Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real-time PCR assay
Article first published online: 21 SEP 2009
© 2009 The Authors. Journal compilation © 2009 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 108, Issue 3, pages 1083–1093, March 2010
How to Cite
Pal, N., Bender, J.S. and Opriessnig, T. (2010), Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real-time PCR assay. Journal of Applied Microbiology, 108: 1083–1093. doi: 10.1111/j.1365-2672.2009.04560.x
- Issue published online: 8 FEB 2010
- Article first published online: 21 SEP 2009
- 2009/0950: received 30 May 2009, revised 4 September 2009 and accepted 5 September 2009
- Erysipelothrix spp;
- multiplex PCR;
Aim: The aim of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens.
Methods and Results: A primer set and three species-specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia-associated non-Erysipelothrix spp. bacterial isolates. Cross-reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection.
Conclusion: The multiplex real-time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive.
Significance and Impact of the Study: The developed real-time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.