Comparative evaluation of DNA extraction methods for detecting mastitis associated bacteria in milk

Bovine milk, a complex biological fluid rich in proteins and fats that can inhibit enzymatic amplification, poses challenges for bacterial DNA extraction. While commercial extraction kits are available, they are often costly and equipment dependent. This study aimed to evaluate a rapid, cost-effective, and centrifuge-free DNA extraction method optimised for use with bovine milk under laboratory conditions. The performance of the optimised HotSHOT (HS) protocol was compared with five commercial spin-column kits and a boiling method using Staphylococcus aureus (S. aureus) and Pasteurella multocida (P. multocida) as Gram-positive and Gram-negative representatives, respectively, spiked into pasteurised milk. Limits of detection (LOD) were determined for each extraction method. DNA extracted by all methods was tested using colourimetric loop-mediated isothermal amplification (LAMP) and PCR. The HS method achieved an LOD of 1.58 × 10 ³ colony-forming units (CFU)/mL for P. multocida and 8.4 × 10⁴ CFU/mL for S. aureus using LAMP, and 1.58 × 10⁵ and 8.4 × 10⁵ CFU/mL, respectively, using PCR. These endpoints fell within the range observed for several commercial kits under the laboratory conditions evaluated. Because the study did not include independent milk-spiking and DNA-extraction replicates, the findings do not establish statistical equivalence or superiority among extraction methods. Further evaluation using independently prepared samples and milk from cows with intramammary infections is required.