A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis.
BACKGROUND
A multiplex qPCR targeting a 128 bp region on the 23S rDNA gene was developed for detection of Brachyspira (B.) hyodysenteriae and B. pilosicoli, the agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), together with a triplet of apathogenic Brachyspira spp. (B. innocens, B. intermedia, B. murdochii) in porcine feces. The multiplex qPCR was evaluated against a duplex PCR (La et al., J Clin Microbiol 41:3372-5, 2003).


RESULTS
Using DNA extracted from fecal culture, the multiplex qPCR showed excellent agreement with the duplex PCR (κ = 0.943 and 0.933). In addition, thanks to the three probes whereof one detecting the apathogenic Brachyspria spp., a more diversified overview of the brachyspiral flora in porcine fecal samples can be delivered as a part of the routine diagnostic. The multiplex qPCR with a limit of detection of 5-10 genomic equivalents (GE) per reaction (6 × 102GE per gram) allows reliable detection of Brachyspira species directly from fecal swab DNA. In line with this, analysis of 202 fecal swabs in comparison with culture-based qPCR showed a high agreement for the causative agents of SD (B.hyodysenteriae: κ = 0.853, sensitivity 87% specificity 98%).


CONCLUSIONS
The novel multiplex qPCR is robust and has a high analytical sensitivity and is therefore suitable for high-throughput screening of porcine fecal swabs for the causative agents of SD. This assay can therefore be used for the direct proof of the pathogenic B. spp. in fecal swabs within the scope of a monitoring program.
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland. anna.borgstroem@vetbakt.uzh.ch.
Anna
A
Borgström
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland.
Simone
S
Scherrer
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland.
Constanze
C
Kirchgässner
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland.
Sarah
S
Schmitt
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland.
Daniel
D
Frei
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, CH 8057, Zurich, Switzerland.
Max M
MM
Wittenbrink
D000818:Animals; D017400:Brachyspira / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D015203:Reproducibility of Results; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology*
D012338:RNA, Ribosomal, 23S
23S rDNA; Brachyspira hyodysenteriae; Multiplex Real-time PCR; Swabs; Swine dysentery