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

Date Published: January 31, 2012

Publisher: BioMed Central

Author(s): Sara Frosth, Jannice S Slettemeås, Hannah J Jørgensen, Øystein Angen, Anna Aspán.

http://doi.org/10.1186/1751-0147-54-6

Abstract

Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR.

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.

Partial Text

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].

Good diagnostic tools are essential to identify the presence of D. nodosus and to study its epidemiology. Such knowledge is important to limit and control footrot, a disease that constitutes a major animal welfare problem. A correct diagnosis is a prerequisite to distinguish footrot from other diseases or conditions that can affect the feet of sheep such as contagious ovine digital dermatitis (CODD), white line disease, granulomas and toe and pedal joint abscesses [13].

The developed real-time PCR assay is a specific and easy-to-interpret method for detection of D. nodosus, and it is more sensitive and faster than either culturing or conventional PCR. There is an advantage that the same detection method is used in the three Scandinavian countries, so that results can be easily compared. A rapid and reliable detection method will aid in diagnosis and efforts to reduce the incidence of, or even to eradicate, virulent footrot in sheep populations. The developed real-time PCR is, however, not intended as a replacement for culturing as isolation of D. nodosus is still required for virulence testing and fingerprinting. However, it is a good complement to the laborious conventional culturing techniques.

The authors declare that they have no competing interests.

The study was designed by SF and AA. SF, JSS and ØA performed the laboratory work and the analysis of results was done by all authors. HJJ performed all statistical calculations. SF drafted the manuscript and all authors revised, read and approved the final manuscript.

 

Source:

http://doi.org/10.1186/1751-0147-54-6