CN105886495B - Method for extracting DNA from feces of cuora flavomarginata - Google Patents
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- 239000002244 precipitate Substances 0.000 claims abstract description 28
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 22
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000002156 mixing Methods 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 15
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 11
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- 238000005406 washing Methods 0.000 claims abstract description 7
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- 238000011002 quantification Methods 0.000 claims abstract description 5
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 abstract description 16
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Abstract
The invention relates to a method for extracting DNA from cistoclemmys flavomarginata excrement, and belongs to the technical field of DNA separation and purification. Collecting the feces of the cuora flavomarginata as a sample, immediately filling the sample into a sterilized centrifugal tube, and soaking the sample in DETs; adding dithiothreitol lysate into a centrifugal tube, uniformly mixing the lysate and excrement, and then carrying out water bath treatment; adding Protease K (PK) for quantification, and standing on ice; centrifuging, taking the supernatant to another sterile centrifuge tube, and discarding the precipitate; adding equal volume of phenol/chloroform/isoamyl alcohol mixed solution into the supernatant fluid for extraction; adding NaAc, mixing uniformly on a vortex mixer, adding precooled absolute ethyl alcohol, mixing uniformly, and standing; centrifuging again, sucking and removing supernatant, and drying the precipitate at room temperature; adding ethanol, and washing the precipitate again; air dried at room temperature, and dissolved by adding TE when the precipitate becomes transparent. The invention is applied to the DNA extraction of the box turtles with the advantages of convenient operation, small damage of the box turtles with the yellow edges and the like.
Description
Technical Field
The invention relates to a method for extracting DNA from cistoclemmys flavomarginata excrement, and belongs to the technical field of DNA separation and purification.
Background
The Yellow-margined Box Turtle (Cistoclemys flavomarginata, Yellow-margined Box Turtle) belongs to the order Testudiniales (Testudinata), the family Rhynchosiales (Geoemydia), the genus Tortoise (Cistoclemys). The dorsum concha margin is yellow in the abdomen and has a golden yellow stripe on the eye socket, so the name is obtained. The yellow box turtles are mainly distributed in Zhejiang, Anhui, Fujian, Jiangsu, Henan, Guangxi, Guangdong, Taiwan and other places in China, and are also distributed in Japan of neighboring countries. Our provinces are mainly distributed in Lin' an, Jian De, an Ji, Long tour, Tiantai, Xinchang, etc. The cuora flavomarginata is a precious material in turtles, is a high-grade tonic, and has high medicinal value and great ornamental value. In recent years, due to artificial transition capture, wild cuora flavomarginata resources and habitats thereof are seriously damaged, and the wild number is sharply reduced. In 1998 the population was listed as a rare or endangered animal in the country and in 2006 as an endangered animal population in the united nations (IUCN, 2006). In addition to human factors, the wild cuora flavomarginata grows slowly, and in addition, the mating environment, the hatching condition and the like of the wild cuora flavomarginata are slightly different from the optimal condition due to frequent occurrence of global warming and severe abnormal weather, so the egg laying amount after sexual maturity is extremely small, the fertility rate, the hatching rate and the survival rate are extremely low, and the sex differentiation and the like of the wild cuora flavomarginata also have great influence, so that the wild population is endangered to be extinct.
At present, germplasm resource investigation of wild box turtles in our province and genetic relationship analysis and identification with wild species in adjacent provinces are not effectively carried out, and the development of pseudoecological domestication and breeding of the box turtles is still in an exploration stage. However, the growth period of the cuora flavomarginata is long, the difference between pure lines and hybrid cuora flavomarginata in the juvenile cuora flavomarginata period is small, and the characters of the rare cuora flavomarginata can be displayed after 6-8 years of culture, so that a large culture risk is generated. The traditional sample for DNA extraction is mainly derived from blood or tissue blocks, but for wild endangered animals, the blood taking and the tissue block obtaining both have damage to the animals and possibly bring about disease infection, and have certain hidden danger for the survival of the animals.
The present application was made based on this.
Disclosure of Invention
In order to overcome the defects existing in the identification of the existing cuora flavomarginata, the application provides the method for extracting the DNA from the cuora flavomarginata excrement, which is simple, convenient and easy to operate and does not need in-vitro dissection.
In order to achieve the purpose, the technical scheme adopted by the application is as follows:
a method for extracting DNA from the feces of box yellow tortoise comprises the following steps: (1) collecting the feces of the cuora flavomarginata as a sample, immediately putting the feces into a sterilized centrifugal tube, and soaking the feces in DETs (DMSO/EDTA/Tris salt solution); (2) putting the soaked excrement into a centrifugal tube, adding Dithiothreitol (DTT) lysate, uniformly mixing the lysate and the excrement, and carrying out water bath treatment; (3) adding Protease K (PK) for quantification, and immediately placing on ice for standing; (4) centrifuging, taking the supernatant to another sterile centrifuge tube, and discarding the precipitate; adding equal volume of phenol/chloroform/isoamyl alcohol mixed solution into the supernatant fluid for extraction; (5) adding NaAc, mixing uniformly on a vortex mixer, adding precooled absolute ethyl alcohol, mixing uniformly, and standing; (6) centrifuging again, sucking and removing supernatant, and drying the precipitate at room temperature; (7) adding ethanol with the volume concentration of 70%, and washing the precipitate again; (8) air dried at room temperature, and dissolved by adding TE when the precipitate becomes transparent.
Further, as preferable:
in the step (1), the sample is collected from the DNA extraction used in the step (2) and the subsequent steps and is stored for 90 to 120 days at normal temperature.
In the step (2), the lysate is composed of: 100mmol/L Tris-HCl, pH8, 1.4mol/L NaCl, 20mmol/L DTA, 0.4mol/L DTT.
In the step (2), the water bath temperature is 40-60 ℃, and the water bath time is 1-4 hours.
And (3) adding protease K for quantification, processing at 50-55 ℃ for 5-15 minutes, and standing on ice for 5-15 minutes.
In the step (4), in the mixed solution of phenol/chloroform/isoamyl alcohol, the ratio of phenol: chloroform: isoamyl alcohol is 25: 24: 1 (volume ratio).
In the step (5), the NaAc concentration is 1.5-5 mol/L, and the addition amount is 0.1-0.3 time of the volume of the solution treated in the step (4); the addition amount of the absolute ethyl alcohol is 1-3 times of the volume of the solution after NaAc is added; after being mixed uniformly, the mixture is kept stand for 0.5 to 1 hour at the temperature of between 10 ℃ below zero and 30 ℃ below zero.
Through the implementation of the technical scheme, by collecting and evaluating wild cuora flavomarginata germplasm resources and adopting an excrement sampling mode, the mode can be used for extracting animal DNA and carrying out PCR amplification by using samples under the condition of not damaging or even not seeing animals, and then by utilizing genetics and molecular biology technologies such as mtDNA and PCR, a cuora flavomarginata germplasm molecular identification and analysis method is established, so that the wild cuora flavomarginata germplasm resources are detected on the premise of not influencing the wild population distribution, the established cuora flavomarginata germplasm molecular identification method is beneficial to detecting the wild cuora flavomarginata germplasm resources, and a cuora flavomarginata simulated ecological domestication and breeding system is constructed through investigating conditions such as living, mating, breeding and the like of the wild cuora flavomarginata, so that the genetic information of biological individuals is analyzed; finally, the endangered species is effectively protected, and a way is provided for protecting the diversity of species and sustainable utilization of natural resources of traditional Chinese medicines.
Drawings
FIG. 1 is a diagram of the agarose nucleic acid electrophoresis detection spectrum of the present application.
Detailed Description
Example 1
Immediately after collection, the fecal samples were placed into sterilized centrifuge tubes, soaked with DETs (DMSO/EDTA/Tris salt solution) and kept at room temperature for 90 days for use.
1) Taking 100mg of the excrement, adding 1mL of Dithiothreitol (DTT) lysate into a 1mL centrifuge tube, uniformly mixing, and carrying out water bath at 40 ℃ for 4h, wherein the lysate comprises: 100mmol/L tris-HCl, pH8, 1.4mol/L nacl, 20mmol/L dta, 0.4mol/L DTT;
2) protease K (PK) was added to 0.02mg at 55 ℃ for 10min, and immediately placed on ice for 15 min.
3) Centrifuge at 12000g for 5min, take the supernatant to another sterile 1.5mL centrifuge tube, discard the precipitate. Equal volume of phenol was added to the supernatant: chloroform: extracting the mixture of isoamyl alcohol in the ratio of 25 to 24 to 1;
4) adding 1.5mol/L NaAc with the volume 0.15 times that of the mixture, uniformly mixing the mixture on a vortex mixer, adding 2 times of precooled absolute ethyl alcohol (the volume calculated after adding salt), fully mixing the mixture, and standing the mixture for 0.5 to 1 hour at the temperature of minus 10 ℃;
5) centrifuging at 12000r/min for 10min, sucking and removing supernatant, and air drying precipitate at room temperature;
6) adding 500 μ L of 70% ethanol, and washing the precipitate for 2 times;
7) air dried at room temperature, and dissolved by adding 100 mu LTE when the precipitate is clear.
Example 2
Immediately after collection, the fecal samples were placed into sterilized centrifuge tubes, soaked with DETs (DMSO/EDTA/Tris salt solution) and kept at room temperature for 100 days for use.
1) Taking 100mg of the excrement, adding 1mL of Dithiothreitol (DTT) lysate into a 1mL centrifuge tube, uniformly mixing, and carrying out water bath at 45 ℃ for 3h, wherein the lysate comprises: 100mmol/L tris-HCl, pH8, 1.4mol/L nacl, 20mmol/L dta, 0.4mol/L DTT;
2) protease K (PK) was added to 0.02mg at 50 ℃ for 15min, and immediately placed on ice for 5 min.
3) Centrifuge at 12000g for 5min, take the supernatant to another sterile 1.5mL centrifuge tube, discard the precipitate. Equal volume of phenol was added to the supernatant: chloroform: extracting the mixture of isoamyl alcohol in the ratio of 25 to 24 to 1;
4) adding 4mol/LNaAc with the volume 0.2 times that of the mixture, uniformly mixing the mixture on a vortex mixer, adding 2 times (calculated volume after adding salt) of precooled absolute ethyl alcohol, fully and uniformly mixing the mixture, and standing the mixture for 0.5 to 1 hour at the temperature of minus 20 ℃;
5) centrifuging at 12000r/min for 10min, sucking and removing supernatant, and air drying precipitate at room temperature;
6) adding 500 μ L of 70% ethanol, and washing the precipitate for 2 times;
7) air dried at room temperature, and dissolved by adding 100 mu LTE when the precipitate is clear.
Example 3
Immediately after collection, fecal samples were loaded into sterilized centrifuge tubes and soaked with DETs (DMSO/EDTA/Tris salt solution).
1) Taking 100mg of the soaked excrement, adding 1mL of Dithiothreitol (DTT) lysate into a 1mL centrifuge tube, uniformly mixing the lysate (100mmol/LTris-HCl, pH8, 1.4mol/LNaCl, 20mmol/LEDTA and 0.4mol/L DTT), and carrying out water bath at 55 ℃ for 2 h;
2) protease K (PK) was added to 0.02mg at 50 ℃ for 8min, and immediately placed on ice for 10 min.
3) Centrifuge at 12000g for 5min, take the supernatant to another sterile 1.5mL centrifuge tube, discard the precipitate. Equal volume of phenol was added to the supernatant: chloroform: extracting the mixture of isoamyl alcohol in the ratio of 25 to 24 to 1;
4) adding 3mol/LNaAc with the volume of 0.1 time, uniformly mixing on a vortex mixer, adding 1.5 times (calculated volume after adding salt) of precooled absolute ethyl alcohol, fully mixing, and standing for 0.5-1 h at-20 ℃;
5) centrifuging at 12000r/min for 10min, sucking and removing supernatant, and air drying precipitate at room temperature;
6) adding 500 μ L of 70% ethanol, and washing the precipitate for 2 times;
7) air dried at room temperature, and dissolved by adding 100 mu LTE when the precipitate is clear.
Example 4
Immediately after collection, the fecal samples were placed into sterilized centrifuge tubes, soaked with DETs (DMSO/EDTA/Tris salt solution) and kept at room temperature for 120 days for use.
1) Taking 100mg of the excrement, adding 1mL of Dithiothreitol (DTT) lysate into a 1mL centrifuge tube, uniformly mixing, and then carrying out water bath at 60 ℃ for 1h, wherein the lysate comprises: 100mmol/L tris-HCl, pH8, 1.4mol/L nacl, 20mmol/L dta, 0.4mol/L DTT;
2) protease K (PK) was added to 0.02mg at 55 ℃ for 5min, and immediately placed on ice for 10 min.
3) Centrifuge at 12000g for 5min, take the supernatant to another sterile 1.5mL centrifuge tube, discard the precipitate. Equal volume of phenol was added to the supernatant: chloroform: extracting the mixture of isoamyl alcohol in the ratio of 25 to 24 to 1;
4) adding 5mol/LNaAc with the volume of 0.3 time, uniformly mixing on a vortex mixer, adding 3 times (calculated volume after adding salt) of precooled absolute ethyl alcohol, fully mixing, and standing for 0.5-1 h at-30 ℃;
5) centrifuging at 12000r/min for 10min, sucking and removing supernatant, and air drying precipitate at room temperature;
6) adding 500 μ L of 70% ethanol, and washing the precipitate for 2 times;
7) air dried at room temperature, and dissolved by adding 100 mu LTE when the precipitate is clear.
The solutions of the above examples were treated as follows:
(a) PCR primer design
TABLE 1 complete sequence amplification primers for mitochondrial genome of box turtle
(b) PCR amplification
PCR amplification used the high fidelity enzyme KOD FX from Toyobo (Shanghai) Biotechnology, Inc.
TABLE 2PCR reaction Components
| Reaction components | Volume (μ l) |
| Upstream primer | 0.3 |
| Downstream primer | 0.3 |
| DNA template | 1 |
| KOD FX(1U/μl) | 0.1 |
| 2×PCR Buffer for KOD FX | 10 |
| 2mM dNTP | 1 |
| ddH2O | Up to 20 |
TABLE 3PCR reaction procedure
Carrying out agarose electrophoresis detection on mitochondrial gene PCR products, and recovering target fragments by using a gel recovery kit (DNASELExtractionkit, Takara); performing polyacrylamide electrophoresis detection on the microsatellite PCR product; purification recovery was performed using a purification kit (e.z.n.acyclepurekit, Omega). Sequencing the recovered product, and using the sequence for comparison analysis in NCBI database to complete the extraction and detection of the whole DNA.
And (3) electrophoresis detection: 1% agarose electrophoresis, combined with FIG. 1, shows that the DNA bands are obvious and the DNA collection purity is high.
Through the implementation of the technical scheme, by collecting and evaluating wild cuora flavomarginata germplasm resources and adopting an excrement sampling mode, the mode can be used for extracting animal DNA and carrying out PCR amplification by using samples under the condition of not damaging or even not seeing animals, and then by utilizing genetics and molecular biology technologies such as mtDNA and PCR, a cuora flavomarginata germplasm molecular identification and analysis method is established, so that the wild cuora flavomarginata germplasm resources are detected on the premise of not influencing the wild population distribution, the established cuora flavomarginata germplasm molecular identification method is beneficial to detecting the wild cuora flavomarginata germplasm resources, and a cuora flavomarginata simulated ecological domestication and breeding system is constructed through investigating conditions such as living, mating, breeding and the like of the wild cuora flavomarginata, so that the genetic information of biological individuals is analyzed; finally, the endangered species is effectively protected, and a way is provided for protecting the diversity of species and sustainable utilization of natural resources of traditional Chinese medicines.
The above description is provided for the purpose of describing the preferred embodiments of the present invention in more detail, and it should not be construed that the embodiments of the present invention are limited to the description above, and it will be apparent to those skilled in the art that the present invention can be implemented in many different forms without departing from the spirit and scope of the present invention.
Claims (3)
1. A method for extracting DNA from the feces of box yellow tortoise is characterized by comprising the following steps:
(1) collecting the feces of the cuora flavomarginata as a sample, immediately filling the sample into a sterilized centrifugal tube, and soaking the sample in DETs;
(2) putting the soaked excrement into a centrifugal tube, adding a lysis solution, uniformly mixing the lysis solution and the excrement, and carrying out water bath treatment;
(3) adding protease K for quantification, and immediately placing on ice for standing;
(4) centrifuging, taking the supernatant to another sterile centrifuge tube, and discarding the precipitate; adding equal volume of phenol/chloroform/isoamyl alcohol mixed solution into the supernatant fluid for extraction;
(5) adding NaAc, mixing uniformly on a vortex mixer, adding precooled absolute ethyl alcohol, mixing uniformly, and standing;
(6) centrifuging again, sucking and removing supernatant, and drying the precipitate at room temperature;
(7) adding ethanol with the volume concentration of 70%, and washing the precipitate again;
(8) drying at room temperature, and adding TE to dissolve when the precipitate is transparent;
in the step (2), the lysate is composed of: 100mmol/L Tris-HCl, pH8, 1.4mol/L NaCl, 20mmol/L DTA, 0.4mol/L DTT;
in the step (4), in the mixed solution of phenol/chloroform/isoamyl alcohol, the ratio of phenol: chloroform: isoamyl alcohol = 25: 24: 1;
in the step (3), adding protease K for quantification, processing at 50-55 ℃ for 5-15 minutes, and standing on ice for 5-15 minutes;
in the step (5), the NaAc concentration is 1.5-5 mol/L, and the addition amount is 0.1-0.3 time of the volume of the solution treated in the step (4); the addition amount of the absolute ethyl alcohol is 1-3 times of the volume of the solution after NaAc is added; after being mixed uniformly, the mixture is kept stand for 0.5 to 1 hour at the temperature of between 10 ℃ below zero and 20 ℃ below zero.
2. The method for extracting DNA from the feces of box yellow turtle as claimed in claim 1, wherein: in the step (1), the sample is collected from the DNA extraction used in the step (2) and the subsequent steps and is stored for 90 to 120 days at normal temperature.
3. The method for extracting DNA from the feces of box yellow turtle as claimed in claim 1, wherein: in the step (2), the water bath temperature is 40-60 ℃, and the water bath time is 1-4 hours.
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| CN1718728A (en) * | 2004-07-07 | 2006-01-11 | 李迪强 | Extraction method of Pu goa feaces DNA |
| CN1904066A (en) * | 2006-07-15 | 2007-01-31 | 安徽大学 | Extraction method of mammal faeces DNA |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
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