Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.Footrot is a contagious bacterial disease that affects the feet of sheep, and it has been reported in many countries [1]. The fastidious and anaerobic bacterium, Dichelobacter nodosus, is the main causative agent of ovine footrot [2].In its mildest form, footrot manifests itself as a slight inflammation of the interdigital skin of sheep, but the disease may also progress to severe necrotic separation of the claw capsule from underlying tissues. Severity of the disease depends on the breed of sheep, climatic conditions, management factors and virulence of the infecting D. nodosus strain.Ovine footrot was first diagnosed in Sweden in 2004 [3], and in 2008 it was detected for the first time in 60 years in Norway [4]. In 2009, the d