title: Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.
abstract: A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.
authors.affiliation: Toshiba Corporation, 1, Komukai-Toshiba-cho, Saiwai-ku, Kawasaki, Kanagawa 212-8582, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Unno
authors.forename: Mika
authors.initials: M
authors.lastname: Inada
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Michie
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Keiko
authors.initials: K
authors.lastname: Ito
authors.forename: Koji
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Masaru
authors.initials: M
authors.lastname: Nikaido
authors.forename: Tomohito
authors.initials: T
authors.lastname: Hayashi
authors.forename: Eiji
authors.initials: E
authors.lastname: Hata
authors.forename: Ken
authors.initials: K
authors.lastname: Katsuda
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kiku
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Tagawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kawai
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis / Q000382:microbiology*; D008892:Milk / Q000737:chemistry* / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial
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