CN108048455A - A kind of fish superficial microbes genome DNA extracting method - Google Patents
A kind of fish superficial microbes genome DNA extracting method Download PDFInfo
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Abstract
A kind of fish superficial microbes genome DNA extracting method, belongs to biological technical field, it comprises the following steps：Acquisition, the pretreatment of body surface sample, the cracking of superficial microbes, the free of superficial microbes DNA, precipitation and the collection of fish superficial microbes sample.Technical solution of the present invention realizes the high efficiency extraction of seawater fish superficial microbes genomic DNA by optimizing the sampling plan of fish superficial microbes flora sample, establishing efficient superficial microbes example enrichment and efficient cleavage method, high-efficiency shielding high concentration foreign protein disturb.
Description
Technical field
The invention belongs to biological technical fields, specifically a kind of fish superficial microbes extracting genome DNA side
Method.
Background technology
Since living environment is special, Fish Surface mulch the rete malpighii secreted by ectoderm globuli cell.And water body
The microorganism enriched as suitable communication media, value volume and range of product of surviving.Scientific investigations showed that in long-term ecology interaction
Under effect, the microorganisms such as fish body surface bacterium, fungi and rete malpighii form metastable micro-ecological environment, become and resist disease
The natural cover for defense of pathogenic microorganism invasion, more and more researchs show that body surface flora has been actively engaged in host immune and had been metabolized
Journey or even the action selection for influencing host.
The research of fish superficial microbes focuses on separation, identification, infection mechanism and the mucus of specific pathogen bacterium for a long time
It is less to the concern of body surface conventional microbiological in terms of adherency and killing mechanism to specific pathogen bacterium.So far, main application
Traditional isolated culture, researcher can cultivate carefully the seawater fish body surfaces such as Atlantic salmon, rainbow trout, grey mullet, whiting, Yong fishes
The value volume and range of product of bacterium identified, isolated about 15 class Pseudomonas.However, the studies above is limited only to what can be cultured
Superficial microbes, far from meeting the needs of we recognize fish body surface micro-ecological environment.
Compared with traditional isolated culture method and Molecular Identification technology, based on high throughput sequencing technologies of new generation
16S/18S rDNA and metagenomics (Metagenomics) research, can obtain more fully data, can deepen the mankind couple
The composition of microbiologic population under various complicated environmental conditions, function and between environment interaction cognition.Carry out above-mentioned
The premise of work be obtain high quality genomic DNA, at this stage the bacteria total DNAs such as excrement, water environment, soil extracting method compared with
For maturation, and there is commercial kit.Yet with the special living environment of seawater fish, body surface is covered with a large amount of mucus
Layer, physical and chemical composition are complicated, a large amount of Microorganism colonizations or are adsorbed on and wherein, form a special micro-ecological environment.Using
The bacteria total DNAs such as traditional excrement, soil, water environment extracting method or commercial kit extract the micro- life of seawater fish body surface
Object Genomic DNA is poor, and efficiency is low or even is difficult to extract.Therefore, it is promotion fish body surface microbial physiology and ecology research
With the exploitation of application technology, there is an urgent need for establish a kind of seawater fish superficial microbes genome DNA extracting method.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of fish superficial microbes genome DNA extracting method.
The method can extract the fish superficial microbes genomic DNA of high quality, based on meeting high throughput sequencing technologies
16S/18S rDNA and metagenomics research.
The present invention is achieved by the following technical solution：
A kind of fish superficial microbes genome DNA extracting method, comprises the following steps：Fish superficial microbes sample
The acquisition of product, the pretreatment of body surface sample, the cracking of superficial microbes, the free of superficial microbes DNA, precipitation and collection.
Further, the acquisition of the fish superficial microbes sample：Fish body table is rinsed with sterile saline 2-3 times,
In body surface vertebra both sides and pectoral fin perturbing area is avoided using rigid plastics thin plate, is mixed away from fecal pollution area scraping fish epidermal mucus
Object is closed, is quickly put into the centrifuge tube for filling distilled water, then is sprayed to body surface with suction pipe absorption 1-2ml physiological saline and scrapes erasure,
It carries out epidermal mucus mixture again to scrape, repeat 3-4 times, fish epidermal mucus volume of mixture distills to be former in every centrifuge tube
Centrifuge tube, is then slowly shaken mixing 30-40 minutes by the 10%-20% of water volume.
Further, the pretreatment of the fish body surface sample：The epidermal mucus sample of acquisition needs freeze thawing 2-3 times, freezing
Dry, subsequent 8000g is centrifuged 15-20 minutes, collects sample.
Further, the cracking of the fish superficial microbes：Pretreated sample is ground in liquid nitrogen, rear plus cracking
Liquid, Proteinase K and SDS solution, vortex mixing 5-8 minutes, 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing 30s, 70 DEG C of thermostatted waters
Bath 4 minutes, vortex mixing 10s, again 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing 10s.
Further, the free of the fish superficial microbes DNA, precipitation and collection：By gained cleavage mixture in
15000g is centrifuged 10 minutes, is collected supernatant in new centrifuge tube, is then added isometric phenol/chloroform/isoamyl alcohol mixing extract,
Mixing, 12000g are centrifuged 15 minutes, are collected supernatant in new centrifuge tube, are then added chloroform/isoamyl alcohol mixing extract, carry out two
Secondary extracting, 12000g are centrifuged 15 minutes, collect supernatant in new centrifuge tube, then add isopropanol, mixing, and -20 DEG C to stand 1-2 small
When, 15000g is centrifuged 15 minutes, abandons supernatant completely, and addition TE buffer solutions are resuspended precipitation, then add Proteinase K, 70 DEG C of constant temperature
Water-bath 5 minutes, then chloroform/isoamyl alcohol mixing extract is added, 12000g is centrifuged 15 minutes, is collected supernatant in new centrifuge tube, is added
Enter the absolute ethyl alcohol of 1.5 times of volumes, mixing, when 4 DEG C of standings 1 are small, subsequent 12000g is centrifuged 15 minutes, is abandoned supernatant completely, is left and taken
Precipitation, the ultra-pure water or TE buffer solutions of rear 40-60 DEG C of preheating of addition collect DNA.
Further, the lysate ingredient and final concentration of 100mmol/L Tris-Hcl, pH 8.0,1.0mmol/L
EDTA, 100mmol/L NaCl, 1%Triton x-100, proteinase K concentration are that 40mg/ml and SDS solution concentrations are 5% (W/
V)。
Further, the volume ratio of the phenol/chloroform/each ingredient of isoamyl alcohol mixing extract is 25：24：1, chloroform/different
The volume ratio of each ingredient of amylalcohol mixing extract is 24：1.
Further, the TE buffer components and final concentration of 10mmol/L Tris-HCl, 1mmol/L pH8.0's
EDTA。
The advantageous effect of the present invention compared with prior art：
Through analysis, there are three difficult points during seawater fish superficial microbes extracting genome DNA, first, sample obtains
Difficulty is taken, sampling method is improper to easily cause the flocculation of epidermal mucus sample；Second is that in view of that sample is sticky, protein content is high etc. is special
Physicochemical property, the enrichment and cracking of microorganism are difficult, and the technical program is by groping number of freezing and thawing, introducing liquid nitrogen grinding technology ring
Section, optimization cracking agent prescription can efficiently concentrating and cracking fish superficial microbes；Third, sample foreign protein is more, for the problem,
The method that the technical program proposes secondary protease K digesting and secondary extracting improves the acquisition efficiency and quality of total DNA.
In conclusion in view of at this stage without the extracting method of ripe referential seawater fish superficial microbes genomic DNA, this skill
Art scheme is by optimizing the sampling plan of fish superficial microbes flora sample, establishing efficient superficial microbes example enrichment
It is disturbed with efficient cleavage method, high-efficiency shielding high concentration foreign protein, realizes the height of seawater fish superficial microbes genomic DNA
Effect extraction.
Description of the drawings
Fig. 1 turbot superficial microbes total DNAs：M:Mark；2,3,4:Bacterium, excrement, water environment microorganism commercial reagents
Box extracts turbot superficial microbes total DNA effect；1,5,6,7,8:Turbot superficial microbes total DNA carries after this experiment Innovatation
Take effect.
Specific embodiment
Technical scheme is further elaborated below by embodiment combination attached drawing, but the protection of the present invention
Limitation of the scope from embodiment in any form.
1 turbot superficial microbes extracting genome DNA of embodiment：
A kind of fish superficial microbes genome DNA extracting method, comprises the following steps：Fish superficial microbes sample
The acquisition of product, the pretreatment of body surface sample, the cracking of superficial microbes, the free of superficial microbes DNA, precipitation and collection.Tool
Body step is as follows：
(1) acquisition of turbot superficial microbes sample：By the turbot deep anaesthesia of weight 300g or so, with sterile life
It manages normal saline washing fish body table 2-3 times, in body surface vertebra both sides and pectoral fin perturbing area is avoided using rigid plastics thin plate, away from excrement
Contaminated area scrapes fish epidermal mucus mixture, is quickly put into the 50ml centrifuge tubes for filling 25ml distilled water, is drawn with suction pipe
1ml physiological saline is sprayed to body surface and scrapes erasure, carries out epidermal mucus mixture again and scrapes, is repeated 3 times, gathers epidermal mucus altogether
Centrifuge tube, is then slowly shaken mixing 30 minutes by sample 5ml.
(2) turbot epidermal mucus sample needs freeze thawing 3 times after mixing, is then freeze-dried, and then 8000g centrifuges 20 points
Clock collects sample.
(3) centrifugation gained sample is ground in liquid nitrogen, collects sample in 1.5ml centrifuge tubes, addition lysate 350ul,
Proteinase K 10ul and SDS solution 40ul, vortex mixing 5-8 minutes, 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing 30s repeat
70 DEG C of operation water bath with thermostatic control 4 minutes, vortex mixing 10s is twice.
(4) cleavage mixture 15000g obtained by above-mentioned steps is centrifuged 10 minutes, collects supernatant in new centrifuge tube, then add
Add isometric phenol/chloroform/isoamyl alcohol (volume ratio 25：24：1) extract, mixing are mixed, 12000g is centrifuged 15 minutes, is collected
Supernatant then adds isometric chloroform/isoamyl alcohol mixing extract (volume ratio 24 in new centrifuge tube：1) secondary pumping, is carried out
It carries, 12000g is centrifuged 15 minutes, collects supernatant in the isopropyl of new centrifuge tube, then supernatant volume obtained by the upper step of 0.6 times of addition
Alcohol, mixing, when -20 DEG C of standings 1 are small, 15000g is centrifuged 15 minutes, abandons supernatant completely, it is heavy that addition 390ul TE buffer solutions are resuspended
It forms sediment, rear to add Proteinase K 10ul, chloroform/isoamyl alcohol mixing extract 400ul is added in 70 DEG C of waters bath with thermostatic control 5 minutes afterwards,
12000g is centrifuged 15 minutes, collects supernatant in new centrifuge tube, adds in the absolute ethyl alcohol of 1.5 times of volumes, mixing, and 4 DEG C to stand 1 small
When, subsequent 12000g is centrifuged 15 minutes, is abandoned supernatant completely, is left and taken precipitation, the ultrapure water dissolution of rear 50ul60 DEG C of preheating of addition.
The lysate ingredient and final concentration of 100mmol/L Tris-Hcl, pH 8.0,1.0mmol/L EDTA,
100mmol/L NaCl, volume fraction are 1%Triton x-100, and proteinase K concentration is that 40mg/ml and SDS solution concentrations are
5% (W/V).
The TE buffer components and final concentration of 10mmol/L Tris-HCl, the EDTA of 1mmol/L pH 8.0.
(5) electrophoresis observation result such as Fig. 1.
Embodiment 2
It is big that the present embodiment takes bacterium commonly used in the market, excrement, the commercial reagents box of water environment microorganism to carry out
Brill superficial microbes extract, and operating procedure is according to corresponding specification.
Conclusion：As shown in Figure 1：1,5,6,7,8 is 5 batch turbot body surface samples using the technical program extraction
Microbe genome DNA, extraction the results show repeatability and stability are preferable, and 2,3,4 be using bacterium, excrement, water environment
Microorganism business kit extraction 3 batches turbot body surface sample microbe genome DNA, extraction the results show without
Microbe genome DNA master tape, gene samples are identified through Guangzhou Ji Diao bio tech ltd after extraction, 1,5,6,7,8
Sample and the technical program extraction sample meet the quality studied applied to subsequent high pass amount 16S rDNA and metagenomics will
It asks, and 2,3,4 samples and application bacterium, excrement, the sample of water environment microorganism business kit extraction can not meet follow-up height
Flux 16S rDNA and the quality requirement of metagenomics research.
Claims (8)
1. a kind of fish superficial microbes genome DNA extracting method, it is characterised in that it comprises the following steps：Seawater fish body
The acquisition of table microbiological specimens, the pretreatment of body surface sample, the cracking of superficial microbes, free, the precipitation of superficial microbes DNA
With collection.
2. a kind of fish superficial microbes genome DNA extracting method according to claim 1, it is characterised in that described
The acquisition of fish superficial microbes sample：Fish body table is rinsed with sterile saline 2-3 times, using rigid plastics thin plate in body
Table vertebra both sides and pectoral fin perturbing area is avoided, scrape fish epidermal mucus mixture away from fecal pollution area, be quickly put into and fill steaming
It in the centrifuge tube of distilled water, then is sprayed to body surface with suction pipe absorption 1-2ml physiological saline and scrapes erasure, carry out epidermal mucus again and mix
Object scrapes, and repeats 3-4 times, and fish epidermal mucus volume of mixture is the 10%-20% of former distilled water volume in every centrifuge tube, with
Centrifuge tube is slowly shaken to mixing afterwards 30-40 minutes.
3. a kind of fish superficial microbes genome DNA extracting method according to claim 1, it is characterised in that described
The pretreatment of fish body surface sample：The epidermal mucus sample of acquisition needs freeze thawing 2-3 times, freeze-drying, subsequent 8000g centrifugations
15-20 minutes, collect sample.
4. a kind of fish superficial microbes genome DNA extracting method according to claim 1, it is characterised in that described
The cracking of fish superficial microbes：Pretreated sample is ground in liquid nitrogen, and afterwards plus lysate, Proteinase K and SDS are molten
Liquid, vortex mixing 5-8 minutes, 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing 30s, 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing
10s, again 70 DEG C of waters bath with thermostatic control 4 minutes, vortex mixing 10s.
5. a kind of fish superficial microbes genome DNA extracting method according to claim 1, it is characterised in that described
The free of fish superficial microbes DNA, precipitation and collection：Gained cleavage mixture in 15000g is centrifuged 10 minutes, is collected
Supernatant then adds isometric phenol/chloroform/isoamyl alcohol mixing extract, mixing, 12000g centrifuges 15 points in new centrifuge tube
Clock collects supernatant in new centrifuge tube, then adds chloroform/isoamyl alcohol mixing extract of volume, carry out secondary extracting, 12000g
Centrifugation 15 minutes collects supernatant in new centrifuge tube, then adds isopropanol, mixing, when -20 DEG C of standing 1-2 are small, 15000g is centrifuged
15 minutes, abandon supernatant completely, precipitation is resuspended in addition TE buffer solutions, then adds Proteinase K, 70 DEG C of waters bath with thermostatic control 5 minutes, then adds
Chlorination imitates/isoamyl alcohol mixing extract, and 12000g is centrifuged 15 minutes, collects supernatant in new centrifuge tube, the nothing of 1.5 times of volumes of addition
Water-ethanol, mixing, when 4 DEG C of standings 1 are small, subsequent 12000g is centrifuged 15 minutes, is abandoned supernatant completely, is left and taken precipitation, add 40-60 afterwards
DEG C preheating ultra-pure water or TE buffer solutions collect DNA.
6. a kind of fish superficial microbes genome DNA extracting method according to claim 4, it is characterised in that described
Lysate ingredient and final concentration of 100mmol/L Tris-Hcl, pH 8.0,1.0mmol/L EDTA, 100mmol/L
NaCl, volume fraction are 1%Triton x-100, and proteinase K concentration is that 40mg/ml and SDS solution concentrations are 5% (W/V).
7. a kind of fish superficial microbes genome DNA extracting method according to claim 5, it is characterised in that described
Phenol/chloroform/isoamyl alcohol mixing extract in phenol/chloroform/isoamyl alcohol volume ratio be 25：24：1, chloroform/isoamyl alcohol mixing is taken out
The volume ratio of chloroform and isoamyl alcohol is 24 in extract：1.
8. a kind of fish superficial microbes genome DNA extracting method according to claim 5, it is characterised in that described
TE buffer components and final concentration of 10mmol/L Tris-HCl, the EDTA of 1mmol/L pH 8.0.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592076A (en) * | 2019-10-25 | 2019-12-20 | 电子科技大学中山学院 | Reagent and method for extracting microbial community DNA of fermented bean curd |
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| CN110592076A (en) * | 2019-10-25 | 2019-12-20 | 电子科技大学中山学院 | Reagent and method for extracting microbial community DNA of fermented bean curd |
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Application publication date: 20180518 |