# US 12,110,544 B2 · Method for extracting total microbial DNA from milk > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,110,544 B2 - Document ID: US12110544B2 - Status: Granted - Kind: B2 - Application number: 17/058,964 - Filed: 2018-07-13 - Published: 2021-09-23 - Granted: 2024-10-08 - Earliest claimed: 2018-05-25 - Assignees: Institute of Animal Sciences of Chinese Academy of Agricultural Sciences - Applicants: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences - Inventors: Jiaqi Wang; Shengguo Zhao; Nan Zheng; Xu Zhou; Songli Li - Web page: https://patentgrep.com/patent/US12110544B2 ## CPC - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 1/689: for bacteria - C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor} - C12Q 1/06: Quantitative determination - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract In a method for extracting microbial DNA from milk, the extracted DNA can be directly used for PCR amplification. The method includes the following steps: treating milk with TE solution, fully disrupting microbial cells by bead milling, immobilizing proteins in the middle layer between the aqueous and organic phases with high-vacuum silicone grease so as to prevent impurities in the middle layer from entering the aqueous phase, etc. The extracted DNA has a high degree of intactness, high concentration and purity. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein, in step (1), the milk sample is centrifuged at 12000-15000 g before and after the addition of TE solution, and the centrifugation time is preferably 3-10 min; the fat and residual fat are removed by using sterile cotton swabs; the amount of the TE solution added is 1-5 ml; the TE solution is 1-20 mM Tris-HCl, 0.5-5 mM EDTA or disodium EDTA, pH 7-9. ### Claim 3 (depends on 1) The method according to claim 1, wherein, in step (2), the volume of the CTAB lysate is 500-1500 μl; the diameter of glass beads is 0.01-0.1 mm; the cell disruption is performed at 10-30 m/s, and the time is 0.5-2 min; the cell disruption is performed 1-3 times, a pause of 30 s-5 min; the proteinase K solution is an aqueous solution of proteinase K; the concentration of the proteinase K solution is 10-50 mg/ml; the amount of the proteinase K solution added is 10-50 μl; the mixture is firstly kept in a 52-58° C. water bath for 20-40 min, and then in a 65-75° C. water bath for 10-30 min; the mixture is mixed every 5-10 min in the water bath; the mixture is centrifugated at 12000-15000 g, and the centrifugation time is 1-10 min; the CTAB lysate contains 1.4 M NaCl, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA or disodium EDTA (pH 8.0) and 2% CTAB. ### Claim 4 (depends on 1) The method according to claim 1, wherein, in step (3), the ratio of each component in the mixed solution of phenol:chloroform:isoamyl alcohol is 25:24:1; the solution is vortex mixed for 2-10 s; the amount of the high-vacuum silicone grease added is 0.1-0.3 g; the centrifugation performed before and after the addition of isopropanol, and after being washed with the ethanol aqueous solution, is carried out at 12000-15000 g; the centrifugation time before the addition of isopropanol is 1-10 min, the centrifugation time after the addition of isopropanol is 10-20 min; the centrifugation is performed preferably at 4° C.; the centrifugation time after being washed with the ethanol aqueous solution is 5-15 min; the amount of isopropanol added is 0.6-1 times the volume of the upper aqueous phase taken; the temperature of isopropanol is 0-20° C.; the standing time is 15 min-1 h, and the standing is carried out at 4° C.; the concentration of the ethanol aqueous solution is 60-80%; the amount of the ethanol aqueous solution added is 500-1000 μl; and the drying time is 15 min-1 h. ### Claim 5 (depends on 1) The method according to claim 1, wherein, in step (4), the TE solution is 10 mM Tris-HCl, 1 mM EDTA or disodium EDTA, pH 8; the amount of the TE solution added is 50-150 μl. ### Claim 6 (depends on 5) The method according to claim 5, wherein, in step (4), the amount of the TE solution added is 100 μl. ### Claim 7 (depends on 2) The method according to claim 2, wherein, the milk sample is centrifuged at 14000 g before and after the addition of TE solution, and the centrifugation time is 5 min; the amount of the TE solution added is 3 ml; and the TE solution is 10 mM Tris-HCl, 1 mM EDTA or disodium EDTA, pH 8. ### Claim 8 (depends on 4) The method according to claim 4, wherein, in step (2), the volume of the CTAB lysate is 1000 μl; the diameter of glass beads is 0.05 mm; the cell disruption is performed at 30 m/s, and the time is 1 min; the cell disruption is performed twice, with a pause of 1 min; the concentration of the proteinase K solution is 20 mg/ml; the amount of the proteinase K solution added is 20 μl; the mixture is firstly kept in a 55° C. water bath for 30 min, and then in a 70° C. water bath for 20 min; the mixture is mixed every 5 min in the water bath; the mixture is centrifugated at 14000 g, and the centrifugation time is 5 min. ### Claim 9 (depends on 1) The method according to claim 1, wherein, in step (3), the solution is vortex mixed for 3-5 s; the amount of the high-vacuum silicone grease added is 0.2 g; the centrifugation performed before and after the addition of isopropanol, and after being washed with the ethanol aqueous solution, is carried out at 14000 g; the centrifugation time before the addition of isopropanol is 5 min, the centrifugation time after the addition of isopropanol is 15 min; the centrifugation time after being washed with the ethanol aqueous solution is 10 min; the amount of isopropanol added is 0.8 times the volume of the upper aqueous phase taken; the temperature of isopropanol is 4° C.; the standing time is 30 min; the concentration of the ethanol aqueous solution is 70%; the amount of the ethanol aqueous solution added is 800 μl; and the drying time is 20 min.