Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

• Published in: Journal of Veterinary Medical Science Vol. 77; no. 8; pp. 1007 - 1009
• Main Authors: UNNO, Hirotaka, INADA, Mika, NAKAMURA, Akiyoshi, HASHIMOTO, Michie, ITO, Keiko, HASHIMOTO, Koji, NIKAIDO, Masaru, HAYASHI, Tomohito, HATA, Eiji, KATSUDA, Ken, KIKU, Yoshio, TAGAWA, Yuichi, KAWAI, Kazuhiro
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 01.01.2015; Japan Science and Technology Agency; The Japanese Society of Veterinary Science
• Subjects: Animals; Bacteriology; Cattle; cow milk; DNA chip; DNA extraction; DNA, Bacterial - isolation & purification; Female; loop-mediated isothermal amplification (LAMP); mastitis; Mastitis, Bovine - diagnosis; Mastitis, Bovine - microbiology; Milk - chemistry; Milk - microbiology; Nucleic Acid Amplification Techniques - veterinary; pathogen; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcal Infections - veterinary; Staphylococcus aureus; Staphylococcus aureus - genetics
• ISSN: 0916-7250; 1347-7439; 1347-7439
• DOI: 10.1292/jvms.14-0159

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.