A multiplex real-time PCR for the simultaneous detection and quantitation of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in pig faeces

A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis, 14 DNA copies for B. pilosicoli and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 10(3) cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.