title: Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus.
abstract: A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.
authors.affiliation: AgResearch, New Zealand. gale.brightwell@agresearch.co.nz
authors.forename: Gale
authors.initials: G
authors.lastname: Brightwell
authors.forename: Eilidh
authors.initials: E
authors.lastname: Mowat
authors.forename: Robyn
authors.initials: R
authors.lastname: Clemens
authors.forename: Jackie
authors.initials: J
authors.lastname: Boerema
authors.forename: David J
authors.initials: DJ
authors.lastname: Pulford
authors.forename: Stephen L
authors.initials: SL
authors.lastname: On
mesh_terms: D000818:Animals; D020640:Arcobacter / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology*; D008460:Meat / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D012756:Sheep
chemical_list: D004269:DNA, Bacterial; D012338:RNA, Ribosomal, 23S