title: Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM).
abstract: Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis.
authors.affiliation: Department of Pathobiology, Nihon University School of Veterinary Medicine.
authors.forename: Hideto
authors.initials: H
authors.lastname: Sobukawa
authors.forename: Masato
authors.initials: M
authors.lastname: Ibaraki
authors.forename: Rui
authors.initials: R
authors.lastname: Kano
authors.forename: Takaaki
authors.initials: T
authors.lastname: Ito
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Suzuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kamata
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Hasegawa
mesh_terms: D000818:Animals; D002417:Cattle; D048028:DNA, Algal / Q000032:analysis / Q000235:genetics; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000209:etiology*; D011525:Prototheca / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
chemical_list: D048028:DNA, Algal; D012337:RNA, Ribosomal, 18S