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

The HotSHOT extraction method shows promise as a low-cost mastitis diagnostic alternative, but requires further clinical validation.

This study evaluated a modified HotSHOT (HS) DNA extraction protocol as a rapid, cost-effective, and centrifuge-free alternative to commercial spin-column kits for detecting mastitis-associated bacteria in bovine milk. Bovine milk presents inherent challenges for molecular diagnostics due to its high protein and fat content, which can inhibit enzymatic amplification. The HS method was compared against five commercial kits and a simple boiling method using Staphylococcus aureus (Gram-positive) and Pasteurella multocida (Gram-negative) spiked into pasteurised milk. Detection was performed using both colourimetric loop-mediated isothermal amplification (LAMP) and conventional PCR. The optimised HS protocol achieved limits of detection of 1.58 × 10³ CFU/mL for P. multocida and 8.4 × 10 CFU/mL for S. aureus via LAMP, with comparable performance using PCR. These results were within the range achieved by several commercial kits under the same laboratory conditions. However, a significant methodological limitation was the absence of independent milk-spiking and DNA-extraction replicates, which prevents statistical comparison of equivalence or superiority among methods. The study was also conducted exclusively under controlled laboratory conditions using spiked pasteurised milk rather than clinical samples from cows with natural intramammary infections. While the HS method shows promise as a low-cost diagnostic tool for bovine mastitis, further validation using independently prepared replicates and clinical milk samples is necessary before field implementation can be recommended.

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