CN113817722A - Method for extracting and purifying DNA from camel milk powder and detection method of camel milk powder applying method - Google Patents
Method for extracting and purifying DNA from camel milk powder and detection method of camel milk powder applying method Download PDFInfo
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Abstract
The invention relates to a method for extracting and purifying DNA from camel milk powder, which extracts and purifies the DNA from the camel milk powder through a column type deep processing product genome DNA extraction kit. The invention also relates to a detection method of camel milk powder, which comprises the steps of obtaining a nucleic acid solution containing purified DNA from milk powder by the method, measuring the concentration and purity of the nucleic acid solution by using a Nanodrop microspectrophotometer, and identifying the species of the total genome of the nucleic acid solution by using a PCR amplification method. The method of the invention has simple operation, can obtain purified nucleic acid from milk powder with low somatic cell content, and is subsequently used in PCR amplification experiments. The method has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of gene purification detection, and particularly relates to a method for extracting and purifying DNA from camel milk powder cells.
Background
Camel milk is considered as a rich nutrient source for people living in arid regions and also as a basic source of livelihood for desert herdsmen. Has certain hypoallergenic, blood sugar lowering, antioxidant, liver protecting, anti-inflammatory and antibacterial effects, is especially suitable for the elderly and diabetic patients, and becomes a popular high-quality health product. In order to maintain its physical, chemical and nutritional properties, camel milk is usually produced and processed into camel milk powder to extend its shelf life.
Compared with other animal milks, camel milk has lower yield and can be extruded only about 1.5-2.5kg per day, so camel milk is a precious milk which is not easy to obtain, and the price of camel milk is continuously increased. Driven by economic benefits, a lot of vendors add low-price milk to camel milk and camel milk powder to reduce cost and improve benefits.
Various detection methods have been established to detect milk and adulteration of milk products, and these methods generally use proteins, lipids and DNA in milk as detection targets. Since milk powder is a product produced by thermal processing, protein and fat are extremely easy to denature, and DNA has higher thermal stability and specificity, DNA molecules become target compounds for species identification of milk powder based on a PCR method.
DNA in camel milk and substrates for PCR reactions are derived from camel somatic cells that drop into the milk during milking, containing white blood cells (98% -99%) and a small number of mammary epithelial cells (about 1% -2%). Compared with tissue samples and blood samples, extraction of DNA from milk and milk powder is extremely difficult because somatic cells in milk are much smaller than those in tissue and blood and contain more fat and protein impurities. Aiming at the problem of low DNA content in milk or milk powder, the DNA extraction method in the field of biomolecular technical research mainly comprises the following steps: NaOH lysis, PBS heating, direct boiling, and EDTA lysis, but these methods are complicated to operate.
Disclosure of Invention
The invention aims to provide a method for extracting and purifying DNA from camel milk powder cells, and the method is applied to detection and identification of camel milk powder.
The idea of the invention is to adopt the optimized column type deep processing product genome DNA extraction kit to extract purified DNA from camel milk powder cells, realize the steps of successfully extracting nucleic acid with high purity and stable quality from camel milk powder cells simply, and confirm that the obtained camel milk powder cell DNA can be used for PCR amplification so as to realize species identification of camel milk powder.
The invention provides a method for extracting and purifying DNA from camel milk powder, which extracts and purifies the DNA from the camel milk powder by a column type deep processing product genome DNA extraction kit, and the method comprises the following steps:
(1) weighing 600-plus 700mg camel milk powder samples into a 2ml centrifuge tube, adding 1ml lysate GMO Buffer and 20 mu l protease, shaking and mixing evenly, and cracking for 1h in a 65 ℃ water bath;
(2) after the water bath is finished, centrifuging and sucking the supernatant, centrifuging at 12000rpm for 3min, transferring the supernatant into a new 2ml centrifuge tube, adding 400 mu l of chloroform into the supernatant, centrifuging and sucking the supernatant after fully mixing, centrifuging at 12000rpm for 5min, transferring the supernatant into a new 2ml centrifuge tube, adding 400 mu l of chloroform again, fully mixing, centrifuging again to take the supernatant, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new 2ml centrifuge tube;
(3) adding the same volume of lysate GMP Buffer into the product obtained in the step (2), fully and uniformly mixing, adding 300 mu l of isopropanol, fully and uniformly mixing, and standing at room temperature for 30 min;
(4) transferring 800 μ l of the product obtained in the step (3) to an adsorption column, standing at room temperature for 2min, centrifuging for 30s at 12000rpm, pouring the filtrate into the adsorption column, centrifuging again to remove the filtrate, centrifuging for 30s at 12000rpm, and returning the adsorption column to the collection tube;
(5) adding 500 μ l of Wash Solution into the adsorption column, centrifuging to remove filtrate, centrifuging at 12000rpm for 30s, and placing the adsorption column back into the collection tube;
(6) repeating the operation step (5);
(7) putting the adsorption column back into the collection tube, centrifuging in an empty tube at 12000rpm for 2min, and discarding filtrate;
(8) taking out the adsorption column, placing into a new 2ml centrifuge tube, opening the cover, and drying in a 56 deg.C drying oven for 10 min;
(9) adding 50 μ l of TE Buffer (pH8.0) as eluent into the center of the adsorption membrane, standing at room temperature for 3-5min to strip nucleic acid from the adsorption membrane sufficiently, and centrifuging at 12000rpm for 2min to obtain nucleic acid solution containing purified DNA.
In the present invention, the centrifugation in each step is carried out at normal temperature.
According to a preferred embodiment, the isopropanol of step (3) is stored at-20 ℃. The function of isopropanol is to protect hydrophilic groups in the DNA strand, equivalent to precipitation, by the hydrophobic interaction of-OH. In the invention, the isopropanol needs to be frozen at-20 ℃ in advance, the precipitation of DNA is promoted by low temperature, and the frozen isopropanol is beneficial to improving the extraction efficiency aiming at the characteristic of low DNA content in the milk powder. Because the content of DNA in the milk powder is very low, if the DRL Buffer in the conventional column type deep processing product genome DNA extraction kit is adopted, the DNA cannot be completely cracked and precipitated, so that the DNA with higher concentration and purity cannot be extracted, and the subsequent PCR amplified species identification experiment cannot be carried out.
Particularly preferably, the Wash Solution of step (4) contains anhydrous ethanol, and the volume ratio of the anhydrous ethanol in the Wash Solution is 80%.
In the invention, the absolute ethyl alcohol is 95 vol% absolute ethyl alcohol, and the addition of the absolute ethyl alcohol is favorable for eluting impurities such as lipids, proteins, salts and the like.
The column type deep processing product genome DNA extraction kit is a product sold by biological engineering limited company.
In the step (1), the camel milk powder cell membrane and nuclear membrane can be cracked by cracking in a water bath at 65 ℃, and the genome DNA is released. As the content of somatic cells in the milk powder is very low, if the cracking time is too short, the somatic cells are not cracked sufficiently, DNA cannot be released, and the effectiveness of an experimental result is influenced.
It should be noted that the supernatant is aspirated at each step without aspiration into the pellet, which would otherwise affect the purity of the final genome.
In the invention, chloroform is added as an organic solvent to denature protein, so that an organic phase and an inorganic phase can be effectively and rapidly separated, protein and DNA are separated, and the DNA enters a water phase.
The steps are fully and uniformly mixed after chloroform is added, but strong oscillation is avoided, and the hydrophilic group of the DNA is easily contacted with the water phase by the strong oscillation.
Preferably, the method is repeated for three times, and the supernatant obtained by the three times of repetition passes through the adsorption column, so that a small amount of somatic cell DNA in camel milk powder can be fully enriched on the adsorption membrane of the adsorption column, and the concentration of the genome is improved.
When the adsorption column is taken out from the drying box and dried, the fact that no washing liquid Wash Solution remains in the adsorption column is fully guaranteed, and the influence on the purity of the final genome is avoided.
Based on the method, the invention also provides a camel milk powder detection method, the nucleic acid solution containing the purified DNA is obtained from the milk powder by the method, then the concentration and the purity of the nucleic acid solution are measured by a Nanodrop spectrophotometer, and the species identification is carried out on the total genome of the nucleic acid solution by a PCR amplification method.
Wherein, in the PCR amplification method, the primer sequences are as follows:
luotuo-F:5'-CATTATCACGGCTCTAGTGGC-3'
luotuo-R:5'-CTGGTGAGAATAATACGAGGATAAG-3'
in the PCR amplification method, an amplification system comprises: template genomic DNA 1. mu.l, 10. mu.M of the upper primer 1. mu.l, 10. mu.M of the lower primer 1. mu.l, 10mM Dntp (mix) 1. mu.l, 10 XTaq Buffer (with MgCl)2)2.5 mul, 5U/mul Taq DNA polymerase 0.2 mul, and sterile enzyme-free water is added to the total volume of the PCR amplification system to be 25 mul;
the PCR amplification procedure is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, cooling at 0.5 ℃ per cycle, extension at 72 ℃ for 30s, and 10 cycles; pre-denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, final repair at 72 ℃ for 106min, and heat preservation at 4 ℃.
The invention has the characteristics and beneficial effects that:
(1) the method has the advantages of simple and clear operation steps, convenient operation, capability of obtaining purified nucleic acid and reducing impurities, and the extracted DNA can be used for PCR amplification.
(2) The invention can obtain the camel milk powder cell genome DNA only by simple operations of centrifuging, taking supernatant and the like.
(3) By selecting optimized conditions, the method can fully enrich camel milk powder cell genome DNA, improve the concentration and purity of the total genome, and effectively overcome the defect of low camel milk powder cell genome DNA content.
(4) It was confirmed from the present invention that the DNA obtained by the method of the present invention had a total genomic concentration and purity as a result of concentration of 39-47 ng/. mu.l, OD260/280Between 1.6 and 1.7, indicating that the DNA purity and quality are high, and the extracted and purified total genomic DNA is enough to be used in the subsequent PCR amplification experiment.
(5) The PCR amplification primer adopted by the invention has good specificity, and after the extracted genome DNA is sequenced, the specific primer is designed based on the conserved region of the mitochondrial Cytb gene, so that the PCR amplification specificity is very strong.
(6) The invention has wide application prospect in the technical field of biomolecules such as DNA extraction from somatic cells of camel milk powder, PCR amplification, species identification and the like.
Drawings
FIG. 1 shows the results of the PCR amplification of example 1% agarose gel electrophoresis;
wherein, the samples 1, 2 and 3 are the glue running result of extracting and purifying the total genome DNA of the somatic cells from the camel milk powder product in the market which is extracted by adding the conventional DRL Buffer cracking in the step of the specific embodiment 1.3; samples 4, 5, and 6 are the results of gel running of purified somatic cell total genomic DNA extracted from commercial camel milk powder products extracted by freezing isopropanol lysis added in the step of embodiment 1.3.
Detailed Description
The invention will now be further illustrated with reference to specific examples.
In the present invention, "%" used for explaining concentrations is a weight percent, ": all the terms "parts" and "parts" are parts by weight.
Example 1 extraction of somatic genomic DNA from Camel milk powder
The total genome DNA is extracted from camel milk powder cells by an optimization method based on the operation of an Ezup column type deep processing product genome DNA extraction kit (purchased from the manufacturer, the product number is B518264).
1.1 weighing 600-700mg camel milk powder samples in 2ml centrifuge tubes, sampling 6 parts, respectively adding 1ml lysis solution GMO Buffer and 20 μ l protease K, shaking and mixing uniformly, and putting into a 65 ℃ water bath for lysis for 1 h.
And (3) after the 1.2 water bath is finished, centrifuging at the room temperature of 12000rpm for 3min, respectively transferring the supernatant to a new 2ml centrifuge tube, adding 400 mu l of chloroform into the supernatant for purification, reversing the upside down and mixing uniformly, then centrifuging at the 12000rpm for 5min, transferring the supernatant to a new 2ml centrifuge tube, adding 400 mu l of chloroform again for purification, reversing the upside down and mixing uniformly, performing secondary operation, centrifuging at the 12000rpm for 5min, and transferring the supernatant to a new 2ml centrifuge tube.
1.3 adding equal volume of lysis solution GMP Buffer into the supernatant respectively, reversing the upper part and mixing the solution evenly, adding 300 mul of DRL Buffer into the No. 1-3 sample, adding 300 mul of isopropanol at the temperature of 20 ℃ into the No. 4-6 sample, reversing the upper part and mixing the solution evenly, and standing the mixture for 30min at room temperature.
1.4 transfer 700 μ l supernatant to adsorption column (purchased from Biotech, Cat. B515115-0100), centrifuge at 12000rpm for 30s after standing at room temperature for 2min, pour filtrate into adsorption column, centrifuge again to remove filtrate, centrifuge at 12000rpm for 30s, place adsorption column back into collection tube, repeat operation until all supernatant in 3 tubes passes adsorption column.
1.5 Add 500. mu.l Wash Solution to the adsorption column and centrifuge the reject filtrate at 12000rpm for 30s, place the adsorption column back into the collection tube.
1.6 Add 500. mu.l Wash Solution to the adsorption column and centrifuge the reject filtrate at 12000rpm for 30s, and place the adsorption column back into the collection tube.
1.7 the column was returned to the collection tube, the filtrate was discarded by centrifugation in an empty tube, and centrifuged at 12000rpm for 2 min.
1.8 take out the adsorption column, put into a new 2ml centrifuge tube, open the cover, put into a 56 ℃ drying oven and dry for 10 min.
1.9 adding 50 μ l of TE Buffer (pH8.0) eluent to the center of the adsorption membrane to elute nucleic acid, standing at room temperature for 5min to strip nucleic acid from the adsorption membrane sufficiently, centrifuging at 12000rpm for 2min to obtain nucleic acid solution.
Example 2 determination of concentration and purity of Total genome of somatic cells
The concentration and purity of camel milk powder cell total genome were measured using a Nanodrop microspectrophotometer, and the results are shown in table 1.
Table 1: concentration and purity results of Camel milk powder cell Total genome
As can be seen from the above results, the concentration of samples 1, 2 and 3 extracted from the DRL Buffer lysis added in step 1.3 was 17.1-23.8 ng/. mu.l, OD260/280Between 0.91 and 1.18, the purity and quality of the DNA are low, and the extracted and purified total genomic DNA cannot be used in the subsequent PCR amplification experiment; the samples 4, 5 and 6 extracted by the above 1.3 step of adding frozen isopropanol were lysed at a concentration of 29.5-47.1 ng/. mu.l, OD260/2801.62-1.71, which is obviously superior to the comparison, shows that the purity and the quality of the DNA are higher, and the extracted and purified total genomic DNA can be used in the subsequent PCR amplification experiment.
Example 3 PCR amplification and validation of somatic genomic DNA
3.1 the following primer sequences are designed and synthesized, and after the extracted genome DNA is sequenced, specific primers are designed based on the conserved region of the mitochondrial Cytb gene, and the sequences are as follows:
luotuo-F:5'-CATTATCACGGCTCTAGTGGC-3'
luotuo-R:5'-CTGGTGAGAATAATACGAGGATAAG-3'
3.2 wherein the PCR amplification system comprises:
template genomic DNA 1. mu.l, 10. mu.M of the upper primer 1. mu.l, 10. mu.M of the lower primer 1. mu.l, 10mM Dntp (mix) 1. mu.l, 10 XTaq Buffer (with MgCl)2)2.5 mul, 5U/mul Taq DNA polymerase 0.2 mul, sterile and enzyme-free water is added to the total volume of the PCR amplification system to be 25 mul.
3.3 wherein the PCR amplification procedure is:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, cooling at 0.5 ℃ per cycle, extension at 72 ℃ for 30s, and 10 cycles; pre-denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, final repair at 72 ℃ for 106min, and heat preservation at 4 ℃.
4.1% agarose gel electrophoresis detection
4.1PCR product 5. mu.l was blown and mixed with 2. mu.l of 6 XRNA/DNA loading buffer (from Bomaide organisms) and detected by 1% agarose gel electrophoresis, electrophoresis parameters: electrophoresis observation is carried out for 20min at 150V and 100 mA.
The results of 4.21% agarose gel electrophoresis are shown in FIG. 1 (M: BM5000 DNA Marker from Bomader).
FIG. 1 shows that the conventional extraction method cannot obtain DNA for PCR amplification due to the low content of somatic cells in camel milk powder. The invention optimizes the cracking step on the basis of the prior art, selects proper cracking temperature and time, and adopts frozen isopropanol to replace the conventional DRL Buffer, thereby finally obtaining DNA with higher purity, and being used for PCR amplification and obtaining effective results.
The method only needs simple operations of centrifuging to take supernatant and the like, can enrich camel milk powder cell genome DNA by combining optimized experimental conditions, overcomes the defects that the content of the somatic cell genome DNA in camel milk powder is too low and camel milk powder species detection is difficult to carry out by a PCR method, and has wide application prospect in the technical field of biological molecules such as PCR amplification, species detection and the like.
Claims (9)
1. A method for extracting purified DNA from camel milk powder is characterized in that the column type deep processing product genome DNA extraction kit is used for extracting the purified DNA from the camel milk powder, and the method comprises the following steps:
(1) weighing 600-plus 700mg camel milk powder samples into a 2ml centrifuge tube, adding 1ml lysate GMO Buffer and 20 mu l protease, shaking and mixing evenly, and cracking for 1h in a 65 ℃ water bath;
(2) after the water bath is finished, centrifuging and sucking the supernatant, centrifuging at 12000rpm for 3min, transferring the supernatant into a new 2ml centrifuge tube, adding 400 mu l of chloroform into the supernatant, centrifuging and sucking the supernatant after fully mixing, centrifuging at 12000rpm for 5min, transferring the supernatant into a new 2ml centrifuge tube, adding 400 mu l of chloroform again, fully mixing, centrifuging again to take the supernatant, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new 2ml centrifuge tube;
(3) adding the same volume of lysate GMP Buffer into the product obtained in the step (2), fully and uniformly mixing, adding 300 mu l of isopropanol, fully and uniformly mixing, and standing at room temperature for 30 min;
(4) transferring 800 μ l of the product obtained in the step (3) to an adsorption column, standing at room temperature for 2min, centrifuging for 30s at 12000rpm, pouring the filtrate into the adsorption column, centrifuging again to remove the filtrate, centrifuging for 30s at 12000rpm, and returning the adsorption column to the collection tube;
(5) adding 500 μ l of Wash Solution into the adsorption column, centrifuging to remove filtrate, centrifuging at 12000rpm for 30s, and placing the adsorption column back into the collection tube;
(6) repeating the operation step (5);
(7) putting the adsorption column back into the collection tube, centrifuging in an empty tube at 12000rpm for 2min, and discarding filtrate;
(8) taking out the adsorption column, placing into a new 2ml centrifuge tube, opening the cover, and drying in a 56 deg.C drying oven for 10 min;
(9) adding 50 μ l of TE Buffer (pH8.0) as eluent into the center of the adsorption membrane, standing at room temperature for 3-5min to strip nucleic acid from the adsorption membrane sufficiently, and centrifuging at 12000rpm for 2min to obtain nucleic acid solution containing purified DNA.
2. The method according to claim 1, wherein the centrifugation in each step is carried out at normal temperature.
3. The method according to claim 1, wherein the isopropyl alcohol of step (3) is stored at-20 ℃.
4. The method according to claim 1, wherein the Wash Solution of step (4) comprises absolute ethanol, and the volume ratio of the absolute ethanol in the Wash Solution is 80%.
5. A detection method of camel milk powder, characterized in that a nucleic acid solution containing purified DNA is obtained from milk powder by the method according to any one of claims 1 to 4, then the concentration and purity of the nucleic acid solution are measured by a Nanodrop microspectrophotometer, and the species of the total genome of the nucleic acid solution is identified by a PCR amplification method.
6. The method of claim 5, wherein in the PCR amplification method, the primer sequences are as follows:
luotuo-F:5'-CATTATCACGGCTCTAGTGGC-3'
luotuo-R:5'-CTGGTGAGAATAATACGAGGATAAG-3'。
7. the method of claim 5, wherein the PCR amplification system comprises: template genomic DNA 1. mu.l, 10. mu.M of the upper primer 1. mu.l, 10. mu.M of the lower primer 1. mu.l, 10mM Dntp (mix) 1. mu.l, 10 XTaq Buffer (with MgCl)2)2.5 mul, 5U/mul Taq DNA polymerase 0.2 mul, and sterile enzyme-free water is added to the total volume of the PCR amplification system to be 25 mul;
the PCR amplification procedure is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, cooling at 0.5 ℃ per cycle, extension at 72 ℃ for 30s, and 10 cycles; pre-denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, final repair at 72 ℃ for 106min, and heat preservation at 4 ℃.
8. The specific primer for detecting camel milk powder is characterized by comprising the following primer sequences:
luotuo-F:5'-CATTATCACGGCTCTAGTGGC-3'
luotuo-R:5'-CTGGTGAGAATAATACGAGGATAAG-3'。
9. the specific primers as claimed in claim 8, wherein the nucleic acid solution containing the purified DNA is obtained from milk powder by the method as claimed in any one of claims 1 to 4, and then the concentration and purity of the nucleic acid solution are measured by Nanodrop spectrophotometer, and the total genome of the nucleic acid solution is subjected to species identification by PCR amplification.
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