Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real-time PCR assay.
OBJECTIVE
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.


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.


CONCLUSIONS
The multiplex real-time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive.


CONCLUSIONS
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.
Pal
N
N
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
Bender
J S
JS
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
Opriessnig
T
T
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification; D004888:Erysipelothrix / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012703:Serotyping; D013045:Species Specificity; D013552:Swine / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial