Development of a multiplex-PCR for rapid detection of the enteric pathogens Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli in porcine faeces.
OBJECTIVE
To develop an assay to simultaneously detect Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig faeces.


RESULTS
A multiplex-polymerase chain reaction (M-PCR) was designed to amplify a 655-base pair (bp) portion of the L. intracellularis 16S rRNA gene, a 354-bp portion of the B. hyodysenteriae NADH oxidase gene, and a 823-bp portion of the B. pilosicoli 16S rRNA gene. Specificity was assessed using 80 strains of Brachyspira spp. and 30 other enteric bacteria. Bacterial DNA was extracted from faeces using the QIAamp DNA Stool Mini Kit. The M-PCR was tested in parallel with culture and/or PCR on 192 faecal samples from eight piggeries. Faeces also were seeded with known cell concentrations of the three pathogenic species, and the limits of detection of the M-PCR tested. The M-PCR was specific, with limits of detection of 10(2)-10(3) cells of the respective species per gram of faeces.


CONCLUSIONS
The M-PCR is a rapid, sensitive and specific test for detecting three important enteric bacterial pathogens of pigs.


CONCLUSIONS
The availability of a new diagnostic M-PCR will allow rapid detection and control of three key porcine enteric pathogens.
La
T
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School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA, Australia.
Collins
A M
AM
Phillips
N D
ND
Oksa
A
A
Hampson
D J
DJ
D000818:Animals; D000829:Animals, Domestic; D001315:Australia; D045824:Desulfovibrionaceae Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D003967:Diarrhea / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005243:Feces / Q000382:microbiology; D020639:Lawsonia Bacteria / Q000235:genetics / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013144:Spirochaetales / Q000235:genetics / Q000302:isolation & purification*; D013145:Spirochaetales Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology
D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases