Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR

Abstract

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.

FIG. 1

Effect of lysis buffer composition and denaturing agent on the detection of Brucella DNA by PCR. Samples in lanes 2 to 7 were sterile bovine milk inoculated with B. abortus (2 × 10⁵ CFU/ml). Lanes: 1, negative control without DNA; 2 and 3, DNA extracted with SDS and TE buffer; 4 and 5, DNA extracted with SDS and NET buffer; 6 and 7, DNA extracted with SDS, NaOH, and NET buffer; 8, positive control with B. abortus DNA; 9, φX174 DNA/HaeIII marker (Boehringer Mannheim). The lysis incubation temperature was 80°C in lanes 2, 4, and 6, and 100°C in lanes 3, 5, and 7. The size of the amplification product is about 905 bp. No amplification was detected when Zw 3-14 was used instead of SDS (data not shown).

FIG. 2

Effect of the DNA extraction with commercial systems on the detection of Brucella DNA by PCR. Samples in lanes 3 to 5 were sterile bovine milk inoculated with B. abortus (2 × 10⁵ CFU/ml). Lanes: 1, φX174 DNA/HaeIII marker (Boehringer Mannheim); 2, positive control with B. abortus DNA; 3, DNA extracted with the Prep-A-Gene system; 4, DNA extracted with the Instagene system; 5, DNA extracted with phenol-chloroform-isoamyl alcohol; 6, negative control without DNA.

FIG. 3

Limit of detection of PCR. Lanes: 1, φX174 DNA/HaeIII marker (Boehringer Mannheim); 2 to 4, 50 B. abortus CFU/ml of milk; 5 to 7, 5 B. abortus CFU/ml of milk; 8, negative control without DNA. Brucella DNA was extracted from bovine milk by the optimized method.