the number of CFU and the CT value.

Figure 1

Standard curve showing the linear relationship between CT and log CFU for serially diluted C. jejuni (●) and C. coli (▲) cells per PCR reaction. Linear regression coefficients were R2=0.99 (C. jejuni) and R2=0.98 (C. coli).

3.2 Pilot study of 60 uncharacterised strains with parallel analyses by phenotype and PCR

The Taqman assay when tested with an initial panel of 60 strains was successful and gave results 100% concordant with the serotyping and phenotypic hippurate tests as well as with the conventional PCR.

3.3 Large-scale routine use of the assay

During the first 6 months of routine use, 5877 out of 6015 (97.6%) cell lysates tested were either mapA-positive or ceuE-positive, therefore identified as being either C. jejuni or C. coli (Table 1). Of these 5336 (88.7%) were identified as C. jejuni (mapA-positive/ceuE-negative) and 541 (9%) identified as C. coli (mapA-negative/ceuE-positive). In all strains the Taqman molecular identification was consistent with the species specificity of the serotype [15] and any non-serotypeable strains were confirmed using conventional PCR [6]. Of the remainder, 123 (2.1%) failed to amplify with either gene. Ninety-five (1.6%) of these samples were identified by the Vandamme PCR [6] to be C. jejuni, 17 (0.3%) were identified by the same technique to be C. coli, which were also confirmed by serotype. Thus 1.9% of strains were identified by conventional PCR to be C. jejuni/coli. This lack of specificity in the Taqman assay is being addressed, in order to determine whether it is failure of the primers or probes, or the absence or variation in the genes, which is responsible for this small proportion of negative results. Eleven strains (0.2%) were identified as non-C. jejuni/coli but as other Campylobacter species (two C. upsaliensis, one C. fetus and eight C. lari), using 16S rRNA RFLP [16].

View this table:
Table 1

Numbers of strains identified from the set of 6015 over 6 months with each set of Taqman primers and probes and the resulting species determination

Gene sequence amplifiedNumber (%)Species identification
mapA+/ceuE5336 (88.7%)C. jejuni
mapA−/ceuE+541 (9%)C. coli
mapA−/ceuE123 (2.1%)95 (1.6%) C. jejuni
17 (0.3%) C. coli
Other species 11 (0.2%)
2 C. upsaliensis
1 C. fetus
8 C. lari
mapA+/ceuE+15 (0.2%)10 (0.16%) true mixed colonies
5 (0.08%) potential ‘hybrids’.
  • Confirmed by serotype.

  • Identified by conventional PCR.

  • Identified by 16S rRNA RFLP.

3.4 Isolates which amplified both genes

The remaining 15 strains (0.2%) amplified both genes. Possible explanations are that either the Taqman assay was non-specific, cells of both species were present or that these single strains possessed both genes. It was considered unlikely to be a result of the assay being non-specific, due to the small proportion of strains (0.2%) involved and the evidence from evaluation studies that the primers and probes were specific. The primers for each gene were also tested independently, and also validated in a conventional PCR. For each ‘mixed’ strain, multiple single colony picks were taken and tested with the Taqman assay. Ten out of 15 were true mixed cultures and resolved into either C. jejuni/coli species following single colony picks, where results were concordant with the serotype species designation. However, in none of the remaining five strains where both genes were detected, was it possible to isolate two species following single colony picks. For these strains results from other speciation tests showed no conclusive designation with a non-serotypeable being reported in each case. For each of the five mixed strains the target genes were amplified and sequenced, it was shown that both mapA and ceuE genes were present in their entirety in these ‘mixed’ strains and these matched the consensus sequences in each case. It has been demonstrated that coinfection can occur with Campylobacter species [17] but these data suggest there are a very small proportion of strains, which are possible ‘hybrids’ with respect to these two genes used.

3.5 Advantages of speciation using the Taqman assay

This assay enabled accurate speciation of 97.6%C. jejuni and C. coli strains tested. ‘Mixed strains’ would previously have been unrecognised by phenotypic testing, as a positive hippurate assay would have identified them as C. jejuni. A proportion of strains failed in this assay, but these were identifiable as C. jejuni or C. coli with other methods and a few strains were identified as other Campylobacter species. Despite this, the throughput and high proportion speciated gave time and cost gains compared to phenotypic or conventional PCR methods. The Taqman system has allowed a greater throughput than would have been possible using conventional PCR, where assay completion would be in excess of 4 h (for a 96-well plate thermal cycler) plus time for gel electrophoresis.

Conventional PCR methods involve amplification of target genes followed by separation of the product on agarose gels. Sequence-specific validation of PCR products is performed by time-consuming hybridisation analysis using blots or gels. Real-time PCR using Taqman technology offers a rapid one-step PCR amplification and sequence-specific validation, within a closed tube. This helps to eliminate contamination problems, reduces hands-on time, and facilitates large-scale sample processing.

Taqman PCR assays use small amplicons, up to 100 bp, compared to the larger 200-bp amplicon required for a standard PCR using separation on agarose gels. Furthermore, all Taqman assays are designed to amplify under the same cycling and reaction conditions making possible the running of more than one assay on one reaction plate. Further primer and probe combinations could be included in the same reaction plate allowing for identification of other Campylobacter species during the same run.

3.6 Application to non-cultured samples

The majority of isolates speciated by the reference laboratory are received as pure culture on charcoal transport swabs. The usual procedure requires culture as described, however in the case of urgent samples requiring a rapid result, direct DNA extraction from the charcoal transport medium has been applied in conjunction with this Taqman assay. With the use of the Roche MagNApure for the DNA extraction approximately 50 isolates have been successfully speciated with this method. Work in this area is ongoing with the applicability of the Taqman assay being tested with other non-culture samples including food and clinical specimens.

In conclusion, this assay has provided a rapid and reliable direct identification assay for C. jejuni and C. coli, which can be applied to crude cell lysates of pure cultures, which is now being used as a primary speciation tool in this laboratory. Major advantages of the Taqman system include comparatively low reagent costs and the potential of integrating the process into automated data capture for laboratory information systems. Application to environmental samples and food samples is being evaluated. The use of the MagNApure for rapid DNA extraction directly from transport swabs or samples linked to real-time PCR makes C. jejuni/coli speciation possible within a single working day.


We wish to thank the HPA for funding through a PhD studentship, the staff of the Campylobacter Reference Unit and Henry Smith for reading the manuscript.


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