title: A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.
abstract: The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA.
authors.forename: S
authors.initials: S
authors.lastname: Boonyayatra
authors.forename: L K
authors.initials: LK
authors.lastname: Fox
authors.forename: T E
authors.initials: TE
authors.lastname: Besser
authors.forename: A
authors.initials: A
authors.lastname: Sawant
authors.forename: J M
authors.initials: JM
authors.lastname: Gay
authors.forename: Z
authors.initials: Z
authors.lastname: Raviv
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis / Q000382:microbiology*; D008892:Milk / Q000382:microbiology; D009174:Mycoplasma / Q000235:genetics*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D045646:Mycoplasma bovigenitalium / Q000235:genetics*; D045682:Mycoplasma bovis / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S