Optimization and validation of a triplex real-time PCR assay for thermotolerant Campylobacter species associated with foodborne disease

The genus Campylobacter is globally recognised as the leading bacterial cause of human foodborne gastroenteritis. Every year around 8000 Swedes are infected by Campylobacter. Most people are infected by thermotolerant Campylobacter species, commonly C. jejuni and C. coli. In this study a triplex real-time PCR has been developed in order to be a last step in a qualitative method for identification of thermotolerant Campylobacter, originating from food and water suspected to have caused human gastroenteritis. PCR is preceded by enrichment and isolation on selective medium and cultivation microaerophilically at 41.5 °C. The developed triplex real-time PCR was based on a combination of previously published primers and probes targeting the hipO, cadF, and 16S rRNA genes. In total 115 strains, representing 17 different Campylobacter species and additionally 10 nonCampylobacter species, genetically related to Campylobacter or commonly causing human gastroenteritis, were tested for inclusivity and exclusivity. Species-specific PCR products were produced to distinguish C. coli and C. jejuni. Thereto an amplicon of the 16S rRNA gene was produced for C. coli, C. jejuni, C. lari, two of seven C. upsaliensis, and C. insulaenigrae. No PCR product was obtained from any non-Campylobacter strains. Thereby, the method sufficiently met the needed requirements. The aim was also to compare two different methods for DNA extraction, where the InstaGene kit with a slight modification was preferred to simple boiling. Also the inhibition of the PCR assay if bacterial colonies were grown on blood agar or modified charcoal cefoperazone deoxycholate agar (mCCDA) was compared, with no differences found between the culturing medias.