Method for extracting total microbial DNA from milk

A method for identifying microbial species in milk, comprising the following steps:

• (1) milk pre-treatment:
• placing milk samples in centrifuge tubes, centrifuging, discarding the supernatant, removing fat, adding TE solution, repeatedly pipetting up and down with a pipetting gun until the precipitate is completely dissolved, centrifuging, discarding the supernatant, removing residual fat, and retaining the precipitate;
• (2) microbial lysis:
• adding CTAB lysate into said precipitate obtained in step (1), repeatedly pipetting up and down with a pipetting gun until said precipitate is completely dissolved, adding glass beads, disrupting cells in a bead mill, adding a proteinase K solution, placing in water bath, centrifuging, and taking the supernatant for use;
• (3) DNA purification:
• adding an equal volume of a mixed solution of phenol:
• chloroform:
• isoamyl alcohol to said supernatant obtained in step (2), vortex mixing until a white emulsion is obtained, adding appropriate amount of high-vacuum silicone grease, centrifuging, taking the upper aqueous phase, adding cold isopropanol, mixing the mixture upside and down, letting it stand, centrifuging, discarding the supernatant, adding an ethanol aqueous solution to wash the precipitate, centrifuging, discarding the supernatant, placing the precipitate on a filter paper, and drying at room temperature to obtain the milk total microbial DNA;
• and
• (4) identification of microbial species:
• dissolving said milk total microbial DNA obtained in step (3) in TE solution, performing PCR amplification, performing recovery and purification with PCR product purification kits, sequencing, and then identifying microbial species in milk;
• the upstream primer for PCR amplification consists of the nucleotide sequence as shown in SEQ ID NO:
• 1 and the downstream primer consists of the nucleotide sequence as shown in SEQ ID NO:
• 2;
• and
• the amount of the milk sample is 7 ml.