CN110016475A - Faeces DNA extracts kit - Google Patents
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- CN110016475A CN110016475A CN201910388938.8A CN201910388938A CN110016475A CN 110016475 A CN110016475 A CN 110016475A CN 201910388938 A CN201910388938 A CN 201910388938A CN 110016475 A CN110016475 A CN 110016475A
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- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
It is extracted the present invention relates to medical detection field more particularly to a kind of faeces DNA and saves liquid and preservation pipe, kit and extracting method.A kind of faeces DNA extraction preservation liquid, which includes 0.20 ~ 0.30M EDTA, 0.2 ~ 0.8% alanine, 20 ~ 30% ethyl alcohol, 0.2 ~ 0.8% lauryl sodium sulfate, 1.0 ~ 5.0% polyvinylpyrrolidones, pH 10.0 ~ 11.0；Above-mentioned percentage is mass percent.The preservation liquid is under conditions of high EDTA/ is alkaline; using alcohol as fungicide; buffer system of the alanine as high alkalinity environment is used simultaneously; and detergent and detergent are used as using lauryl sodium sulfate and polyvinylpyrrolidone collaboration; the excrement holding time can be extended; protection is instantly available after excrement is in vitro, it is ensured that the integrality of gene.
Description
Technical field
The present invention relates to medical detection field more particularly to a kind of faeces DNA extract save liquid and save pipe, kit and
Extracting method.
Background technique
Colorectal cancer is the most common malignant tumor of digestive tract, and incidence is only second to gastric cancer and cancer of the esophagus.In the latest 20 years
The disease incidence of colorectal cancer is gradually increasing, meanwhile, age of onset tends to aging.In western developed country, colorectal cancer
It is the second malignant neoplasm for being only second to lung cancer.In recent years some research shows that some genes such as K-ras, P53, APC, MMR
Mutation will lead to normal colonic mucosa epithelial cell be converted to tumour cell (Vogelstein, B.and Kinzler, K.W.,
1993), to the progress of these gene mutations, so that becoming a kind of possibility by faeces DNA checkout and diagnosis colorectal cancer.And
Human gene group DNA it can become the key factor for influencing diagnostic accuracy in high efficiency extraction excrement.
Excrement complicated component, containing a large amount of polysaccharide, cholic acid etc. to human gene group DNA detection can produce inhibiting effect at
Point, and the faeces DNA overwhelming majority derives from enteric bacteria, source of people DNA content is seldom.So the fecal specimens that acquisition comes need as early as possible
It saves, currently used store method is usually low-temperature treatment, i.e. freezen protective.Freezing method is complicated for operation, at high cost, can not
Rapid and convenient collecting sample.
In addition, RNA isolation kit is that current clinical application is extensive and the simplest method, the faeces DNA of commercialization extract reagent
Box is many kinds of, and performance is totally different, but excrement human gene group DNA extraction efficiency is different.In addition, since clinical fecal sample amount is big,
DNA extraction can not be usually carried out after acquisition in time to be stored, a large amount of nucleases contained in excrement can cause faeces DNA sternly
It degrades again, therefore, influence of the storage time to human gene group DNA's extraction efficiency is very big.
As Chinese invention patent (publication number CN106967712A, publication date: 2017.07.21) faeces DNA rapidly extracting is tried
Agent box, the extraction reagent principle are as follows: pre-treatment is carried out with night soil-treatment liquid to extracted excrement, be centrifuged off excrement residue and
The impurity such as part albumen are removed in absorption, are released nucleic acid cleavage with magnetic bead lysate and Proteinase K, the nucleic acid released
Specificity is integrated on the magnetic bead for being mixed with specific probe, after the washing that cleaning solution 2 is taken turns by 1 wheel cleaning solution 1 and 2, is used
Nucleic acid is eluted from magnetic bead and the magnetic bead after elution is discarded by eluent, finally obtains excrement genome.The patent is also
Be focus in how rapidly extracting, for how effectively to save DNA, then no longer limit of consideration.
Also the technology saved about faeces DNA, such as Chinese invention patent (CN108866045A, publication date
2018.11.23 a kind of human feces DNA extraction method) is disclosed, this method acquires 2-5g excrement after excrement is discharged in people immediately
Just sample is put into collection tube, is saved in collection tube equipped with 10-15mL excrement, and the excrement keeps liquid ingredient: 0.3-0.6M
EDTA, 3-6M NaCl, 0.3-1.5M Tris.Chinese invention patent (publication number CN107980763A publication date 2018.05.04)
It discloses excrement and saves reagent, including Cell Buffer, cell fixative, metal ion chelation agent and preservative；Cell buffering
Liquid is Tris-HCl, and work pH range is 8-10；Cell fixative in SDS, Triton X-100 and PEG-2000 extremely
Few one kind；Metal ion chelation agent is EDTA and/or EDTA-Na2；Preservative is NaCl.The two technologies are all using Tris-
HCl buffer system, NaCl is as preservative, and DNA is still easy degradation in practical applications, to human gene group DNA's extraction efficiency
Influence it is very big.
Summary of the invention
It is extracted it is an object of the present invention to provide a kind of faeces DNA and saves liquid, item of the preservation liquid in high EDTA/ alkalinity
Under part, using alcohol as fungicide, while the buffer system using alanine as high alkalinity environment, and use dodecyl
Sodium sulphate and polyvinylpyrrolidone collaboration are used as detergent and detergent, can extend the excrement holding time, excrement is stood afterwards in vitro
It is protected, it is ensured that the integrality of gene.
In order to achieve the above purpose, a kind of faeces DNA of the invention, which extracts, saves liquid, and which includes 0.20~
0.30M EDTA, 0.2~0.8% alanine, 20~30% ethyl alcohol, 0.2~0.8% lauryl sodium sulfate, 1.0~5.0%
Polyvinylpyrrolidone, pH10.0~11.0；Above-mentioned percentage is mass percent.
The present invention saves the function of each component in liquid: 1.EDTA: as the inhibitor of nuclease and as stabilizer.2.
Alanine: the buffer system of high alkalinity environment is provided.3. alcohol: there is stronger bactericidal effect, fixed sample material can be played.
4.SDS: detergent, anionic surfactant, detergent.5.PVP: detergent.Each component of the invention simultaneously has
Synergistic effect, can extend the excrement holding time, make to be instantly available protection after excrement is in vitro, it is ensured that the integrality of gene.
As a specific embodiment, which includes 0.25M EDTA, 0.5% alanine, 25% ethyl alcohol,
0.5% lauryl sodium sulfate, 2.0% polyvinylpyrrolidone, pH 10.5；Above-mentioned percentage is mass percent.
Another object of the present invention is to provide a kind of faeces DNA extraction preservation pipe, and the preservation pipe is containing the preservation
Liquid.
Another object of the present invention is to provide a kind of faeces DNA extracts kit, which includes that excrement saves
Liquid, lysate, combination buffer, cleaning solution and eluent, the excrement save liquid and use the preservation liquid.
As A scheme, the cracking combination liquid contains: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M
GuSCN, 1.0% lauryl sodium sulfate, pH 8.9；Combination buffer is the isopropanol that mass percent concentration is 80%；Institute
The cleaning solution stated includes cleaning solution I and cleaning solution II, and cleaning solution I contains: 3M GuHCl, 10mM Tris, 50% ethyl alcohol, pH
8.0；Cleaning solution II includes: 10mM Tris, 80% ethyl alcohol, pH 7.5；Eluent contains: 10mM Tris, 0.1mM EDTA, pH
9.0。
As B scheme, the cracking combination liquid contains: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M
GuSCN, 1.0% dodecyl-N- glycine betaine, pH 8.9；Combination buffer is the isopropanol that mass percent concentration is 80%；
The cleaning solution contains: 3M GuHCl, 10mM Tris, 50% ethyl alcohol, sodium bicarbonate 35mM, pH 9.0；Eluent contains:
10mM Tris, 0.1mM EDTA, pH 9.0.Currently, paramagnetic particle method extracts the washing for being intended to not wait in DNA by 2-4 times, not only
Elapsed time, and more fine operation is needed when absorption supernatant after washing, it is easy to produce existing outside pollution/unsuitable laboratory
Operation) and extraction effect on also have much places to be modified, washing times, it is found by the applicant that being difficult to reduce to washing times
Reason essentially consists in the weaker alkalinity of cleaning solution and passes through to the washability deficiency and elution efficiency problem, the application of mucoprotein
Prepare the sodium bicarbonate (alkalinity be slightly better than sodium acetate and sodium chloride) containing higher concentration cleaning solution combine ionic equilibrium and
The effect of alkalinity is provided, and is used cooperatively amphoteric ion Orvus Gardinol dodecyl-N- glycine betaine, step washing may be implemented
(patent of applicant: application number: 2019102093130, carry out detailed record).
Another object of the present invention is to provide the method that the kit extracts faeces DNA.
As a specific embodiment, the kit of this method A scheme, this method includes the following steps:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) centrifuge tube 12000rpm is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, be vortexed concussion
For suspension；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by centrifuge tube 12000rpm centrifuging and taking supernatant, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) cleaning solution I is added to wash twice；
8) cleaning solution II is added to wash twice；
9) elution is added, is shaken 5 minutes at a temperature of 60 DEG C, centrifuge tube is then placed on magnetic separation frame 2 points
Clock is moved to supernatant in another clean centrifuge tube with pipette tips；Obtained DNA is recycled, is stored in -20 DEG C.
As a specific embodiment, the kit of this method B scheme includes the following steps:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) centrifuge tube 12000rpm is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, be vortexed concussion
For suspension；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by centrifuge tube 12000rpm centrifuging and taking supernatant, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) centrifuge tube is taken out from magnetic separation frame, 1000ul cleaning solution is added, be vortexed concussion centrifuge tube 5-10 times, makes
Magnetic bead mixes well, and is then placed in centrifuge tube on magnetic separation frame 2 minutes, so that magnetic bead is adsorbed completely in managing, carefully
Whole supernatants are removed in the case where not touching precipitating using liquid-transfering gun；
8) elution is added, is shaken 5 minutes at a temperature of 60 DEG C, centrifuge tube is then placed on magnetic separation frame 2 points
Clock is moved to supernatant in another clean centrifuge tube with pipette tips；Obtained DNA is recycled, is stored in -20 DEG C.
Another object of the present invention is to provide the preservation liquid or the preservation pipe or faeces DNA extracts reagent
Application of the box in gene screening and forensic identification.
The present invention due to the adoption of the above technical solution, has the characteristics that following:
(1) the preservation liquid of high EDTA/ alkalinity can extend the excrement holding time, and protection is instantly available after excrement is in vitro, it is ensured that
The integrality of gene；
(2) lysate lytic effect fast and stable can release sample nucleic acid under room temperature, while to releasing
DNA is combined, and a step completes cracking and combines two big functions；It is time saving and energy saving, it improves efficiency, convenient for operating the computer；
(3) be not only suitable for manual extraction, also can mating instrument for extracting nucleic acid uses in an automated manner in microwell plate, mention
Purity is high and easy to operate and safe is taken, effectively avoids degradable in excrement extraction process, extracts ropy problem；
(4) high concentration, purity: combining with magnetic bead, and the DNA concentration of extraction, purity are very high, can be directly used for downstream reality
It tests；
(5) kit is with good stability；
(6) reagent is without the toxic solvents such as phenol, chloroform, safe operation environmental protection.
Detailed description of the invention
Fig. 1 is that the fecal sample of 1 method of embodiment, five people extracts the electrophoresis picture of DNA.
Fig. 2 is the DNA concentration figure that 1 method of embodiment, five fecal samples extract.
Fig. 3 is the electrophoresis picture of the sample DNA of embodiment 1 method excrement room temperature 1d, 3d, 7d, 14d.
Fig. 4 is the DNA concentration figure that 1 side of embodiment extracts the different holding times.
Fig. 5 is that the fecal sample of 2 method of embodiment, five people extracts the electrophoresis picture of DNA.
Fig. 6 is the electrophoresis picture of the sample DNA of embodiment 2 method excrement room temperature 1d, 3d, 7d, 14d.
Fig. 7 is 1 method part amplified production electrophoresis picture of embodiment.
Specific embodiment
Main agents and instrument
Tris-HCl, Tris, EDTA, Alanine, NaCl, SDS, PVP and GuHCl are provided by sigma；
Ethyl alcohol, isopropanol are produced by Chinese medicines group；
Magnetic bead used (is derived from Magattract DNA Kit, enriching by Qiagen production in the application detection method
Magnetic bead in kit is included for kit).
Detect samples sources
The fecal sample source used when verifying the application kit DNA extraction effect is coming from cooperative medical service testing agency just
The sample often acquired.
Embodiment 1
One, it is formulated:
1, excrement preservation liquid: 0.25M EDTA, 0.5% alanine, 25% ethyl alcohol, 0.5% lauryl sodium sulfate, 2%
PVP；pH10.5；
2, lysate: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1% lauryl sodium sulfate,
20% isopropanol；pH 8.9；
3, wash I:3M GuHCl, 10mM Tris, 50% ethyl alcohol；pH 8.0；
4, wash II:10mM Tris, 80% ethyl alcohol；pH 7.5；
5, elution:10mM Tris, 0.1mM EDTA；pH 9.0.
Two, the method for taking faeces DNA, this method include the following steps:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) centrifuge tube 12000rpm is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, be vortexed concussion
For suspension；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by centrifuge tube 12000rpm centrifuging and taking supernatant, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) cleaning solution I is added to wash twice；
8) cleaning solution II is added to wash twice；
9) elution is added, is shaken 5 minutes at a temperature of 60 DEG C, centrifuge tube is then placed on magnetic separation frame 2 points
Clock is moved to supernatant in another clean centrifuge tube with pipette tips；Obtained DNA is recycled, is stored in -20 DEG C.
Embodiment 2
One, it is formulated:
1, excrement preservation liquid: 0.25M EDTA, 0.5% alanine, 25% ethyl alcohol, 0.5% lauryl sodium sulfate, 2%
PVP；pH10.5；
2, lysate: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1% lauryl sodium sulfate,
20% isopropanol；pH 8.9；
3, wash:3M GuHCl, 10mM Tris, 50% ethyl alcohol, sodium bicarbonate 35mM, pH 9.0；
4, elution:10mM Tris, 0.1mM EDTA；pH 9.0.
Two, the method for taking faeces DNA, this method include the following steps:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) centrifuge tube 12000rpm is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, be vortexed concussion
For suspension；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by centrifuge tube 12000rpm centrifuging and taking supernatant, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) centrifuge tube is taken out from magnetic separation frame, 1000ul cleaning solution is added, be vortexed concussion centrifuge tube 5-10 times, makes
Magnetic bead mixes well, and is then placed in centrifuge tube on magnetic separation frame 2 minutes, so that magnetic bead is adsorbed completely in managing, carefully
Whole supernatants are removed in the case where not touching precipitating using liquid-transfering gun；
8) elution is added, is shaken 5 minutes at a temperature of 60 DEG C, centrifuge tube is then placed on magnetic separation frame 2 points
Clock is moved to supernatant in another clean centrifuge tube with pipette tips；Obtained DNA is recycled, is stored in -20 DEG C.
Test example
1, DNA solution concentration and purity testing
Take DNA2ul to be measured that cuvette is added, mixed well after adding distilled water to 100ul, through UV-7504 is ultraviolet can
Light-exposed photometer detects OD260, OD280 and OD260 of DNA and the ratio of OD280.
DNA concentration calculation formula: DNA concentration=OD260 × extension rate × 50/1000 (ug/u1)；
DNA purity: OD260/OD280 ratio is 1.8-2.0, it is believed that DNA purity is relatively good；OD260/OD280 ratio is bright
It is aobvious to be greater than 2.0, it is believed that may to have RNA pollution or DNA fragmentation fracture in DNA；OD260/OD280 ratio is significantly lower than 1.8, it is believed that
There may be phenol when protein or remaining extracting in DNA.
2,2% agarose gel electrophoresis judges the concentration and integrality of extracted DNA
The Loading buffer (total volume 6u1) for the DNA+lul for taking 5ul to extract, is added in 2% Ago-Gel,
Electrophoresis 30min, observes the purity and estimated concentration of band in KH-UVI type ultraviolet transmission analyzer, and shoots photo, makees to remember
Record.
3. the confirmation of human genome DNA
Because human genome DNA only accounts for 1% or so of DNA total amount in excrement, experiment flow provided by embodiment 1 is only
It is to improve human genome as far as possible to say the ratio in the DNA extracted, but non-human DNA cannot be excluded completely.General expansion
Increase the distinctive gene of the mankind to judge, according to the literature, this experiment β-actin gene.
1) β-actin gene magnification:
Human genome β-actin gene amplification reaction liquid is prepared and reaction condition setting see the table below 1, table 2, primer, annealing
Temperature and product are shown in Table 3.
1 β-actin gene amplification reaction liquid of table composition
2 β-actin gene amplification reaction condition of table
Primer, annealing temperature and the primer size of 3 PCR of table reaction
2) detection of PCR amplification result:
Amplified production observes amplified band through 2% agarose gel electrophoresis in KH-UVI type ultraviolet transmission analyzer
Purity and estimated concentration, and photo is shot, it notes down.
4, result
1) the extraction result of faeces DNA
It extracts faeces DNA and presses 1 step operation of embodiment, products therefrom uses OD under spectrophotometer measurement 260rim wavelength
Value, then it is converted into dsDNA concentration.Fecal sample 100 are extracted altogether, and the extracted amount of DNA fluctuation of 200mg excrement is in 20-
105ug, average 56ug.The OD260/OD280 ratio of DNA is 8 less than 18, and 1.8~2.0 M is 90, and being greater than 2.0 is 2
Example.
Five groups are taken, uses 2% agarose gel electrophoresis to gained faeces DNA is extracted, electrophoresis picture is shown in Fig. 1, and DNA concentration is such as
Shown in Fig. 2.Wherein the sample of one group of excrement room temperature 1d, 3d, 7d, 14d use 2% agarose gel electrophoresis, electrophoresis picture
See that Fig. 3, DNA concentration are as shown in Figure 4.
It extracts faeces DNA and presses 2 step operation of embodiment, products therefrom uses OD under spectrophotometer measurement 260rim wavelength
Value, then it is converted into dsDNA concentration.Fecal sample 100 are extracted altogether, the extracted amount of DNA of 200mg excrement is fluctuated in 30-85ug,
Average 45ug.The OD260/OD280 ratio of DNA is 11 less than 18, and 1.8~20 M is 86, and being greater than 2.0 is 3.
Five groups are taken, uses 2% agarose gel electrophoresis to gained faeces DNA is extracted, electrophoresis picture is shown in Fig. 5.Wherein one group
The sample of excrement room temperature 1d, 3d, 7d, 14d use 2% agarose gel electrophoresis, and electrophoresis picture is shown in Fig. 6.
2) confirmation of human gene group DNA
Faeces DNA to 1 sample extraction of embodiment is the presence for confirming human genome, carries out PCR reaction amplification people's gene
β-actin segment, 15% agarose gel electrophoresis of amplified production carry out next step sulfurous acid oxonium salt after amplifying target fragment
Processing and MS-PCR reaction.100 faeces DNAs can all amplify-actin sections of people's gene β, part amplified production electrophoresis knot
Fruit sees Fig. 7.
The above are the descriptions to the embodiment of the present invention to keep this field special by the foregoing description of the disclosed embodiments
Industry technical staff can be realized or using the present invention.Various modifications to these embodiments carry out those skilled in the art
Saying will be apparent.The general principles defined herein can be the case where not departing from the spirit or scope of the present invention
Under, it realizes in other embodiments.Therefore, the present invention is not intended to be limited to these implementation columns shown in this article, but to accord with
Close the widest scope consistent with principles disclosed herein and novel point.
Claims (10)
1. a kind of faeces DNA, which extracts, saves liquid, which is characterized in that the preservation liquid includes 0.20 ~ 0.30M EDTA, and 0.2 ~ 0.8% the third
Propylhomoserin, 20 ~ 30% ethyl alcohol, 0.2 ~ 0.8% lauryl sodium sulfate, 1.0 ~ 5.0% polyvinylpyrrolidones, pH 10.0 ~ 11.0；
Above-mentioned percentage is mass percent.
2. preservation liquid according to claim 1, which is characterized in that the preservation liquid includes 0.25M EDTA, 0.5% alanine,
25% ethyl alcohol, 0.5% lauryl sodium sulfate, 2.0% polyvinylpyrrolidone, pH 10.5；Above-mentioned percentage is quality percentage
Than.
3. a kind of faeces DNA, which extracts, saves pipe, the preservation pipe is containing preservation liquid described in as claimed in claim 1 or 22.
4. a kind of faeces DNA extracts kit, the kit include excrement save liquid, lysate, combination buffer, cleaning solution and
Eluent, which is characterized in that the excrement saves liquid and uses preservation liquid of any of claims 1 or 2.
5. kit according to claim 3, which is characterized in that cracking contains in conjunction with liquid: 0.1M Tris-HCl, 15mM
EDTA, 1M NaCl, 3M GuSCN, 1.0% lauryl sodium sulfate, 20% isopropanol, pH 8.9；The cleaning solution includes washing
Liquid I and cleaning solution II are washed, cleaning solution I contains: 3 M GuHCl, 10mM Tris, 50% ethyl alcohol, pH 8.0；Cleaning solution II includes:
10mM Tris, 80% ethyl alcohol, pH 7.5；Eluent contains: 10mM Tris, 0.1 mM EDTA, pH 9.0.
6. kit according to claim 3, which is characterized in that cracking contains in conjunction with liquid: 0.1M Tris-HCl, 15mM
EDTA, 1M NaCl, 3M GuSCN, 1.0% dodecyl-N- glycine betaine, 20% isopropanol, pH 8.9；The cleaning solution contains
Have: 3 M GuHCl, 10mM Tris, 50% ethyl alcohol, sodium bicarbonate 35mM, pH 9.0；Eluent contains: 10mM Tris, and 0.1
MM EDTA, pH 9.0.
7. the method for the extraction faeces DNA of kit described in claim 4 or 5 or 6.
8. the method according to the description of claim 7 is characterized in that kit described in the claim 5 that this method uses, packet
Include following step:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) 12000 rpm of centrifuge tube is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, the concussion that is vortexed is outstanding
Supernatant liquid；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by 12000 rpm centrifuging and taking supernatant of centrifuge tube, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) cleaning solution I is added to wash twice；
8) cleaning solution II is added to wash twice；
9) elution is added, shakes 5 minutes at a temperature of 60 DEG C, is then placed in centrifuge tube on magnetic separation frame 2 minutes, uses
Pipette tips move to supernatant in another clean centrifuge tube；Obtained DNA is recycled, is stored in -20 DEG C.
9. according to the method described in claim 6, it is characterized in that, the kit as claimed in claim 6 that this method uses, packet
Include following step:
1) excrement containing 1ml saves middle addition 0.2g excrement in liquid centrifuge tube, and concussion mixes；
2) centrifuge tube is heated into 70 DEG C of vortexs and shakes 20min；
3) 12000 rpm of centrifuge tube is centrifuged 5min, abandons supernatant and stays precipitating；
4) lysate of 1ml is added in precipitating, 10ul Enhancer, 15ul Inhibitor Remover, the concussion that is vortexed is outstanding
Supernatant liquid；
5) centrifuge tube is heated into 55 DEG C of concussion 20min；
6) by 12000 rpm centrifuging and taking supernatant of centrifuge tube, 10ul magnetic bead, 65 DEG C of concussion 5min；
7) centrifuge tube is taken out from magnetic separation frame, 1000ul cleaning solution is added, be vortexed concussion centrifuge tube 5-10 times, makes magnetic
Pearl mixes well, and is then placed in centrifuge tube on magnetic separation frame 2 minutes, so that magnetic bead is adsorbed completely in managing, carefully makes
Whole supernatants are removed in the case where not touching precipitating with liquid-transfering gun；
8) elution is added, shakes 5 minutes at a temperature of 60 DEG C, is then placed in centrifuge tube on magnetic separation frame 2 minutes, uses
Pipette tips move to supernatant in another clean centrifuge tube；Obtained DNA is recycled, is stored in -20 DEG C.
10. preservation liquid according to claim 1 or 2 or preservation as claimed in claim 3 pipe or claim 4 or 5 or 6 institutes
Application of the faeces DNA extracts kit stated in gene screening and forensic identification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388938.8A CN110016475B (en) | 2019-05-10 | 2019-05-10 | Excrement DNA extraction kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388938.8A CN110016475B (en) | 2019-05-10 | 2019-05-10 | Excrement DNA extraction kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110016475A true CN110016475A (en) | 2019-07-16 |
| CN110016475B CN110016475B (en) | 2021-07-06 |
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| CN201910388938.8A Active CN110016475B (en) | 2019-05-10 | 2019-05-10 | Excrement DNA extraction kit |
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| CN115627297A (en) * | 2022-12-05 | 2023-01-20 | 北京吉检医疗科技有限公司 | Kit for detecting human excrement helicobacter pylori nucleic acid |
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