CN116240206A - Kit and method for extracting total DNA of microorganisms in animal milk sample - Google Patents

Abstract

The invention discloses a kit and a method for extracting total DNA of microorganisms in an animal milk sample, and belongs to the technical field of molecular biology. The kit comprises a lysis reagent, wherein the lysis reagent comprises a lysis solution, lysozyme and proteinase K, the lysis reagent further comprises cross-linked or uncrosslinked polyvinylpyrrolidone, and the lysis solution comprises 2% of CTAB,1.4M NaCl,20mM EDTA and 100mM Tris-HCl. The kit and the method can effectively remove milk fat and protein in the extracted total DNA, are very suitable for extracting the microbial total DNA in animal milk samples, and the obtained total DNA has good quality and high purity and can obtain more and more comprehensive microorganism type and diversity information.

Description

Kit and method for extracting total DNA of microorganisms in animal milk sample

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a kit and a method for extracting total DNA of microorganisms in an animal milk sample.

Background

Animal milk is a raw material for processing dairy products commonly used in our lives, the content of microorganisms in the milk is an important factor affecting the quality and safety of food such as dairy products, and the detection of the microorganism types of the animal milk is particularly important. Since all microorganisms in the animal milk cannot be completely cultured by in vitro culture, all microorganism species contained in the animal milk cannot be identified by pure culture. At present, PCR amplification is used for identifying microorganism types, so that a large number of uncultured microorganisms can be effectively detected, however, the method needs to extract the microorganism DNA with higher quantity and purity, so that the extraction of the microorganism DNA in milk is an important link for detecting microorganisms in animal milk.

There are various methods for extracting microorganism total DNA from animal milk, and at present, the methods for extracting microorganism total DNA from animal milk at home and abroad mainly comprise: (1) The phenol-chloroform method has complex operation steps and long time consumption, causes sample loss, and also easily causes pollution among milk samples, and residual organic matters such as phenol have a certain inhibition effect on polymerase; (2) Nucleic acid purification columns are difficult to use in the detection of large quantities of milk samples due to their complexity and high cost. Because of the different milk fat and protein contents in the milk of different species of animals, if the milk is directly extracted according to the existing extraction method of total DNA of microorganisms in the milk of animals, the concentration is low, the milk fat and protein contents are high, and the DNA in the milk of animals cannot be completely and effectively extracted.

Disclosure of Invention

In order to solve at least one of the technical problems, the invention adopts the following technical scheme:

the invention provides a kit for extracting total DNA of microorganisms in an animal milk sample, which comprises a lysis reagent, wherein the lysis reagent comprises a lysis solution, lysozyme and proteinase K, and the lysis reagent also comprises cross-linked or uncrosslinked polyvinylpyrrolidone.

In the present invention, the uncrosslinked polyvinylpyrrolidone is polyvinylpyrrolidone.

In some embodiments of the invention, the lysate comprises 2% CTAB,1.4M NaCl,20mM EDTA and 100mM Tris-HCl.

In some embodiments of the invention, the kit further comprises a rinse solution and an eluent.

In some embodiments of the invention, the rinse solution is an organic alcohol solution, such as ethanol, butanol, and the like. In some preferred embodiments of the invention, the rinse solution is 80% ethanol.

In some embodiments of the invention, the eluent is selected from TE buffer, tris-HCl buffer and water. In some embodiments of the invention, the eluent is Tris-HCl at ph=8.0.

In a second aspect, the present invention provides a method for extracting total DNA from an animal milk sample by using the kit for extracting total DNA from an animal milk sample according to the first aspect of the present invention, comprising the steps of:

s1, respectively centrifuging 200 mu L of animal milk samples in a 2mL sterilizing centrifuge tube at 7000-10000 Xg and 4 ℃ for 8-15 min, and removing upper suspension;

s2, adding the same volume of the lysate into the sterilization centrifuge tube, adding crosslinked polyvinylpyrrolidone with the mass system fraction of 1-5%, 30-80 mu L of lysozyme with the mass system fraction of 100mg/mL and 15-20 mu L of proteinase K with the mass system fraction of 20mg/mL, and grinding for 5-15 min;

s3, placing the ground sample in a water bath kettle at 60-70 ℃ for cracking for 0.8-1.5 h;

s4, taking out the sample from the water bath kettle, adding 1-3 mu L of 10mg/mL RNase, and carrying out water bath at 35-40 ℃ for 25-60 min;

s5, adding chloroform with the volume of 0.5 times, reversing and uniformly mixing until the solution is completely emulsified into white, and centrifuging for 8-15 min at the temperature of 12000-15000 Xg and the temperature of 4 ℃;

s6, transferring 800 mu L of supernatant obtained in the sucking step into a new 1.5mL sterilizing centrifuge tube;

s7, adding magnetic beads with the volume of 0.6 times of that of the supernatant fluid, uniformly mixing by blowing for more than 10 times, and standing for 3-8 min at room temperature;

s8, placing the centrifuge tube in a magnetic rack until the solution is clear, and sucking and discarding the supernatant;

s9, keeping the centrifuge tube on a magnetic rack, adding 200 mu L of rinsing liquid into the tube, standing at room temperature for 20-40S, and then sucking and discarding the supernatant;

s10, performing instantaneous centrifugation, discarding redundant rinsing liquid in the centrifuge tube, and drying the magnetic beads at room temperature for 2-5 min;

s11, taking down the centrifuge tube from the magnetic rack, adding 65 mu L of eluent, lightly blowing and mixing by using a pipettor, and standing for 3-8 min at room temperature;

s12, placing the centrifuge tube on a magnetic rack, standing at room temperature for 3-8 min until the solution is clarified, sucking supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified total DNA of microorganisms in the milk sample.

In some embodiments of the invention, step S9 is repeated one time before step S10.

In some embodiments of the invention, the rinse solution is 80% ethanol.

In some embodiments of the invention, the eluent is selected from TE buffer, tris-HCl buffer and water. In some preferred embodiments of the invention, the eluent is 10mM Tris-HCl, pH=8.0.

In some embodiments of the invention, the animal is a pig, cow, sheep, or dog. In some preferred embodiments of the invention, the animal is a cow. In other preferred embodiments of the invention, the animal is a dairy goat.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following beneficial effects:

the kit and the method can effectively remove the milk fat and the protein in the total DNA obtained by extraction, and are very suitable for extracting the microbial total DNA in animal milk samples.

The kit and the method have the advantages that the obtained total DNA has good quality and high purity, and more comprehensive microorganism types and diversity information can be obtained.

Drawings

FIG. 1 shows the gel detection results of the total DNA samples of microorganisms in each of the milk samples extracted in examples 2 to 5 of the present invention. Wherein M is a DNA Marker, and 1-6 are detection results of total DNA gel of microorganisms in milk samples of cows extracted in example 2; 7-12 are the detection results of the total DNA gel of the microorganisms in the milk samples of the cows extracted in the example 3; 13-18 are the results of detecting the total DNA gel of the microorganisms in the milk sample of the dairy goat extracted in example 4; 19-24 are the results of the detection of the total DNA gel of the microorganisms in the milk sample of the dairy goat extracted in example 5.

FIG. 2 shows the gel detection results of PCR amplification using the total DNA samples of microorganisms in each of the milk samples extracted in examples 2 to 5 of the present invention as templates to obtain PCR products. Wherein M is a DNA Marker, and 1-6 are gel detection results of PCR products obtained by using total microorganism DNA in the milk sample extracted in example 2 as a template for amplification; 7-12 are gel detection results of PCR products obtained by using the total DNA of microorganisms in the milk samples extracted in the example 3 as a template for amplification; 13-18 are gel detection results of PCR products obtained by using the microorganism total DNA in the milk sample of the dairy goat extracted in the example 4 as a template for amplification; 19-24 are gel detection results of PCR products obtained by amplification using total DNA of microorganisms in the milk sample of the dairy goat extracted in example 5 as a template.

FIG. 3 shows Venn diagrams of microorganism species obtained by PCR amplification and library-building sequencing using the total DNA samples of microorganisms in milk samples of cows extracted in examples 2 and 3 of the present invention as templates. Wherein T represents a microorganism species amplified and sequenced using the total DNA of the microorganism of the milk sample extracted in example 2 as a template; c represents the microorganism species amplified and sequenced using the total DNA of the cow milk sample extracted in example 3 as a template.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.

Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The molecular biology experiments described in the following examples, which are not specifically described, were performed according to the specific methods listed in the "guidelines for molecular cloning experiments" (fourth edition) (j. Sambrook, m.r. Green, 2017) or according to the kit and product specifications. Other experimental methods, unless otherwise specified, are all conventional. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

EXAMPLE 1 microbial Total DNA extraction reagent for milk samples

1. Cleavage reagent

Solution a: including 2% CTAB,1.4M NaCl,20mM EDTA,100mM Tris-HCl.

Also included are crosslinked polyvinylpyrrolidone, lysozyme (100 mg/mL) and proteinase K (20 mg/mL).

2. Rinsing liquid

80% ethanol.

3. Eluent (eluent)

10mM Tris-HCl(pH＝8.0)。

The reagent can be used for preparing a kit for extracting total DNA of microorganisms in milk samples. The method is characterized in that the cracking reagent comprises crosslinked polyvinylpyrrolidone, so that the cracking effect can be remarkably improved, and the quality of DNA extraction can be improved.

Example 2 method for extracting microbial Total DNA from milk samples of cows (I)

The method comprises the following specific steps:

(1) Taking 1mL samples of milk produced by 6 healthy cows in a 2mL sterilizing tube, respectively, wherein the sample numbers are 1, 2, 3, 4, 5 and 6, centrifuging for 10min at 8000 Xg and 4 ℃, and removing the upper suspension;

(2) Adding an equal volume of solution A to the sterilization tube in the step (1), adding 2% (w/v) of cross-linked polyvinylpyrrolidone, 50 mu L of lysozyme (100 mg/mL) and 20 mu L of proteinase K (20 mg/mL), and grinding for 10min at 50Hz by using a grinder;

(3) Placing the sample ground in the step (2) in a water bath kettle at 65 ℃ for cracking for 1h;

(4) Taking the sample in the step (3) out of the water bath kettle, adding 2 mu L of RNase (10 mg/mL), and carrying out water bath at 37 ℃ for 30min;

(5) Adding chloroform with the volume of 0.5 times to the sample in the step (4), reversing and uniformly mixing the sample for 30s until the solution is completely emulsified into white, and centrifuging for 10min at the temperature of 13000 Xg and the temperature of 4 ℃;

(6) Pipetting 800 μl of supernatant from step (5) and transferring to a new 1.5mL centrifuge tube;

(7) Adding magnetic beads (Hangzhou Liangchuan biotechnology Co., ltd.) with 0.6 times of volume and mixing uniformly into the supernatant in the step (6), lightly blowing and mixing by a pipette for more than 10 times, and standing for 5min at room temperature;

(8) Placing the centrifuge tube in the step (7) on a magnetic rack for 5min until the solution is clear, carefully sucking and discarding the supernatant;

(9) Holding the centrifuge tube on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, carefully sucking and discarding the supernatant;

(10) Repeating the step (9) once;

(11) Instantly centrifuging, discarding redundant ethanol in the centrifuge tube, and then drying the magnetic beads at room temperature for 2-5 min;

(12) Taking down the centrifuge tube in the step (11) from the magnetic rack, adding 65 mu L of 10mM Tris-HCl eluent, lightly blowing and mixing by using a pipettor, and standing for 5min at room temperature;

(13) Placing the centrifuge tube in the step (12) on a magnetic rack, standing at room temperature for 5min until the solution is clear, carefully sucking 60 mu L of supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified DNA.

Example 3 method for extracting microbial Total DNA from milk samples of cows (II)

The embodiment provides another method for extracting total DNA of microorganisms in milk samples of cows, which comprises the following specific steps:

(1) Taking 1mL samples of milk produced by 6 healthy cows in a 2mL sterilizing tube, wherein the sample numbers are 7, 8, 9, 10, 11 and 12 respectively;

(2) Adding an equal volume of solution A to the sterilization tube in the step (1), and grinding 50 mu L of lysozyme (100 mg/mL) and 20 mu L of proteinase K (20 mg/mL) for 10min by using a grinder at 50 Hz;

(3) Placing the sample ground in the step (2) in a water bath kettle at 65 ℃ for cracking for 1h;

(4) Taking the sample in the step (3) out of the water bath kettle, adding 2 mu L of RNase (10 mg/mL), and carrying out water bath at 37 ℃ for 30min;

(5) Adding chloroform with the volume of 0.5 times to the sample in the step (4), reversing and uniformly mixing the sample for 30s until the solution is completely emulsified into white, and centrifuging for 10min at the temperature of 13000 Xg and the temperature of 4 ℃;

(6) Pipetting 800 μl of supernatant from step (5) and transferring to a new 1.5mL centrifuge tube;

(7) Adding magnetic beads (Hangzhou Liangchuan biotechnology Co., ltd.) with 0.6 times of volume and mixing uniformly into the supernatant in the step (6), lightly blowing and mixing by a pipette for more than 10 times, and standing for 5min at room temperature;

(8) Placing the centrifuge tube in the step (7) on a magnetic rack for 5min until the solution is clear, carefully sucking and discarding the supernatant;

(9) Holding the centrifuge tube on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, carefully sucking and discarding the supernatant;

(10) Repeating the step (9) once;

(11) Instantly centrifuging, discarding redundant ethanol in the centrifuge tube, and then drying the magnetic beads at room temperature for 2-5 min;

(12) Taking down the centrifuge tube in the step (11) from the magnetic rack, adding 65 mu L of 10mM Tris-HCl eluent, lightly blowing and mixing by using a pipettor, and standing for 5min at room temperature;

(13) Placing the centrifuge tube in the step (12) on a magnetic rack, standing at room temperature for 5min until the solution is clear, carefully sucking 60 mu L of supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified DNA.

This embodiment differs from embodiment 2 in that: (1) before lysis, centrifugation was not performed; (2) during cleavage, crosslinked polyvinylpyrrolidone was not added.

Example 4 method for extracting microbial Total DNA from milk samples of milk goats (I)

The method comprises the following specific steps:

(1) Taking 1mL samples of milk produced by 6 healthy dairy goats in a 2mL sterilizing tube, respectively, wherein the sample numbers are 13, 14, 15, 16, 17 and 18, centrifuging for 10min at 8000 Xg and 4 ℃, and removing the upper suspension;

(2) Adding an equal volume of solution A to the sterilization tube in the step (1), adding 2% (w/v) of cross-linked polyvinylpyrrolidone, 50 mu L of lysozyme (100 mg/mL) and 20 mu L of proteinase K (20 mg/mL), and grinding for 10min at 50Hz by using a grinder;

(3) Placing the sample ground in the step (2) in a water bath kettle at 65 ℃ for cracking for 1h;

(4) Taking the sample in the step (3) out of the water bath kettle, adding 2 mu L of RNase (10 mg/mL), and carrying out water bath at 37 ℃ for 30min;

(5) Adding chloroform with the volume of 0.5 times to the sample in the step (4), reversing and uniformly mixing the sample for 30s until the solution is completely emulsified into white, and centrifuging for 10min at the temperature of 13000 Xg and the temperature of 4 ℃;

(6) Pipetting 800 μl of supernatant from step (5) and transferring to a new 1.5mL centrifuge tube;

(7) Adding magnetic beads (Hangzhou Liangchuan biotechnology Co., ltd.) with 0.6 times of volume and mixing uniformly into the supernatant in the step (6), lightly blowing and mixing by a pipette for more than 10 times, and standing for 5min at room temperature;

(8) Placing the centrifuge tube in the step (7) on a magnetic rack for 5min until the solution is clear, carefully sucking and discarding the supernatant;

(9) Holding the centrifuge tube on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, carefully sucking and discarding the supernatant;

(10) Repeating the step (9) once;

(11) Instantly centrifuging, discarding redundant ethanol in the centrifuge tube, and then drying the magnetic beads at room temperature for 2-5 min;

(12) Taking down the centrifuge tube in the step (11) from the magnetic rack, adding 65 mu L of 10mM Tris-HCl eluent, lightly blowing and mixing by using a pipettor, and standing for 5min at room temperature;

(13) Placing the centrifuge tube in the step (12) on a magnetic rack, standing at room temperature for 5min until the solution is clear, carefully sucking 60 mu L of supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified DNA.

Example 5 method for extracting microbial Total DNA from milk samples of milk goats (II)

The embodiment provides another method for extracting microorganism total DNA of a milk sample of a dairy goat, which comprises the following specific steps:

(1) Taking 1mL samples of milk produced by 6 healthy dairy goats in a 2mL sterilizing tube, wherein the sample numbers are 19, 20, 21, 22, 23 and 24 respectively;

(2) Adding an equal volume of solution A to the sterilization tube in the step (1), and grinding 50 mu L of lysozyme (100 mg/mL) and 20 mu L of proteinase K (20 mg/mL) for 10min by using a grinder at 50 Hz;

(3) Placing the sample ground in the step (2) in a water bath kettle at 65 ℃ for cracking for 1h;

(4) Taking the sample in the step (3) out of the water bath kettle, adding 2 mu L of RNase (10 mg/mL), and carrying out water bath at 37 ℃ for 30min;

(5) Adding chloroform with the volume of 0.5 times to the sample in the step (4), reversing and uniformly mixing the sample for 30s until the solution is completely emulsified into white, and centrifuging for 10min at the temperature of 13000 Xg and the temperature of 4 ℃;

(6) Pipetting 800 μl of supernatant from step (5) and transferring to a new 1.5mL centrifuge tube;

(7) Adding magnetic beads (Hangzhou Liangchuan biotechnology Co., ltd.) with 0.6 times of volume and mixing uniformly into the supernatant in the step (6), lightly blowing and mixing by a pipette for more than 10 times, and standing for 5min at room temperature;

(8) Placing the centrifuge tube in the step (7) on a magnetic rack for 5min until the solution is clear, carefully sucking and discarding the supernatant;

(9) Holding the centrifuge tube on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, carefully sucking and discarding the supernatant;

(10) Repeating the step (9) once;

(11) Instantly centrifuging, discarding redundant ethanol in the centrifuge tube, and then drying the magnetic beads at room temperature for 2-5 min;

(12) Taking down the centrifuge tube in the step (11) from the magnetic rack, adding 65 mu L of 10mM Tris-HCl eluent, lightly blowing and mixing by using a pipettor, and standing for 5min at room temperature;

(13) Placing the centrifuge tube in the step (12) on a magnetic rack, standing at room temperature for 5min until the solution is clear, carefully sucking 60 mu L of supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified DNA.

This embodiment differs from embodiment 4 in that: (1) before lysis, centrifugation was not performed; (2) during cleavage, crosslinked polyvinylpyrrolidone was not added.

EXAMPLE 6 detection of total DNA quality of milk sample microorganism

The total DNA of the microorganisms in the milk samples of the cows extracted by electrophoresis in example 2 and example 3 and the total DNA of the microorganisms in the milk samples of the cows extracted by electrophoresis in example 4 and example 5 were detected, and the electrophoresis results are shown in FIG. 1. As can be seen from FIG. 1, the total DNA of microorganisms (lanes 1 6) in the milk sample of the dairy cow extracted in example 2 and the total DNA of microorganisms (lanes 13-18) in the milk sample of the dairy goat extracted in example 4 are bright and clear, which indicates that the quality of the total DNA extracted is better; whereas the total DNA of the microorganisms (lanes 7-24) in the milk samples of the cows extracted in example 3 and the total DNA of the microorganisms (lanes 7-12) in the milk samples of the cows extracted in example 5 were blurred, indicating that the total DNA extracted was of poor quality.

The inventors further measured the concentration of total DNA of microorganisms and OD260/OD280 in the milk samples of cows extracted in examples 2 and 3 and the milk samples of goats extracted in examples 4 and 5. The measurement results are shown in Table 1:

TABLE 1 DNA concentration and OD260/OD280 of each milk sample

Note that: OD260/OD280 represents the ratio of UV light absorption at 260nm and 280nm for detecting the purity of the extracted DNA, the appropriate ratio being between 1.8-2.0, <1.8 indicating protein contamination, >2.0 indicating RNA contamination.

As can be seen from Table 1, the OD260/OD280 of the total microorganism DNA of the milk sample extracted from the dairy cow in example 2 and the total microorganism DNA of the milk sample extracted from the dairy goat in example 4 are all between 1.8 and 2.0; the OD260/OD280 of the total DNA of the milk sample microorganism extracted in example 3 and the total DNA of the milk sample microorganism extracted in example 5 are lower than 1.8, which indicates that the total DNA of the milk sample microorganism extracted in example 3 and the total DNA of the milk sample microorganism extracted in example 5 have protein pollution, and the total DNA of the milk sample microorganism extracted in example 2 and example 4 can effectively remove protein pollution.

EXAMPLE 7 microbiological detection of milk samples

The 16S r RNA gene was amplified using bacterial universal primers using the microbial DNA of the milk sample obtained in example 2 and example 3 and the microbial DNA of the milk sample obtained in example 4 and example 5 as templates, respectively, to detect the kind and diversity of microorganisms.

Wherein, the primer sequences are respectively as follows:

341F：5’-CCTACGGGNGGCWGCAG-3’

806R：5’-GACTACHVGGGTATCTAATCC-3’

the PCR reaction system is as follows:

the reaction solution (2X Phanta Max master mix, novamat Biotech Co., ltd.) was 12.5. Mu.L, the upstream primer (1. Mu.M) was 2.5. Mu.L, the downstream primer (1. Mu.M) was 2.5. Mu.L, and the template DNA was 5-10. Mu.L, and was filled up to 25. Mu.L with sterilized deionized water.

The amplification conditions were: 98 ℃ for 1min;98℃10s,52℃30s,72℃45s,20 cycles; and at 72℃for 5min.

The gel electrophoresis diagram of the PCR products is shown in FIG. 2.

As can be seen from FIG. 2, the product (band 1 6) obtained by amplification using the microorganism total DNA of the milk sample extracted in example 2 as a template has a bright and clear band, which indicates that the DNA template has high purity. The amplification product (band 7 12) obtained by using the total DNA of the microorganisms of the milk sample extracted in example 3 as a template was blurred, indicating that the DNA template was of lower purity.

Similarly, the product (lanes 13-18) obtained by amplifying the microbial total DNA of the milk sample of the dairy goat extracted in example 4, which is taken as a template, has bright and clear bands, which shows that the DNA template has high purity. The products (lanes 19-24) obtained by amplification using the microbial total DNA of the milk sample of the dairy goat extracted in example 5 as a template were blurred, indicating that the DNA template was of lower purity.

The total DNA of the microorganisms extracted in examples 2 to 5 was used for library construction, and since the concentration of the product obtained by amplifying the total DNA of the microorganism extracted in example 5 as a template was very low, the conditions for library construction could not be satisfied, and further on-machine sequencing analysis could not be performed.

The products obtained by amplifying the microbial total DNA of the milk samples extracted in the example 2 and the example 3 are subjected to library construction and on-machine sequencing, and the analysis result of sequencing data is shown in figure 3.

As can be seen from FIG. 3, at the seed level, 269 bacteria were detected in the 2 DNA groups, of which only 92 bacteria overlapped. There were 108 species detected only in the total DNA (T) of the sample microorganisms of the milk of the dairy cows extracted in example 2, and there were 69 species detected only in the total DNA (C) of the sample microorganisms of the milk of the dairy cows extracted in example 3. From this, it was found that the total DNA of the microorganisms extracted from the milk samples of the cows in example 2 was used as a template for amplification and sequencing, and more strains were detected than in example 3, indicating that the extraction method of example 2 can obtain DNA of a larger variety of microorganisms.

The results show that the effect of extracting the total DNA of the microorganisms of the milk sample by adding the crosslinked polyvinylpyrrolidone is obviously improved, protein pollution in the total DNA can be effectively removed, the obtained total DNA has good quality and high purity, and more comprehensive microorganism types and diversity information can be obtained. The inventors have further developed that similar effects can be achieved by adding polyvinylpyrrolidone.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A kit for extracting total DNA of microorganisms in an animal milk sample, which comprises a lysis reagent, wherein the lysis reagent comprises a lysis solution, lysozyme and proteinase K, and is characterized in that the lysis reagent also comprises cross-linked or uncrosslinked polyvinylpyrrolidone.

2. The kit for extracting total DNA of microorganisms in an animal milk sample according to claim 1, wherein the lysate comprises 2% CTAB,1.4M NaCl,20mM EDTA and 100mM Tris-HCl.

3. The kit for extracting total DNA from microorganisms in an animal milk sample according to claim 1, further comprising a rinse solution and an eluent.

4. The kit for extracting total DNA from microorganisms in an animal milk sample according to claim 2, wherein said rinsing liquid is an organic alcohol solution.

5. The kit for extracting total DNA from microorganisms in an animal milk sample according to claim 2, wherein said eluent is selected from the group consisting of TE buffer, tris-HCl buffer and water.

6. A method for extracting microbial total DNA from an animal milk sample using a microbial total DNA extraction kit in an animal milk sample according to claim 1, comprising the steps of:

s1, respectively centrifuging 200 mu L of animal milk samples in a 2mL sterilizing centrifuge tube at 7000-10000 Xg and 4 ℃ for 8-15 min, and removing upper suspension;

s2, adding the same volume of the lysate into the sterilization centrifuge tube, adding crosslinked polyvinylpyrrolidone with the mass system fraction of 1-5%, 30-80 mu L of lysozyme with the mass system fraction of 100mg/mL and 15-20 mu L of proteinase K with the mass system fraction of 20mg/mL, and grinding for 5-15 min;

s3, placing the ground sample in a water bath kettle at 60-70 ℃ for cracking for 0.8-1.5 h;

s4, taking out the sample from the water bath kettle, adding 1-3 mu L of 10mg/mL RNase, and carrying out water bath at 35-40 ℃ for 25-60 min;

s5, adding chloroform with the volume of 0.5 times, reversing and uniformly mixing until the solution is completely emulsified into white, and centrifuging for 8-15 min at the temperature of 12000-15000 Xg and the temperature of 4 ℃;

s6, transferring 800 mu L of supernatant obtained in the sucking step into a new 1.5mL sterilizing centrifuge tube;

s7, adding magnetic beads with the volume of 0.6 times of that of the supernatant fluid, uniformly mixing by blowing for more than 10 times, and standing for 3-8 min at room temperature;

s8, placing the centrifuge tube in a magnetic rack until the solution is clear, and sucking and discarding the supernatant;

s9, keeping the centrifuge tube on a magnetic rack, adding 200 mu L of rinsing liquid into the tube, standing at room temperature for 20-40S, and then sucking and discarding the supernatant;

s10, performing instantaneous centrifugation, discarding redundant rinsing liquid in the centrifuge tube, and drying the magnetic beads at room temperature for 2-5 min;

s11, taking down the centrifuge tube from the magnetic rack, adding 65 mu L of eluent, lightly blowing and mixing by using a pipettor, and standing for 3-8 min at room temperature;

s12, placing the centrifuge tube on a magnetic rack, standing at room temperature for 3-8 min until the solution is clarified, sucking supernatant, and transferring the supernatant into a corresponding new sample preservation tube to obtain purified total DNA of microorganisms in the milk sample.

7. The method according to claim 6, characterized in that step S9 is repeated one time before step S10.

8. The method of claim 6, wherein the rinse solution is 80% ethanol.

9. The method of claim 6, wherein the eluent is selected from the group consisting of TE buffer, tris-HCl buffer and water.

10. The method of any one of claims 6 to 9, wherein the animal is a pig, cow, sheep or dog.

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