CN106754874A - The kit and its application method of DNA are extracted in a kind of sample from human excrement and urine - Google Patents
The kit and its application method of DNA are extracted in a kind of sample from human excrement and urine Download PDFInfo
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- CN106754874A CN106754874A CN201611113440.3A CN201611113440A CN106754874A CN 106754874 A CN106754874 A CN 106754874A CN 201611113440 A CN201611113440 A CN 201611113440A CN 106754874 A CN106754874 A CN 106754874A
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Abstract
The invention discloses kit and its application method that DNA is extracted in a kind of sample from human excrement and urine, containing lysate I, lysate II, protein precipitation liquid I, protein precipitation liquid II, DNA sedimentation liquids, rinsing liquid, wash-out lysate and 0.1 1mm sterilizing beades in the kit, application method includes that vibration cracking, albumen precipitation, DNA precipitations, cold drift, wash-out dissolving are standby.The kit and its application method of DNA are extracted in a kind of sample from human excrement and urine of the invention has extraction rapid, better extract effect, and safety and environmental protection obtains the characteristics of amount of DNA is big, and quality is high.
Description
Technical field
The present invention relates to biology field, the kit of DNA is extracted in more particularly to a kind of sample from human excrement and urine
And its application method.
Background technology
It is colonized that a group is sufficiently complex and metastable microorganism in human body intestinal canal, these microorganisms are in human body intestinal canal
Form a complexity and dynamic microflora, have very important influence to the health of human body.Microorganism in enteron aisle
The slight change of system all can produce tremendous influence to human organism.Shown according to research, microorganism is to human body in human body intestinal canal
Health is most important.Microorganism in enteron aisle is closely related with the various diseases of human body, and correlative study shows：Some common diseases
It is low including diabetes, asthma, chronic diarrhea, intestinal irritable syndrome (IBS), inflammatory bowel disease (IBD), obesity, immunity
The inferior imbalance for being all found to be balanced with Multiple drug resistance has close association.As research deepens continuously, scientists also found many
The generation for planting cancer is closely bound up with the trickle change of gut flora.
Because enteron aisle length is long, it is difficult to directly be detected to intestinal flora, the microorganism in excrement is analyzed,
Flora situation in human body intestinal canal can indirectly be reflected, help to understand the change of human body intestinal canal Tiny ecosystem.So, to micro- in fecal sample
Biological detection has turned into the Main Means for understanding intestinal microecology.
Colorectal cancer (carcinoma of colon and rectum) is common malignant tumor of digestive tract.China
The people of the annual new cases of colorectal cancer about 33.1 ten thousand, the incidence of disease is number four position in whole malignant tumours；Die from this every year
The patient of disease has 15.9 ten thousand people, and the death rate then occupies cancer mortality reason the 5th.Adenoma is before the carcinoma of the rectum is formed
In the stage, developing into cancer from adenoma will typically experience 5-15 or longer, and this is just that the examination of colorectal cancer and early diagnosis early control offer
May.Therefore, it is a row for controlling colorectal cancer case fatality rate to find a kind of simple, quick, sensitive early detection method
Effective method.At present, the diagnosis to colorectal cancer is mainly colonoscopy, but colonoscopy belongs to intrusive mood inspection
Look into, it is easy to cause and do not accommodate complication.In recent years, detected by the gut flora to colorectal cancer patients and Healthy People and found
Flora has notable difference in distribution, and gut flora may take part in colorectal cancer development.If the excrement to subject is carried out
Analysis, is just capable of determining whether to be possible to colorectal cancer, just can carry out examination to general population well, so as to accomplish to knot
The carcinoma of the rectum early finds early treatment.
Due to the particularity of intestinal environment, enteric microorganism only has the little part can to cultivate in vitro, this serious resistance
Structure and multifarious cognition of the people to enteric microorganism are hindered.With continuing to develop for Protocols in Molecular Biology, people
Gut flora can be studied from DNA level directly.Because excrement complicated component (contains polysaccharide, cholate, humic
The PCR such as matter, cholic acid mortifiers), and bacterial species are various in enteron aisle, and different extracting method susceptibilitys is differed, and result in
The extraction effect and efficiency of Different Extraction Method differ.And then cause subsequent analysis result unsatisfactory, or even produce deviation.
The method that current fecal microorganism DNA is extracted has a lot, common are Mechanical Method, such as paramagnetic particle method, freeze-thaw method；Chemistry
Method, such as SDS methods, phynol method, enzyme process (such as lysozyme, Proteinase K) and RNA isolation kit.The combination of these methods is that laboratory carries
Take the conventional method of faeces DNA.It is general with cell lysis released dnas such as SDS, lysozyme, ultrasonic waves, then with phenol-chloroform, magnetic bead
Etc. being stripped, last absolute ethyl alcohol precipitation.RNA isolation kit is simple to operate, efficiency high, but the STb gene concentration extracted is low, place
Manage large quantities of sample high costs.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of quick, effective and economical from human excrement and urine's sample
The kit of middle extraction DNA, and simply, quickly, high-quality, high efficiency and the suitable application method of price.
In a kind of sample from human excrement and urine of the invention extract DNA kit, in the kit containing lysate I,
Lysate II, protein precipitation liquid I, protein precipitation liquid II, DNA sedimentation liquids, rinsing liquid, wash-out lysate and 0.1-1mm go out
Bacterium bead, wherein
The lysate I is the NaCl of 0.1-2M comprising substance withdrawl syndrome, and substance withdrawl syndrome is 10-150mM, PH=
8 Tris-HCl, substance withdrawl syndrome is the EDTA of 10-150mM, PH=8, and mass concentration is the SDS and mass concentration of 1-5%
It is the PVP of 0.5-2%；
The lysate II is the Proteinase K Solution of 5-30mg/ml comprising mass concentration；
The protein precipitation liquid I is the potassium acetate solution of 1-5M comprising substance withdrawl syndrome；
The protein precipitation liquid II is 4-6M NaCl solutions comprising substance withdrawl syndrome；
The DNA sedimentation liquids include absolute ethyl alcohol；
The rinsing liquid is the ethanol solution of 60-80% comprising volume fraction；
1 × TE Buffer of the wash-out lysate comprising PH=8 used.
Further, the lysate I comprising substance withdrawl syndrome for 0.5M NaCl, substance withdrawl syndrome be 50mM,
The Tris-HCl of PH=8, substance withdrawl syndrome is the EDTA of 50mM, PH=8, and mass concentration is 4% SDS and mass concentration is
1% PVP.
Further, the lysate II is the Proteinase K Solution of 20mg/ml comprising mass concentration.
Further, the protein precipitation liquid I is the potassium acetate solution of 2M comprising substance withdrawl syndrome.
Further, the protein precipitation liquid II is 6M NaCl solutions comprising substance withdrawl syndrome.
Further, the rinsing liquid includes the ethanol solution that volume fraction is 70%.
Further, 1 × TE Buffer of the PH=8 of the wash-out lysate are 10mM, PH=by substance withdrawl syndrome
8 Tris-HCl and substance withdrawl syndrome are made up of the EDTA of 1mM, PH=8.
In this kit, lysate I provides cell cracking environment, and contains Dnase mortifiers.Lysate II can disappear
Change protein release DNA.Protein precipitation liquid I, II is used for the impurity such as precipitating proteins.DNA sedimentation liquids can make DNA molecular sink
Get off in shallow lake.Rinsing liquid is used to rinse out the impurity remained on DNA, in certain density alcoholic environment, the damage that DNA will not be larger
Lose.Wash-out lysate provides a less salt stabilization PH environment, for eluted dna.
The application method of the kit of DNA is extracted in a kind of sample from human excrement and urine, is comprised the following steps：
1) 0.1-0.2g human excrement and urine's samples are taken, the 0.1-1mm sterilizing beades of 0.1-0.3g add 300-600 μ l cracking
Liquid I is shaken using oscillator and mixes 3-5min；
2) to step 1) 5-20 μ l lysates II are added in resulting solution, oscillator concussion mixes 5-20s, 60-75 DEG C of water
Bath 10-50min, oscillator concussion 5-20s centrifuging and taking supernatants are taken out every 10min；
3) to step 2) 50-200 μ l protein precipitations liquid I is added in gained supernatant, turn upside down and fully mix, on ice
It is incubated 10-30min, centrifuging and taking supernatant；
4) to step 3) the protein precipitation liquid II of 1/4 volume is added in gained supernatant, turn upside down and fully mix, from
The heart takes supernatant；
5) to step 4) 2 times of DNA precipitated liquids of volume are added in gained supernatant, mixing of turning upside down shifts solution
To adsorption column, centrifugation, removal centrifugation liquid in pipe；
6) to 0.5-1.5ml 2-8 DEG C precooling rinsing liquids are added in adsorption column, 1-5min is stood, centrifugation goes that intraluminal fluid is centrifuged
Body, repeats 1-2 times；
7) adsorption column is removed, 3-8min is stood, is fully dried；
8) to the wash-out lysate for adding 40-70 μ l 60-75 DEG C to preheat in adsorption column, dissolving 2-8min, centrifugation are stood；
9) by step 8) resulting solution -20 DEG C of Refrigerator stores of placement.
In the present invention, the characteristics of make use of microbial cell film and cell membrane in human feces sample, and DNA and egg
The relevant nature of white matter, creates a kind of kit of new extraction human feces microorganism total DNA.Kit similar with other
Compare, the present invention has following advantage：
1st, extract rapid, can be extracted in the human feces DNA that better quality was completed in 4 hours using this kit, with
Other similar kits are compared and greatly improve fecal sample DNA extraction efficiencies；
2nd, with better extract effect, this kit can effectively crack each quasi-microorganism in human feces sample, can be effective
Extract all kinds of microorganism total DNAs in human feces sample；
3rd, compared with traditional DNA extraction method, this kit does not use the toxic reagents such as phenol, chloroform, but uses vinegar
The impurity, safety and environmental protection such as protein, the polysaccharide contained in the nontoxic reagent removal sample such as sour potassium, sodium chloride；
4th, amount of DNA is obtained greatly using this kit, quality is high, this kit is for microorganism in human feces sample
Cell membrane and cell membrane feature, the suitable cracking liquid energy of selection effectively crack each quasi-microorganism in human feces sample, for
All kinds of PCR mortifiers remove all kinds of PCR mortifiers from suitable mortifier remover in human feces sample, using effective
DNA precipitates liquid precipitate DNA, and repeatedly washing DNA using suitable cleaning solution precipitates, and finally giving that more and purity is higher can be very
The good DNA product for follow-up test.
Specific embodiment
The technical scheme in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
Embodiment 1
The kit and its application method of DNA are extracted in a kind of sample from human excrement and urine of the present embodiment, is contained in kit
Have lysate I, lysate II, protein precipitation liquid I, protein precipitation liquid II, DNA sedimentation liquids, rinsing liquid, wash-out lysate and
0.1-1mm sterilizing beades, wherein
Lysate I is the NaCl of 0.5M comprising substance withdrawl syndrome, and substance withdrawl syndrome is the Tris- of 50mM, PH=8
HCl, substance withdrawl syndrome is the EDTA, the PVP that mass concentration is 4% SDS and mass concentration is 1% of 50mM, PH=8.
Lysate II is the Proteinase K Solution of 20mg/ml comprising mass concentration.
Protein precipitation liquid I is the potassium acetate solution of 2M comprising substance withdrawl syndrome.
Protein precipitation liquid II is 6M NaCl solutions comprising substance withdrawl syndrome.
DNA sedimentation liquids include absolute ethyl alcohol；
Rinsing liquid includes the ethanol solution that volume fraction is 70%.
Wash-out lysate is 1 × TE Buffer of PH=8, is the Tris-HCl of 10mM, PH=8 by substance withdrawl syndrome
With substance withdrawl syndrome by the EDTA of 1mM, PH=8 is made.
The application method of the kit of DNA is extracted in a kind of sample from human excrement and urine, is comprised the following steps：
1) 0.1g human excrement and urine's fresh samples are taken, 0.2g sterilizing beades add 500 μ l lysates I to be shaken using oscillator
Mix 5min；
2) to step 1) 10 μ l lysates II are added in resulting solution, oscillator concussion mixes 10s, 70 DEG C of water-bath 30min,
Oscillator concussion 10s, 10000rpm centrifugation 5min is taken out every 10min take supernatant；
3) to step 2) gained supernatant in add 100 μ l protein precipitations liquid I, turn upside down fully mix, incubate on ice
10min is educated, 10000rpm centrifugations 5min takes supernatant；
4) to step 3) the protein precipitation liquid II of 1/4 volume is added in gained supernatant, turn upside down and fully mix,
10000rpm centrifugations 5min takes supernatant；
5) to step 4) 2 times of DNA precipitated liquids of volume are added in gained supernatant, mixing of turning upside down shifts solution
To adsorption column, 10000rpm centrifugations 5min takes supernatant, removal centrifugation liquid in pipe；
6) to 4 DEG C of precooling rinsing liquids of 1ml are added in adsorption column, 2min is stood, 10000rpm centrifugations 5min is gone in centrifuge tube
Liquid, repeats 1-2 times；
7) adsorption column is removed, 5min is stood, is fully dried；
8) to 60 μ l, 70 DEG C of wash-out lysates of preheating are added in adsorption column, dissolving 5min, 10000rpm centrifugation is stood
5min；
9) by step 8) resulting solution -20 DEG C of Refrigerator stores of placement.
Embodiment 2
The kit and its application method of DNA are extracted in a kind of sample from human excrement and urine of the present embodiment, is contained in kit
Have lysate I, lysate II, protein precipitation liquid I, protein precipitation liquid II, DNA sedimentation liquids, rinsing liquid, wash-out lysate and
0.1-1mm sterilizing beades, wherein
Lysate I is the NaCl of 0.5M comprising substance withdrawl syndrome, and substance withdrawl syndrome is the Tris- of 50mM, PH=8
HCl, substance withdrawl syndrome is the EDTA, the PVP that mass concentration is 4% SDS and mass concentration is 1% of 50mM, PH=8.
Lysate II is the Proteinase K Solution of 20mg/ml comprising mass concentration.
Protein precipitation liquid I is the potassium acetate solution of 2M comprising substance withdrawl syndrome.
Protein precipitation liquid II is 6M NaCl solutions comprising substance withdrawl syndrome.
DNA sedimentation liquids include absolute ethyl alcohol；
Rinsing liquid includes the ethanol solution that volume fraction is 70%.
Wash-out lysate is 1 × TE Buffer of PH=8, is the Tris-HCl of 10mM, PH=8 by substance withdrawl syndrome
With substance withdrawl syndrome by the EDTA of 1mM, PH=8 is made.
The application method of the kit of DNA is extracted in a kind of sample from human excrement and urine, is comprised the following steps：
1) -80 DEG C of refrigeration samples of 0.1g human excrement and urines are taken, 0.2g sterilizing beades add 500 μ l lysates I to use vibration
Device concussion mixes 5min；
2) to step 1) 10 μ l lysates II are added in resulting solution, oscillator concussion mixes 10s, 70 DEG C of water-bath 30min,
Oscillator concussion 10s, 10000rpm centrifugation 5min is taken out every 10min take supernatant；
3) to step 2) gained supernatant in add 100 μ l protein precipitations liquid I, turn upside down fully mix, incubate on ice
10min is educated, 10000rpm centrifugations 5min takes supernatant；
4) to step 3) the protein precipitation liquid II of 1/4 volume is added in gained supernatant, turn upside down and fully mix,
10000rpm centrifugations 5min takes supernatant；
5) to step 4) 2 times of DNA precipitated liquids of volume are added in gained supernatant, mixing of turning upside down shifts solution
To adsorption column, 10000rpm centrifugations 5min takes supernatant, removal centrifugation liquid in pipe；
6) to 4 DEG C of precooling rinsing liquids of 1ml are added in adsorption column, 2min is stood, 10000rpm centrifugations 5min is gone in centrifuge tube
Liquid, repeats 1-2 times；
7) adsorption column is removed, 5min is stood, is fully dried；
8) to 60 μ l, 70 DEG C of wash-out lysates of preheating are added in adsorption column, dissolving 5min, 10000rpm centrifugation is stood
5min；
9) by step 8) resulting solution -20 DEG C of Refrigerator stores of placement.
The kit and its application method that DNA is extracted in a kind of sample from human excrement and urine of above example have：
1st, extract rapid, can be extracted in the human feces DNA that better quality was completed in 4 hours using this kit, with
Other similar kits are compared and greatly improve fecal sample DNA extraction efficiencies；
2nd, with better extract effect, this kit can effectively crack each quasi-microorganism in human feces sample, can be effective
Extract all kinds of microorganism total DNAs in human feces sample；
3rd, compared with traditional DNA extraction method, this kit does not use the toxic reagents such as phenol, chloroform, but uses vinegar
The impurity, safety and environmental protection such as protein, the polysaccharide contained in the nontoxic reagent removal sample such as sour potassium, sodium chloride；
4th, amount of DNA is obtained greatly using this kit, quality is high, this kit is for microorganism in human feces sample
Cell membrane and cell membrane feature, the suitable cracking liquid energy of selection effectively crack each quasi-microorganism in human feces sample, for
All kinds of PCR mortifiers remove all kinds of PCR mortifiers from suitable mortifier remover in human feces sample, using effective
DNA precipitates liquid precipitate DNA, and repeatedly washing DNA using suitable cleaning solution precipitates, and finally giving that more and purity is higher can be very
The good DNA product for follow-up test.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each implementation method is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Specification an as entirety, the technical scheme in each embodiment can also be formed into those skilled in the art through appropriately combined
May be appreciated other embodiment.
Claims (8)
1. the kit of DNA is extracted in a kind of sample from human excrement and urine, it is characterised in that：In the kit containing lysate I,
Lysate II, protein precipitation liquid I, protein precipitation liquid II, DNA sedimentation liquids, rinsing liquid, wash-out lysate and 0.1-1mm go out
Bacterium bead, wherein
The lysate I is the NaCl of 0.1-2M comprising substance withdrawl syndrome, and substance withdrawl syndrome is 10-150mM, PH=8
Tris-HCl, substance withdrawl syndrome is the EDTA of 10-150mM, PH=8, and mass concentration is for the SDS and mass concentration of 1-5%
The PVP of 0.5-2%；
The lysate II is the Proteinase K Solution of 5-30mg/ml comprising mass concentration；
The protein precipitation liquid I is the potassium acetate solution of 1-5M comprising substance withdrawl syndrome；
The protein precipitation liquid II is 4-6M NaCl solutions comprising substance withdrawl syndrome；
The DNA sedimentation liquids include absolute ethyl alcohol；
The rinsing liquid is the ethanol solution of 60-80% comprising volume fraction；
1 × TE Buffer of the wash-out lysate comprising PH=8 used.
2. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
Lysate I is the NaCl of 0.5M comprising substance withdrawl syndrome, and substance withdrawl syndrome is the Tris-HCl of 50mM, PH=8, material
Amount concentration is the EDTA, the PVP that mass concentration is 4% SDS and mass concentration is 1% of 50mM, PH=8.
3. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
Lysate II is the Proteinase K Solution of 20mg/ml comprising mass concentration.
4. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
Protein precipitation liquid I is the potassium acetate solution of 2M comprising substance withdrawl syndrome.
5. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
Protein precipitation liquid II is 6M NaCl solutions comprising substance withdrawl syndrome.
6. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
Rinsing liquid includes the ethanol solution that volume fraction is 70%.
7. the kit of DNA is extracted in a kind of sample from human excrement and urine according to claim 1, it is characterised in that：It is described
1 × TE the Buffer for eluting the PH=8 of lysate are the Tris-HCl of 10mM, PH=8 and the amount of material by substance withdrawl syndrome
Concentration is made up of the EDTA of 1mM, PH=8.
8. the application method of the kit of DNA, its feature are extracted in a kind of sample from human excrement and urine according to claim 1
It is：Comprise the following steps：
1) 0.1-0.2g human excrement and urine's samples are taken, the 0.1-1mm sterilizing beades of 0.1-0.3g add 300-600 μ l lysates I
Shaken using oscillator and mix 3-5min；
2) to step 1) 5-20 μ l lysates II are added in resulting solution, oscillator concussion mixes 5-20s, 60-75 DEG C of water-bath 10-
50min, oscillator concussion 5-20s centrifuging and taking supernatants are taken out every 10min；
3) to step 2) 50-200 μ l protein precipitations liquid I is added in gained supernatant, turn upside down and fully mix, it is incubated on ice
10-30min, centrifuging and taking supernatant；
4) to step 3) the protein precipitation liquid II of 1/4 volume is added in gained supernatant, turn upside down and fully mix, centrifuging and taking
Supernatant；
5) to step 4) 2 times of DNA precipitated liquids of volume are added in gained supernatant, solution is transferred to suction by mixing of turning upside down
Attached column, centrifugation, removal centrifugation liquid in pipe；
6) to 0.5-1.5ml 2-8 DEG C precooling rinsing liquids are added in adsorption column, 1-5min is stood, centrifugation goes that liquid in pipe is centrifuged,
Repeat 1-2 times；
7) adsorption column is removed, 3-8min is stood, is fully dried；
8) to the wash-out lysate for adding 40-70 μ l 60-75 DEG C to preheat in adsorption column, dissolving 2-8min, centrifugation are stood；
9) by step 8) resulting solution -20 DEG C of Refrigerator stores of placement.
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| CN107760675A (en) * | 2017-11-07 | 2018-03-06 | 泰州达安达瑞医学检验有限公司 | Cast-off cells DNA extracts kit and extracting method in a kind of human faecal mass sample |
| CN108866220A (en) * | 2018-08-15 | 2018-11-23 | 博奥生物集团有限公司 | A kind of method and detection kit for nasal cavity bacteria detection |
| CN109897906A (en) * | 2019-03-04 | 2019-06-18 | 福建西陇生物技术有限公司 | A kind of detection method and its application of intestinal flora 16S rRNA gene |
| CN111197073A (en) * | 2019-12-19 | 2020-05-26 | 武汉艾米森生命科技有限公司 | Method for extracting DNA sample from excrement and methylation detection method of colorectal cancer related gene |
| CN112553077A (en) * | 2020-12-14 | 2021-03-26 | 南通伊仕生物技术股份有限公司 | Novel coronavirus lysate and application thereof |
| CN114323850A (en) * | 2021-12-22 | 2022-04-12 | 上海锐翌生物科技有限公司 | Sample pretreatment device and pretreatment method of nucleic acid and fecal sample |
| CN115992128A (en) * | 2021-10-28 | 2023-04-21 | 深圳吉因加医学检验实验室 | Composition, kit and purification method thereof |
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