CN110592071A - Method for extracting shellfish DNA from shellfish excrement - Google Patents
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Abstract
The invention discloses a method for extracting shellfish DNA from shellfish excrement, which comprises collecting shellfish excrement, placing on a bolting silk net, and adding ddH2Slowly flushing with O, adding ddH2Blowing and beating the mixture into a drainage homogenate, and centrifuging to obtain a supernatant; adding SDS and proteinase K, mixing uniformly and then carrying out water bath; adding RNA enzyme, mixing, water bathing, centrifuging, and collecting supernatant; adding ice-cold saturated phenol, mixing, water bathing, centrifuging, and collecting supernatant; adding chloroform, mixing, water bathing, centrifuging, and collecting supernatant; adding isopropanol to precipitate DNA, mixing, centrifuging, and removing supernatant; adding ethanol to rinse and precipitate DNA, mixing uniformly, centrifuging, taking precipitate, and drying to obtain shellfish DNA. The invention can be applied to the nondestructive extraction of shellfish DNA, has no stimulation and no damage to shellfish, has simple operation and high detection accuracy, and is suitable for popularization and application.
Description
Technical Field
The invention relates to a DNA extraction method, in particular to a method for extracting shellfish DNA from shellfish excrement.
Background
The shellfish farming industry plays an important role in supplying high-quality protein, increasing fisherman income, improving offshore environment and the like. Meanwhile, shellfish culture also faces the problem of resource failure, more and more scholars obtain shellfish DNA through molecular biology technology and research the genetic information thereof, and the molecular assisted breeding method is used for achieving the purposes of improving shellfish quality and breeding shellfish stress-resistant new products and solving the problem of shellfish resource exhaustion. However, the traditional sampling method mostly focuses on the destructive sampling or the non-destructive sampling, such as direct dissection, shell opening by using a mouth expander, drilling on the shell and the like, which damages the integrity of the internal tissue structure of the shellfish, causes strong mechanical damage to the shellfish and even causes death, and is not beneficial to the shellfish later-stage seed preservation, genetic breeding and the protection of shellfish resources, especially to endangered precious shellfish. Therefore, how to extract the shellfish DNA without damage becomes a technical problem to be solved urgently in the field.
Disclosure of Invention
In order to solve the problems, the invention provides a method for extracting shellfish DNA from excrement so as to realize nondestructive DNA extraction of shellfish, which is realized by the following steps:
a method for nondestructive extraction of shellfish DNA from excrement comprises the following steps:
1. collecting shellfish excrement and pretreating:
(1) starving the shellfish for 2 days to completely discharge impurities in the shellfish. Feeding a proper amount of bait, and collecting excrement from the bottom of the pool by using a siphon method after defecation.
(2) Moving the collected feces to 20On a 0-mesh screen cloth, using ddH2O carefully and slowly rinse the fecal surface to remove surface impurities. Approximately 200mg of feces were transferred to a 1.5mL centrifuge tube and approximately 200. mu.L of ddH was added2And O, blowing and beating the mixture into excrement homogenate by using a suction pipe, centrifuging the excrement homogenate for 3min at the speed of 800r/min, and taking supernatant.
2. DNA extraction:
(1) adding 400 μ L10% SDS and 10 μ L of protease K into the supernatant, performing water bath at 65 deg.C for 60min until complete digestion, mixing uniformly every 10min, adding 10 μ L20mg/mLRNase, performing water bath at 37 deg.C for 10min, centrifuging at 12000r/min for 3min, and collecting the supernatant;
(2) adding ice-cold saturated phenol with the same volume, reversing the upper part and the lower part, uniformly mixing, standing at room temperature for 5min, centrifuging at 12000r/min for 12min, and taking supernatant; adding equal volume of chloroform, turning upside down, mixing, centrifuging at 12000r/min for 10min, and collecting supernatant; adding isovolumetric isopropanol, reversing for 8 times, mixing well, standing at room temperature for 3min, centrifuging at 12000r/min for 5min, and removing supernatant;
(3) adding 1mL of 70% ice-cold ethanol for rinsing, centrifuging at 12000r/min for 3min, and removing the supernatant; repeatedly rinsing once, centrifuging and discarding the supernatant; the precipitate was dried at room temperature and dissolved in 30. mu.L of TE Buffer.
The 'shellfish' in the invention refers to marine shellfish including clam, scallop, oyster and the like.
Has the advantages that: the invention can be applied to the nondestructive extraction of shellfish DNA, has no stimulation and no damage to shellfish, has simple operation and high detection accuracy, and is suitable for popularization and application.
Drawings
FIG. 1 is a schematic diagram showing the electrophoresis result of the method of the present invention for extracting DNA in example 1;
FIG. 2 is a schematic diagram of the detection result of the clam DNA extracted from the clam feces by 4 pairs of primers PCR amplification electrophoresis in example 2;
FIG. 3 is a graph showing the morphological changes of the clam feces in example 3 at different soaking times and ambient temperatures;
FIG. 4 is a schematic diagram showing the electrophoresis results of the DNA of the clam feces in example 4 at different soaking times and ambient temperatures;
table 1 shows 2 pairs of clam mitochondrial DNA primers and 2 pairs of clam nuclear genomic DNA primers.
Detailed Description
Example 1 extraction of DNA from clam feces
1. Sample collection and pretreatment
Experimental groups:
(1) randomly selecting 24 clams, and starving for 2 days to completely discharge impurities in the clams. Feeding a proper amount of chlorella, and collecting clam excrement from the bottom of the pool by using a siphon method after the clam defecates.
(2) Transferring the collected feces to 200-mesh silk screen, and screening with ddH2O carefully and slowly rinse the fecal surface to remove surface impurities.
(3) About 200mg of feces were aspirated with a pipette, transferred to a centrifuge tube, and about 200. mu.L of ddH was added2And O, blowing and beating the mixture into excrement homogenate by using a suction pipe, centrifuging the excrement homogenate for 3min at the speed of 800r/min, and taking supernatant.
6 clam stool supernatant samples were obtained.
Positive control group:
(1) approximately 25mg of the clam feet were cut with sterilized dissecting scissors and ground on ice with a high speed tissue grinder to a fine powder.
2. Extraction of Cyclina sinensis DNA
(1) Adding 400 μ L10% SDS and 10 μ L protease K, water bathing at 65 deg.C for 60min to completely digest, mixing once every 10min, adding 10 μ L20mg/mL RNase, water bathing at 37 deg.C for 10min, centrifuging at 12000r/min for 3min, and collecting supernatant;
(2) adding ice-cold saturated phenol with the same volume, reversing the upper part and the lower part, uniformly mixing, standing at room temperature for 5min, centrifuging at 12000r/min for 12min, and taking supernatant; adding equal volume of chloroform, turning upside down, mixing, centrifuging at 12000r/min for 10min, and collecting supernatant; adding isovolumetric isopropanol, reversing for 8 times, mixing well, standing at room temperature for 3min, centrifuging at 12000r/min for 5min, and removing supernatant;
(3) adding 1mL of 70% ice-cold ethanol for rinsing, centrifuging at 12000r/min for 3min, and removing the supernatant; repeatedly rinsing once, centrifuging and discarding the supernatant; the precipitate was dried at room temperature and dissolved in 30. mu.L of TE Buffer.
3. Electrophoretic detection
Carrying out electrophoresis detection on the DNA sample extracted in the step 2, wherein the buffer solution is 1 XTAE, the power is 50W, and the time is 25 min; 1% agarose, loading amount 6. mu.L, electrophoresis detection result is shown in figure 1, wherein M: DNA Marker; n: negative control (ddH)2O); f: positive control (clam foot DNA); 1-6: the method is adopted to extract the clam DNA from the clam excrement.
As can be seen from FIG. 1, the DNA template bands extracted by the method are clear, and the 2, 4, 5 and 6 bands have high brightness, and are similar to positive control (foot DNA), thus proving the reliability of the method for extracting the DNA of the clam.
Example 2 Cyclina sinensis mitochondrial DNA and Nuclear genomic DNA amplification detection assay
The DNA extracted in example 1 was amplified by PCR products using 2 pairs of clam mitochondrial DNA primers and 2 pairs of clam nuclear genomic DNA primers in Table 1, respectively.
Reaction system: template DNA 1.0. mu.L, upstream and downstream 0.3. mu.L each, 2 XTAQUACRMSTER Mix 7.5. mu.L, ddH2O 5.9μL。
Reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 53 deg.C for 30sec, extension at 72 deg.C for 10min after 30 cycles, and storing at 4 deg.C.
The buffer solution is 1 XTAE, the power is 50W, and the time is 15 min; 1.5% agarose, loading amount of 6. mu.L, 4 pairs of primers, electrophoresis detection results are shown in FIG. 2, wherein a: CsCOX I; b: cs 16S; c: cs 18S; d: cspds; m: DNA Marker; n: negative control (ddH)2O); f: positive control (clam foot DNA); 1-6: the method is adopted to extract the clam DNA from the clam excrement.
The detection result of fig. 2 shows that the clam feces and the foot extraction amplification result are consistent, and the validity and the reliability of the method are verified.
Example 3 Effect of soaking time and ambient temperature on stool morphology of Cyclina sinensis
1. Collecting clam feces
When the clams discharge the excrement, the excrement is immediately collected, and excrement samples are soaked in clean experimental seawater and are respectively stored in the environment of 28 ℃, 15 ℃ and 4 ℃.
2. Observing stool morphology
A small amount of feces were aspirated at 0, 5, 10, 15, 20 and 25d, respectively, and placed on a clean glass slide, and the feces morphology was observed with a stereoscope and photographed, and the result of the morphological change is shown in FIG. 3, where a: group 28 ℃; b: 15 ℃ group; c: group 4 ℃.
The results in figure 3 show the effect of stool morphology with soaking time and ambient temperature. The fresh fecal pellets (0d) were yellow-green cylinders. In the group at 28 ℃ (fig. 3a) and 5d, the exogenous part of the excrement slightly breaks, filamentous fungi grow in a large amount and gradually form zoogloea, and more bacteria are attached to the zoogloea; at 10d and 15d, obvious fracture of loose fecal particles occurs, and obvious decomposition of local structures occurs; at 20 days, the broken parts of the excrement particles are increased, and the particles become looser; at 25 days, the fibers are completely converted into loose floccules. In the group at 15 ℃ (fig. 3b), the 10d feces form is not obviously broken, and the exogenous site of the feces is slightly decomposed to gradually form zoogloea; from 15d to 20d, the broken parts of the fecal particles are increased, and the obvious breakage phenomenon appears at 25 d. No significant fracture occurred in 10d, 15d was slightly loose, and slight fracture occurred at the outer edge of 20d and 25d feces in the 4 ℃ group (FIG. 3 c).
Example 4 Effect of soaking time and ambient temperature on the quality of DNA extraction from clam feces
1. Collecting clam feces
When the clams discharge the excrement, the excrement is immediately collected, and excrement samples are soaked in clean experimental seawater and are respectively stored in the environment of 28 ℃, 15 ℃ and 4 ℃.
2. Extraction of Cyclina sinensis DNA
About 200mg of fecal samples were taken at 0, 5, 10, 15, 20 and 25d, respectively, for extraction, and clam feet DNA was used as positive control, ddH2O is a negative control, and the DNA extraction adopts the same extraction method as the embodiment 1 to extract the clam feces DNA and the clam foot DNA.
3. Electrophoretic detection
Carrying out electrophoresis detection on the DNA sample extracted in the step 2, wherein the buffer solution is 1 XTAE, the power is 50W, and the time is 25 min; 1% agarose, loading 6. mu.L, electrophoresis detection results are shown in FIG. 4, wherein a: group 28 deg.C(ii) a b: 15 ℃ group; c: 4 ℃ group; m: DNAmarker; n: negative control (ddH)2O); f: positive control (clam foot DNA); 1-12: the method is adopted to extract the clam DNA from the clam excrement.
The detection results in fig. 4 show that the quality of the extracted DNA of the stool sample is significantly different under different environmental temperatures and soaking times. Under the environment of 28 ℃, the fecal samples begin to degrade to different degrees after being soaked for 5 days, but some samples can extract DNA with higher quality, while the fecal samples soaked for 15 and 20 days extract DNA with poorer quality and more serious degradation phenomenon (figure 4 a). Under the environment of 15 ℃, DNA with better quality can be extracted after 10 days of the excrement, and degradation with different degrees begins to appear after 15 days (figure 4 b). Higher quality DNA was obtained when the stool samples were soaked for 15 days at 4 deg.C (FIG. 4 c).
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions of the above embodiments or substitute some technical features of the above embodiments. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A method for extracting shellfish DNA from shellfish excrement comprises the following steps:
(1) placing shellfish excrement on 200 mesh bolting-silk net, adding appropriate amount of ddH2O, slowly washing the excrement to remove impurities on the surface of the excrement; about 200mg of fecal material was placed in a 1.5mL centrifuge tube and 200. mu.L ddH was added2O, blowing and beating the mixture into excrement homogenate by a suction pipe, centrifuging the excrement homogenate for 3min at the speed of 800r/min, and taking supernatant;
(2) adding 400 μ L10% SDS and 10 μ L proteinase K, completely digesting in 65 deg.C water bath for 60min, mixing once every 10min, adding 10 μ L20mg/mLRNase, water bath at 37 deg.C for 10min, centrifuging at 12000r/min for 3min, and collecting supernatant;
(3) adding ice-cold saturated phenol with the same volume, reversing the upper part and the lower part, uniformly mixing, standing at room temperature for 5min, centrifuging at 12000r/min for 12min, and taking supernatant; adding equal volume of chloroform, turning upside down, mixing, centrifuging at 12000r/min for 10min, and collecting supernatant; adding isovolumetric isopropanol, reversing for 8 times, mixing well, standing at room temperature for 3min, centrifuging at 12000r/min for 5min, and removing supernatant;
(4) adding 1mL of 70% ice-cold ethanol for rinsing, centrifuging at 12000r/min for 3min, and removing the supernatant; repeatedly rinsing once, centrifuging and discarding the supernatant; the precipitate was dried at room temperature and dissolved in 30. mu.LTE Buffer.
2. The method for extracting shellfish DNA from shellfish excretion according to claim 1, wherein the shellfish excretion of step 1) is 0-5d (summer)/0-10 d (spring, autumn)/0-25 d (winter) excretion from shellfish; ddH is necessary for rinsing2O slowly washes the surface of the excrement without destroying the excrement structure.
3. The method for extracting shellfish DNA from shellfish excrement according to claim 1 or 2, wherein said shellfish comprises marine shellfish such as Cyclina sinensis, Meretrix meretrix Linnaeus, scallop, oyster, etc.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586234A (en) * | 2012-03-12 | 2012-07-18 | 云南师范大学 | Method for extracting high-molecular-weight genome from animal feces |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586234A (en) * | 2012-03-12 | 2012-07-18 | 云南师范大学 | Method for extracting high-molecular-weight genome from animal feces |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
Non-Patent Citations (3)
| Title |
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| ARIEL LEVI SIMONS等: "High turnover of faecal microbiome from algal feedstock experimental manipulations in the Pacific oyster (Crassostrea gigas)", 《MICROB BIOTECHNOL》 * |
| 刘艳华等: "野生动物粪便在濒危物种遗传结构研究中的应用", 《野生动物》 * |
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