Improved method for rapid DNA extraction of Mastitis pathogens directly from milk

Efficient control against bovine mastitis requires sensitive, rapid and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen; therefore, an efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive and fast method to extract DNA from the main Gram positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis) directly from milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of six total basic steps (three of which were done twice) was developed and applied directly to milk samples. The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.