Development of a multiplex-PCR for rapid detection of the enteric pathogens Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli in porcine faeces

Abstract

Aims: To develop an assay to simultaneously detect Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig faeces. Methods and 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 102–103 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. Significance and Impact of the Study: The availability of a new diagnostic M‐PCR will allow rapid detection and control of three key porcine enteric pathogens.

Detection of Brachyspira hyodysenteriae, Lawsonia intracellularis and Brachyspira pilosicoli in feral pigs

“…The 655-bp 16S rDNA products amplified from six faecal samples which were PCR positive for L. intracellularis (two from each study area) also were sequenced in duplicate, as previous described ( La et al, 2006).…”

“…The purified DNA was amplified by a hot-start multiplex PCR (M-PCR) for L. intracellularis, B. hyodysenteriae and B. pilosicoli, as previously described ( La et al, 2006) HotStarTaq DNA polymerase activation step at 95 °C, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 90 s, and primer extension at 68 °C for 2 min, with a final 10 min at 68 °C. The PCR products were subjected to electrophoresis in 1.5% (wt/vol) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA), stained with ethidium bromide, and viewed over UV light.…”

“…The three individual species-specific PCRs were conducted independently on the DNA extracted from the positive samples, as previously described (La et al, 2006).…”

Hibridização in situ fluorescente para diagnóstico de Brachyspira hyodysenteriae e B. pilosicoli em suínos

“…Foi utilizada a técnica de amplificação dupla (dPCR) para B. hyodysenteriae e B. pilosicoli de acordo com o protocolo de La et al (2006), adaptada por Viott (2013b). Os pares de primers utilizados na PCR foram respectivamente: H1 (FWH) (5'-ACTAAAGATCCTGATGTATTTG-3') e H2 (REV) (5'-CTAATA-AACGTCTGCTGC-3') que tem como alvo a amplificação de um fragmento de 354 pb no gene nox da B. hyodysenteriae; P1 (FWH) (5'-AGAGGAAAGTTTTTTCGCTTC-3') e P2 (REV) (5'-GCACCTA-TGTTAAACGTCCTTG-3') amplificando uma região de 823 pb do segmento 16S do rRNA da B. pilosicoli.…”

A preliminary study of the molecular epidemiology of Brachyspira hyodysenteriae isolates in Australia

“…In this study, a multiplex PCR for B. hyodysenteriae, B. pilosicoli, L. intracellularis and Salmonella spp. including primers described by Elder et al (1997) was compared with the PCR targeting NADH oxidase (nox) as described by La et al (2006). Multi-locus sequence typing (MLST) was used to determine the relatedness of the B. hyodysenteriae isolates (La et al 2009).…”