CN111235142A - Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition - Google Patents
Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition Download PDFInfo
- Publication number
- CN111235142A CN111235142A CN202010064011.1A CN202010064011A CN111235142A CN 111235142 A CN111235142 A CN 111235142A CN 202010064011 A CN202010064011 A CN 202010064011A CN 111235142 A CN111235142 A CN 111235142A
- Authority
- CN
- China
- Prior art keywords
- microorganisms
- dna
- buffer solution
- substrate
- total dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 69
- 244000005700 microbiome Species 0.000 title claims abstract description 59
- 239000002253 acid Substances 0.000 title abstract description 29
- 239000007853 buffer solution Substances 0.000 claims abstract description 36
- 238000005406 washing Methods 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 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 13
- 239000011159 matrix material Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 37
- 230000002378 acidificating effect Effects 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 20
- 238000005119 centrifugation Methods 0.000 claims description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- SDRZXZKXVBHREH-UHFFFAOYSA-M potassium;dihydrogen phosphate;phosphoric acid Chemical compound [K+].OP(O)(O)=O.OP(O)([O-])=O SDRZXZKXVBHREH-UHFFFAOYSA-M 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 238000013329 compounding Methods 0.000 claims 1
- 229920002477 rna polymer Polymers 0.000 abstract description 24
- 230000000813 microbial effect Effects 0.000 abstract description 20
- 238000012163 sequencing technique Methods 0.000 abstract description 16
- 238000000246 agarose gel electrophoresis Methods 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 53
- 238000000605 extraction Methods 0.000 description 24
- 239000000945 filler Substances 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 16
- 241001052560 Thallis Species 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 15
- 238000011010 flushing procedure Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 13
- 238000006477 desulfuration reaction Methods 0.000 description 13
- 230000023556 desulfurization Effects 0.000 description 13
- 108010067770 Endopeptidase K Proteins 0.000 description 11
- 239000000499 gel Substances 0.000 description 11
- 230000006872 improvement Effects 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 239000003292 glue Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- 102000016943 Muramidase Human genes 0.000 description 9
- 108010014251 Muramidase Proteins 0.000 description 9
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 9
- 239000004325 lysozyme Substances 0.000 description 9
- 229960000274 lysozyme Drugs 0.000 description 9
- 235000010335 lysozyme Nutrition 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 238000007400 DNA extraction Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 229910001385 heavy metal Inorganic materials 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 6
- 239000013614 RNA sample Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 5
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000007613 environmental effect Effects 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000003546 flue gas Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000004021 humic acid Substances 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000012160 loading buffer Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000001360 synchronised effect Effects 0.000 description 4
- 238000002137 ultrasound extraction Methods 0.000 description 4
- 238000003287 bathing Methods 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011066 ex-situ storage Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 238000007429 general method Methods 0.000 description 2
- 239000003864 humus Substances 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 230000007064 DNA hydrolysis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000589651 Zoogloea Species 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000013590 bulk material Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- -1 hydrogen ions Chemical class 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting total DNA of microorganisms in a substrate-attached biomembrane under a strong acid condition, and belongs to the technical field of molecular biology. The method comprises the following steps: 1) eluting microorganisms from the attachment matrix by using a buffer solution 1 with the pH value of 4-5 under proper water pressure, and centrifugally collecting a thallus sample; 2) washing with a buffer solution 2 containing EDTA and having a pH value of 7-7.5, and centrifuging to collect a thallus sample; 3) extracting DNA from the thallus sample collected in the step 2). The microbial total ribonucleic acid extracted by the method has good quality, the total band obtained by detecting the method by adopting 1 percent agarose gel electrophoresis is bright and clear, the length and the concentration of the DNA chain meet the requirements of sequencing and library building when the DNA is subjected to on-machine sequencing, and the method lays a technical foundation for microbial ecology and molecular biology research under the strong acid condition.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for extracting microbial total DNA attached to a substrate under a strong acid solution condition.
Background
In recent years, with the development of sequencing technology, the metagenomic technology for sequencing the total DNA of microorganisms in a certain environment is widely used in the analysis of microbial diversity and functions, and the metagenomic technology is recognized as the most comprehensive and accurate method for researching the structure and functional genes of the microbial community in the environment at present because it has no special selectivity (such as pure culture/enrichment) and simultaneously reduces the preference of the sequencing technology (such as the PCR amplification step of 16S rRNA or iTS gene).
However, the analysis of microbial communities under extremely acidic conditions using molecular biology techniques presents technical difficulties, mainly due to the following: the extraction and purification of the total genome DNA of the environmental sample are difficult, the extreme acid environment (pH <2) can bring serious survival pressure to the microorganism, and once the microbial thallus is broken, the strong acid environment can cause the DNA or RNA to be degraded into short-chain small fragments in a very short time. In order to adapt to the complex habitat, the microorganisms can generate special polymerase by themselves, and the special polymerase constitutes a biological membrane, a bacterial colony and the like to resist the survival pressure under the complex habitat. However, various inhibitors (hydrogen ions, humic acid, phenolic compounds, heavy metal ions and the like) existing in the environment can affect the enzyme activity of microorganisms under the condition of low pH value, and cause instability of ribonucleic acid molecules, so that denaturation of protein and hydrolysis of DNA are directly triggered, humic acid substances widely existing in an environment sample are tightly combined with the ribonucleic acid, so that the humic acid substances and the ribonucleic acid are co-extracted, and the difficulty of downstream experiments is further increased.
At present, many researchers adopt different methods to pretreat microorganisms under acidic conditions so as to meet the requirement of total DNA extraction. These methods can be largely classified into two types, one is an in situ extraction method in which total DNA is extracted at the original location where the microorganism grows by sequentially adding different reagents. In "extraction of microbial DNA in acidic drainage sediments by multiple methods", the yellow sea adopts four methods to extract ribonucleic acid of a sample (pH 2.47-2.78): high salt freeze thawing method, low salt protease K method, high salt protease K method and ZR Soil Microbe ribonucleic acid KitTM method. Finally, it was concluded that a good effect could be achieved by pre-washing the sample one to two times in a short time with a strong dispersant, then lysing the cells in an extraction buffer with low salt content using the extraction step of the low-salt proteinase K method (lysozyme + proteinase K + glass bead impact lysis), purifying the extracted crude ribonucleic acid using the common gel electrophoresis-low melting gel bath method followed by PCR amplification.
Another method is ex-situ extraction in which the entire microbial community is washed from the attachment substrate with a reagent and then extracted. In the Aneffective method of DNA extraction for bioleaching microbial genomic ribonucleic acid, Zeng et al concluded that the method for extracting RNA from acidic mine waste water (pH 4.0) was to first wash AMD sediment samples with PBS solution, then place the collected cells in An extraction buffer solution containing SDS and CTAB, incubate them in boiling water bath at 60 + -5 deg.C and 72 deg.C, and finally extract and precipitate nucleic acids with phenol/chloroform/ethanol and glacial ethanol.
Although there are various extraction methods developed above for different researchers, these methods are directed to a medium strong acid environment with an environmental solution pH of 2 or more, and a general method capable of extracting total DNA of an environmental sample in a strong acid solution environment (pH <2) has not been found. And different from a pure solution environment, in an environment sample attached to a solid matrix, the content of impurities such as heavy metal, humic acid and the like is high, the components are complex, and the seeking of a general method is more difficult.
At present, the patent related to extracting ribonucleic acid of microbial genome under acidic condition also discloses related applications, such as chinese patent application No. 200910237938.4, published date is 2011, 6 months and 1 days, the inventive name is: the invention relates to a method for extracting acid soil genome with high quality, which comprises the steps of taking 10g of soil sample, mixing with sterile sodium pyrophosphate solution uniformly, incubating for 30min, and extracting genome ribonucleic acid.
Based on the defects of the prior art, it is necessary to research a targeted and suitable ribonucleic acid extraction method for microorganisms on a biofilm packed tower under the condition of an acid solution.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems that microorganisms are easy to inactivate after being separated from an attached matrix and a bacterial colony and a biomembrane environment under a strong acid condition (pH is less than 2), and the chromosome of the microorganisms is quickly degraded due to the rupture of the cell membrane/wall after inactivation, so that the fragmentation of DNA long chain molecules is caused, and the quality requirement of sequencing cannot be met by an extract, the total ribonucleic acid of the microorganisms extracted by the method disclosed by the invention is better in quality, a total strip obtained by adopting 1% agarose gel electrophoresis detection is bright and clear, the length and the concentration of the DNA chain accord with the requirements of sequencing and library building during on-machine sequencing, and a technical basis is laid for the microbial ecology and molecular biology research under the strong acid condition.
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for extracting total DNA of microorganisms in a substrate-attached biomembrane under a strong acid condition, which comprises the following steps:
1) eluting microorganisms from the attachment matrix by using a buffer solution 1 with the pH value of 4-5 under proper water pressure, and centrifugally collecting a thallus sample;
2) washing with a buffer solution 2 containing EDTA and having a pH value of 7-7.5, and centrifuging to collect a thallus sample;
3) extracting DNA from the thallus sample collected in the step 2).
The substrate surface comprises various substrate material surfaces for the attachment and growth of microorganisms and also comprises inner surfaces which can be exposed to the attachment and growth of microorganisms by crushing the material; the total DNA is the sum of DNA contained by all microorganisms attached to the surface of the substrate.
As a further improvement of the invention, the suitable water pressure is 30-120 MPa.
In a further improvement of the present invention, the molar concentration of EDTA contained in the buffer solution 2 is 50 to 100 mM.
As a further improvement of the invention, the buffer solution 2 is a PBS-Tris buffer solution, and the buffer solution contains a Tris-HCl solution with a molar concentration of 50-100 mM.
The buffer solution 2 is prepared by mixing a PBS solution and a Tris-HCl buffer solution, and then EDTA is added, wherein the molar concentration of the EDTA is 50-100 mM.
As a further improvement of the invention, the elution in step 1) is carried out in no more than 90 seconds.
As a further improvement of the invention, in the step 2), the washing times of the buffer solution 2 are 2-3 times, the washing time is 2-5 minutes, and the volume used for each washing is 20-50 mL, preferably 40 mL.
As a further improvement of the invention, the buffer solution 1 comprises an isotonic buffer solution with the pH value of 4-5 or a simulated solution which is compounded by other chemical components in the environmental liquid of the microorganism and is adjusted to be acidic to the pH value of 4-5 by sulfuric acid,
as a further improvement of the invention, the buffer solution 1 comprises a phosphate buffer solution or a phosphate-potassium dihydrogen phosphate buffer solution, and the volume of the buffer solution 1 is 50-100 mL.
As a further improvement of the invention, the conditions for centrifugally collecting the thallus sample in the step 1) and the step 2) are as follows: the speed is 5000-6000 g, the centrifugation time is 2-5 minutes, and the centrifugation temperature is 4 ℃.
As a further improvement of the invention, the method for extracting the total DNA in the step 3) comprises a freeze-thaw method or an ultrasonic method.
As a further development of the invention, the strongly acidic condition is an environmental condition with a pH < 2.
As a further improvement, the invention provides a method for extracting the total DNA of microorganisms attached to a substrate under the condition of a strong acid solution, which comprises the following steps:
step A, collecting a functional flora sample: if the substrate attached to the microorganisms is in a shape which is easy to obtain, such as a small block shape, the substrate material with a good microorganism growth state and the volume of which is not more than 0.5L is put into a beaker with 1L, the part attached with the bacteria on the substrate material is washed by buffer solution 1 and high-strength water pressure (30-120MPa, and pipelines need acid corrosion resistance) to be quickly separated, all the parts are collected in the beaker, the substrate material is discarded, and the bacteria are collected after centrifugation at the temperature of 5000-6000 g for 2-5 minutes; if the microbial community needing to be extracted also grows in the substrate, the substrate can be firstly crushed, the inner surface of the growing microbial community is exposed, then washing is carried out, attached microorganisms are quickly stripped, and centrifugation collection is carried out (5000 g-6000 g); if the matrix is a bulk material which is difficult to obtain, or the surface of a rock wall which is not easy to damage and the like, the water pressure and the flushing direction are carefully controlled, and after a beaker, a test tube or other containers for holding the thalli are contacted with the surface of the matrix, the containers are connected to the bacterial colony when the bacterial colony is washed and fallen off, so that the thalli under washing are prevented from falling off, and sampling failure is caused.
Under the condition of adjusting the water pressure to be 30-120MPa, the washing time is not more than 90 seconds, and the microbial flora attached to the surface of the substrate can fall off from the substrate. Through the step, the environment of the thallus is transited from the extreme acid condition to the moderate acid condition.
Step B, pretreatment of the sample: washing the thalli obtained in the step A by using a buffer solution 2(PBS buffer solution 1 multiplied by PBS + 50-100 mM Tris-HCl + 50-100 mM EDTA) for 2-3 times, wherein the washing time is 2-5 minutes, and the volume used for washing each time is 20-50 mL; after each washing, the cells were collected in a 1.5mL centrifuge tube by centrifugation (5000g) for 2 minutes at 4 ℃ and the supernatant was discarded, and the cell pellet was completely immersed in 70% ethanol. Selecting appropriate storage conditions according to the time interval of further processing, and storing in-80 deg.C refrigerator if long-term storage is required.
Step C, extracting ribonucleic acid of the sample genome: and (3) thawing the thalli in the step (2), breaking the thalli by using an ultrasonic method, a repeated freezing and thawing method or other methods, and extracting total DNA by using a soil genome total DNA extraction kit to obtain the microbial total ribonucleic acid in the biofilm packed tower for flue gas desulfurization and denitrification under the strong acid condition.
Step D, detecting ribonucleic acid of the sample genome: and D, taking trace of the total ribonucleic acid obtained in the step C, using a trace ultraviolet visible spectrophotometer to determine the content of the ribonucleic acid, and using agarose gel electrophoresis with the mass percentage concentration of 1% to detect the quality of the microbial total ribonucleic acid.
As a further improvement of the invention, in the step D, the genomic ribonucleic acid extraction kit is 6560-200 type Fast produced by MPBiomedia company
SPIN kit for Soil kit.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) compared with the traditional ex-situ extraction method, the extraction method of the total DNA of the microorganisms attached to the substrate under the strong acid condition adopts the medium-strong acid buffer solution and the high-strength water pressure, can quickly wash the microorganisms attached to the substrate, and has higher extraction speed, wherein the microorganisms comprise living microorganisms which are easy to strip and tightly combined with the substrate or are positioned in a biological membrane or a bacterial colony; the traditional ectopic extraction method is easier to obtain dead bacteria separated from the matrix, and the extraction time is long, so that the viable bacteria ratio is further reduced;
(2) compared with the existing in-situ extraction method, the method disclosed by the invention avoids the influence of a large amount of impurities introduced during in-situ extraction, particularly easily soluble humic acid and heavy metal ions, on microbial DNA polymerase, shortens the extraction time caused by impurity cleaning, causes the death of microorganisms under the extremely strong acidic condition, and causes the serious degradation phenomenon of DNA.
(3) The method for extracting the total DNA of the microorganisms attached to the substrate under the strong acid condition comprises the steps of adjusting the pH of the bacterial liquid in two steps, so that death and breakage of flushed bacteria caused by severe pH change in the environment can be avoided, meanwhile, the buffer solution 2 adopts a buffer solution containing EDTA with appropriate concentration, so that heavy metal chelate ions in the bacteria and humic substances can be well adsorbed, inhibitors such as heavy metals, humic substances and the like can be separated from ribonucleic acid, the risk of inhibitor pollution is reduced to the greatest extent, and the extraction efficiency in downstream operation is remarkably improved.
(4) The extraction method of the invention obviously improves the extraction efficiency and quality of the genome DNA of the matrix-attached microorganism under the strong acid condition, the total band obtained by gel electrophoresis detection is over 500bp, the band is bright and clear, basically has no degradation, and the determination on a second-generation sequencing computer is accurate, thus the growth and evolution conditions of the matrix-attached microorganism under the extreme acid condition can be more accurately and comprehensively reflected, and the invention lays a technical foundation for the subsequent researches on microorganism species composition, gene function, signal path and the like, in particular for the whole genome splicing assembly and functional gene prediction of dominant species.
Drawings
FIG. 1 is a graph showing the ratio of 260/280 UV OD values of genomic ribonucleic acid samples extracted in examples 1 and 2;
FIG. 2 is a gel diagram of the genomic ribonucleic acid samples extracted in examples 1 and 2;
FIG. 3 is a bacterial genus distribution diagram of the extracted microbial DNA of example 2 after metagenomic sequencing compared with NCBI database;
FIG. 4 is a graph showing the ratio of 260/280 UV OD values of the genomic ribonucleic acid sample extracted in comparative example 1;
FIG. 5 is a gel diagram of a genomic ribonucleic acid sample extracted in comparative example 1;
FIG. 6 is a graph showing the ratio of 260/280 UV OD values of the genomic ribonucleic acid sample extracted in comparative example 2;
FIG. 7 is a gel diagram of a genomic ribonucleic acid sample extracted in comparative example 2;
FIG. 8 is a graph showing the ratio of 260/280 UV OD values of the genomic ribonucleic acid sample extracted in comparative example 3;
FIG. 9 is a gel diagram of the genomic ribonucleic acid sample extracted in comparative example 3.
Detailed Description
In order to further understand the content of the present invention, the following examples are provided to further describe the method for extracting total DNA of microorganisms in a flue gas desulfurization and denitrification biomembrane packed tower under strong acidic conditions.
Example 1
The embodiment is directed to a method for extracting total DNA of microorganisms attached to the surface of a filler under the condition of super-strong acid circulating liquid in a flue gas desulfurization and denitrification biomembrane packed tower, which comprises the following steps:
step one, collecting a functional flora sample: from a stably operating biofilm packed tower with synchronous desulfurization and denitrification, the biofilm packed tower is the same as the device in the Chinese patent application No. CN 2019101044777.
Taking about 0.5L of ceramsite filler with good microorganism growth state in a circulating nutrient solution, placing the ceramsite filler in a beaker with the volume ratio of 1L, wherein the pH of the nutrient solution is 0-2, the diameter of the ceramsite filler in the embodiment is 50-200 mm, and the bulk density is 200kg/m3A specific surface area of 170 to 200m2/m3And a porosity of 53%. And (3) filling 100mL of the prepared phosphoric acid buffer solution with the pH value of 4-5 into a water pressure container, flushing the filler by adopting the pressure of 90MPa for no more than 90s, flushing the thalli attached to the filler, and transitioning from the extreme acidic condition to the moderate acidic condition. The filling was removed from the beaker, the bacterial suspension was poured into a 50mL centrifuge tube and centrifuged at 5,000g at 4 ℃ for 2 minutes until the bacterial suspension was completely centrifuged.
Step two, pretreatment of the sample: and (2) washing the thalli obtained in the first step by adopting a PBS (1 XPBS + 50-100 mM Tris-HCl + 50-100 mM EDTA) buffer solution with the pH value of 7-7.5, wherein the volume for washing is 40mL, slightly reversing and shaking the thalli in a centrifuge tube for 2 minutes, after washing, centrifuging the thalli for 2 minutes at the rotating speed of 5,000g and the temperature of 4 ℃, collecting the thalli, washing twice, removing a supernatant, and eluting heavy metal ions and humus on the thalli as much as possible. Re-dissolving the thalli by using 70% ethanol solution to obtain a sample S1-1; taking the same amount of the filler with good growth vigor of the biomembrane of the same layer, repeating the operation, adjusting the washing pressure and the washing volume, repeatedly washing by adopting 500mL of phosphate buffer solution at 150MPa, and completely stripping the biomembrane to obtain a sample S1-2; the above operation was repeated, and a volume was washed with 10mL of phosphate buffer solution at 30MPa to obtain sample S1-3. So far, the next experiment can be carried out, and if the next experiment is not carried out immediately, the sample needs to be stored in a refrigerator at minus 80 ℃.
Step three, extracting the genomic DNA of the sample: thawing the thallus obtained in the step two, crushing by using an ultrasonic crusher, performing ultrasonic treatment with 60% energy for 3s, pausing for 2s, performing ultrasonic treatment for 2 minutes totally, and then using 6560-type 200-type food additive manufactured by MP Biomedicals company to completely crack the thallus
The SPIN kit for Soil kit is used for carrying out DNA extraction operation according to the flow steps suggested by the specification to obtain the microbial total DNA in the biomembrane packed tower for flue gas desulfurization and denitrification under the strong acid condition.
Step four, detecting the genomic DNA of the sample: and taking 2 microliters of DNA obtained in the step three, using a trace ultraviolet-visible spectrophotometer to measure the DNA content, and reading the DNA concentration and the 260/280nm ultraviolet OD value. And then 4 microliters of DNA is taken and mixed with 1 microliter of loading buffer uniformly, and the mass of the total DNA of the microorganism is detected by using agarose gel electrophoresis with the mass percentage concentration of 1 percent, wherein the concentration of the agarose used is 1 percent, the electrophoresis voltage is 120V, and the electrophoresis time is 20 min.
Wherein the ratio of the 260/280nm ultraviolet OD values of S1-1, S1-2 and S1-3 is shown in FIG. 1, and the glue maps of S1-1, S1-2 and S1-3 are shown in FIG. 2, according to the graph, the DNA quality of the sample S1-1 is best, the glue maps show bright bands with uniformly distributed long tails with high molecular weight, and the ratio of the 260/280 ultraviolet OD values is about 1.78, which indicates that the protein pollution is less. And after the washing liquid amount is increased in S1-2, protein pollution is increased, the ratio of the 260/280 ultraviolet OD value is reduced to about 1.58, and a glue pattern shows obvious bright spots at the front end of a small molecular weight, which shows that DNA is degraded, probably because dead bacteria are increased after washing and the content of the DNA after the introduced dead bacteria are degraded is increased after the washing pressure is increased, or because the death of the bacteria caused by the separation of microorganisms from the attached matrix and the protection structure of a biological membrane or a bacterial colony is caused by the extension of the washing time, therefore, the excessive increase of the washing amount and the extension of the washing time cannot increase the total amount of DNA extraction, influence the extraction quality and cause that the extracted DNA sample cannot be applied to on-machine sequencing. On the other hand, the S1-3 sample is more seriously polluted by protein, the ratio of the 260/280 ultraviolet OD value is below 1.5, and the DNA gel graph shows fuzzy bright bands concentrated at the two ends of the maximum molecular weight and the minimum molecular weight, and the result shows that when the impact water pressure and the impact volume are too small, the bacteria quantity washed down is insufficient, and the protein protective layer and the surface layer dead bacteria on the surface of the biomembrane or the zoogloea are contained in the washed down part, so that the extraction quality is influenced.
Example 2
The embodiment is directed to a method for extracting total DNA of microorganisms attached to the interior of a filler under the condition of super-strong acid circulating liquid in a flue gas desulfurization and denitrification biomembrane packed tower, which comprises the following steps:
step one, collecting a functional flora sample: from a biomembrane packed tower for synchronous desulfurization and denitrification, about 0.5L of high polymer acid-resistant plastic packing with good microorganism growth state is placed in a 1L beaker, the diameter of the plastic packing in the embodiment is about 50mm, and the bulk density is 500kg/m3Specific surface area of 700m2/m3Porosity 75%, the surface of the filler was physically broken to expose the internal porous surface. And (3) filling 80mL of prepared phosphoric acid-potassium dihydrogen phosphate buffer solution with the pH value of 4-5 (the flushing volume is determined according to the number and the area of the broken surfaces) into a hydraulic container, flushing the filler by adopting the pressure of 80MPa, flushing the thalli attached to the filler, and transitioning from the extreme acidic condition to the moderate acidic condition. The filling was removed from the beaker, the bacterial suspension was poured into a 50mL centrifuge tube and centrifuged at 6,000g at 4 ℃ for 5 minutes until the bacterial suspension was completely centrifuged. Obtaining a sample I1-1, taking the same amount of filler with good growth vigor of the biomembrane of the same layer, repeating the operation, adjusting the flushing time and the flushing volume, adopting 300mL and 121MPa for repeated flushing, completely stripping the biomembrane from the inside of the sphere, obtaining a sample I1-2, adopting 10mL of flushing volume and 20MPa of water pressure, and flushing the inner pores to obtain a sample I1-3.
The subsequent steps were the same as in example 1. Wherein the ratio of 260/280nm ultraviolet OD values of I1-1, I1-2 and I1-3 is shown in FIG. 1, the glue maps of I1-1, I1-2 and I1-3 are shown in FIG. 2, and in the glue map of FIG. 2, M represents Marker.
As shown, the flushing volume and water pressure of the I1-1 sample and the corresponding flushing time are suitable, so that the obtained results are better, and the reasons for the poor results of I1-2 and I1-3 are similar to those of S1-2 and S1-3 respectively.
After the DNA samples obtained according to the step are subjected to on-machine sequencing, sequencing data of 0-200G/sample can be obtained according to requirements, the sequencing requirements are completely met, 3 samples are taken for parallel experiments to perform sequencing, the sequencing depth of each sample is about 20G, the species information is annotated to the genus after the results are processed by bioinformatics to obtain a figure 3, the relative abundance of the level of the bacteria in the figure 3 is compared, the repeatability of microorganisms attached to the surface or the inner matrix is better, and the extraction method is stable and reliable.
After the obtained DNA is processed by bioinformatics, a large number of key node genes on a sulfur metabolism path can be found by carrying out annotation analysis on the obtained DNA and a KEGG database, and the DNA extracted by the method meets the requirement of molecular signal path analysis. And performing splicing assembly and single bacterium box separation analysis on the obtained macro gene data to construct 18 single bacteria, wherein 10 single bacteria have a completeness degree of more than 94% after the pollution rate is deducted, which indicates that the length of the DNA chain extracted by the method is enough to meet the requirement of deep excavation of macro gene information.
Comparative example 1
In this example, a strong soil DNA extraction kit from QIAGEN, USA was used
The DNASsolalation Kit ectopically extracts the genome DNA under the extreme acid condition, and the steps are as follows:
1. about 0.5L of ceramsite filler with good microorganism growth state is put into a beaker with 1L from a biofilm filler tower with synchronous desulfurization and denitrification, independent desulfurization and independent denitrification, and the filler is immersed by using circulating nutrient solution used by a reactor. And (3) putting the beaker in an ultrasonic cleaner, carrying out ultrasonic treatment at the power of 300W for 5min at 50kHz, taking out the filler, pouring the bacterial suspension into eight 50mL centrifuge tubes, centrifuging at 6000g for 5min, removing supernatant, and combining precipitated bacteria to obtain a sample R1. The same amount of filler is taken and the steps are repeated, only the ultrasonic extraction is changed into the process of placing the beaker on a shaker for 100 revolutions per minuteCarrying out oscillation extraction for half an hour, and then centrifuging the bacterial suspension to obtain a sample R2; or extracting with high speed oscillation instead of ultrasonic extraction, and using cell homogenizer (MP)
MP Biomedicals, usa) at a parameter setting speed of 5.0, and the bacterial suspension was transferred to a 50mL centrifuge tube for centrifugation in three cycles of 15 seconds each to obtain sample R3.
2. Adding 0.25g of sample into 2ml of Bead Solution Tubes;
3. add 60 μ L C1 solution, invert several times, or vortex mix gently for a short time.
4. The lysis tube was horizontally mounted on a vortexer clamp, (instrument model 13000-V1-24).
5. Vortex at maximum speed for 10 min.
6. The tube was centrifuged at 10000g for 30 s.
7. The supernatant was transferred to another clean 2ml Collection Tube.
8. Add 250 μ L C2 solution into Collection Tube, vortex for 5s, and let stand at 4 deg.C for 5 min.
9. Centrifugation is carried out for 1min at 10000 g.
10. To avoid clumping, 600. mu.L of supernatant was transferred to another clean 2ml Collectiontube.
11. Add 200. mu. L C3 solution to the supernatant and vortex for 5s and incubate at 4 ℃ for 5 min.
12. Centrifugation is carried out for 1min at 10000 g.
13. To avoid clumping, 750. mu.L of supernatant was transferred to a clean 2ml Collection Tube.
14. The C4 solution was shaken, 12000. mu.L added to the supernatant and vortexed for 5 s.
15. Transferring 675 μ L to MB Spin Column, centrifuging at 10000g for 1min, and discarding the effluent.
16. Repeating the 14 steps twice until the sample is transferred.
17. Add 500 u L C5 solution, 10000g centrifugation 30 s.
18. The effluent was discarded and centrifuged again at 10000g for 1 min.
19. MB Spin columns were carefully placed into a clean 2ml Collection Tube, avoiding the C5 solution from splashing onto the columns.
20. The 100. mu. L C6 solution was added to the center of the white filter, and sterile DNA-free PCR-grade ultrapure water was used instead.
21. Centrifuge at 10000g for 30s at room temperature, discard MB Spin Column. The DNA can be used for downstream operations. Stored in a-80 ℃ refrigerator.
22. Detection of sample genomic DNA: taking 2 microliter of extracted DNA, using a micro ultraviolet visible spectrophotometer to perform DNA content determination, and reading the ratio of the DNA concentration to the 260/280nm ultraviolet OD value. And then 4 microliters of DNA is uniformly mixed with 1 microliter of loadingbuffer, and the mass of the total DNA of the microorganism is detected by using agarose gel electrophoresis with the mass percentage concentration of 1 percent, wherein the concentration of the agarose is 1 percent, the electrophoresis voltage is 120V, and the electrophoresis time is 20 min.
FIGS. 4 and 5 are graphs showing the ratio of the UV OD values at 260/280nm of the samples extracted from comparative example 1, R1, R2 and R3, respectively, and a glue map; in the glue pattern of fig. 5, M denotes Marker, 1 denotes R1, 2 denotes R2, and 3 denotes R3. As shown in the figure, 3 pretreatment methods can not extract DNA in thalli, probably because the specificity of the kit for DNA molecules is poor, the ratio of the ultraviolet OD value at 260/280nm is less than 1.5, and serious protein pollution is shown.
Comparative example 2
In this example, the genomic DNA under the extremely acidic condition is ectopically extracted by a portable method of SDS-CTAB, which comprises the following steps:
step one, collecting a functional flora sample: about 0.5L of ceramsite filler with good microorganism growth state is put into a beaker with 1L from a biofilm filler tower with synchronous desulfurization and denitrification, independent desulfurization and independent denitrification, and the filler is immersed by nutrient solution used by a reactor. The beaker was placed in an ultrasonic cleaner and sonicated at 50kHz and 300W for 5min to remove the filler, and the bacterial suspension was poured into eight 50mL centrifuge tubes and centrifuged at 6000g for 5min to obtain sample R1. The same amount of filler is taken and the steps are repeated, only the ultrasonic extraction is changed into the process of placing the beaker on a shaker for 100 revolutions per minuteCarrying out oscillation extraction for half an hour, and then centrifuging the bacterial suspension to obtain a sample R2; or extracting with high speed oscillation instead of ultrasonic extraction, and using cell homogenizer (MP)
MPBiomedicals, usa), setting the speed at 5.0, transferring the bacterial suspension to a 50mL centrifuge tube for centrifugation in three cycles of 15 seconds each, and obtaining a sample R3.
Step two, extracting the genomic DNA of the sample: adding 1mL of buffer solution (50mM Tris-HCl, pH 8.0,50mM EDTA) into the centrifugal tube for collecting the thalli in the first step to suspend the thalli, adding 1mg/mL (final concentration, the same below) of lysozyme solution, and carrying out water bath at 37 ℃ for 30min without shaking and shaking uniformly; adding 100. mu.L proteinase K, 1.25% SDS and 60mM lysis solution (2% Triton X-100, 2.5mg/mL sodium azide, dissolved in 0.1mol/L Tris-HCl solution with pH 8.0), bathing at 37 deg.C for 30min and shaking uniformly; adding CTAB/NaCl solution (final concentration of 0.5% CTAB and 50mM NaCl), and water-bathing at 65 deg.C for 30min without shaking; add an equal volume of 24: 1, chloroform/isoamylol, evenly mixing, carrying out ice bath for 5min, centrifuging for 5min at 12,000g, transferring supernatant into a new centrifugal tube, and repeatedly leaching for a plurality of times; transferring the supernatant into a new centrifuge tube, adding isovolumetric isopropanol and 1/10 volumes of 3M sodium acetate solution, and standing overnight at-20 ℃; centrifuge at 12,000g for 5min, wash the precipitate twice with pre-cooled 70% ethanol, air dry at room temperature, and dissolve the precipitate with 50 μ L of TE.
Step three, detecting the genomic DNA of the sample: and taking 2 microliters of DNA obtained in the step two, using a trace ultraviolet-visible spectrophotometer to measure the DNA content, and reading the ratio of the DNA concentration to the 260/280nm ultraviolet OD value. And then 4 microliters of DNA is taken and mixed with 1 microliter of loading buffer uniformly, and the mass of the total DNA of the microorganism is detected by using agarose gel electrophoresis with the mass percentage concentration of 1 percent, wherein the concentration of the agarose used is 1 percent, the electrophoresis voltage is 120V, and the electrophoresis time is 20 min.
FIGS. 6 and 7 are a graph showing the ratio of the UV OD values at 260/280nm of the extracted genomic ribonucleic acids R1, R2 and R3 of comparative example 2, and a gel plot, respectively; in the glue pattern of fig. 7, the first column indicates Marker, 1 is denoted R1, 2 is denoted R2, and 3 is denoted R3.
The observation of the glue pattern and the 260/280nm ultraviolet OD value ratio shows that DNA cannot be extracted in other two ways except that the high-speed oscillation method can extract DNA molecules which are partially degraded into small fragments, and the 260/280nm ultraviolet OD value ratio is less than 1.4, so that the protein pollution is serious.
Comparative example 3
In this embodiment, a CTAB freeze-thaw method is used to extract genomic DNA in situ under an extremely acidic condition, and the steps are as follows:
step one, collecting a functional flora sample: from a biofilm-packed column for simultaneous desulfurization and denitrification, single desulfurization and single denitrification, about 0.5L of a ceramsite packing having a good growth state of microorganisms was placed in a 1L beaker and mixed with a buffer solution A (pH 8.0,0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH)2PO4And 0.1M Na2HPO4) Mixing, incubating at 55 ℃ for 20min, removing supernatant, adding 3mL of buffer solution A and lysozyme, incubating at 37 ℃ for 1h, treating at 37 ℃ for 30min by using 20mg/mL proteinase K and 10% CTAB, adding SDS with the concentration of 20%, lysing cells at 65 ℃ for 2h, centrifuging to obtain DNA-containing supernatant and precipitate, and transferring the supernatant to a new centrifuge tube; to the precipitate obtained above, 3mL of buffer B (pH 8.0,0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH) was added2PO4，0.1M Na2HPO4And 1 wt% CTAB) and SDS at a concentration of 20%, and water bath at 65 ℃ for 15 min. Freezing at 80 deg.C for 15min, water bathing at 65 deg.C for 5min, repeatedly freezing and thawing for three times, centrifuging to obtain supernatant, and mixing with the supernatant; equal volume of phenol was added: chloroform: extracting protein with isoamyl alcohol (25: 24: 1), adding equal volume of chloroform and isoamyl alcohol (24: 1) to extract protein, and precipitating with 0.6 volume times of isopropanol for 2 h; and adding the mixed solution into a centrifugal purification column, centrifugally washing 50 mu L of TE solution, eluting, and collecting liquid in a centrifugal tube, namely a genome DNA solution sample R1. We adopted different processing time of proteinase K and lysozyme to explore the influence of different processing time of proteinase K and lysozyme on DNA extraction, and adopted the method to shorten half of the processing time of proteinase K (15min) to obtain a sample R2; shortening half of lysozyme treatment time (30min) to obtain sampleR3; simultaneously shortening half of the processing time of the proteinase K and the lysozyme to obtain a sample R4; the lysozyme treatment time was shortened to 20min, and the proteinase K treatment time was kept unchanged to obtain a sample R5.
Step two, detecting the genomic DNA of the sample: and (3) taking 2 microliters of DNA obtained in the step one, using a micro ultraviolet-visible spectrophotometer to measure the DNA content, and reading the ratio of the DNA concentration to the 260/280 ultraviolet OD value. And then 4 microliters of DNA is taken and mixed with 1 microliter of loading buffer uniformly, and the mass of the total DNA of the microorganism is detected by using agarose gel electrophoresis with the mass percentage concentration of 1 percent, wherein the concentration of the agarose used is 1 percent, the electrophoresis voltage is 120V, and the electrophoresis time is 20 min.
FIGS. 8 and 9 are a graph showing the ratio of the 260/280 UV OD values of the genomic ribonucleic acid samples R1 to R5 extracted in comparative example 3, and a gel chart, respectively; in the glue pattern of fig. 9, M denotes Marker.
The gel separation result shows that a large amount of DNA can be extracted and obtained by the method, the purity is high, the ratio of 260/280 ultraviolet OD values is 1.75-1.85, less protein impurities are displayed, however, in samples R1-R5, the DNA is degraded in a large amount, the generated fragment molecules are mainly concentrated below 200bp, the DNA degradation of the R3, R4 and R5 samples obtained by shortening the lysozyme treatment time is slightly improved, the R5 samples have the best effect, but the gel separation result is observed, and the extracted DNA is mainly concentrated on the degradation fragments with the molecular weight less than 200 bp. Therefore, this method is not suitable for DNA extraction under extremely acidic conditions.
Through comparative analysis of the different implementation cases, it can be found that only in examples 1 and 2, long and complete DNA fragments can be obtained through flushing with high-pressure buffer solution, pH adjustment and washing of heavy metal ions and humus with buffer solution, and downstream experiments can be carried out. In the comparative examples 3-5, no matter the DNA is extracted in situ or ex situ, the high-quality total DNA of the sample cannot be extracted under the extreme acid condition, and even if the DNA sample is extracted, a large amount of pollution and serious degradation exist, so that the subsequent high-throughput sequencing experiment cannot be carried out.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. A method for extracting total DNA of microorganisms in a substrate-attached biomembrane under strong acidic condition is characterized by comprising the following steps: the method comprises the following steps:
1) eluting microorganisms from the attachment matrix by using a buffer solution 1 with the pH value of 4-5 under proper water pressure, and centrifugally collecting a thallus sample;
2) washing with a buffer solution 2 containing EDTA and having a pH value of 7-7.5, and centrifuging to collect a thallus sample;
3) extracting DNA from the thallus sample collected in the step 2).
2. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 1, wherein the method comprises the following steps: the suitable water pressure is 30-120 MPa.
3. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 1 or 2, wherein the method comprises the steps of: the molar concentration of EDTA contained in the buffer solution 2 is 50-100 mM.
4. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 3, wherein the method comprises the following steps: the buffer solution 2 is a PBS-Tris buffer solution, and the buffer solution contains a Tris-HCl solution with the molar concentration of 50-100 mM.
5. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 3, wherein the method comprises the following steps: the elution in the step 1) is not washed for more than 90 seconds.
6. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 4, wherein the method comprises the following steps: the buffer solution 1 comprises an isotonic buffer solution with the pH value of 4-5 or a simulated solution which is prepared by compounding other chemical components in an environment liquid where microorganisms are located and adjusting the acidity to the pH value of 4-5 by using sulfuric acid.
7. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 1 or 2, wherein the method comprises the steps of: the conditions for centrifugally collecting the thallus samples in the step 1) and the step 2) are as follows: the speed is 5000-6000 g, the centrifugation time is 2-5 minutes, and the centrifugation temperature is 4 ℃.
8. The method for extracting total DNA of microorganisms in a substrate-attached biofilm according to claim 3, wherein the method comprises the following steps: the method for extracting the total DNA in the step 3) comprises a freeze-thawing method or an ultrasonic method.
9. The method for extracting total DNA of microorganisms in a biofilm adhered to a substrate under strongly acidic conditions as claimed in claim 8, wherein: the buffer solution 1 comprises a phosphate buffer solution or a phosphate-potassium dihydrogen phosphate buffer solution, and the volume of the buffer solution 1 is 50-100 mL.
10. The method for extracting total DNA of microorganisms in a biofilm adhered to a substrate under strongly acidic conditions as claimed in claim 9, wherein: the strongly acidic condition is a condition with a pH value of < 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010064011.1A CN111235142A (en) | 2020-01-20 | 2020-01-20 | Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010064011.1A CN111235142A (en) | 2020-01-20 | 2020-01-20 | Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111235142A true CN111235142A (en) | 2020-06-05 |
Family
ID=70874764
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010064011.1A Pending CN111235142A (en) | 2020-01-20 | 2020-01-20 | Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111235142A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112071366A (en) * | 2020-10-13 | 2020-12-11 | 南开大学 | Metagenome data analysis method based on second-generation sequencing technology |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103221550A (en) * | 2010-12-06 | 2013-07-24 | 3M创新有限公司 | Microorganism concentration process and device |
-
2020
- 2020-01-20 CN CN202010064011.1A patent/CN111235142A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103221550A (en) * | 2010-12-06 | 2013-07-24 | 3M创新有限公司 | Microorganism concentration process and device |
Non-Patent Citations (4)
| Title |
|---|
| LEPING ZENG等: "An effective method of DNA extraction for bioleaching bacteria from acid mine drainage", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 刘守华等: "《矿井测尘工》", 31 May 2007, 中国矿业大学出版社 * |
| 石慧等: "《食品分子微生物》", 31 May 2019, 中国农业大学出版社 * |
| 黄海: "用多种方法提取酸性排水沉积物中微生物DNA", 《万方数据知识服务平台》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112071366A (en) * | 2020-10-13 | 2020-12-11 | 南开大学 | Metagenome data analysis method based on second-generation sequencing technology |
| CN112071366B (en) * | 2020-10-13 | 2024-02-27 | 南开大学 | Metagenome data analysis method based on second-generation sequencing technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rajendhran et al. | Strategies for accessing soil metagenome for desired applications | |
| EP1756136B1 (en) | Kits and processes for removing contaminants from nucleic acids in environmental and biological samples | |
| CN101475987B (en) | Rapid molecule detecting method for microflora composition in waste water biological treatment reactor | |
| CN103993007A (en) | Simple method for efficiently extracting DNA (deoxyribonucleic acid) from soil samples | |
| CN101712953A (en) | DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals | |
| CN114107286A (en) | Universal soil genome DNA extraction kit and use method thereof | |
| CN106399533A (en) | Method and composition for removal of ribosomal nucleic acid rRNA from total RNA sample | |
| CN111235142A (en) | Method for extracting total DNA of microorganisms in substrate-attached biomembrane under strong acid condition | |
| CN104830880B (en) | A kind of alginate lyase SHA I genes and its expression vector | |
| WO2019180519A1 (en) | Method and kit for isolation of nucleic acids from a sample | |
| Gülay et al. | Neutrophilic iron-oxidizing bacteria: occurrence and relevance in biological drinking water treatment | |
| CN107151666A (en) | The extracting method of microbial DNA in a kind of water body | |
| CN111269800B (en) | Device and method for extracting total DNA of matrix-attached microorganisms | |
| Hernandez-Leon et al. | Construction and characterization of a metagenomic DNA library from the rhizosphere of wheat (Triticum aestivum) | |
| CN103333883A (en) | Method for efficiently extracting underground water microbial DNA for PCR amplification | |
| CN111254139A (en) | Method for extracting total RNA and total protein of matrix microorganisms under strong acid condition | |
| CN114085830A (en) | Method for extracting microbial genome DNA (deoxyribonucleic acid) in complex sample | |
| Debler et al. | High-molecular weight DNA extraction and small fragment removal of Ascochyta lentis | |
| CN110511977B (en) | Genomic DNA extraction method, sequencing method and kit | |
| CN112175941B (en) | Efficient soil microorganism total RNA extraction method | |
| CN103146742A (en) | Method of repairing environmental pollutions by using RhlA and RhlB transgenic plants | |
| JP5105237B2 (en) | Compositions and kits for nucleic acid extraction and use thereof | |
| CN115896161A (en) | Method for enhancing cadmium resistance of rice by using gene overexpression | |
| Jones et al. | DNA clean-up and size selection for long-read sequencing | |
| CN1970753A (en) | Method for extracting whole genome of abyssal sediment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |
|
| RJ01 | Rejection of invention patent application after publication |