CN107904230A - A kind of extracting method of termite alcohol sample enteric microorganism genome DNA - Google Patents
A kind of extracting method of termite alcohol sample enteric microorganism genome DNA Download PDFInfo
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- CN107904230A CN107904230A CN201711129984.3A CN201711129984A CN107904230A CN 107904230 A CN107904230 A CN 107904230A CN 201711129984 A CN201711129984 A CN 201711129984A CN 107904230 A CN107904230 A CN 107904230A
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Abstract
The invention discloses a kind of extracting method of termite alcohol sample enteric microorganism genome DNA.The present invention softens the termite tissue of alcohol immersion using Tris EDTA solution again, broken intestinal tissue epidermis release enteric microorganism is shaken using tissue homogenizer low speed, use bacteriolyze enzymatic treatment bacteria cell wall, microbial cell is fully cracked in conjunction with Proteinase K and SDS, abundant released dna, finally it is combined with DNA extraction universal kits, so that extracting method of the present invention solves existing method and termite alcohol sample is difficult to obtain complete intestinal tissue, microbial cell cracking is not exclusively caused by intestinal microflora diversity and DNA release efficiencies are low, the extracting method of the present invention is by utmost retaining the abundance and diversity of termite alcohol sample enteric microorganism, from the quality and integrality for improving group's Genome DNA extraction.
Description
Technical field：
The invention belongs to DNA to extract field, and in particular to a kind of termite alcohol sample enteric microorganism genome DNA
Extracting method.
Background technology：
Termite is most important carbon cycle organism in ball ecological environment, its utilization rate to lignocellulosic can reach
To more than 97%.Termite can so efficient lignocellulosic digestive system, the microbial consortia of its hindgut played to pass
Important effect.The research of Microorganisms in Termite Gut group it is digested mechanism illustrate and the structure of external Bionic digestion system
The only way which must be passed.These enteric microorganism overwhelming majority can not carry out purification culture under prior art conditions, therefore with
Protocols in Molecular Biology based on DNA analysis is the important means for studying Microorganisms in Termite Gut group.
The enteric microorganism integrated distribution of usual termite in hindgut, cover protokaryon bacterium (gram-positive bacterium and
Gram-negative bacteria), archeobacteria and protozoan (flagellate), up to thousands of kinds.Wherein, gram positive bacterial cell wall
It is generally difficult to directly be crushed with cell pyrolysis liquid, and gramnegative bacterium largely colonizes in the internal of proterknot worm and causes
Common genome DNA extracting reagent kit can not be applicable in.In addition, field acquisition termite sample use alcohol immersion, enteron aisle without
Method is directly peeled off, this extraction to enteric microorganism STb gene adds very big difficulty.
The content of the invention：
The object of the present invention is to provide a kind of extracting method of termite alcohol sample enteric microorganism STb gene, the extraction side
Method can effectively solve the problem that the interference of alcohol immersion and enteric microorganism broad categories to Microorganisms in Termite Gut Genome DNA extraction,
The abundance and diversity of enteric microorganism are at utmost kept, technical support is provided for the research of follow-up intestinal microflora.
The extracting method of the termite alcohol sample enteric microorganism STb gene of the present invention, it is characterised in that including following step
Suddenly：
The termite soaked in alcohol is taken out, surface alcohol is blotted with filter paper, is put into after air-drying in Tris-EDTA buffer solutions,
Ice bath until between termite epidermis and enteron aisle gap full of buffer solution and present it is translucent, thus obtain immersion softening after it is white
Ant；
The termite after softening will be soaked and cut off cephalothorax in the position of second pair of foot first under anatomical lens, then opened from belly
Epidermis is torn at end, is finally peeled away complete middle intestines and is cured tissue block with hindgut, this tissue block is transferred to Tris-EDTA buffer solutions
In, with steel ball concussion homogenizer rupture enteron aisle epidermis releasing microbe, obtain intestinal microflora extracting solution；
Lysozyme is added in intestinal microflora extracting solution to be reacted, and SDS and Proteinase K are added after reaction,
Reaction cracking is carried out, thus obtains lysate, DNA extractions are carried out using lysate as material, thus obtain the micro- life of termite gut
Thing STb gene.
It is preferred that the extracting method of the termite alcohol sample enteric microorganism STb gene, comprises the following steps that：
The termite soaked in alcohol is taken, filter paper blots surface alcohol, and 20mM pH8.0Tris-EDTA bufferings are transferred to after air-drying
In liquid, ice bath until between termite epidermis and enteron aisle gap full of buffer solution and present it is translucent, thus obtain immersion softening
Termite afterwards；
The termite after softening will be soaked and cut off cephalothorax in the position of second pair of foot first under anatomical lens, then opened from belly
Epidermis is torn at end, is finally peeled away complete middle intestines and is cured tissue block with hindgut, this tissue block is transferred to 20mM pH8.0Tris-
In edta buffer liquid, with steel ball concussion homogenizer rupture enteron aisle epidermis releasing microbe, steel ball is taken out, obtains intestinal microbiota
Fall extracting solution；
Lysozyme is added to 200 μ g/mL in intestinal microflora extracting solution, when 37 DEG C of reactions 1 are small；Then, SDS is added
To mass fraction 1%, Proteinase K to 250 μ g/mL, gentle inversion mixing, when 55 DEG C of reactions 3 are small, then 70 DEG C fully crack 20 points
Clock, obtains lysate, reuses the extraction and purification that general pillar genome extracts kit carries out DNA, obtains termite gut
Microorganism total DNA；
The steel ball concussion homogenizer rupture enteron aisle epidermis releasing microbe, its steel ball concussion homogenizer use parameter
For, 500 beats/min of vibration frequency, duration 20 seconds.
It is described until gap presents translucent full of buffer solution between termite epidermis and enteron aisle, its soft condition is leaching
The bubble time is directly proportional to the alcohol Sample preservation time, when ice bath immersion 30 minutes~6 is small, soaks the judgement of terminal：Until termite
Gap is presented translucent full of buffer solution between epidermis and enteron aisle.
The present invention softens the termite tissue of alcohol immersion using Tris-EDTA solution again, utilizes tissue homogenizer low speed
The broken intestinal tissue epidermis release enteric microorganism of concussion, using bacteriolyze enzymatic treatment bacteria cell wall, in conjunction with Proteinase K and
SDS fully cracks microbial cell, and abundant released dna, is finally combined with DNA extraction universal kits, so that the present invention carries
Take method to solve existing method termite alcohol sample is difficult to obtain complete intestinal tissue, intestinal microflora diversity is made
Into microbial cell cracking not exclusively and DNA release efficiencies it is low, extracting method of the invention is white by utmost retaining
The abundance and diversity of ant alcohol sample enteric microorganism, from the quality and integrality for improving group's Genome DNA extraction.
Brief description of the drawings：
Fig. 1 is Workers of Coptotermes formosanus Shiraki alcohol sample hindgut tissue block
Fig. 2 is Workers of Coptotermes formosanus Shiraki intestinal microflora STb gene electrophoretogram：M1 is 15K marker, 2 μ L of loading；It is designated as
Standard sample, 5 μ L of loading, 1,2,3 be sample sequence number, is respectively the Microorganisms in Termite Gut STb gene extracted in embodiment 1, on
2 μ L of sample.
Fig. 3 is various using the 16s rna genes library alpha- of Workers of Coptotermes formosanus Shiraki intestinal microflora STb gene structure
Property (species), square, circular, triangle curve represent three sample microbial specific diversity saturation curves respectively, and curve connects
Closely parallel with abscissa, i.e., microbe species are close to saturation in sample.
Embodiment：
Following embodiments are to further explanation of the invention, rather than limitation of the present invention.
Embodiment 1：
1st, 30 1 term Workers of Coptotermes formosanus Shiraki worker ants soaked in 95% alcohol of volume fraction are taken, filter paper blots surface wine
Essence, and air-dry 5 minutes, 2mL 20mM Tris-EDTA (20mM Tris-HCl, 1mM EDTA, pH8.0) buffer solution is transferred to afterwards
In, when ice bath immersion 2 is small, until gap is translucent full of buffer solution presentation between termite epidermis and enteron aisle, obtain soaking soft
Worker ant after change；
2nd, by the worker ant after immersion softening in Tris-EDTA solution, cut first in the position of second pair of foot under anatomical lens
Epidermis is torn except cephalothorax, then from the belly beginning, complete middle intestines is finally peeled away and cures tissue block (Fig. 1) with hindgut, by this group
Block is knitted to be transferred in 700 μ L 20mM Tris-EDTA (20mM Tris-HCl, 1mM EDTA, pH8.0) buffer solution；
3rd, with steel ball concussion homogenizer rupture enteron aisle epidermal cell, 500 beats/min of vibration frequency, duration 20 seconds, takes out steel
Pearl, obtains about 630 μ L of intestinal microflora extracting solution；
4th, it is small to about 200 μ g/mL, 37 DEG C of reactions 1 in 3 μ L of intestinal microflora extracting solution addition 50mg/mL lysozymes
When；Then, 70 μ L of 10%SDS are added to mass fraction 1%, 2 μ L of 100mg/m Proteinase Ks to about 250 μ g/mL, gentle inversion
Mixing, when 55 DEG C of reactions 3 are small, then 70 DEG C fully crack 20 minutes, obtain lysate；
5th, the lysate that will be obtained in step 4, is century general pillar genome extracts kit (catalog number (Cat.No.) using health
CW2298S) to the further extraction purifications of DNA, the DNA adsorbed on 80 μ L distilled waters dissolving purification column film is eventually adding, obtains platform
Gulf formosanes enteric microorganism STb gene aqueous solution.
6th, using micro-ultraviolet-visible spectrophotometer, the measure of DNA content is carried out；
7th, the agarose gel electrophoresis that use quality percentage concentration is 1% detects to obtain Workers of Coptotermes formosanus Shiraki alcohol sample enteron aisle
The quality (Fig. 2) of microorganism total DNA, wherein 1,2,3 represent what 3 termites by alcohol immersion obtained according to the method described above respectively
Workers of Coptotermes formosanus Shiraki enteric microorganism STb gene.
8th, the use of the Workers of Coptotermes formosanus Shiraki alcohol sample enteric microorganism STb gene obtained in this example is template, with bacterium 16s
RNAV4 regions are amplification target fragment, build intestinal bacterial population 16s rna genes library.With the thing in Alpha- diversity
Phylogenetic diversity of bacteria saturation degree (Fig. 3) in kind quantity saturation curve detection sample
The Workers of Coptotermes formosanus Shiraki enteric microorganism STb gene concentration that the present embodiment obtains is 268~375ng/ μ L, and 260/280 is
1.78~1.88.Test result indicates that the present invention can extract a large amount of high-purities from the termite alcohol sample preserved for a long time
Microbiologic population's STb gene, at utmost retains the abundance and diversity of termite alcohol sample enteric microorganism, from improving group
The quality and integrality of Genome DNA extraction.
Claims (3)
1. a kind of extracting method of termite alcohol sample enteric microorganism STb gene, it is characterised in that comprise the following steps：
The termite soaked in alcohol is taken out, surface alcohol is blotted with filter paper, is put into after air-drying in Tris-EDTA buffer solutions, ice bath
Until between termite epidermis and enteron aisle gap full of buffer solution and present it is translucent, thus obtain immersion softening after termite；
The termite after softening will be soaked and cut off cephalothorax in the position of second pair of foot first under anatomical lens, then torn from the belly beginning
Game clock skin, is finally peeled away complete middle intestines and cures tissue block with hindgut, this tissue block is transferred in Tris-EDTA buffer solutions, is used
Steel ball concussion homogenizer rupture enteron aisle epidermis releasing microbe, obtains intestinal microflora extracting solution；
Lysozyme is added in intestinal microflora extracting solution to be reacted, and SDS and Proteinase K are added after reaction, is carried out
Reaction cracking, thus obtains lysate, and DNA extractions are carried out using lysate as material, it is total thus to obtain Microorganisms in Termite Gut
DNA。
2. extracting method according to claim 1, it is characterised in that comprise the following steps that：
The termite soaked in alcohol is taken, filter paper blots surface alcohol, and 20mM pH8.0 Tris-EDTA buffer solutions are transferred to after air-drying
In, ice bath until between termite epidermis and enteron aisle gap full of buffer solution and present it is translucent, thus obtain immersion softening after
Termite；
The termite after softening will be soaked and cut off cephalothorax in the position of second pair of foot first under anatomical lens, then torn from the belly beginning
Game clock skin, is finally peeled away complete middle intestines and cures tissue block with hindgut, this tissue block is transferred to 20mM pH8.0 Tris-EDTA
In buffer solution, with steel ball concussion homogenizer rupture enteron aisle epidermis releasing microbe, steel ball is taken out, intestinal microflora is obtained and carries
Take liquid；
Lysozyme is added to 200 μ g/mL in intestinal microflora extracting solution, when 37 DEG C of reactions 1 are small；Then, SDS is added to matter
Fraction 1%, Proteinase K to 250 μ g/mL are measured, gentle inversion mixing, when 55 DEG C of reactions 3 are small, then 70 DEG C fully crack 20 minutes,
Lysate is obtained, the extraction and purification that general pillar genome extracts kit carries out DNA is reused, obtains the micro- life of termite gut
Thing STb gene.
3. extracting method according to claim 1 or 2, it is characterised in that the steel ball concussion homogenizer rupture enteron aisle
Epidermis releasing microbe, its steel ball concussion homogenizer is 500 beats/min of vibration frequency using parameter, duration 20 seconds.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852608A (en) * | 2019-02-01 | 2019-06-07 | 广东省农业科学院动物科学研究所 | A kind of extracting method of insect gut microbial DNA |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748118A (en) * | 2009-09-25 | 2010-06-23 | 中国科学院海洋研究所 | A kind of extracting method of prawn intestinal microbial DNA |
| CN102827833A (en) * | 2012-09-24 | 2012-12-19 | 四川国际旅行卫生保健中心 | Extraction method of fly intestinal microorganism total DNA (deoxyribonucleic acid) |
| CN102978198A (en) * | 2012-11-29 | 2013-03-20 | 上海市农业科学院 | Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora |
| CN104560951A (en) * | 2014-12-03 | 2015-04-29 | 复旦大学泰州健康科学研究院 | Extraction method of metagenome DNA and kit for extraction method |
| CN104651508A (en) * | 2015-02-12 | 2015-05-27 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescent quantitative PCR molecule detection method of Coptotermestravians Haviland based on COII genes |
| CN104673909A (en) * | 2015-02-12 | 2015-06-03 | 珠海出入境检验检疫局检验检疫技术中心 | C. sjostedti fluorescent quantitative PCR (Polymerase Chain Reaction) detection method based on COII gene |
| CN105483120A (en) * | 2016-02-16 | 2016-04-13 | 天津农学院 | High-temperature combined method for extracting genomic DNA of fish enteric microorganisms |
-
2017
- 2017-11-15 CN CN201711129984.3A patent/CN107904230A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748118A (en) * | 2009-09-25 | 2010-06-23 | 中国科学院海洋研究所 | A kind of extracting method of prawn intestinal microbial DNA |
| CN102827833A (en) * | 2012-09-24 | 2012-12-19 | 四川国际旅行卫生保健中心 | Extraction method of fly intestinal microorganism total DNA (deoxyribonucleic acid) |
| CN102978198A (en) * | 2012-11-29 | 2013-03-20 | 上海市农业科学院 | Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora |
| CN104560951A (en) * | 2014-12-03 | 2015-04-29 | 复旦大学泰州健康科学研究院 | Extraction method of metagenome DNA and kit for extraction method |
| CN104651508A (en) * | 2015-02-12 | 2015-05-27 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescent quantitative PCR molecule detection method of Coptotermestravians Haviland based on COII genes |
| CN104673909A (en) * | 2015-02-12 | 2015-06-03 | 珠海出入境检验检疫局检验检疫技术中心 | C. sjostedti fluorescent quantitative PCR (Polymerase Chain Reaction) detection method based on COII gene |
| CN105483120A (en) * | 2016-02-16 | 2016-04-13 | 天津农学院 | High-temperature combined method for extracting genomic DNA of fish enteric microorganisms |
Non-Patent Citations (3)
| Title |
|---|
| ERIC MATSON等: "Extracting DNA from the Gut Microbes of the Termite (Zootermopsis nevadensis)", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| 张迎春等: "不同保藏处理的昆虫标本DNA 提取及其随机扩增多态DNA反应", 《昆虫学报》 * |
| 陈镈尧等: "六种白蚁消化***解剖比较研究", 《白蚁科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852608A (en) * | 2019-02-01 | 2019-06-07 | 广东省农业科学院动物科学研究所 | A kind of extracting method of insect gut microbial DNA |
| CN109852608B (en) * | 2019-02-01 | 2021-05-07 | 广东省农业科学院动物科学研究所 | Method for extracting DNA of insect intestinal microorganisms |
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