Campylobacters isolated from mussels and oysters in The Netherlands were analysed by a novel assay, based on DNA amplification with primers, based on semiconserved GTP‐binding sites of a putative GTPase gene. Polymerase chain reaction (PCR) was followed by a single step reverse hybridization line probe assay (PCR‐LiPA). This permits identification of Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. Among a group of 44 isolates, three C. jejuni, one C. upsaliensis, one double infection with C. jejuni and C. coli, and 38 C. lari strains were identified. These results were in complete agreement with conventional identification methods and whole cell protein analysis. One C. hyointestinalis isolate was not identified by the PCR‐LiPA, since the reverse hybridization assay does not comprise specific probes for this particular species. PCR products from 36 C. lari isolates were sequenced and phylogenetic analysis revealed the presence of two major C. lari subgroups: one comprised 11 highly homologous sequences, whereas the 25 sequences in the other subgroup were more heterogeneous. This confirmed earlier findings that C. lari isolates comprise a more heterogeneous group of isolates as compared with C. jejuni, C. coli and C. upsaliensis. Based on the sequence information, a novel PCR‐LiPA was developed that permits specific and rapid detection of the different C. lari variants.

Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerantCampylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species.