CN108949750A - Extract the method and kit of faeces DNA - Google Patents
Extract the method and kit of faeces DNA Download PDFInfo
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Abstract
Kit the invention proposes the method for extracting faeces DNA and for extracting faeces DNA.The described method includes: the coated magnetic bead of streptavidin is contacted with the probe of biotin modification, to obtain magnetic bead-probe complex；Excrement to be measured is pre-processed, to obtain pretreatment fluid；And contact the pretreatment fluid with the magnetic bead-probe complex, to extract faeces DNA.Extracting faeces DNA using method of the invention at least has at least one of following advantages: high specificity, easy to operate, faeces DNA yield is high, purity is high and is suitable for scale application.
Description
Technical field
The present invention relates to biological fields.In particular it relates to extract the method and kit of faeces DNA.
Background technique
The multiple target point faeces DNA detection of rising in recent years is formed because having many advantages, such as that non-invasive, convenient and simple, accuracy is high
For one of the research hotspot of clinical diagnosis, the basic principle is that normal adult can all have daily epithelial cell shedding to enteric cavity and with
Excrement excretes, and intestinal canal tumour cell is due to the change of biological behaviour, the abnormal apoptosis of cell, metabolism quickening etc.
Factor, it is more easy to fall off than normal epithelium cell.Abnormal DNA by collecting and detecting intestinal canal tumour cell in excrement can be realized
The early screening of cancer.
However, the method for extraction faeces DNA and kit still require study at present.
Summary of the invention
The present invention is directed to solve at least one the technical problems existing in the prior art at least to a certain extent.
It should be noted that the present invention is the following discovery based on inventor and completes:
Currently, host cell DNA content is less in fecal sample, and interfering substance type and content is more, such as various enzymes
Co-inhibitor (mucus, bacterium and swill etc.) and cholate etc. cause the amount of DNA extracted in excrement very low or extract
DNA mass out is lower to be difficult to use in PCR amplification.Moreover, most cumbersome, time-consuming, the nothing of existing faeces DNA extracting method
Method is widely applied.
In view of this, inventor using the coated magnetic bead combination biotin modification of streptavidin probe specificity catch
Target gene is obtained, while playing the purpose of purifying and enrichment DNA, improves DNA recovery rate and purity.As a result, according to this hair
The method of the extraction faeces DNA of bright embodiment at least has at least one of following advantages: high specificity, easy to operate, excrement
DNA yield is high, purity is high and is suitable for scale application.
For this purpose, in one aspect of the invention, the invention proposes a kind of methods for extracting faeces DNA.According to the present invention
Embodiment, which comprises the coated magnetic bead of streptavidin is contacted with the probe of biotin modification, to obtain magnetic
Pearl-probe complex；Excrement to be measured is pre-processed, to obtain pretreatment fluid；And by the pretreatment fluid with it is described
Magnetic bead-probe complex contact, to extract faeces DNA.Inventor utilizes the coated magnetic bead combination biotin of streptavidin
Target gene is captured to the probe specificity of modification, while playing the purpose of purifying and enrichment DNA, improves DNA recovery rate
And purity.The method according to an embodiment of the present invention for extracting faeces DNA, which extracts faeces DNA, as a result, at least has following advantages
At least one: high specificity, easy to operate, faeces DNA yield be high, purity is high and is suitable for scale application.
According to an embodiment of the invention, the method for extracting faeces DNA can also have following additional technical feature:
According to an embodiment of the invention, the coated magnetic bead of streptavidin is provided in the form of magnetic bead solution, institute
The probe for stating biotin modification is provided in the form of probe solution, and in the magnetic bead-probe complex, the magnetic bead concentration is
80nM~120nM, the concentration and probe concentration are 10nM~15nM.The method according to an embodiment of the present invention for extracting faeces DNA as a result,
Further specific, easier operation, higher faeces DNA yield or purity with higher.
According to an embodiment of the invention, when the coated magnetic bead of streptavidin is with the contact of the probe of biotin modification
Between be 1~2 hour.As a result, it is according to an embodiment of the present invention extract faeces DNA method further it is with higher specificity,
Easier operation, higher faeces DNA yield or purity.
According to an embodiment of the invention, magnetic bead-the probe complex obtains in the following manner: 1) taking containing chain
The mould coated magnetic bead solution of avidin abandons supernatant in standing on magnetic frame；2) buffering is added into the obtained magnetic bead of step 1)
Liquid cleans magnetic bead, mixes, and carries out magnetic-adsorption, abandons supernatant；3) buffer is added into the obtained magnetic bead of step 2), institute is resuspended
Magnetic bead is stated, the probe solution containing biotin modification, incubation at room temperature, to obtain the magnetic bead-probe complex is added.By
This, the method according to an embodiment of the present invention for extracting faeces DNA further specific, easier operation with higher, compared with
High faeces DNA yield or purity.
Preferred embodiment in accordance with the present invention, the magnetic bead-probe complex obtain in the following manner: 1) taking 15
The coated magnetic bead solution of μ L streptavidin abandons supernatant in standing 4min on magnetic frame；2) into the obtained magnetic bead of step 1)
1mL Binding Buffer is added and cleans magnetic bead, concussion abandons supernatant in standing on magnetic frame, this step of repetition is primary,
In, the Binding Buffer includes: 5mmol/L Tris-Hcl, 0.5mmol/L EDTA and 1mol/L NaCL；3) to step
200 μ L Binding Buffer are added in rapid 2) obtained magnetic bead, magnetic bead is resuspended, the probe that 10 μ L biotin modifications are added is molten
Liquid is incubated at room temperature 1h；4) 1mLBinding Buffer is added into the resulting mixed liquor of step 3), it is soft to shake, in magnetic frame
Upper standing abandons supernatant, this step of repetition is primary, washes away unbonded probe, adds 200 μ L Binding Buffer that magnetic bead-is resuspended
Probe complex, wherein the magnetic bead concentration is 98nM~119nM, and the concentration and probe concentration is 14.5nM.As a result, according to this hair
Method further specific, the easier operation with higher, higher faeces DNA of the extraction faeces DNA of bright embodiment
Yield or purity.
It is saved in liquid according to an embodiment of the invention, the excrement to be measured is pre-stored in, contains second in the preservation liquid
Edetate disodium, Tris, NaCl or dehydrated alcohol.As a result, it is according to an embodiment of the present invention extract faeces DNA method into
Specific, the easier operation with higher of one step, higher faeces DNA yield or purity.
According to an embodiment of the invention, reagent used by the pretreatment includes: crosslinked polyvinylpyrrolidone, different sulphur
Cyanic acid guanidine solution and Proteinase K.The method according to an embodiment of the present invention for extracting faeces DNA is further with higher as a result,
Specific, easier operation, higher faeces DNA yield or purity.
According to an embodiment of the invention, the pretreatment includes: by the excrement to be measured and crosslinked polyvinylpyrrolidone
It is incubated at room temperature, is centrifuged, collect supernatant；At room temperature by the supernatant, guanidine isothiocyanate solution and Proteinase K Solution
It is incubated for, to obtain mixed liquor；And be incubated for the mixed liquor, end is placed on cooled on ice, described to obtain
Pretreatment fluid, wherein final concentration of 30~60mg/mL of the crosslinked polyvinylpyrrolidone, the guanidine isothiocyanate solution
Final concentration of 2~3mol/L, final concentration of 70~120 μ g/mL of the Proteinase K.It is according to an embodiment of the present invention as a result,
Extract method further specific, easier operation, higher faeces DNA yield or the purity with higher of faeces DNA.
Preferred embodiment in accordance with the present invention, the pretreatment includes: the preservation liquid for 1) taking 5mL to contain excrement, with 60HZ
Frequency homogenization 5min, be centrifuged 6min under the conditions of 3200 × g, collect the first supernatant；2) to first supernatant
Middle addition crosslinked polyvinylpyrrolidone, final concentration of 50mg/mL, room temperature rotation are incubated for 15min, are centrifuged under the conditions of 3200 × g
6min collects the second supernatant；3) guanidine isothiocyanate solution that concentration is 6mol/L is added into second supernatant, it is dense eventually
Degree is 2.4mol/L, and the Proteinase K that concentration is 20mg/mL is added, and final concentration of 90 μ g/mL after mixing, is incubated at room temperature 30min；
And mixed liquor obtained by step 3) 4) is subjected to 90 DEG C of metal bath 10min, after in cooled on ice, to obtain the pre- place
Manage liquid.The method according to an embodiment of the present invention for extracting faeces DNA is further with higher specific, easier as a result,
Operation, higher faeces DNA yield or purity.
According to an embodiment of the invention, the pretreatment fluid contacted with the magnetic bead-probe complex be at room temperature into
Row 1~2 hour.As a result, it is according to an embodiment of the present invention extract faeces DNA method further it is with higher specificity, compared with
Easy operation, higher faeces DNA yield or purity.
According to an embodiment of the invention, further comprising after the pretreatment fluid is contacted with the magnetic bead-probe complex
Following steps: (1) obtained mixed liquor carries out magnetic force suction after contacting the pretreatment fluid with the magnetic bead-probe complex
It is attached, abandon supernatant；(2) it will be added in step (1) obtained magnetic bead without enzyme water, be incubated for, end is placed on cooled on ice；(3) will
The obtained magnetic bead of step (2) carries out magnetic-adsorption, supernatant is collected, to extract faeces DNA.Implement according to the present invention as a result,
The method of the extraction faeces DNA of example further specific, easier operation with higher, higher faeces DNA yield or
Purity.
Preferred embodiment in accordance with the present invention, comprising: (1) it is compound that the magnetic bead-probe is added into the pretreatment fluid
Object is incubated at room temperature 1~2h；(2) step (1) obtained mixed liquor is placed in 5~10min of standing adsorption on magnetic frame, in abandoning
Clearly；(3) 1mL wash buffer will be added in step (2) obtained precipitating and magnetic bead is resuspended, be transferred in 1.5mL centrifuge tube,
Three times using 1mL wash buffer cleaning, in standing adsorption on magnetic frame, supernatant is abandoned；(4) obtained heavy to step (3)
40 μ L are added in shallow lake without enzyme water, mix well, 90 DEG C of metal bath 10min, after in cooled on ice；And (5) by step (4)
Obtained solution is placed in standing adsorption 4min on magnetic frame, and centrifugation collects supernatant, to obtain the DNA in excrement.By
This, the method according to an embodiment of the present invention for extracting faeces DNA further specific, easier operation with higher, compared with
High faeces DNA yield or purity.
In still another aspect of the invention, the invention proposes a kind of for extracting the kit of faeces DNA.According to the present invention
Embodiment, the kit includes: the coated magnetic bead of streptavidin；The probe of biotin modification；Crosslinked polyethylene pyrroles
Alkanone；Guanidine isothiocyanate solution；Proteinase K Solution, the kit are used to implement the method noted earlier for extracting faeces DNA.
Extracting faeces DNA using kit according to an embodiment of the present invention as a result, at least has at least one of following advantages: specificity
By force, easy to operate, faeces DNA yield is high, purity is high and is suitable for scale application.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the flow diagram of the method according to an embodiment of the invention for extracting faeces DNA；And
Fig. 2 shows electrophoretogram according to an embodiment of the invention.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Explicitly or implicitly include one or more of the features.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
A kind of kit the invention proposes method for extracting faeces DNA and for extracting faeces DNA, below will respectively
It is described in greater detail.
The method for extracting faeces DNA
In one aspect of the invention, the invention proposes a kind of methods for extracting faeces DNA.Implementation according to the present invention
Example, referring to Fig. 1, this method comprises: S100 obtains magnetic bead-probe complex, S200 pretreatment and S300 pretreatment fluid and magnetic
Pearl-probe complex contact.The method according to an embodiment of the present invention for extracting faeces DNA is extracted faeces DNA and is at least had as a result,
At least one of following advantages: high specificity, easy to operate, faeces DNA yield be high, purity is high and is suitable for scale application.Under
Face will be described in detail.
According to an embodiment of the invention, this method comprises:
S100 obtains magnetic bead-probe complex
In this step, the coated magnetic bead of streptavidin is contacted with the probe of biotin modification, to obtain magnetic bead-
Probe complex.
By the way that the coated magnetic bead of Streptavidin is contacted with the probe of biotin modification, Streptavidin meeting and biotin
Specific binding forms magnetic bead-Streptavidin-biotin-probe complex, will using the probe of target gene sequence design
It is specifically bound with the target gene in excrement, to extract the target gene in excrement.
According to an embodiment of the invention, magnetic bead concentration is 80nM~120nM in magnetic bead-probe complex, concentration and probe concentration is
10nM~15nM.According to a particular embodiment of the invention, the concentration of magnetic bead and probe ratio is (5~10): 1, preferably (6~9): 1.
Inventors have found that the volume ratio of the probe of the coated magnetic bead of streptavidin and biotin modification will affect the yield of DNA and pure
Degree, under the above conditions, DNA yield and purity are higher.
It should be noted that " magnetic bead concentration " and " concentration and probe concentration " refer to containing in magnetic bead-probe complex mixed liquor,
Total volume based on mixed liquor, the concentration of magnetic bead and the concentration of probe on magnetic bead-probe complex.
According to an embodiment of the invention, the time of contact of the probe of the coated magnetic bead of streptavidin and biotin modification is
1~2 hour.Inventors have found that Streptavidin and biotin can be combined sufficiently with this condition, to obtain magnetic bead-chain
Mould Avidin-Biotin-probe complex.
According to an embodiment of the invention, magnetic bead-probe complex obtains in the following manner: 1) taking containing strepto- parent
The coated magnetic bead solution of element is closed, in standing on magnetic frame, abandons supernatant；2) it is clear that buffer is added into the obtained magnetic bead of step 1)
Magnetic bead is washed, is mixed, magnetic-adsorption is carried out, abandons supernatant；3) buffer is added into the obtained magnetic bead of step 2), magnetic bead is resuspended,
The probe solution containing biotin modification, incubation at room temperature, to obtain magnetic bead-probe complex is added.As a result, according to the present invention
Further specific, easier operation with higher, higher faeces DNA obtain the method for the extraction faeces DNA of embodiment
Rate or purity.
Preferred embodiment in accordance with the present invention, magnetic bead-probe complex obtain in the following manner: 1) taking 15 μ L chains
The mould coated magnetic bead solution of avidin abandons supernatant in standing 4min on magnetic frame；2) it is added into the obtained magnetic bead of step 1)
1mL Binding Buffer cleans magnetic bead, and concussion abandons supernatant in standing on magnetic frame, it is primary to repeat this step, wherein institute
Stating Binding Buffer includes: 5mmol/L Tris-Hcl, 0.5mmol/L EDTA and 1mol/L NaCL；3) to step 2)
200 μ L Binding Buffer are added in obtained magnetic bead, magnetic bead are resuspended, the probe solution of 10 μ L biotin modifications is added,
It is incubated at room temperature 1h；4) 1mLBinding Buffer is added into the resulting mixed liquor of step 3), it is soft to shake, on magnetic frame
It stands, abandons supernatant, this step of repetition is primary, washes away unbonded probe, adds 200 μ L Binding Buffer that magnetic bead-spy is resuspended
Needle compound, wherein magnetic bead concentration is 98nM~119nM, and the concentration and probe concentration is 14.5nM.Implement according to the present invention as a result,
The method of the extraction faeces DNA of example further specific, easier operation with higher, higher faeces DNA yield or
Purity.
S200 pretreatment
In this step, excrement to be measured is pre-processed, to obtain pretreatment fluid.It is pre- by being carried out to excrement to be measured
Processing avoids it from influencing the extraction and utilization of target gene to remove impurity therein.
It is saved in liquid according to an embodiment of the invention, excrement to be measured is pre-stored in, saves and contain ethylenediamine tetrem in liquid
Acid disodium, Tris, NaCl or dehydrated alcohol.Inventor obtains above-mentioned more excellent composition by many experiments, thus so that in excrement
DNA keep stablize, avoid its degradation as far as possible.
According to an embodiment of the invention, reagent used by pre-processing includes: crosslinked polyvinylpyrrolidone, isothiocyanic acid
Guanidine solution and Proteinase K.Since the ingredient of excrement is more complex, inventor obtains above-mentioned compounding pretreatment examination by many experiments
Agent, triplicity use, and can sufficiently remove impurity, avoid it that DNA is interfered to extract and its apply.
Specifically, crosslinked polyvinylpyrrolidone (abbreviation PVPP) can be adsorbed, the substances such as phenol, quinone and carbohydrate are complexed, and keep away
Exempt from its influence to operations such as DNA extraction, PCR, improves the yield and purity of DNA.Why select PVPP rather than PVP, be by
It is not soluble in water in PVPP, it can be directly centrifuged removal, improve treatment effeciency.Cell is cleaved under guanidinium isothiocyanate denaturation,
Protein denaturation on ribosome simultaneously, discharges nucleic acid.Proteinase K also functions to the effect of enzymolysis protein, discharges nucleic acid.
According to an embodiment of the invention, pretreatment include: by excrement to be measured and crosslinked polyvinylpyrrolidone at room temperature
It is incubated for, supernatant is collected in centrifugation；Supernatant, guanidine isothiocyanate solution and Proteinase K Solution are incubated at room temperature, to obtain
Obtain mixed liquor；And mixed liquor is incubated for, after be immediately placed on cooled on ice, to obtain pretreatment fluid, wherein hand over
Join final concentration of 30~60mg/mL of polyvinylpyrrolidone, final concentration of 2~3mol/L of guanidine isothiocyanate solution, albumen
Final concentration of 70~120 μ g/mL of enzyme K.Since chaff interferent is more in excrement, and DNA content is less, and inventor is by a large amount of real
It tests to obtain more excellent crosslinked polyvinylpyrrolidone, guanidine isothiocyanate solution and Proteinase K dosage, both can effectively remove as a result,
Chaff interferent is removed, guarantees DNA purity, can also be improved DNA yield.
Preferred embodiment in accordance with the present invention, pretreatment includes: the preservation liquid for 1) taking 5mL to contain excrement, with the frequency of 60HZ
Rate homogenization 5min is centrifuged 6min under the conditions of 3200 × g, collects the first supernatant；2) add into first supernatant
Entering crosslinked polyvinylpyrrolidone, final concentration of 50mg/mL, room temperature rotation is incubated for 15min, is centrifuged 6min under the conditions of 3200 × g,
Collect the second supernatant；3) guanidine isothiocyanate solution that concentration is 6mol/L is added into second supernatant, it is final concentration of
2.4mol/L, is added the Proteinase K that concentration is 20mg/mL, and final concentration of 90 μ g/mL after mixing, is incubated at room temperature 30min；And
4) mixed liquor obtained by step 3) is subjected to 90 DEG C of metal bath 10min, after immediately in cooled on ice, to obtain the pre- place
Manage liquid.The method further faeces DNA yield or pure with higher according to an embodiment of the present invention for extracting faeces DNA as a result,
Degree.
S300 pretreatment fluid is contacted with magnetic bead-probe complex
In this step, pretreatment fluid is contacted with magnetic bead-probe complex, to extract faeces DNA.Excrement will be contained
The pretreatment fluid of DNA is contacted with magnetic bead-probe complex, using target gene sequence design probe by in pretreatment fluid
Target gene specific binding, extracts target gene by magnetic-adsorption magnetic bead from pretreatment fluid.Meanwhile passing through magnetic
Target gene in pearl-probe complex and pretreatment fluid is specifically bound, and eliminates DNA purification step, simplifies extraction stream
Journey highly shortened detection time, while reduce the degradation of DNA in purification process, improve DNA yield.Also, it reduces
The use of experiment reagent especially toxic reagent is suitable for scale application.
In addition, inventors have found that obtaining biotin-probe-with first contacting the probe of biotin modification with pretreatment fluid
Target gene compound, then the compound is contacted with the coated magnetic bead of Streptavidin and obtains magnetic bead-Streptavidin-biology
Element-probe-target gene composite extracting mode is compared, and the present invention is using first by the coated magnetic bead of streptavidin and biology
The probe contact of element modification obtains magnetic bead-Streptavidin-biotin-probe complex, then by the compound and pretreatment fluid
Contact obtains the target gene yield that magnetic bead-Streptavidin-biotin-probe-target gene composite mode extracts
It is higher with purity.Inventor speculates that, since excrement ingredient is more complex, chaff interferent is excessive, and Cucumber may result in biology
Element-probe-target gene composite and Streptavidin non-specific binding.
According to an embodiment of the invention, it is that progress 1~2 is small at room temperature that pretreatment fluid is contacted with magnetic bead-probe complex
When.Inventor obtains the more excellent reaction time by many experiments, and thus, it is possible to make target gene and spy in pretreatment fluid
Needle sufficiently combines, and recycles magnetic-adsorption magnetic bead to extract the target gene, improves the yield and purity of target gene.
According to an embodiment of the invention, further comprising as follows after pretreatment fluid is contacted with the magnetic bead-probe complex
Step: (1) obtained mixed liquor carries out magnetic-adsorption after contacting the pretreatment fluid with the magnetic bead-probe complex,
Abandon supernatant；(2) will be added in step (1) obtained magnetic bead without enzyme water, be incubated for, after be immediately placed on cooled on ice；(3) will
The obtained magnetic bead of step (2) carries out magnetic-adsorption, supernatant is collected, to extract faeces DNA.Implement according to the present invention as a result,
The method of the extraction faeces DNA of example further specific, easier operation with higher, higher faeces DNA yield or
Purity.
Preferred embodiment in accordance with the present invention, comprising: (1) magnetic bead-probe complex is added into pretreatment fluid, room temperature is incubated
Educate 1~2h；(2) step (1) obtained mixed liquor is placed in 5~10min of standing adsorption on magnetic frame, abandons supernatant；It (3) will step
Suddenly 1mL wash buffer is added in (2) obtained precipitating and magnetic bead is resuspended, be transferred in 1.5mL centrifuge tube, use 1mL
Wash buffer is cleaned three times, in standing adsorption on magnetic frame, abandons supernatant；(4) it is added into step (3) obtained precipitating
40 μ L are mixed well without enzyme water, 90 DEG C of metal bath 10min, after immediately in cooled on ice；And (5) will be obtained by step (4)
To solution be placed in standing adsorption 4min on magnetic frame, be centrifuged, collect supernatant, to obtain the DNA in excrement.Root as a result,
According to method further specific, the easier operation with higher, higher excrement of the extraction faeces DNA of the embodiment of the present invention
Just DNA yield or purity.
For extracting the kit of faeces DNA
In still another aspect of the invention, the invention proposes a kind of for extracting the kit of faeces DNA.According to the present invention
Embodiment, which includes: the coated magnetic bead of streptavidin；The probe of biotin modification；Crosslinked polyethylene pyrrolidines
Ketone；Guanidine isothiocyanate solution；Proteinase K Solution, the kit are used to implement the method noted earlier for extracting faeces DNA.
By the way that the coated magnetic bead of Streptavidin is contacted with the probe of biotin modification, Streptavidin meeting and biotin
Specific binding forms magnetic bead-Streptavidin-biotin-probe complex, will using the probe of target gene sequence design
It is specifically bound with the target gene in excrement, to extract the target gene in excrement.PVPP can adsorb, phenol is complexed,
The substances such as quinone and carbohydrate avoid it from improving the yield and purity of DNA to the influence of the operations such as DNA extraction, PCR.Why select
PVPP rather than PVP are since PVPP is not soluble in water, and centrifugation can remove, and improve treatment effeciency.Cell is denaturalized in guanidinium isothiocyanate to be made
It is cleaved under, while the protein denaturation on ribosome, discharges nucleic acid.Proteinase K also functions to the effect of enzymolysis protein, releases
Put nucleic acid.As a result, using kit according to an embodiment of the present invention extract faeces DNA at least have following advantages at least it
One: high specificity, easy to operate, faeces DNA yield be high, purity is high and is suitable for scale application.
It will be appreciated to those of skill in the art that above for feature and excellent described in the method for extracting faeces DNA
Point is equally applicable to the kit, and details are not described herein.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
In this embodiment, the KRAS gene in excrement is extracted in following manner:
1, the acquisition and preservation of fecal specimens
" dung collection box " is put into defecation after sitting posture or squatting pot, it is ensured that excrement is discharged into box.Collection tube is taken out, it will
Thief rod is inserted perpendicularly into excrement, rotates thief rod so that excrement fills up sampling cavity.Thief rod is turned back into collection tube after sampling, is used
Power tightens lid, with liquid-leakage preventing, and weak vibrations, it is ensured that fecal sample and 10mL save liquid (disodium ethylene diamine tetraacetate,
Tris, NaCl or dehydrated alcohol) contact.It is transported at room temperature, after receiving sample, is placed in -20 DEG C of preservations.
2, magnetic bead and probe covalent bond
1) tenderness mixes well Streptavidin coating magnetic bead solution (avoiding acutely shaking), takes 15 μ L magnetic bead solution, in
4min is stood on magnetic frame, abandons supernatant；
2) 1mL Binding Buffer (5mmol/L Tris-Hcl, 0.5mmol/L EDTA and 1mol/L is added
NaCL magnetic bead) is cleaned, it is soft to shake, in standing on magnetic frame, supernatant is abandoned, it is primary to repeat this step；
3) 200 μ L Binding Buffer are added and magnetic bead is resuspended, the probe of 10 μ L KRAS biotin modifications is then added
Solution is incubated at room temperature 1h；
4) after being incubated for, 1mL Binding Buffer is added, it is soft to shake, in standing on magnetic frame, abandon supernatant, weight
This multiple step is primary, washes away unbonded probe, adds 200 μ L Binding Buffer that magnetic bead-probe complex is resuspended,
In, magnetic bead concentration is 98-119nM, concentration and probe concentration 14.5nM.
3, DNA is extracted
1) experiment sample is taken out in thaw at RT from refrigerator；
2) excrement is mixed well, takes 5mL sample in homogeneous pipe, homogenization (60HZ/5min)；
3) above-mentioned processed sample is centrifuged under the conditions of 3200 × g 6min, takes supernatant into new 15mL centrifuge tube；
4) PVPP processing (final concentration of 50mg/mL) is added, room temperature rotation is incubated for 15min, is centrifuged under the conditions of 3200 × g
6min takes supernatant；
5) it is (whole that the guanidinium isothiocyanate that concentration is 6mol/L is added into new 15mL centrifuge tube in the supernatant after transfer centrifugation
Concentration is 2.4mol/L), the Proteinase K (90 μ g/mL of final concentration) that concentration is 20mg/mL is added, after mixing, is placed at room temperature for
30min；
6) be incubated for after, centrifuge tube is placed on metal bath, 90 DEG C of denaturation 10min, after be immediately placed on it is cold on ice
But；
7) magnetic bead (coating Streptavidin) of step 2 bonding probes (biotin modification) is added, is incubated at room temperature 1h；
8) centrifuge tube is placed in 5~10min of standing adsorption on magnetic frame, abandons supernatant；
9) 1mL wash buffer (containing MOPS and NaCl) resuspension magnetic bead is added, is transferred in 1.5mL centrifuge tube, adds
Enter 1mL wash buffer cleaning three times；
10) in standing adsorption on magnetic frame, supernatant is abandoned；
11) be added 40 μ L without enzyme water, mix well, 90 DEG C of metal bath 10min, after be immediately placed on cooled on ice；
12) centrifuge tube is placed in standing adsorption 4min on magnetic frame, be centrifuged, shifted on supernatant to new centrifuge tube, 4 DEG C
Or -20 DEG C store for future use.
The DNA that said extracted is obtained carries out PCR amplification, using agarose gel electrophoresis observation as a result, acquired results such as
Shown in Fig. 2, the faeces DNA template of all extractions can be expanded successfully, obtain product identical with expected fragments size, be surveyed
Sequence result also demonstrates reliability of the invention.
Comparative example 1
In this embodiment, faeces DNA is extracted in following manner:
(1) the 1 of the step 3 of embodiment 1 is carried out)~6) operation, to pre-process to excrement, obtain pretreatment fluid；
(2) the 10 μ L probe solution for containing KRAS biotin modification is added in pretreatment fluid, is incubated at room temperature 1 hour；
(3) in the pretreatment fluid by step (2) after being incubated for be added 1 step 2 of embodiment 1) and 2) operate it is obtained
The coated magnetic bead of streptavidin is incubated at room temperature 1 hour；
(4) pretreatment fluid of the step (3) after being incubated for is placed in 5~10min of standing adsorption on magnetic frame, abandons supernatant, so
The 9 of 1 step 3 of embodiment is carried out afterwards)~12) operation.
The result of 1 two kinds of catching methods of embodiment 1 and comparative example is as shown in the table.As can be seen that using first by magnetic bead with
Probe combines, then the DNA total amount that the method in conjunction with target gene is captured will be apparently higher than probe and target gene knot first
It closes, then the DNA total amount that the method in conjunction with magnetic bead is captured.
1 two kinds of catching methods of table compare
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (10)
1. a kind of method for extracting faeces DNA characterized by comprising
The coated magnetic bead of streptavidin is contacted with the probe of biotin modification, to obtain magnetic bead-probe complex；
Excrement to be measured is pre-processed, to obtain pretreatment fluid；And
The pretreatment fluid is contacted with the magnetic bead-probe complex, to extract faeces DNA.
2. the method according to claim 1, wherein in the magnetic bead-probe complex, the magnetic bead concentration is
80nM~120nM, the concentration and probe concentration are 10nM~15nM.
3. the method according to claim 1, wherein the coated magnetic bead of the streptavidin and biotin modification
Probe time of contact be 1~2 hour.
4. the method according to claim 1, wherein the magnetic bead-probe complex is to obtain in the following manner
:
1) it takes containing the coated magnetic bead solution of streptavidin, in standing on magnetic frame, abandons supernatant；
2) buffer solution for cleaning magnetic bead is added into the obtained magnetic bead of step 1), mixes, carries out magnetic-adsorption, abandons supernatant；
3) buffer is added into the obtained magnetic bead of step 2), the magnetic bead is resuspended, the probe containing biotin modification is added
Solution, incubation at room temperature, to obtain the magnetic bead-probe complex；
Preferably, the magnetic bead-probe complex obtains in the following manner:
1) the 15 coated magnetic bead solution of μ L streptavidin are taken, in standing 4min on magnetic frame, abandon supernatant；
2) 1mL Binding Buffer is added into the obtained magnetic bead of step 1) and cleans magnetic bead, concussion, in quiet on magnetic frame
Set, abandon supernatant, repeat this step it is primary, wherein the Binding Buffer include: 5mmol/L Tris-Hcl,
0.5mmol/L EDTA and 1mol/L NaCL；
3) 200 μ L Binding Buffer are added into the obtained magnetic bead of step 2), magnetic bead is resuspended, 10 μ L biotins are added and repair
The probe solution of decorations is incubated at room temperature 1h；
4) 1mL Binding Buffer is added into the resulting mixed liquor of step 3), it is soft to shake, in being stood on magnetic frame, abandon
Supernatant, this step of repetition is primary, washes away unbonded probe, adds 200 μ L Binding Buffer that magnetic bead-probe is resuspended compound
Object, wherein the magnetic bead concentration is 98nM~119nM, and the concentration and probe concentration is 14.5nM.
5. it is saved in liquid the method according to claim 1, wherein the excrement to be measured is pre-stored in, it is described
It saves in liquid containing disodium ethylene diamine tetraacetate, Tris, NaCl or dehydrated alcohol；
Optionally, reagent used by the pretreatment includes: crosslinked polyvinylpyrrolidone, guanidine isothiocyanate solution and albumen
Enzyme K.
6. the method according to claim 1, wherein the pretreatment includes:
The excrement to be measured is incubated at room temperature with crosslinked polyvinylpyrrolidone, is centrifuged, supernatant is collected；
The supernatant, guanidine isothiocyanate solution and Proteinase K Solution are incubated at room temperature, to obtain mixed liquor；And
The mixed liquor is incubated for, end is placed on cooled on ice, to obtain the pretreatment fluid,
Wherein, final concentration of 30~60mg/mL of the crosslinked polyvinylpyrrolidone, the end of the guanidine isothiocyanate solution are dense
Degree be 2~3mol/L, final concentration of 70~120 μ g/mL of the Proteinase K,
Preferably, described pre-process includes:
1) the preservation liquid for taking 5mL to contain excrement is centrifuged 6min with the frequency homogenization 5min of 60HZ under the conditions of 3200 × g,
Collect the first supernatant；
2) crosslinked polyvinylpyrrolidone, final concentration of 50mg/mL are added into first supernatant, room temperature rotation is incubated for
It is centrifuged 6min under the conditions of 15min, 3200 × g, collects the second supernatant；
3) guanidine isothiocyanate solution that concentration is 6mol/L is added into second supernatant, final concentration of 2.4mol/L adds
Enter the Proteinase K that concentration is 20mg/mL, final concentration of 90 μ g/mL after mixing, is incubated at room temperature 30min；And
4) mixed liquor obtained by step 3) is subjected to 90 DEG C of metal bath 10min, after in cooled on ice, to obtain the pre- place
Manage liquid.
7. the method according to claim 1, wherein the pretreatment fluid connects with the magnetic bead-probe complex
Touching is to carry out 1~2 hour at room temperature.
8. the method according to the description of claim 7 is characterized in that the pretreatment fluid connects with the magnetic bead-probe complex
After touch, further comprise following steps:
(1) obtained mixed liquor carries out magnetic-adsorption after contacting the pretreatment fluid with the magnetic bead-probe complex, abandons
Supernatant；
(2) it will be added in step (1) obtained magnetic bead without enzyme water, be incubated for, end is placed on cooled on ice；
(3) the obtained magnetic bead of step (2) is subjected to magnetic-adsorption, supernatant is collected, to extract faeces DNA.
9. the method according to the description of claim 7 is characterized in that including:
(1) magnetic bead-probe complex is added into the pretreatment fluid, is incubated at room temperature 1~2h；
(2) step (1) obtained mixed liquor is placed in 5~10min of standing adsorption on magnetic frame, abandons supernatant；
(3) 1mL wash buffer will be added in step (2) obtained precipitating and magnetic bead is resuspended, be transferred to 1.5mL centrifuge tube
In, three times using 1mL wash buffer cleaning, in standing adsorption on magnetic frame, abandon supernatant；
(4) 40 μ L are added into step (3) obtained precipitating without enzyme water, mix well, 90 DEG C of metal bath 10min, after
In cooled on ice；And
(5) step (4) obtained solution is placed in standing adsorption 4min on magnetic frame, be centrifuged, supernatant is collected, to obtain
DNA in excrement.
10. a kind of for extracting the kit of faeces DNA characterized by comprising
The coated magnetic bead of streptavidin；
The probe of biotin modification；
Crosslinked polyvinylpyrrolidone；
Guanidine isothiocyanate solution；
Proteinase K Solution,
The kit is used to implement any one of claim 1~9 method for extracting faeces DNA.
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| CN114507667A (en) * | 2022-04-20 | 2022-05-17 | 天津欧德莱生物医药科技有限公司 | Probe for specifically enriching miRNA in excrement, application and method |
| CN114507667B (en) * | 2022-04-20 | 2022-08-02 | 天津欧德莱生物医药科技有限公司 | Probe for specifically enriching miRNA in excrement, application and method |
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