CN104531680A - Kit and extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms - Google Patents
Kit and extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms Download PDFInfo
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Abstract
The invention discloses a kit and an extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms. The extraction method for extracting microbial genome DNA from animal fecal microorganisms comprises the following five steps: pre-treatment of feces; splitting decomposition of microbial cells in feces; dissociation of DNA and abstraction of impure proteins; DNA adsorption by a DNA adsorption column and removal of impurities; and elution of DNA. The kit disclosed by the invention is capable of absorbing the genome DNA by a silicone mold adsorption column and removing impurities such as proteins by using special deproteinization liquid and wash liquid. Compared with conventional extraction methods for extracting microbial genome DNA from animal fecal microorganisms, the microbial genome DNA from animal fecal microorganisms can be extracted within 4 hours, so that not only is a lot of extraction time shortened, but also the experimental steps are simplified. Therefore, the microbial genome DNA from animal fecal microorganisms can be effectively, quickly and economically extracted.
Description
Technical field
The invention belongs to biological technical field, be specifically related to test kit and the extracting method of rapid extraction animal excrement microbe genome DNA.
Background technology
The very complicated and metastable microorganism of a group in animal intestinal field planting, and these microorganisms form an extremely complicated microecosystem, have very important impact to animal health.The change of intestinal microecology has great importance and impact on body, the research of intestinal microecology especially dominant microflora is just more and more subject to people's attention, become the focus of current research, because enteron aisle length is long, under scope, segmentation obtains intestinal microflora sample has wound (causing wound with mechanical sampling) because having, difficulty is large, the factor such as to waste time and energy fails to carry out clinical, flora in ight soil is analyzed, can indirectly reflect intestinal microflora situation, contribute to understanding intestinal microecology change, become the main path understanding intestinal microecology change in recent years.Differentiate that the method for bacterium has a lot in ight soil, conventional has: bacteriology separation and Culture authenticate technology, biochemical test qualification, Morphological Identification and analysis etc., and these methods need a large amount of work and time, and sometimes can not the bacterium of diagnostic species level.Therefore by being parsed into a kind of important method in current research to the genome in ight soil.
The genome of microorganism in ight soil is analyzed and first needs to extract the microbe genome DNA obtained in ight soil, because in ight soil, impurities is too many, not only containing animal intestinal cast-off cells, and the food, digestive ferment, mucus, choline, bilirubin, vegetable polysaccharides etc. containing enteric microorganism, end digestion, these impurity bring very large impact to the extraction of microbial genome in ight soil.Therefore, study one animal excrement microbial genome easily and efficiently extraction test kit and seem most important.
Summary of the invention
The object of the present invention is to provide a kind of speed to extract test kit and the extracting method of animal excrement microbe genome DNA, described test kit is easy to operate, quick, can obtain animal excrement microbe genome DNA fast.
For achieving the above object, the present invention adopts following technical scheme:
The test kit of rapid extraction animal excrement microbe genome DNA, described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.25-0.37mol/L potassium primary phosphate and final concentration 0.175-0.2mol/L sodium hydroxide, pH=7-8;
Working fluid B: be made up of final concentration 10-20mmol/L Tutofusin tris (Tris) and final concentration 1-10mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), pH=5.0-8.0;
Working fluid C:10-50mg/ml N,O-Diacetylmuramidase;
Working fluid D:20-50mg/ml Proteinase K;
The sodium lauryl sulphate (SDS) of working fluid E: mass concentration 10-20%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 70-80% ethanol;
Protein liquid removal: be made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 10-30mmol/L Tutofusin tris (Tris), pH=6.0-7.0, adds ethanol before using, makes ethanol mass concentration in protein liquid removal be 30-40%;
Rinsing liquid: be made up of final concentration 20-50mmol/L NaCl and final concentration 2-5mmol/L Tutofusin tris (Tris), pH=7.0-8.0, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Elutriant: 10-20mmol/L Tutofusin tris (Tris), pH=8-9.
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Final concentration of the present invention refers to the final concentration of solution in working fluid, such as working fluid A: be made up of final concentration 0.25-0.37mol/L potassium primary phosphate and final concentration 0.175-0.2mol/L sodium hydroxide, pH=7-8; Refer to that the concentration of potassium primary phosphate in working fluid A is 0.25-0.37mol/L, the concentration of sodium hydroxide in working fluid A is the pH=7-8 of 0.175-0.2mol/L, working fluid A.
Further, described protein liquid removal is made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20mmol/L Tutofusin tris, and pH=6.6, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 38%.
Further, described rinsing liquid is made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tutofusin tris, and pH=7.5, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 85%.
The extracting method of described test kit, comprises the following steps:
1) pre-treatment of ight soil: take 0.3-0.5g fresh excreta in 2ml centrifuge tube, add 1-2ml working fluid A vibration 1-2min, the centrifugal 10-20min of 3000-5000rpm/min, transfer supernatant is in the centrifuge tube of sterilizing, the centrifugal 10-20min of 10000-12000rpm/min, abandon supernatant, retain precipitation;
2) cracking of microorganism cells in ight soil: add 450-600 μ l working fluid B and 50-60 μ l working fluid C in above-mentioned precipitation, after mixing, 37-40 DEG C of water-bath 1-2h, adds 65-70 μ l working fluid D and 65-70 μ l working fluid E, after mixing, 37-60 DEG C of water-bath 1-2h;
3) dissociating of DNA and extracting of foreign protein: add 600-800 μ l working fluid F in above-mentioned solution, after vibrator concussion 30-60s, the centrifugal 10-20min of 12000-13000rpm/min, a supernatant liquor is moved in the centrifuge tube of sterilizing, add and an isopyknic working fluid G of supernatant liquor, the centrifugal 10-20min of 12000-13000rpm/min, moves in the centrifuge tube of sterilizing by secondary supernatant liquor;
4) removal of DNA adsorption column adsorption of DNA and impurity: the precipitation agent adding secondary supernatant liquor two volumes in the centrifuge tube containing secondary supernatant liquor, after mixing, add in DNA adsorption column, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 500-600 μ l protein liquid removal is added, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 300-500 μ l rinsing liquid is added, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, and then add 500-600 μ l rinsing liquid, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
5) DNA wash-out: add 30-60 μ l elutriant in DNA adsorption column, the centrifugal 1-2min of 12000-13000rpm/min, gained liquid is fecal microorganism genomic dna; Follow-uply get 3-6 μ l aqueous dna, electrophoresis detection in 1-2% sepharose.
Further, described step 4) add rinsing liquid, centrifugal, after abandoning waste liquid, again by centrifugal for adsorption column 12000-13000rpm/min 1-2min, abandon waste liquid, then DNA adsorption column is left standstill and dry up.The object of recentrifuge is mainly in order to out centrifugal from adsorption column by liquid thoroughly.
The present invention adopts above technical scheme, comprise the cracking of microorganism cells in the pre-treatment of ight soil, ight soil, dissociating of DNA and extracting of foreign protein, the removal of DNA adsorption column adsorption of DNA and impurity, DNA wash-out five steps carries animal excrement microbe genome DNA.Conventional animal excrement microbe genome DNA mainly adopts dehydrated alcohol to precipitate genome, and the operating time is longer, needs more than 14h, and processing ease causes genome to degrade for a long time.Test kit of the present invention mainly utilizes Silicon moulds adsorption column to adsorb genomic dna, distinctive protein liquid removal and rinsing liquid is adopted to wash away the impurity such as albumen again, compared with the animal excrement microbe genome DNA extracting method of routine, the present invention only needs 4h just can extract animal excrement microbe genome DNA.Not only save a large amount of extraction time, simplify experimental procedure simultaneously, animal excrement microbe genome DNA can be extracted more effectively, fast, economically.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure of animal excrement microbe genome DNA in 1% sepharose utilizing test kit of the present invention to extract:
Swimming lane 1,2 is the animal excrement microbe genome DNAs utilizing test kit of the present invention to extract;
Swimming lane 3 is the DL4500marker of TAKARA company.
Embodiment
The test kit of rapid extraction animal excrement microbe genome DNA, described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.25-0.37mol/L potassium primary phosphate and final concentration 0.175-0.2mol/L sodium hydroxide, pH=7-8;
Working fluid B: by final concentration 10-20mmol/L Tutofusin tris (Tris) and final concentration
1-10mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) forms, pH=5.0-8.0;
Working fluid C:10-50mg/ml N,O-Diacetylmuramidase;
Working fluid D:20-50mg/ml Proteinase K;
The sodium lauryl sulphate (SDS) of working fluid E: mass concentration 10-20%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 70-80% ethanol;
Protein liquid removal: be made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 10-30mmol/L Tutofusin tris (Tris), pH=6.0-7.0, adds ethanol before using, makes ethanol mass concentration in protein liquid removal be 30-40%;
Rinsing liquid: be made up of final concentration 20-50mmol/L NaCl and final concentration 2-5mmol/L Tutofusin tris (Tris), pH=7.0-8.0, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Elutriant: 10-20mmol/L Tutofusin tris (Tris), pH=8-9;
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
The extracting method of described test kit, comprises the following steps:
1) pre-treatment of ight soil: take 0.3-0.5g fresh excreta in 2ml centrifuge tube, add 1-2ml working fluid A vibration 1-2min, the centrifugal 10-20min of 3000-5000rpm/min, transfer supernatant is in the centrifuge tube of sterilizing, the centrifugal 10-20min of 10000-12000rpm/min, abandon supernatant, retain precipitation;
2) cracking of microorganism cells in ight soil: add 450-600 μ l working fluid B and 50-60 μ l working fluid C in above-mentioned precipitation, after mixing, 37-40 DEG C of water-bath 1-2h, adds 65-70 μ l working fluid D and 65-70 μ l working fluid E, after mixing, 37-60 DEG C of water-bath 1-2h;
3) dissociating of DNA and extracting of foreign protein: add 600-800 μ l working fluid F in above-mentioned solution, after vibrator concussion 30-60s, the centrifugal 10-20min of 12000-13000rpm/min, a supernatant liquor is moved in the centrifuge tube of sterilizing, add and an isopyknic working fluid G of supernatant liquor, the centrifugal 10-20min of 12000-13000rpm/min, moves in the centrifuge tube of sterilizing by secondary supernatant liquor;
4) removal of DNA adsorption column adsorption of DNA and impurity: the precipitation agent adding secondary supernatant liquor two volumes in the centrifuge tube containing secondary supernatant liquor, after mixing, add in DNA adsorption column, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 500-600 μ l protein liquid removal is added, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 300-500 μ l rinsing liquid is added, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, and then add 500-600 μ l rinsing liquid, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, and then the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
5) DNA wash-out: add 30-60 μ l elutriant in DNA adsorption column, the centrifugal 1-2min of 12000-13000rpm/min, gained liquid is fecal microorganism genomic dna; Follow-uply get 3-6 μ l aqueous dna, electrophoresis detection in 1-2% sepharose.
Embodiment 1
The test kit of rapid extraction animal excrement microbe genome DNA, described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.25mol/L potassium primary phosphate and final concentration 0.175mol/L sodium hydroxide, pH=7.2;
Working fluid B: be made up of final concentration 10mmol/L Tutofusin tris (Tris) and final concentration 1mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), pH=8.0;
Working fluid C:10mg/ml N,O-Diacetylmuramidase;
Working fluid D:20mg/ml Proteinase K;
Working fluid E: the sodium lauryl sulphate (SDS) of mass concentration 10%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 70% ethanol;
Protein liquid removal: be made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20mmol/L Tutofusin tris (Tris), pH=6.6, adds ethanol before using, makes ethanol mass concentration in protein liquid removal be 38%;
Rinsing liquid: be made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tutofusin tris (Tris), pH=7.5, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 85%;
Elutriant: 10mmol/L Tutofusin tris (Tris), pH=8.5;
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Embodiment 2
The test kit of rapid extraction animal excrement microbe genome DNA, described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.30mol/L potassium primary phosphate and final concentration 0.185mol/L sodium hydroxide, pH=7;
Working fluid B: be made up of final concentration 15mmol/L Tutofusin tris (Tris) and final concentration 5mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), pH=6.5;
Working fluid C:30mg/ml N,O-Diacetylmuramidase;
Working fluid D:35mg/ml Proteinase K;
The sodium lauryl sulphate (SDS) of working fluid E: mass concentration 10-20%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 75% ethanol;
Protein liquid removal: be made up of final concentration 3mol/L Guanidinium hydrochloride and final concentration 10mmol/L Tutofusin tris (Tris), pH=6.0, adds ethanol before using, makes ethanol mass concentration in protein liquid removal be 30%;
Rinsing liquid: be made up of final concentration 35mmol/L NaCl and final concentration 3mmol/L Tutofusin tris (Tris), pH=7.0, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80%;
Elutriant: 15mmol/L Tutofusin tris (Tris), pH=8;
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Embodiment 3
The test kit of rapid extraction animal excrement microbe genome DNA, described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.37mol/L potassium primary phosphate and final concentration 0.2mol/L sodium hydroxide, pH=8;
Working fluid B: be made up of final concentration 20mmol/L Tutofusin tris (Tris) and final concentration 10mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), pH=5.0;
Working fluid C:50mg/ml N,O-Diacetylmuramidase;
Working fluid D:50mg/ml Proteinase K;
The sodium lauryl sulphate (SDS) of working fluid E: mass concentration 10-20%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 80% ethanol;
Protein liquid removal: be made up of final concentration 6mol/L Guanidinium hydrochloride and final concentration 30mmol/L Tutofusin tris (Tris), pH=7.0, adds ethanol before using, makes ethanol mass concentration in protein liquid removal be 40%;
Rinsing liquid: be made up of final concentration 50mmol/L NaCl and final concentration 5mmol/L Tutofusin tris (Tris), pH=8.0, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 90%;
Elutriant: 20mmol/L Tutofusin tris (Tris), pH=9;
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Embodiment 4
Utilize the test kit of embodiment 1, extract animal excrement microbe genome DNA, specifically comprise the following steps:
1) pre-treatment of ight soil: take the fresh dog ight soil of two pipe 0.3g respectively in 2ml centrifuge tube, adds the centrifugal 10min of 1ml working fluid A vibration 1min, 3000rpm/min, transfer supernatant is in the centrifuge tube of sterilizing, the centrifugal 10min of 10000rpm/min, abandons supernatant, retains precipitation;
2) cracking of microorganism cells in ight soil: add 450 μ l working fluid B and 50 μ l working fluid C in above-mentioned precipitation, after mixing, 37 DEG C of water-bath 1h, add 65 μ l working fluid D and 65 μ l working fluid E, after mixing, 37 DEG C of water-bath 2h;
3) dissociating of DNA and extracting of foreign protein: add 600 μ l working fluid F in above-mentioned solution, after vibrator concussion 60s, the centrifugal 10min of 13000rpm/min, a supernatant liquor is moved in the centrifuge tube of sterilizing, add and an isopyknic working fluid G of supernatant liquor, the centrifugal 10min of 13000rpm/min, moves in the centrifuge tube of sterilizing by secondary supernatant liquor;
4) removal of DNA adsorption column adsorption of DNA and impurity: the precipitation agent adding secondary supernatant liquor two volumes in the centrifuge tube containing secondary supernatant liquor, after mixing, add in DNA adsorption column, the centrifugal 2min of 13000rpm/min, abandon waste liquid, then 500 μ l protein liquid removals are added, the centrifugal 1min of 13000rpm/min, abandon waste liquid, then 500 μ l rinsing liquids are added, the centrifugal 2min of 13000rpm/min, abandon waste liquid, and then add 500 μ l rinsing liquids, 13000rpm/min 2min, abandon waste liquid, and then the centrifugal 2min of 13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
5) DNA wash-out: add 30 μ l elutriants in DNA adsorption column, the centrifugal 2min of 13000rpm/min, gained liquid is fecal microorganism genomic dna; Follow-uply get 3-6 μ l aqueous dna, electrophoresis detection in 1-2% sepharose.
Embodiment 5
The test kit of extraction animal excrement microbe genome DNA of the present invention, its extracting method comprises the following steps:
1) pre-treatment of ight soil: take 0.5g fresh excreta in 2ml centrifuge tube, add the centrifugal 20min of 2ml working fluid A vibration 2min, 5000rpm/min, transfer supernatant is in the centrifuge tube of sterilizing, and the centrifugal 20min of 12000rpm/min, abandons supernatant, retains precipitation;
2) cracking of microorganism cells in ight soil: add 600 μ l working fluid B and 60 μ l working fluid C in above-mentioned precipitation, after mixing, 40 DEG C of water-bath 2h, add 70 μ l working fluid D and 70 μ l working fluid E, after mixing, 60 DEG C of water-bath 1h;
3) dissociating of DNA and extracting of foreign protein: add 800 μ l working fluid F in above-mentioned solution, after vibrator concussion 30s, the centrifugal 20min of 12000rpm/min, a supernatant liquor is moved in the centrifuge tube of sterilizing, add and an isopyknic working fluid G of supernatant liquor, the centrifugal 20min of 12000rpm/min, moves in the centrifuge tube of sterilizing by secondary supernatant liquor;
4) removal of DNA adsorption column adsorption of DNA and impurity: the precipitation agent adding secondary supernatant liquor two volumes in the centrifuge tube containing secondary supernatant liquor, after mixing, add in DNA adsorption column, the centrifugal 1min of 10000rpm/min, abandon waste liquid, then 600 μ l protein liquid removals are added, the centrifugal 2min of 10000rpm/min, abandon waste liquid, then 300 μ l rinsing liquids are added, the centrifugal 2min of 12000rpm/min, abandon waste liquid, and then add 600 μ l rinsing liquids, the centrifugal 1min of 12000rpm/min, abandon waste liquid, and then the centrifugal 1min of 12000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
5) DNA wash-out: add 60 μ l elutriants in DNA adsorption column, the centrifugal 1min of 12000rpm/min, gained liquid is fecal microorganism genomic dna; Follow-uply get 3-6 μ l aqueous dna, electrophoresis detection in 1-2% sepharose.
Utilize the test kit of embodiment 1, the electrophoresis detection of animal excrement microbe genome DNA in 1% sepharose extracted according to the extracting method of embodiment 4 the results are shown in Figure 1, wherein swimming lane 1,2 is all the animal excrement microbe genome DNAs utilizing test kit of the present invention to extract, and swimming lane 3 is the DL4500marker of TAKARA company.As can be seen from Figure 1 present method can extract animal excrement microbe genome DNA comparatively rapidly.
The animal excrement microbe genome DNA extracted, its purity OD
260nm/ OD
280nmratio is weighed, and purity is in table 1.
The purity of table 1 animal excrement microbe genome DNA
| Swimming lane 1 | Swimming lane 2 | |
| OD ratio (260:280) | 1.79 | 1.80 |
As can be seen from Table 1, OD (260:280) scope of DNA is at 1.79-1.80.Therefrom can find out, utilize test kit of the present invention can extract the high animal excrement microbe genome DNA of purity rapidly.
Claims (5)
1. the test kit of rapid extraction animal excrement microbe genome DNA, is characterized in that: described test kit is made up of DNA adsorption column and following working fluid:
Working fluid A: be made up of final concentration 0.25-0.37 mol/L potassium primary phosphate and final concentration 0.175-0.2 mol/L sodium hydroxide, pH=7-8;
Working fluid B: be made up of final concentration 10-20mmol/L Tutofusin tris and final concentration 1-10mmol/L ethylenediamine tetraacetic acid (EDTA), pH=5.0-8.0;
Working fluid C:10-50mg/ml N,O-Diacetylmuramidase;
Working fluid D:20-50mg/ml Proteinase K;
The sodium lauryl sulphate of working fluid E: mass concentration 10-20%;
Working fluid F: volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Working fluid G: volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Precipitation agent: mass concentration 70-80% ethanol;
Protein liquid removal: be made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 10-30 mmol/L Tutofusin tris, pH=6.0-7.0, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 30-40%;
Rinsing liquid: be made up of final concentration 20-50mmol/L NaCl and final concentration 2-5mmol/L Tutofusin tris, pH=7.0-8.0, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Elutriant: 10-20mmol/L Tutofusin tris, pH=8-9.
2. the test kit of rapid extraction animal excrement microbe genome DNA according to claim 1, it is characterized in that: described protein liquid removal is made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20 mmol/L Tutofusin tris, pH=6.6, add ethanol before using, make ethanol mass concentration in protein liquid removal be 38%.
3. the test kit of rapid extraction animal excrement microbe genome DNA according to claim 1, it is characterized in that: described rinsing liquid is made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tutofusin tris, pH=7.5, add ethanol after having adjusted pH, make the ethanol mass concentration in rinsing liquid be 85%.
4. the extracting method of test kit as described in one of claims 1 to 3, is characterized in that: it comprises the following steps:
1) pre-treatment of ight soil: take ight soil in centrifuge tube, 1-2ml working fluid A vibration 1-2min is added in every 0.3-0.5g ight soil, the centrifugal 10-20min of 3000-5000rpm/min, transfer supernatant is in the centrifuge tube of sterilizing, the centrifugal 10-20min of 10000-12000rpm/min, abandon supernatant, retain precipitation;
2) cracking of microorganism cells in ight soil: add 450-600 μ l working fluid B and 50-60 μ l working fluid C in above-mentioned precipitation, after mixing, 37-40 ° of C water-bath 1-2h, adds 65-70 μ l working fluid D and 65-70 μ l working fluid E, after mixing, 37-60 ° of C water-bath 1-2h;
3) dissociating of DNA and extracting of foreign protein: add 600-800 μ l working fluid F in above-mentioned solution, after vibrator concussion 30-60s, the centrifugal 10-20min of 12000-13000rpm/min, a supernatant liquor is moved in the centrifuge tube of sterilizing, add and an isopyknic working fluid G of supernatant liquor, the centrifugal 10-20min of 12000-13000rpm/min, moves in the centrifuge tube of sterilizing by secondary supernatant liquor;
4) removal of DNA adsorption column adsorption of DNA and impurity: the precipitation agent adding secondary supernatant liquor two volumes in the centrifuge tube containing secondary supernatant liquor, after mixing, add in DNA adsorption column, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 500-600 μ l protein liquid removal is added, the centrifugal 1-2min of 10000-13000rpm/min, abandon waste liquid, then 300-500 μ l rinsing liquid is added, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, and then add 500-600 μ l rinsing liquid, the centrifugal 1-2min of 12000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
5) DNA wash-out: add 30-60 μ l elutriant in DNA adsorption column, the centrifugal 1-2min of 12000-13000rpm/min, gained liquid is fecal microorganism genomic dna.
5. the extracting method of test kit according to claim 4, is characterized in that: described step 4) adds rinsing liquid, centrifugal, after abandoning waste liquid, again by the centrifugal 1-2min of DNA adsorption column 12000-13000rpm/min, abandon waste liquid, then DNA adsorption column is left standstill and dry up.
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| CN108410858A (en) * | 2018-04-27 | 2018-08-17 | 四川大学 | A kind of application of the extracting method of waste phage DNA, the kit and the kit of extraction waste phage DNA |
| CN109486809A (en) * | 2018-12-29 | 2019-03-19 | 厦门艾德生物医药科技股份有限公司 | A kind of method of a large amount of extraction conversion faeces DNAs |
| CN109486809B (en) * | 2018-12-29 | 2020-08-21 | 厦门艾德生物医药科技股份有限公司 | Method for extracting and transforming DNA of excrement in large quantity |
| 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 |
| CN110592076B (en) * | 2019-10-25 | 2022-03-08 | 电子科技大学中山学院 | Reagent and method for extracting microbial community DNA of fermented bean curd |
| CN110819624A (en) * | 2019-11-13 | 2020-02-21 | 湖南融健基因生物科技有限公司 | Lysate and method for rapidly extracting bacterial DNA from excrement |
| CN112501157A (en) * | 2020-11-30 | 2021-03-16 | 广东军融科创科技有限公司 | Kit for extracting DNA from human excrement sample, extraction method and application |
| CN113186250A (en) * | 2021-05-13 | 2021-07-30 | 厦门承葛医学检验实验室有限公司 | Impurity removing reagent for extracting microbial genome DNA of excrement |
| CN113186250B (en) * | 2021-05-13 | 2022-09-13 | 厦门承葛医学检验实验室有限公司 | Impurity removal reagent for extracting microbial genome DNA of excrement |
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