CN107227345A - A kind of microbiological specimens DNA preserves liquid - Google Patents
A kind of microbiological specimens DNA preserves liquid Download PDFInfo
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- CN107227345A CN107227345A CN201710347085.4A CN201710347085A CN107227345A CN 107227345 A CN107227345 A CN 107227345A CN 201710347085 A CN201710347085 A CN 201710347085A CN 107227345 A CN107227345 A CN 107227345A
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- 239000007788 liquid Substances 0.000 title claims abstract description 43
- 230000002906 microbiologic effect Effects 0.000 title claims abstract description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000011187 glycerol Nutrition 0.000 claims abstract description 7
- 239000002480 mineral oil Substances 0.000 claims abstract description 6
- 235000010446 mineral oil Nutrition 0.000 claims abstract description 6
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 5
- 239000011780 sodium chloride Substances 0.000 abstract description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 abstract description 5
- 235000011152 sodium sulphate Nutrition 0.000 abstract description 5
- 235000002639 sodium chloride Nutrition 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 21
- 238000004321 preservation Methods 0.000 description 14
- 244000005700 microbiome Species 0.000 description 13
- 230000002550 fecal effect Effects 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 238000012163 sequencing technique Methods 0.000 description 8
- 230000009514 concussion Effects 0.000 description 7
- 238000012545 processing Methods 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000003672 processing method Methods 0.000 description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 238000007622 bioinformatic analysis Methods 0.000 description 2
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
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- 239000010871 livestock manure Substances 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000006101 laboratory sample Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- -1 sulfuric acid cyanic acid guanidine Chemical compound 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Liquid is preserved the present invention relates to a kind of biological specimen, more particularly to a kind of microbiological specimens DNA preserves liquid；The microbiological specimens DNA of the present invention preserves liquid, the mineral oil of 0.5~2mM/L EDTA, saturated sodium-chloride (NaCl), the methanol that mass fraction is 2~5%, the glycerine that mass fraction is 5 15%, the AEO that mass fraction is 5 10%, the lauryl sodium sulfate (SDS) that mass fraction is 1 5% and mixing is wherein dissolved with pure water, and adjusts pH to 6.0 9.0；The microbiological specimens DNA of the present invention preserves liquid, it is adaptable to microbiological specimens are preserved under normal temperature, simple to operate, applicability is preferable, can be applied to extensive sample detection.
Description
Technical field
Liquid is preserved the present invention relates to a kind of biological specimen, more particularly to a kind of microbiological specimens DNA preserves liquid.
Background technology
Polymerase chain reaction (PCR) is a kind of isothermal DNA amplification, is obtained extensively in molecular biology and medical domain
General application.The first step of PCR detection techniques is the extraction of DNA in the preparation of template DNA, i.e. sample, directly affects PCR reactions
As a result.With the development of round pcr, there is the method for many extraction genomic DNAs.But will be from live environment sample such as
In soil, dust and sewage, or in human tissue sample such as excrement, quick detection pathogenic microorganisms, because template amount is few, then
Depart from original living environment plus microorganism, the abundance for often leading to original microorganism is changed, DNA is damaged, and makes experiment
Sensitiveness is substantially reduced, wherein especially the most difficult with the preservation of enteric microorganism.
After excrement is in vitro, its environment is also changed, and for example oxygen-free environment is transformed into aerobic environment, wherein providing micro- life
The energy source of thing metabolism also enters only state of the consumption without supplement.At the same time, part bacterium therein also maintains work
Property, continue to be metabolized and grow, and due to the change of environment, its growth of different bacterium is different from defecation with dead speed
Before.Accordingly, it would be desirable to carry out some preservation measures to excrement, as far as possible by the stable shape when just in vitro of the composition of wherein microorganism
State, so that the symbiotic microorganism in best representative large intestine.After feces collection, by extracting the STb gene of bacterium, ribose is expanded
Body 16S rRNA variable regions, sequencing and a series of bioinformatic analysis, it will be appreciated that the bacterium in excrement is constituted, further
Study its relation between health disease.
In the prior art for the preservation of fecal microorganism, many methods using cryogenic freezing, such as Chinese patent are announced
Number be CN102864138A patent of invention, disclose a kind of human feces DNA extraction method, i.e., using Cryopreservation of Viable, adopt
The fecal sample of collection is immediately placed in -20 DEG C~-80 DEG C, but the method for cryogenic freezing needs large scale equipment to use cooperatively, and is applicable
Property it is poor, operate it is also complex, be unsuitable for extensive clinical detection；Common DNA is preserved in liquid, usually contains EDTA, though
It can so use at normal temperatures, but this DNA preserves liquid is not particularly suited for the DNA of fecal microorganism sample and preserves, with making
Use limitation.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide one kind be applied to normal temperature under preserve microbiological specimens,
Simple to operate, applicability is preferable, can be applied to the microbiological specimens DNA preservation liquid of extensive sample detection.
The microbiological specimens DNA of the present invention preserves liquid, wherein being dissolved with 0.5~2mM/L EDTA, saturation chlorine in pure water
Change sodium (NaCl), mass fraction be 2~5% methanol, mass fraction be 5-15% glycerine, mass fraction be 5-10% fat
Fat alcohol APEO, mass fraction are 1-5% lauryl sodium sulfate (SDS) and the mineral oil of mixing, and adjust pH
To 6.0-9.0.
Further, in addition to mass fraction be 5-10% dimethyl sulfoxide (DMSO) (DMSO).
Specifically, the mass fraction of the dimethyl sulfoxide (DMSO) (DMSO) is 8%.
Further, in addition to mass fraction be 2-5% ethanol.
Specifically, the mass fraction of the ethanol is 3%.
Specifically, the microbiological specimens DNA preserves the EDTA that 1mM/L is dissolved with liquid.
Specifically, the mass fraction of the methanol is 4%.
Specifically, the mass fraction of the glycerine is 10%.
Specifically, the mass fraction of the AEO is 8%.
Specifically, the mass fraction of the lauryl sodium sulfate (SDS) is 3%.
Specifically, the preservation liquid regulation pH to 8.0.
It should be noted that 1) EDTA can make calcium become chelate as cation chelating agent, so as to expeditiously prevent
Only biological precursor reactant or enzyme reaction；2) methanol can prevent the generation of hydroxyl to dissociate as antioxidant；3) glycerine, not only has
There is the effect of the anti-oxidation as caused by hydroxyl (OH yls), stably protected at -20 DEG C~4 DEG C as anti-icing fluid while also having
The effect of microbiological specimens is deposited, protein breakdown is also prevented, suppresses the effect of enzyme reaction；4) AEO can be strengthened
Surface-active, with scattered, osmosis, is further added after ethanol, and point that can increase other compositions in water is coordinated with it
The ability of dissipating, and reduce the volatilization effusion of the materials such as methanol；5) ethanol and SDS be as denaturant, can will be in microbiological specimens it is viscous
The organic substance such as albumen and globulin carries out denaturation treatment, as bacteriostatic agent, can prevent the abundance of microorganism from changing；6)
Dimethyl sulfoxide (DMSO) is a kind of important permeabilized cells protective agent, DMSO can quick penetration cell membrane enter in cell, reduction
Freezing point, delay to freeze process, while improving intracellular ion concentration, reduce the formation of intracellular ice crystal, so as to reduce cell damage
Wound, result of study shows that when DMSO concentration is 10% in nutrient solution, inhibitory rate of cell growth nearly 100% suppresses during 1 ‰ concentration
Rate is 35%, even 0.04 ‰ concentration, and growths of the DMSO to cell also has detrimental effect, can avoid the rich of microorganism
Degree changes；7) mineral oil is also known as paraffin oil, is the mixture from the colorless and odorless obtained by crude cut, and it is segmented into
Two kinds of light mineral oil and general mineral oil, and the proportion of light mineral oil and stickiness are relatively low, it has low irritability and not
Wrong closure, microbiological specimens is mixed into after preservation liquid, mineral oil floats on mixed liquor upper surface, can effectively obstruct air,
The metabolism of microorganism is reduced, can also be prevented effectively from for anerobe and change its living environment, while can keep away
Non-volatile material is escaped.
By such scheme, the present invention at least has advantages below：Formula, which is prepared, more than preserves liquid, by the micro- of collection
Biological sample, is placed directly within preservation liquid, without freezing, you can reach the effect that stable wherein microorganism is constituted, also applicable
In long-distance transport.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the gut flora Shannon diversity index result figure under 4 kinds of processing modes of volunteer of the present invention.
Embodiment
With reference to the accompanying drawings and examples, the embodiment to the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
The sample source of embodiment one and packet preserving type
Because biological preserve of fecal microorganism sample requires higher, a preferred embodiment of the present invention uses the micro- life of human faecal mass
Thing sample is tested as follows as laboratory sample.
S1, the fecal sample for gathering 5 volunteers, 18 parts of the sample collection of each volunteer do 6 kinds of different places respectively
Reason, every kind of processing repeats experiment 3 times；
S2, Different treatments
A, freeze one week (label 0)：After collection at least 0.2g fecal specimens, liquid nitrogen flash freezer, sealing preserve at -80 DEG C is protected
Progress DNA extractions after one week are deposited, 16S rRNA gene V3/V4 variable regions amplification, library construction, sequencing etc. is handled；
B, preservation liquid preserve one week (label 1-3)：Collection at least 0.2g fecal specimens, use 3 kinds of different formulations of the invention
(composition is as shown in table 1) (25 DEG C) sealings at room temperature, are kept in dark place one week, then carry out DNA extractions, 16S rRNA genes V3/
V4 variable regions are expanded, the processing such as library construction, sequencing；
C, preservation liquid preserve one week (label 4)：Collection at least 0.2g fecal specimens, use preservation common in the art
Formula of liquid (composition is as shown in table 2) (25 DEG C) sealings at room temperature, are kept in dark place one week, then carry out DNA extractions, 16S rRNA bases
Because of the amplification of V3/V4 variable regions, the processing such as library construction, sequencing；
D, preservation without any processing one week (label 5)：Collection at least 0.2g fecal specimens, (25 DEG C) sealings at room temperature,
It is kept in dark place one week, then carries out DNA extractions, 16S rRNA gene V3/V4 variable regions amplification, library construction, sequencing etc. is handled.
The microbiological specimens DNA of the present invention of table 1 preserves formula of liquid
| Composition/label | 1 | 2 | 3 |
| EDTA(mM/L) | 2 | 1 | 0.5 |
| NaCl | Saturation | Saturation | Saturation |
| Methanol (%) | 2 | 4 | 5 |
| Glycerine (%) | 5 | 10 | 15 |
| AEO (%) | 5 | 8 | 10 |
| SDS (%) | 1 | 3 | 5 |
| DMSO (%) | 5 | 8 | 10 |
| Ethanol (%) | 2 | 3 | 5 |
| pH | 9 | 8 | 6 |
The prior art of table 2 preserves liquid component list
| Disodium ethylene diamine tetraacetate (EDTA-2Na) | 2% |
| Different sulfuric acid cyanic acid guanidine | 0.47% |
| Sodium chloride (NaCl) | 0.9% |
| pH | 7.4 |
The fecal sample processing method of embodiment two
The present embodiment provides a kind of processing method to above-mentioned fecal sample：
S1, control sample pretreatment：
Solid manure (counter sample is marked as 0 and 5)：
200mg solid manures are weighed in 2ml centrifuge tubes, 800ul stool DNA Buffer A, fully shaking is added
Mix 5min or so, 1800g centrifugations 1min；
50ul re-suspension liquids are taken out in clean 1.5ml centrifuge tubes, 800ul Lysis-Binding are added
The concussion of Buffer vortexs is mixed, 70 DEG C of cracking 5min.Centrifuge and supernatant is shifted after 5min into clean 1.5ml centrifuge tubes.
S2, experimental group sample pretreatment
Excrement preserves liquid (counter sample is marked as 1-4)：
The stool DNA Buffer A for taking the excrement of 200ul semi-liquid stages to add no more than 10% (w/v) carry out dilute
Release, fully shaking 5min, be taken out 50ul re-suspension liquids to clean 1.5ml centrifuge tubes.
S3, DNA are extracted
A, the magnetic bead 20ul for adding mixing, vortex concussion 20s are stored at room temperature 4min, vortex concussion 20s, are stored at room temperature
4min.
B, it is placed on magnet stand, static 20s, draws supernatant.
C, addition 500ulWash Buffer W1, vortex concussion mix magnetic bead 20s.
D, it is placed on magnet stand, stands 20s, abandon supernatant.
E, repeat step d once, and remove all liq as far as possible.
F, addition 750ul Wash Buffer W2, vortex concussion mix magnetic bead 20s.
G, it is placed on magnet stand, stands 20s, abandon supernatant.
H, repeat step f-g once, and remove all liq as far as possible.
I, the dry 7-8min that uncaps is placed on magnetic frame, is inhaled with rifle and abandon all liquid.
J, addition 50ul Elution Buffer or ddH2O, vortex concussion mix magnetic bead 15s.
K, 65 DEG C, 7min (period vortex shakes once, shakes 10s).Vortex concussion mixes magnetic bead 15s.
L, it is placed on magnetic frame, stands 2min, sucts clear in collecting pipe.
S4, DNA sequencing
DNA after extraction is sent to sequencing company and carries out building storehouse sequencing, obtains and subsequent bio information point is carried out after sequencing data
Analysis, obtains flora result.
The different preserving type sample bacterial diversity of embodiment three compares
By above-mentioned experimental implementation, the gut flora sequence information under 5 volunteers, 6 kinds of processing modes is obtained, is passed through
Bioinformatic analysis, calculates flora Shannon diversity index, is compared.Each volunteer after testing after shannon index as scheme
Shown in 1, as shown in table 3, the higher abundant degree for representing bacterium of Shannon diversity index numerical value is higher in table for the result after statistics,
P value are the Analysis on confidence that label 0-4 sample and the sample of label 5 compare obtained otherness, are as a result shown, label
0-4 sample can meet the preservation of fecal microorganism sample DNA substantially, however with preservation liquid (label 4) phase of prior art
Than microbiological specimens of the invention preserve the treatment effect of liquid closer to the processing method of liquid nitrogen flash freezer, and especially label 2 is formulated
Preservation liquid.
Shannon index result under the Different treatments of table 3
| Composition/label | 0 | 1 | 2 | 3 | 4 | 5 |
| Shannon index | 9.5 | 7.5 | 9.4 | 8.5 | 6.3 | 3.2 |
| P value | ＜ 0.01 | ＜ 0.01 | ＜ 0.01 | ＜ 0.01 | ＜ 0.05 | - |
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of microbiological specimens DNA preserves liquid, it is characterised in that：Wherein be dissolved with pure water 0.5~2mM/L EDTA,
The fat that glycerine that methanol that saturated sodium-chloride, mass fraction are 2~5%, mass fraction are 5-15%, mass fraction are 5-10%
Fat alcohol APEO, mass fraction are 1-5% lauryl sodium sulfate and the mineral oil of mixing, and adjust pH to 6.0-
9.0。
2. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：It is 5- also to include mass fraction
10% dimethyl sulfoxide (DMSO).
3. microbiological specimens DNA according to claim 2 preserves liquid, it is characterised in that：The quality of the dimethyl sulfoxide (DMSO)
Fraction is 8%.
4. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：It is 2-5% also to include mass fraction
Ethanol.
5. microbiological specimens DNA according to claim 4 preserves liquid, it is characterised in that：The mass fraction of the ethanol is
3%.
6. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：The microbiological specimens DNA is preserved
1mM/L EDTA is dissolved with liquid.
7. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：The mass fraction of the methanol is
4%.
8. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：The mass fraction of the glycerine is
10%.
9. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：The AEO
Mass fraction be 8%.
10. microbiological specimens DNA according to claim 1 preserves liquid, it is characterised in that：The lauryl sodium sulfate
Mass fraction is 3%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710347085.4A CN107227345A (en) | 2017-05-17 | 2017-05-17 | A kind of microbiological specimens DNA preserves liquid |
Applications Claiming Priority (1)
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108209979A (en) * | 2018-01-04 | 2018-06-29 | 福建朗盛生物科技股份有限公司 | A kind of Medical oral cavity cast-off cells harvester |
| CN108546646A (en) * | 2018-04-18 | 2018-09-18 | 成都知己基因生物科技有限公司 | Micro-biological samples for genetic test preserve liquid and preparation method thereof, kit and application |
| CN109609600A (en) * | 2019-01-31 | 2019-04-12 | 浙江大学 | DNA preservation solution for stool samples at room temperature and preparation method and use thereof |
| CN110004212A (en) * | 2019-04-23 | 2019-07-12 | 康美华大基因技术有限公司 | The method that excrement saves liquid and preparation method thereof and saves excrement |
| CN110257369A (en) * | 2019-05-29 | 2019-09-20 | 杭州高六博生物科技有限公司 | A kind of room-temperature extender of enteric microorganism sample DNA |
| CN111631758A (en) * | 2020-06-09 | 2020-09-08 | 温州芳植生物科技有限公司 | Structure and method for preserving sampling material in gastrointestinal tract |
| CN112522362A (en) * | 2020-12-25 | 2021-03-19 | 南京申友生物技术有限公司 | Preservation solution for preserving bacterial DNA in fecal sample at normal temperature |
| WO2021248779A1 (en) * | 2020-06-12 | 2021-12-16 | 广州达安基因股份有限公司 | Swab sample nucleic acid releaser and application thereof |
| CN115011671A (en) * | 2022-07-26 | 2022-09-06 | 郑州标源生物科技有限公司 | Preparation method of respiratory tract nucleic acid composite quality control product |
| CN115728490A (en) * | 2022-09-22 | 2023-03-03 | 大连博格林生物科技有限公司 | Application of sealant in protein-free sealant |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844368A (en) * | 2006-04-27 | 2006-10-11 | 清华大学 | Process for storing methane oxidizing bacteria |
| CN101984804A (en) * | 2010-11-03 | 2011-03-16 | 西安医学院 | Corpse preservation solution and preparation method thereof |
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
| CN106596211A (en) * | 2015-10-15 | 2017-04-26 | 深圳华大基因研究院 | Stool sample preservation liquid, preparation method and application thereof |
-
2017
- 2017-05-17 CN CN201710347085.4A patent/CN107227345A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844368A (en) * | 2006-04-27 | 2006-10-11 | 清华大学 | Process for storing methane oxidizing bacteria |
| CN101984804A (en) * | 2010-11-03 | 2011-03-16 | 西安医学院 | Corpse preservation solution and preparation method thereof |
| CN106596211A (en) * | 2015-10-15 | 2017-04-26 | 深圳华大基因研究院 | Stool sample preservation liquid, preparation method and application thereof |
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108209979A (en) * | 2018-01-04 | 2018-06-29 | 福建朗盛生物科技股份有限公司 | A kind of Medical oral cavity cast-off cells harvester |
| CN108546646A (en) * | 2018-04-18 | 2018-09-18 | 成都知己基因生物科技有限公司 | Micro-biological samples for genetic test preserve liquid and preparation method thereof, kit and application |
| CN109609600A (en) * | 2019-01-31 | 2019-04-12 | 浙江大学 | DNA preservation solution for stool samples at room temperature and preparation method and use thereof |
| CN110004212A (en) * | 2019-04-23 | 2019-07-12 | 康美华大基因技术有限公司 | The method that excrement saves liquid and preparation method thereof and saves excrement |
| CN110257369A (en) * | 2019-05-29 | 2019-09-20 | 杭州高六博生物科技有限公司 | A kind of room-temperature extender of enteric microorganism sample DNA |
| CN111631758A (en) * | 2020-06-09 | 2020-09-08 | 温州芳植生物科技有限公司 | Structure and method for preserving sampling material in gastrointestinal tract |
| WO2021248779A1 (en) * | 2020-06-12 | 2021-12-16 | 广州达安基因股份有限公司 | Swab sample nucleic acid releaser and application thereof |
| US11753672B2 (en) | 2020-06-12 | 2023-09-12 | Da An Gene Co., Ltd. | Swab sample nucleic acid releaser and use thereof |
| CN112522362A (en) * | 2020-12-25 | 2021-03-19 | 南京申友生物技术有限公司 | Preservation solution for preserving bacterial DNA in fecal sample at normal temperature |
| CN115011671A (en) * | 2022-07-26 | 2022-09-06 | 郑州标源生物科技有限公司 | Preparation method of respiratory tract nucleic acid composite quality control product |
| CN115728490A (en) * | 2022-09-22 | 2023-03-03 | 大连博格林生物科技有限公司 | Application of sealant in protein-free sealant |
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