CN102911934B - DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria - Google Patents
DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria Download PDFInfo
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Abstract
The invention discloses a DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria. The DNA extraction method comprises the following steps of: performing ultra-low temperature refrigeration on a sample to reduce the viscosity of the sample so as to facilitate dispersion of bacteria in the sample into eluent I with buffer and viscosity reducing functions; removing pasty polysaccharide and protein in the sample through multiple low-speed centrifugation in a thallus collection step; adding CTAB (Cetyl Trimethyl Ammonium Bromide) solution into lysate to remove mucopolysaccharide in a polysaccharide and miscellaneous protein removal step and adding protease K to further remove interference of soluble protein; and finally obtaining bacteria DNA through an extraction method of eluent II. The DNA extraction method has the advantages of adoption of the conventional test reagents, no need of special experimental conditions, easiness in operation, high repeatability and high bacteria DNA extraction quality. The method is suitable for extracting the bacteria DNA from the polysaccharide and protein-rich sample and has broad application range and application prospect.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of sea cucumber body surface DNA of bacteria extracting method.
Background technology
Along with the develop rapidly of China's aquaculture, processing industry, imitated apostichopus japonicus culture and processing quantity cumulative year after year in recent years.Carry out imitative stichopus japonicus body surface, intestinal microflora diversity analysis, can provide fabulous starting material and data for the Microecology of sea cucumber, for better understanding bioinformation that sea cucumber carries, providing good help for the sound development of China's holothruian cultures and processing industry.
Bacteria total DNA extracting method is more, mainly contains the methods such as CTAB, test kit, the extracting of phenol chloroform, practical in actually operating.Imitative stichopus japonicus epidermis contains a large amount of mucosubstance, and its rich in proteins and mucopolysaccharide, according to general bacteria total DNA extracting method, cannot normal extraction arrive sea cucumber body surface bacteria total DNA at all, and the DNA of bacteria electrophorogram hangover of extraction is serious.Grope and repeatable operation according to experiment for many years, on CTAB method basis, increase sample pre-treatments to reduce the viscosity of sample, and adopted increase proteolytic enzyme amount and CTAB solution removal foreign protein and mucopolysaccharide material, can obtain desirable sea cucumber body surface DNA of bacteria.
Microorganism is extensive in distributed in nature, and the extraction of DNA of bacteria is very easily subject to the impact of its environment of living in.Conventionally the bacterium in air, water body, soil can adopt conventional DNA of bacteria extracting method.And grow nonparasitically upon another plant, parasitic bacterium is conventionally in the body surface or body in plant or animal, living environment is rich in the nutritive substance such as polysaccharide and protein, adopt ordinary method to be difficult to them to separate with host, therefore ordinary method cannot obtain this type of DNA of bacteria, and this problem is perplexing the investigator in this field always.
Summary of the invention
For solving the problems of the technologies described above adopted technical scheme be: in to sea cucumber body surface DNA of bacteria leaching process, main purpose of the present invention is to remove foreign protein, the interference of mucopolysaccharide to sample, and reduction sample viscosity is beneficial to the stripping of body surface bacterium.First sample is carried out to superfreeze to reduce sample viscosity, be beneficial to bacterium in sample and be distributed to and there is buffering, fall in low viscous elutriant I; In microorganism collection step, remove polysaccharide and the albumen of pasty state in sample by low-speed centrifugal repeatedly; Increase the step that removes of mucopolysaccharide and foreign protein, in lysate, added CTAB solution to remove mucopolysaccharide, added the interference that Proteinase K is further removed solubility foreign protein; Final by the extracting of elutriant II, obtain DNA of bacteria.
The extracting method of a kind of sea cucumber body surface DNA of bacteria of the present invention, it comprises the following steps,
(1) sea cucumber sample preparation:
Getting fresh and alive sea cucumbers stroke-physiological saline solution rinses three times, after sterile packaged, place rapidly-80 ℃ and preserve 24h, take out sea cucumber under aseptic condition, dig and scrape sea cucumber body surface by sterile razor blade, plane is scraped to thing and be put in aseptic centrifuge tube, 50mL centrifuge tube fills 6 ~ 10g (weight in wet base) sea cucumber body surface plane and scrapes thing;
(2) sea cucumber body surface microorganism collection:
1., in the centrifuge tube obtaining to step (1), add 15 ~ 20mL elutriant I, 300rpm concussion 5 ~ 10min under normal temperature, the sea cucumber body surface dope that plane is scraped fully mixes with elutriant I;
2. the centrifugal 5 ~ 8min of 1000rpm at 4 ℃, removes lower floor sea cucumber and organizes throw out and upper strata mashed prod, and remaining liq is transferred in another aseptic centrifuge tube;
3. repeating step is 2. until remove throw out and mashed prod completely;
4. by step, 3. gained liquid subpackage is in 1.5mL centrifuge tube, and in 4 ℃ of centrifugal 1 ~ 2min of 10000rpm, collecting precipitation, is sea cucumber body surface bacterial sediment thing;
(3) removing of mucopolysaccharide and foreign protein:
4. in gained sea cucumber body surface bacterial sediment thing, add 500uL lysate in step, piping and druming to precipitation suspends, and then hatches concussion 1 ~ 3h for 55 ℃, until suspension clarification is brief centrifugal to remove the cap wall globule.
(4) bacterium broken wall
In aforesaid liquid, add 180uL N,O-Diacetylmuramidase (20mg/mL), in 37 ℃ hatch enzymolysis 30min more than, interval concussion during this time until the white precipitate producing disappear, brief centrifugal to remove the cap wall globule.
(5) extracting, purifying:
5. in the enzymolysis solution obtaining to step (4), add 300uL phenol and 300uL elutriant II, fully mix rear 4 ℃ of centrifugal 10 ~ 15min of 12000rpm;
6. draw 5. middle supernatant liquor 500uL of step, add elutriant II 500uL in 1.5mLEppendorf test tube, mix rear 4 ℃ of centrifugal 10min of 12000rpm;
7. draw step 6. in supernatant liquor 400uL, add dehydrated alcohol (in advance in-20 ℃ of precoolings) 800uL in 1.5mLEppendorf test tube ,-20 ℃ of standing 20min after mixing, the centrifugal 10min of 12000rpm at 4 ℃;
8. 70% washing with alcohol 2 ~ 3 times for white precipitate, is dissolved in after drying in the aseptic ultrapure water of 100uL, and light shaking is until resolution of precipitate, or is positioned in 50uL damping fluid TE, under-20 ℃ of conditions, stores.
The compound method of the part reagent wherein, using in technique scheme of the present invention is as follows:
Elutriant I: potassium primary phosphate: 0.27g, Sodium phosphate dibasic: 1.42g, sodium-chlor: 8g, Repone K: 0.2g, adds the abundant stirring and dissolving of the about 700mL of deionized water, adjusts pH to 7.4 with hydrochloric acid, and last constant volume is to 1L.Then add the CTAB solution of 200mL, room temperature preservation after autoclave sterilization.
In the CTAB solution of lysate: 3mL, add beta-mercaptoethanol and the 30uL Proteinase K (50mg/mL) of 6uL.
CTAB solution: 100mmol/L Tris-Hcl, the EDTA of 20mmol/L, the NaCl of 1.4mol/L.
5 × TE damping fluid: by Tris 54g, boric acid 25g, 0.5mol/L EDTA(pH8.0) 20mL, add deionized water and be settled to after 1L, after mixing, be placed in 4 ℃ of refrigerators and preserve.
Elutriant II: primary isoamyl alcohol: the volume ratio of chloroform is 1:24.
Character of innovation of the present invention is:
Adopt conventional test reagent, without special experiment condition, simple to operate, reproducible, it is high that DNA of bacteria is extracted quality.The method is applicable to extract DNA of bacteria from be rich in polysaccharide, protein sample, is with a wide range of applications and application prospect.
Accompanying drawing explanation
Fig. 1 is the bacteria total DNA electrophorogram that utilizes test kit method to extract, and wherein swimming lane 1 is DL15000MARK, and swimming lane 2,3 is DNA of bacteria, and hangover is serious.
Fig. 2 is the bacteria total DNA electrophorogram that utilizes CTAB method to extract, and wherein swimming lane 1 is DL2000MARK, and swimming lane 2,3 is DNA of bacteria, has hangover to disturb.
Fig. 3 is the bacteria total DNA electrophorogram that utilizes phenol chloroform method to extract, and wherein swimming lane 1 is DL15000MARK, and swimming lane 2,3 is DNA of bacteria, and hangover is serious.
Fig. 4: be the bacteria total DNA electrophorogram that utilizes this experimental technique to extract, wherein swimming lane 1 is DL15000MARK, and swimming lane 2,3 is DNA of bacteria, and it is high that DNA of bacteria is extracted quality.
Fig. 5: bacterial 16 S rRNA electrophorogram, wherein the Marker of swimming lane 1,4 is DL2000MARK, middle swimming lane 2,3 is PCR product, big or small about 1500bp.
Fig. 6: positive colony carries out colony PCR amplification electrophorogram, wherein swimming lane 1 and 12 is DL2000MARKplus5,000bp 3,000bp, swimming lane 2 ~ 11 is pcr amplification product, size is about 1650bp.
Fig. 7: imitative stichopus japonicus body surface BACTERIAL PHYLOGENY tree.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
The solution relating in method described in the embodiment of the present invention, as follows preparation:
Elutriant I: potassium primary phosphate: 0.27g, Sodium phosphate dibasic: 1.42g, sodium-chlor: 8g, Repone K: 0.2g, adds the abundant stirring and dissolving of the about 700mL of deionized water, adjusts pH to 7.4 with hydrochloric acid, and last constant volume is to 1L.Then add the CTAB solution of 200mL, room temperature preservation after autoclave sterilization.
In the CTAB solution of lysate: 3mL, add beta-mercaptoethanol and the 30uL Proteinase K (50mg/mL) of 6uL.
CTAB solution: 100mmol/L Tris-Hcl, the EDTA of 20mmol/L, the NaCl of 1.4mol/L.
5 × TE damping fluid: by Tris 54g, boric acid 25g, 0.5mol/L EDTA(pH8.0) 20mL, add deionized water and be settled to after 1L, after mixing, be placed in 4 ℃ of refrigerators and preserve.
Elutriant II: primary isoamyl alcohol: the volume ratio of chloroform is 1:24.
1, sea cucumber sample preparation:
Getting the fresh and alive imitative stichopus japonicus stroke-physiological saline solution just having salvaged rinses three times, after sterile packaged, place rapidly-80 ℃ and preserve 24h, take out sea cucumber under aseptic condition, dig and scrape sea cucumber body surface by sterile razor blade, plane is scraped to thing and be put in aseptic centrifuge tube, each 50mL centrifuge tube fills about 6g sea cucumber body surface plane and scrapes thing;
2, sea cucumber body surface microorganism collection:
1., in the centrifuge tube obtaining to step (1), add 15mL elutriant I, 300rpm concussion 5min under normal temperature, the sea cucumber substance on body surface that plane is scraped fully mixes with elutriant I;
2. the centrifugal 5min of 1000rpm at 4 ℃, removes lower floor sea cucumber and organizes throw out and upper strata mashed prod, and residue clear liquid is transferred in another aseptic centrifuge tube;
3. repeating step is 2. until remove throw out and mashed prod completely;
4. by step 3. gained clear liquid be sub-packed in 1.5mL centrifuge tube, and in 4 ℃ of centrifugal 1min of 10000rpm, collecting precipitation, is sea cucumber body surface bacterial sediment thing;
3, removing of mucopolysaccharide and foreign protein:
4. in gained sea cucumber body surface bacterial sediment thing, add 500uL lysate in step, piping and druming to precipitation suspends, and then hatches concussion 1h for 55 ℃, until suspension clarification is brief centrifugal to remove the cap wall globule.
4, bacterium broken wall
In aforesaid liquid, add 180uL N,O-Diacetylmuramidase (20mg/mL), hatch enzymolysis 30min in 37 ℃, interval concussion during this time until the white precipitate producing disappear, brief centrifugal to remove the cap wall globule.
5, extracting, purifying:
5. in the enzymolysis solution obtaining to step 4, add 300uL phenol and 300uL elutriant II, fully mix latter 4 ℃, the centrifugal 10min of 12000rpm;
6. draw 5. middle supernatant liquor 500uL of step, add elutriant II 500uL in 1.5mLEppendorf test tube, mix rear 4 ℃ of centrifugal 10min of 12000rpm;
7. draw step 6. in supernatant liquor 400uL, add dehydrated alcohol (in advance in-20 ℃ of precoolings) 800uL in 1.5mLEppendorf test tube ,-20 ℃ of standing 20min after mixing, the centrifugal 10min of 12000rpm at 4 ℃;
8. 70% washing with alcohol 3 times for white precipitate, is dissolved in after drying in the aseptic ultrapure water of 100uL, and light shaking is until resolution of precipitate, or is positioned in 50uL damping fluid TE, under-20 ℃ of conditions, stores.
Embodiment 2
1, sea cucumber sample preparation:
Getting the fresh and alive sea cucumbers stroke-physiological saline solution just having salvaged rinses three times, after sterile packaged, place rapidly-80 ℃ and preserve 24h, take out sea cucumber under aseptic condition, dig and scrape sea cucumber body surface by sterile razor blade, plane is scraped to thing and be put in aseptic centrifuge tube, each 50mL centrifuge tube fills about 7g sea cucumber body surface plane and scrapes thing;
2, sea cucumber body surface microorganism collection:
1., in the centrifuge tube obtaining to step (1), add 18mL elutriant I, 300rpm concussion 6min under normal temperature, the sea cucumber substance on body surface that plane is scraped fully mixes with elutriant I;
2. the centrifugal 6min of 1000rpm at 4 ℃, removes lower floor sea cucumber and organizes throw out and upper strata mashed prod, and residue clear liquid is transferred in another aseptic centrifuge tube;
3. repeating step is 2. until remove throw out and mashed prod completely;
4. by step 3. gained clear liquid be sub-packed in 1.5mL centrifuge tube, and in 4 ℃ of centrifugal 2min of 10000rpm, collecting precipitation, is sea cucumber body surface bacterial sediment thing;
3, removing of mucopolysaccharide and foreign protein:
4. in gained sea cucumber body surface bacterial sediment thing, add 500uL lysate in step, piping and druming to precipitation suspends, and then hatches concussion 2h for 55 ℃, until suspension clarification is brief centrifugal to remove the cap wall globule.
4, bacterium broken wall
In aforesaid liquid, add 180uL N,O-Diacetylmuramidase (20mg/mL), in 37 ℃ hatch enzymolysis 40min more than, interval concussion during this time until the white precipitate producing disappear, brief centrifugal to remove the cap wall globule.
5, extracting, purifying:
5. in the enzymolysis solution obtaining to step 4, add 300uL phenol and 300uL elutriant II, fully mix latter 4 ℃, the centrifugal 15min of 12000rpm;
6. draw 5. middle supernatant liquor 500uL of step, add elutriant II 500uL in 1.5mLEppendorf test tube, mix latter 4 ℃, the centrifugal 10min of 12000rpm;
7. draw step 6. in supernatant liquor 400uL, add dehydrated alcohol (in advance in-20 ℃ of precoolings) 800uL in 1.5mLEppendorf test tube ,-20 ℃ of standing 20min after mixing, the centrifugal 10min of 12000rpm at 4 ℃;
8. 70% washing with alcohol 2 times for white precipitate, is dissolved in after drying in the aseptic ultrapure water of 100uL, and light shaking is until resolution of precipitate, or is positioned in 50uL damping fluid TE, under-20 ℃ of conditions, stores.
Embodiment 3
1, sea cucumber sample preparation:
Getting the fresh and alive sea cucumbers stroke-physiological saline solution just having salvaged rinses three times, after sterile packaged, place rapidly-80 ℃ and preserve 24h, take out sea cucumber under aseptic condition, dig and scrape sea cucumber body surface by sterile razor blade, plane is scraped to thing and be put in aseptic centrifuge tube, each 50mL centrifuge tube fills about 10g sea cucumber body surface plane and scrapes thing;
2, sea cucumber body surface microorganism collection:
1., in the centrifuge tube obtaining to step (1), add 15mL elutriant I, 300rpm concussion 10min under normal temperature, the sea cucumber substance on body surface that plane is scraped fully mixes with elutriant I;
2. the centrifugal 8min of 1000rpm at 4 ℃, removes lower floor sea cucumber and organizes throw out and upper strata mashed prod, and residue clear liquid is transferred in another aseptic centrifuge tube;
3. repeating step is 2. until remove throw out and mashed prod completely;
4. by step 3. gained clear liquid be sub-packed in 1.5mL centrifuge tube, and in 4 ℃ of centrifugal 2min of 10000rpm, collecting precipitation, is sea cucumber body surface bacterial sediment thing;
3, removing of mucopolysaccharide and foreign protein:
4. in gained sea cucumber body surface bacterial sediment thing, add 500uL lysate in step, piping and druming to precipitation suspends, and then hatches concussion 3h for 55 ℃, until suspension clarification is brief centrifugal to remove the cap wall globule.
4, bacterium broken wall
In aforesaid liquid, add 180uL N,O-Diacetylmuramidase (20mg/mL), in 37 ℃ hatch enzymolysis 50min more than, interval concussion during this time until the white precipitate producing disappear, brief centrifugal to remove the cap wall globule.
5, extracting, purifying:
5. in the enzymolysis solution obtaining to step 4, add 300uL phenol and 300uL elutriant II, fully mix rear 4 ℃ of centrifugal 15min of 12000rpm;
6. draw 5. middle supernatant liquor 500uL of step, add elutriant II 500uL in 1.5mLEppendorf test tube, mix rear 4 ℃ of centrifugal 10min of 12000rpm;
7. draw step 6. in supernatant liquor 400uL, add dehydrated alcohol (in advance in-20 ℃ of precoolings) 800uL in 1.5mLEppendorf test tube ,-20 ℃ of standing 20min after mixing, the centrifugal 10min of 12000rpm at 4 ℃;
8. 70% washing with alcohol 2 ~ 3 times for white precipitate, is dissolved in after drying in the aseptic ultrapure water of 100uL, and light shaking is until resolution of precipitate, or is positioned in 50uL damping fluid TE, under-20 ℃ of conditions, stores.
Embodiment 4
Adopt following four kinds of methods to extract total DNA of sea cucumber body surface bacterium, and carry out electrophoresis, thereby detect the quality of the extraction bacteria total DNA of the method for the invention: method described in test kit method, CTAB method, phenol chloroform method, the embodiment of the present invention 1;
1. the DNA purification kit specification sheets of test kit Fa Jian biotech firm.Fig. 1 is the bacteria total DNA electrophorogram that utilizes test kit method to extract, and wherein swimming lane 1 is DL15000MARK, and swimming lane 2,3 is DNA of bacteria, and hangover is serious.
2. the concrete operation step of conventional CTAB method, referring to " marine organisms Ecological Investigation technical regulation "
[1], special office of National Bureau of Oceanography 908 compiles, Maritime Press, 2006.Fig. 2 is the bacteria total DNA electrophorogram that utilizes CTAB method to extract, and wherein swimming lane 1 is DL2000MARK, and swimming lane 2,3 is DNA of bacteria, has hangover to disturb.
3. the operation steps of phenol chloroform extraction method, referring to " fine works molecular biology experiment guide "
[2], Science Press, 2008.Fig. 3 is the bacteria total DNA electrophorogram that utilizes phenol chloroform method to extract, and wherein swimming lane 1 is DL15000MARK, and swimming lane 2,3 is DNA of bacteria, and hangover is serious.
4. the bacteria total DNA electrophoresis that utilizes the method described in the embodiment of the present invention 1 to extract, its electrophorogram is shown in Fig. 4: wherein swimming lane 1 is DL15000MARK, swimming lane 2,3 is DNA of bacteria, can be determined by the electrophoretic band size in figure and brightness: it is high that DNA of bacteria is extracted quality.
In sum: from Fig. 1 ~ 4, can find out, seriously, although the visible DNA of bacteria of common CTAB method, still having trails disturbs for test kit method, the hangover of phenol chloroform method.And employing present method, the DNA of bacteria band of acquisition is clear.
5. purity and the concentration of utilizing nucleic acid-protein analyser Detection and Extraction DNA, its result is as table 1;
Purity and the concentration of table 1 nucleic acid-protein analyser Detection and Extraction DNA:
| ? | Test kit | Phenol chloroform | CTAB | Embodiment | 1 method |
| 260nm | 0.024 | 0.031 | 0.027 | 0.296 | |
| 280nm | 0.013 | 0.023 | 0.017 | 0.165 | |
| 260/280 | 2.000 | 1.571 | 1.653 | 1.819 | |
| Concentration (ng/ul) | 2.2 | 11 | 17.2 | 29.1 |
260nm is nucleic acid absorption peak, and 280nm is albumen absorption peak, when extracting DNA in the time that the ratio of the light absorption value at this two place is between 1.7-1.9, shows that the DNA quality of extracting is better.Can find out from the data of table 1: adopting the DNA260/280 ratio of test kit method, phenol chloroform method, the extraction of CTAB method not in this scope, is 1.8 and adopt the ratio of embodiment 1 method, meets extraction requirement.
Embodiment 5
Adopt the inventive method to extract sea cucumber body surface DNA and carry out the analysis of sea cucumber body surface bacteria flora, follow-up test result:
1, bacterial 16 S rDNA gene amplification
The imitative stichopus japonicus body surface bacteria total DNA of preparing take method described in embodiment 1 is masterplate, carries out bacterial 16 S rDNA gene amplification.For the Auele Specific Primer of the bacterial 16 S rDNA gene of pcr amplification with reference to Lane, the document of DJ 1991
[3]primer sequence is 27F (5 '-AGA GTT TGA TCC TGG CTCAG-3 ') and 1492R (5 '-TAC GGT TAC CTT GTTACGACT T-3 '), synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, use German Eppendorf pcr amplification instrument, reaction cumulative volume 50ul, comprising 31.5 μ L without bacterium molecule dedicated water, 2.5mM dNTP mixture, 1 × LAPCR BufferII (Mg2+Plus), the each 20pmol/l of primer, 2.5UTaKaRa LA Taq DNA polymerase and 50ng template DNA.Amplified reaction program is as follows: 94 ℃ of denaturation 4min; 94 ℃ of sex change 40s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations, then 72 ℃ of extension 10min.Pcr amplification product loading to 1% agarose gel electrophoresis detects, and in gel imaging system observation analysis, the results are shown in Figure 5.Amplifying bacterial 16 S rDNA electrophorogram both sides Marker is DL2000, and intermediate strap is PCR product, big or small about 1500bp.Selecting positive bacteria falls behind for subsequent use.
2, bacterial 16 S rRNA construction of gene library
By reclaiming after purifying through the PCR product of amplification, with PMD-19
tafter connecting, import in JM109 competent cell and carry out blue hickie screening, and then positive colony is carried out to colony PCR amplification checking, the primer is RV-MF and M13-47R(T carrier universal primer), PCR product size is about 1650bp, see Fig. 6 bacterium colony PCR electrophorogram, wherein: Marker is DL2,000plus, middle electrophoretic band is amplified production.The bacterium colony that PCR product size is about to 1650bp is selected rear for subsequent use.
3,16S rDNA sequencing result
Carry out 16S rDNA amplification take bacterium colony PCR product as template, reaction conditions and system are constant, PCR product uses restriction enzyme Hae III and Hinf I to carry out double digestion, with checking bacterial classification otherness, different differential fragment samples are delivered to Hua Da gene company limited to check order, result is carried out to Blast analysis, and comparison result uses the phylogenetic tree of Clustal X and MEGA3.0 software building.Result is as table 2:
Table 2 is imitated stichopus japonicus body surface bacterial 16 S rDNA sequence alignment result
Comparison result uses the phylogenetic tree of Clustal X and MEGA5.0 software building, and imitative stichopus japonicus body surface BACTERIAL PHYLOGENY tree result is as Fig. 7.
The imitative stichopus japonicus body surface bacteria total DNA obtaining take this experimental technique is as masterplate, amplify the total 16S rDNA of bacterium, then build 16S rRNA gene library, positive colony is carried out to bacterium colony PCR, obtain respectively the 16S rDNA amplified production of different thalline, through order-checking and gene bank comparison, learn that the wild imitative stichopus japonicus body surface bacterium in Dalian is mainly divided into three major types, cannot not culturing bacterium, the microorganism of Rhodopseudomonas and Vibrio.
This result proves, method described in the embodiment of the present invention 1 ~ 4, adopt conventional test reagent, without special experiment condition, simple to operate, reproducible, can be high-quality the DNA of bacteria of extraction sea cucumber body surface, the method is applicable to extract DNA of bacteria from be rich in polysaccharide, protein sample, is with a wide range of applications and application prospect.
Reference:
[1] special office of National Bureau of Oceanography 908 compiles, marine organisms Ecological Investigation technical regulation, Maritime Press, 2006,4, p23-26.
[2] chief editor such as F.M. Ao Sibai, Jin Youxin etc. translate school, fine works molecular biology experiment guide, Science Press, 2008,5, p55.
[3]Lane,D.J.1991.16S/23S?rRNA?sequencing.In?E.Stackebrandt?and?M.Goodfellow(eds.),Nucleic?Acid?Techniques?in?Bacterial?Systematics,pp.115-175.John?Wiley?&?Sons,New?York.
Claims (1)
1. an extracting method for sea cucumber body surface DNA of bacteria, is characterized in that: comprises the following steps,
(1) sea cucumber sample preparation:
Get fresh and alive sea cucumbers stroke-physiological saline solution and rinse three times, after sterile packaged, place rapidly-80 ℃ and preserve 24 h, take out sea cucumber under aseptic condition, scrape sea cucumber body surface with sterile razor blade plane, plane is scraped to thing and be put in aseptic centrifuge tube, the sea cucumber body surface plane of 50 mL centrifuge tube dress weight in wet base 6 ~ 10g is scraped thing;
(2) sea cucumber body surface microorganism collection:
1., in the centrifuge tube obtaining to step (1), add 15 ~ 20 mL elutriant I, 300rpm concussion 5 ~ 10 min under normal temperature, the sea cucumber body surface dope that plane is scraped fully mixes with elutriant I;
2. centrifugal 5 ~ 8 min of 1000 rpm at 4 ℃, remove lower floor sea cucumber and organize throw out and upper strata mashed prod, and remaining liq is transferred in another aseptic centrifuge tube;
3. repeating step is 2. until remove throw out and mashed prod completely;
4. by step, 3. gained liquid subpackage is in 1.5mL centrifuge tube, and in 4 ℃ of centrifugal 1 ~ 2min of 10000rpm, collecting precipitation, is sea cucumber body surface bacterial sediment thing;
(3) removing of mucopolysaccharide and foreign protein:
4. in gained sea cucumber body surface bacterial sediment thing, add 500 μ L lysates in step, piping and druming to precipitation suspends, and then hatches concussion 1 ~ 3h for 55 ℃, until suspension clarification is brief centrifugal to remove the cap wall globule;
(4) bacterium broken wall
The concentration that adds 180 μ L in aforesaid liquid is 20mg/ mL N,O-Diacetylmuramidase, hatches enzymolysis at least 30 min in 37 ℃, interval concussion during this time until the white precipitate producing disappear, brief centrifugal to remove the cap wall globule;
(5) extracting, purifying:
5. in the enzymolysis solution obtaining to step (4), add 300 μ L phenol and 300 μ L elutriant II, fully mix rear 4 ℃ of centrifugal 10 ~ 15min of 12000rpm;
6. draw 5. middle supernatant liquor 500 μ L of step, add elutriant II 500 μ L in 1.5mLEppendorf pipe, mix rear 4 ℃ of centrifugal 10min of 12000rpm;
7. draw step 6. in supernatant liquor 400 μ L, be incorporated in the dehydrated alcohol of-20 ℃ of precoolings, 800 μ L in 1.5mL Eppendorf pipe ,-20 ℃ of standing 20min after mixing, the centrifugal 10min of 12000rpm at 4 ℃;
8. 70% washing with alcohol 2 ~ 3 times for white precipitate, is dissolved in after drying in the aseptic ultrapure water of 100 μ L, and light shaking is until resolution of precipitate, or is positioned in 50 μ L damping fluid TE, under-20 ℃ of conditions, stores
Wherein,
The compound method of CTAB solution is: 100mmol/L Tris-Hcl, the EDTA of 20mmol/L, the NaCl of 1.4mol/L;
The compound method of elutriant I is: potassium primary phosphate: 0.27g, and Sodium phosphate dibasic: 1.42g, sodium-chlor: 8g, Repone K: 0.2g, adds the abundant stirring and dissolving of deionized water 700 mL, adjusts pH to 7.4 with hydrochloric acid, and last constant volume is to 1L; Then add the CTAB solution of 200 mL, room temperature preservation after autoclave sterilization;
The compound method of lysate is: in the CTAB solution of 3 mL, add the Proteinase K that the beta-mercaptoethanol of 6 μ L and the concentration of 30 μ L are 50mg/mL;
The compound method of 5 × TE damping fluid is: by Tris 54g, and boric acid 25g, the EDTA20 mL of 0.5mol/L, pH8.0, adds deionized water and is settled to 1L;
The compound method of elutriant II is: primary isoamyl alcohol: the volume ratio of chloroform is 1:24.
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