Rapid and effective method for separation of Staphylococcus aureus from somatic cells in mastitis milk.
Quantitative PCR can be an effective method for identifying the bacteria causing mastitis. However, PCR detection is hampered by the presence of inflammatory somatic cells. To eliminate this problem, we attempted to establish methods that allow the effective separation of bacterial cells from somatic cells in mastitis milk with amino-silica. Somatic cells and Staphylococcus aureus cells have different sizes, surface structures, and overall electrical charges; therefore, their adsorption and desorption behavior on amino-silica was also different. We found that although amino-silica could efficiently adsorb both somatic cells and Staph. aureus, somatic cells were adsorbed much more strongly than bacterial cells. We identified conditions under which most of the somatic cells adsorbed and only Staph. aureus desorbed from amino-silica upon addition of a desorption solution. We demonstrated that this procedure effectively eliminated somatic cells in heavily contaminated milk samples, which resulted in improved clarity of the PCR band. These results indicate that pretreatment of the samples with amino-silica made the PCR-based strategy for identifying and quantifying disease-causing bacteria applicable for all milk samples.
Kubota
M
M
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Hayashi
T
T
Iwasaki
K
K
Ohtsuka
H
H
Kohiruimaki
M
M
Kawamura
S
S
Sakaguchi
K
K
Abe
R
R
D000327:Adsorption; D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial; D005260:Female; D007958:Leukocyte Count; D007962:Leukocytes; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000166:cytology* / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012822:Silicon Dioxide; D013211:Staphylococcus aureus / Q000302:isolation & purification*
D012822:Silicon Dioxide