A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis

Abstract

BackgroundA 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).ResultsUsing 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 × 102 GE 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%).ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0939-6) contains supplementary material, which is available to authorized users.

Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

“…The concentration of the obtained DNA samples was in the range of 100-400 ng/μl. DNA samples were identified by multiplex qPCR targeting 23 s rDNA (Borgström et al, 2017). For further identification, DNA samples of a subset of epidemiologically non-linked clinical samples originating from different farms were chosen for a genus-specific PCR using primers targeting the Brachyspira nox gene (Rohde et al, 2002).…”

“…Channels for measuring the different fluorophores calculated PCR efficiencies measured in the linear range, and r 2 values are indicated. Borgström et al (2017) To examine the intra-and inter-assay variability of the novel qPCR assay representing its repeatability, the above mentioned four reference strains were tested using tenfold dilution series in the linear range between 10 7 and 100 GE. The variability assays were performed in triplicates in three experiments.…”

The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans

“…Duplex and multiplex PCRs, detecting different Brachyspira species and sometimes also detecting other enteric pathogens, and quantitative PCR assays that allow spirochete quantitation also have been described for use in samples from pigs (188)(189)(190)(191)(192), chickens (189,193), and humans (194).…”

Aportaciones al diagnóstico y control de la disentería porcina = Contributions to the dianosis and control of swine dysentery

“…Más recientemente, se han introducido técnicas de PCR a tiempo real que han demostrado una sensibilidad mayor que la mostrada por la PCR convencional o por el uso de cultivo y pruebas bioquímicas en la identificación de B. hyodysenteriae (Akase et al 2009). Adicionalmente, el empleo de PCR a tiempo real ha permitido desarrollar técnicas cuantitativas que permiten la detección y cuantificación de espiroquetas en concentraciones muy bajas en heces y que se ven afectadas mínimamente por factores de inhibición presentes en las mismas (Borgström et al 2017). Cuando estas técnicas se emplean para el diagnóstico de varias especies de espiroquetas simultáneamente nos referimos a PCR cuantitativas multiplex que permiten disminuir al máximo el tiempo necesario para obtener los resultados del diagnóstico de diferentes especies del género Brachyspira.…”

“…Desde mediados de los años 2000 algunos laboratorios utilizan únicamente este tipo de técnicas para el diagnóstico de DP, evitando el cultivo rutinario de muestras de heces (Song & Hampson 2009b;Hampson et al 2015). De hecho, actualmente ya existen PCRs multiplex y a tiempo real que permiten detectar directamente en las heces casi todas las especies del género Brachyspira que colonizan el intestino del cerdo (Borgström et al 2017). Sin embargo y como principal limitación, cabe destacar que estas técnicas al no asociarse al aislamiento de la cepa implicada no permiten llevar a cabo estudios de sensibilidad antimicrobiana o estudios de caracterización molecular o bioquímica de los aislados.…”