Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk.
Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 microL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.
Graber
H U
HU
Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse-Faculty, University of Berne, Bremgartenstrasse 109a, PO Box 8466, 3001 Berne, Switzerland. hans.graber@knp.unibe.ch
Casey
M G
MG
Naskova
J
J
Steiner
A
A
Schaeren
W
W
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000662:veterinary; D001483:Base Sequence; D002417:Cattle; D015169:Colony Count, Microbial; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000382:microbiology*; D008836:Micrococcal Nuclease / Q000235:genetics; D008892:Milk / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000302:isolation & purification*
D001426:Bacterial Proteins; D017931:DNA Primers; C074002:nuc protein, staphylococcus; D008836:Micrococcal Nuclease