Technical note: 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, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in 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 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and 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.
Cremonesi
P
P
Department of Biomedical Sciences and Technologies, University of Milan, Segrate, Milan, Italy. paola.cremonesi@unimi.it
Castiglioni
B
B
Malferrari
G
G
Biunno
I
I
Vimercati
C
C
Moroni
P
P
Morandi
S
S
Luzzana
M
M
D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005260:Female; D015511:Goat Diseases / Q000382:microbiology; D006041:Goats; D008413:Mastitis / Q000382:microbiology / Q000662:veterinary; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000166:cytology / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics; D013291:Streptococcus / Q000235:genetics; D013292:Streptococcus agalactiae / Q000235:genetics
D004269:DNA, Bacterial