title: Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.
abstract: As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngμL(-1) to 100ngμL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.
authors.affiliation: Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
authors.forename: Jenni
authors.initials: J
authors.lastname: Whiteduck-Léveillée
authors.affiliation: Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Cloutier
authors.affiliation: London Research and Development Centre (LRDC), Agriculture and Agri-Food Canada, London, ON, Canada.
authors.forename: Edward
authors.initials: E
authors.lastname: Topp
authors.affiliation: Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
authors.forename: David R
authors.initials: DR
authors.lastname: Lapen
authors.affiliation: Sherbrooke Research and Development Centre (SRDC), Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada.
authors.forename: Guylaine
authors.initials: G
authors.lastname: Talbot
authors.affiliation: INRS-Institute Armand-Frappier Research Centre, Laval, QC, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Villemur
authors.affiliation: Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada. Electronic address: izhar.khan@agr.gc.ca.
authors.forename: Izhar U H
authors.initials: IU
authors.lastname: Khan
mesh_terms: D000818:Animals; D020640:Arcobacter / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D005353:Fibronectins / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D005353:Fibronectins; D037521:Virulence Factors
keywords: A. cryaerophilus; A. skirrowii; Arcobacter butzleri; Fecal matter; Multiplex PCR assays; Virulence-associated genes