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

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bovine milk's composition presents challenges for bacterial DNA extraction due to inhibitory substances.
• Commercial DNA extraction kits are often expensive and require specialized equipment.
• There is a need for efficient, affordable, and accessible bacterial DNA extraction methods for milk samples.

Purpose of the Study:

• To evaluate a rapid, cost-effective, and centrifuge-free DNA extraction method (HotSHOT protocol) for bovine milk.
• To compare the performance of the optimized HotSHOT method against commercial kits and a boiling method.
• To determine the limits of detection (LOD) for bacterial DNA extracted using different methods.

Main Methods:

• Optimized HotSHOT (HS) DNA extraction protocol development.
• Comparison with five commercial spin-column kits and a boiling method.
• Spiking pasteurized milk with Staphylococcus aureus (Gram-positive) and Pasteurella multocida (Gram-negative).
• Limit of Detection (LOD) determination using colorimetric loop-mediated isothermal amplification (LAMP) and PCR.

Main Results:

• The HS method achieved LODs of 1.58 × 10³ CFU/mL for P. multocida and 8.4 × 10⁴ CFU/mL for S. aureus using LAMP.
• Using PCR, the HS method yielded LODs of 1.58 × 10⁵ CFU/mL for P. multocida and 8.4 × 10⁵ CFU/mL for S. aureus.
• The performance of the HS method was comparable to several commercial kits under the evaluated laboratory conditions.

Conclusions:

• The optimized HotSHOT (HS) protocol offers a promising rapid, cost-effective, and centrifuge-free alternative for bacterial DNA extraction from bovine milk.
• Further validation with independent replicates and samples from infected cows is necessary to establish statistical significance and superiority.
• The HS method demonstrates potential for routine laboratory use in milk bacterial analysis.