Method for efficiently extracting nucleic acid from fresh milk

Abstract

The invention relates to a method for efficiently extracting nucleic acid from fresh milk, which belongs to the technical field of bioengineering application, and is characterized by comprising the following steps: (1) adding normal saline into a fresh milk sample for the second time, mixing and centrifuging, and discarding a fat layer and supernatant liquid to obtain a precipitate A; (2) adding a TES solution, a guanidine hydrochloride solution and a protease K solution into the precipitate A; cooling at room temperature after water bath, adding Tris saturated phenol, extracting and centrifuging, and taking supernate B; and (3) adding a sodium acetate solution and isopropanol into the supernate B, mixing and centrifuging, discarding the supernate to obtain nucleic acid, dissolving the nucleic acid in double distilled water, and preserving at-20 DEG C for later use. The method can effectively eliminate the influence of fat and protein on nucleic acid extraction, and has the characteristics that nucleic acid is conveniently and effectively extracted from various fresh milk, the concentration and purity of the extracted nucleic acid are high, and the method is simple.

Description

Technical Field

[0001] The present invention relates to a method for efficiently extracting nucleic acids from fresh milk, belonging to the technical field of bioengineering applications. Background Art

[0002] In recent years, the development of molecular biology has promoted the depth and breadth of research on animals. For a long time, the sources and extraction of livestock nucleic acids mainly originated from the blood of livestock. However, the collection of livestock blood has a certain stress response on livestock and also has a certain degree of difficulty, while the collection of fresh milk is relatively easy. The nucleic acids extracted from samples are necessary materials for research such as pathogenic microorganism detection, species identification, species origin, species diversity assessment and their genetic relationships, and species phylogenetic evolution. However, fresh milk contains a large amount of fat and rich proteins, which cause great difficulties in extracting nucleic acids from fresh milk, resulting in insufficient purity and low concentration of the extracted nucleic acids. Whether high-quality nucleic acid molecules can be extracted is the key to nucleic acid molecular biology experiments, and the sensitivity and specificity of the extraction method will directly affect the success or failure of subsequent experiments. Traditional nucleic acid extraction methods mainly include guanidine isothiocyanate-phenol-chloroform extraction method, alkaline extraction method, cetyltrimethylammonium bromide extraction method, ethidium bromide-caesium chloride density gradient centrifugation method, and oligo-deoxythymidine-cellulose chromatography method, etc. These methods are difficult to effectively remove the fat and proteins in fresh milk, resulting in insufficient purity of the obtained nucleic acids, seriously affecting the progress of subsequent research, and even making subsequent research impossible to carry out. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for efficiently extracting nucleic acids from fresh milk, which can effectively eliminate the influence of fat and proteins on nucleic acid extraction, and has the characteristics of conveniently and effectively extracting nucleic acids from various fresh milks, high concentration and purity of the extracted nucleic acids, and simple method.

[0004] The present invention realizes the above purpose through the following technical solutions.

[0005] A method for efficiently extracting nucleic acids from fresh milk provided by the present invention includes the following steps:

[0006] (1) Take a fresh milk sample, add physiological saline with a volume of 50% of the fresh milk sample thereto, mix evenly, centrifuge at 13,000 revolutions per minute at 4°C for 10 minutes, and discard the fat layer and the upper liquid; then add physiological saline with the same volume as the fresh milk sample to the remaining liquid, mix evenly, centrifuge at 13,000 revolutions per minute at 4°C for 10 minutes, and discard the fat layer and the upper liquid to obtain precipitate A;

[0007] The fresh milk sample is cow fresh milk, buffalo fresh milk or goat fresh milk;

[0008] (2), Add TES solution with a volume of 86% of the fresh milk sample, guanidine hydrochloride solution with a volume of 10% of the fresh milk sample, and proteinase K solution with a volume of 4% of the fresh milk sample to precipitate A; incubate in a water bath at 50 °C for 12 hours, cool to room temperature, then add Tris-saturated phenol with a volume of 50% of the fresh milk sample, extract for 10 minutes, centrifuge at 13,000 rpm and 4 °C for 5 minutes, take the supernatant B, and discard the precipitate;

[0009] For the TES solution described above, the final concentration of Tris-HCl is 45 mmol / L; the final concentration of EDTA is 12 mmol / L; the final concentration of SDS is 1.3%; the pH value is 7.8; the concentration of the guanidine hydrochloride solution is 4.5 mol / L; the concentration of the proteinase K solution is 20 mg / mL;

[0010] The Tris-saturated phenol is a commercially available product;

[0011] (3), Take the supernatant B, add sodium acetate solution with a volume of 10% of the supernatant B and isopropanol with a volume of 80% of the supernatant B to the supernatant B, mix evenly to precipitate nucleic acids, centrifuge at 13,000 rpm and 4 °C for 10 minutes, discard the supernatant, dissolve the nucleic acids in double-distilled water with a volume of 10% of the fresh milk sample, and store at -20 °C for later use;

[0012] The concentration of the sodium acetate solution described above is 3 mol / L and the pH value is 5.2;

[0013] The isopropanol is a commercially available product.

[0014] The present invention has the following beneficial effects compared with the existing technology:

[0015] 1. The present invention uses the method of adding normal saline twice and high-speed refrigerated centrifugation, so that the fat in fresh milk has no influence on the nucleic acid extraction process, and the purity of nucleic acid extraction is improved.

[0016] 2. The present invention uses guanidine hydrochloride and proteinase K to decompose and digest proteins and Tris-saturated phenol to separate proteins, so that only extremely trace amounts of proteins are contained in the subsequent nucleic acid precipitation process, and the concentration and purity of the extracted nucleic acids are very high.

[0017] 3. The present invention can effectively exclude the influence of fat and proteins on nucleic acid extraction, and has the characteristics of conveniently and effectively extracting nucleic acids from various fresh milks, high concentration and purity of the extracted nucleic acids, and simple method. Specific embodiments

[0018] The present invention will be further described below in conjunction with specific embodiments:

[0019] 1. Reagents (all reagents are of analytical grade or biological grade)

[0020] Normal saline; TES solution (final concentration of Tris-HCl is 45 mmol / L; final concentration of EDTA is 12 mmol / L; final concentration of SDS is 1.3%; pH value is 7.8); guanidine hydrochloride solution (4.5 mol / L); proteinase K solution (20 mg / mL); sodium acetate solution (3 mol / L, pH value is 5.2); Tris-saturated phenol; isopropanol.

[0021] 2. Instrumentation and Materials

[0022] High-speed refrigerated centrifuge (at least meeting 13,000 revolutions per minute, 4°C); ultrapure water generator (molecular biology level); constant temperature water bath; refrigerator (2°C - 8°C, -20°C); micropipette with adjustable volume and matching pipette tips (10 μl - 100 μl, 50 μl - 200 μl, 100 μl - 1000 μl); centrifuge tubes (1.5 mL, 2 mL).

[0023] 3. Experimental Methods

[0024] (1) Take 1000 μl of fresh buffalo milk sample, add 500 μl of normal saline, mix evenly, centrifuge at 13,000 revolutions per minute and 4°C for 10 minutes, discard the fat layer and the upper liquid, then add 1000 μl of normal saline, mix evenly, centrifuge at 13,000 revolutions per minute and 4°C for 10 minutes, discard the fat layer and the upper liquid to obtain precipitate A.

[0025] (2) Add 860 μl of TES solution, 100 μl of guanidine hydrochloride solution with a concentration of 4.5 mol / L, and 40 μl of proteinase K solution with a concentration of 20 mg / mL to precipitate A in step (1), incubate in a water bath at 50°C for 12 hours, cool to room temperature, then add 500 μl of Tris-saturated phenol, extract for 10 minutes, centrifuge at 13,000 revolutions per minute and 4°C for 5 minutes, take the supernatant B, and discard the precipitate.

[0026] (3) Take the supernatant B in step (2), add 10% of the volume of supernatant B of sodium acetate solution with a concentration of 3 mol / L and a pH value of 5.2 and 80% of the volume of supernatant B of isopropanol, mix evenly to precipitate nucleic acid, centrifuge at 13,000 revolutions per minute and 4°C for 10 minutes, discard the supernatant, dissolve the nucleic acid in 100 μl of double-distilled water, and store at -20°C for later use.

[0027] 4. Results

[0028] The somatic cell count (SCC) of fresh buffalo milk and the purity and concentration of the extracted nucleic acid are shown in Table 1.

[0029] SCC range: 16,000 / ml to 1,934,000 / ml, concentration range: 2789.1 ng / μl to 7280.7 ng / μl, A260 / A280 range: 1.56 to 2.17.

[0030] After SPSS statistical analysis, the correlation coefficient between SCC and nucleic acid concentration was 0.716.

[0031] Table 1 Nucleic acid extraction results of fresh buffalo milk

[0032]

[0033]

Claims

1. A method for efficiently extracting nucleic acid from fresh milk, characterized in that It includes the following steps: (1) Take fresh milk samples, add physiological saline with a volume of 50% of the fresh milk samples thereto, mix evenly, centrifuge at 13,000 revolutions per minute at 4°C for 10 minutes, and discard the fat layer and the upper liquid; then add physiological saline with the same volume as the fresh milk samples to the remaining liquid, mix evenly, centrifuge at 13,000 revolutions per minute at 4°C for 10 minutes, and discard the fat layer and the upper liquid to obtain precipitate A; (2) Add a TES solution with a volume of 86% of the fresh milk samples, a guanidine hydrochloride solution with a volume of 10% of the fresh milk samples, and a proteinase K solution with a volume of 4% of the fresh milk samples to precipitate A; incubate in a water bath at 50°C for 12 hours, cool to room temperature, then add Tris-saturated phenol with a volume of 50% of the fresh milk samples, extract for 10 minutes, centrifuge at 13,000 revolutions per minute at 4°C for 5 minutes, take the supernatant B, and discard the precipitate; For the TES solution described above, the final concentration of Tris-HCl is 45 mmol / L; the final concentration of EDTA is 12 mmol / L; the final concentration of SDS is 1.3%; the pH value is 7.8; the concentration of the guanidine hydrochloride solution is 4.5 mol / L; the concentration of the proteinase K solution is 20 mg / mL; the concentration of the sodium acetate solution is 3 mol / L and the pH value is 5.2; (3) Take supernatant B, add a sodium acetate solution with a volume of 10% of supernatant B and isopropanol with a volume of 80% of supernatant B thereto, mix evenly to precipitate nucleic acid, centrifuge at 13,000 revolutions per minute at 4°C for 10 minutes, discard the supernatant, dissolve the nucleic acid in double-distilled water with a volume of 10% of the fresh milk samples, and store at -20°C for standby.

2. The method for efficiently extracting nucleic acid from fresh milk according to claim 1, wherein, The fresh milk samples described above are cow fresh milk, buffalo fresh milk or goat fresh milk.

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