title: Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.
abstract: Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.
authors.affiliation: Department of Production Animal Medicine,Faculty of Veterinary Medicine,University of Helsinki,Paroninkuja 20,04920 Saarentaus,Finland.
authors.forename: Heidi
authors.initials: H
authors.lastname: Hiitiö
authors.affiliation: Veterinary Bacteriology Research Unit,Finnish Food Safety Authority Evira,Neulaniementie 4,70210 Kuopio,Finland.
authors.forename: Rauna
authors.initials: R
authors.lastname: Riva
authors.affiliation: Veterinary Bacteriology Research Unit,Finnish Food Safety Authority Evira,Neulaniementie 4,70210 Kuopio,Finland.
authors.forename: Tiina
authors.initials: T
authors.lastname: Autio
authors.affiliation: Veterinary Bacteriology Research Unit,Finnish Food Safety Authority Evira,Neulaniementie 4,70210 Kuopio,Finland.
authors.forename: Tarja
authors.initials: T
authors.lastname: Pohjanvirta
authors.affiliation: Thermo Fisher Scientific,Ratastie 2,01620 Vantaa,Finland.
authors.forename: Jani
authors.initials: J
authors.lastname: Holopainen
authors.affiliation: Department of Production Animal Medicine,Faculty of Veterinary Medicine,University of Helsinki,Paroninkuja 20,04920 Saarentaus,Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Pyörälä
authors.affiliation: Veterinary Bacteriology Research Unit,Finnish Food Safety Authority Evira,Neulaniementie 4,70210 Kuopio,Finland.
authors.forename: Sinikka
authors.initials: S
authors.lastname: Pelkonen
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification; D001431:Bacteriological Techniques / Q000662:veterinary*; D002417:Cattle; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis*; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*
keywords: bacteriological culture