LU506015B1 - Method for screening active microbiota in soil using propidium monoazide - Google Patents
Method for screening active microbiota in soil using propidium monoazide Download PDFInfo
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- LU506015B1 LU506015B1 LU506015A LU506015A LU506015B1 LU 506015 B1 LU506015 B1 LU 506015B1 LU 506015 A LU506015 A LU 506015A LU 506015 A LU506015 A LU 506015A LU 506015 B1 LU506015 B1 LU 506015B1
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- 239000002689 soil Substances 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012216 screening Methods 0.000 title claims abstract description 16
- 241000736262 Microbiota Species 0.000 title claims abstract description 6
- DXHWIAMGTKXUEA-UHFFFAOYSA-O propidium monoazide Chemical compound C12=CC(N=[N+]=[N-])=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 DXHWIAMGTKXUEA-UHFFFAOYSA-O 0.000 title claims abstract 7
- 238000011534 incubation Methods 0.000 claims abstract description 20
- 230000001580 bacterial effect Effects 0.000 claims abstract description 18
- 239000000725 suspension Substances 0.000 claims abstract description 17
- 238000006303 photolysis reaction Methods 0.000 claims abstract description 15
- 230000015843 photosynthesis, light reaction Effects 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 33
- 239000002953 phosphate buffered saline Substances 0.000 claims description 18
- 238000002474 experimental method Methods 0.000 claims description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 239000000975 dye Substances 0.000 abstract description 11
- 230000000813 microbial effect Effects 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 239000000980 acid dye Substances 0.000 abstract description 5
- 108020004707 nucleic acids Proteins 0.000 abstract description 5
- 102000039446 nucleic acids Human genes 0.000 abstract description 5
- -1 PMA nucleic acid Chemical class 0.000 abstract description 3
- 201000008752 progressive muscular atrophy Diseases 0.000 description 47
- 210000004027 cell Anatomy 0.000 description 24
- 239000000872 buffer Substances 0.000 description 12
- 210000000170 cell membrane Anatomy 0.000 description 12
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 12
- 239000000523 sample Substances 0.000 description 10
- ZLSOONVQLWLPMF-UHFFFAOYSA-M 3-(3,8-diamino-6-phenylphenanthridin-5-ium-5-yl)propyl-diethyl-methylazanium;dibromide Chemical compound [Br-].[Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZLSOONVQLWLPMF-UHFFFAOYSA-M 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- GHUXAYLZEGLXDA-UHFFFAOYSA-N 8-azido-5-ethyl-6-phenylphenanthridin-5-ium-3-amine;bromide Chemical compound [Br-].C12=CC(N=[N+]=[N-])=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 GHUXAYLZEGLXDA-UHFFFAOYSA-N 0.000 description 6
- ZXFDSSZCWQFXAT-UHFFFAOYSA-N Br.[N-]=[N+]=[N-] Chemical compound Br.[N-]=[N+]=[N-] ZXFDSSZCWQFXAT-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 230000004544 DNA amplification Effects 0.000 description 4
- 230000002503 metabolic effect Effects 0.000 description 4
- IVRMZWNICZWHMI-UHFFFAOYSA-N Azide Chemical compound [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 3
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 244000000000 soil microbiome Species 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- LLJZKKVYXXDWTB-UHFFFAOYSA-N acetic acid;sodium Chemical compound [Na].[Na].CC(O)=O LLJZKKVYXXDWTB-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- ZDWVWKDAWBGPDN-UHFFFAOYSA-O propidium Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZDWVWKDAWBGPDN-UHFFFAOYSA-O 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The present invention provides a method for screening active microbiota in soil using propidium monoazide, which belongs to the technical field of microbiology. The method includes the steps of mixing a soil sample with a PBS solution and performing shaking to obtain a soil bacterial suspension; and mixing the soil bacterial suspension with a propidium monoazide solution and performing dark incubation obtain a dark incubated product. Experimental steps for applying PMA nucleic acid dyes to the screening of active microbiota in complex soil samples are embodied. The PMA-soil solution is fully shaken during the processes of dark incubation and exposure reaction, to avoid local soil adhesion resulting in PMA not being able to fully contact dead cell DNA, or disruption of active microbial cells caused by local dye overflow. The temperature of a PMA-LiteTM LED photolysis device is maintained below 37°C during exposure, which avoids damage to active cells caused by excessively high temperature during photolysis.
Description
METHOD FOR SCREENING ACTIVE MICROBIOTA IN SOIL USING PROPIDIUM 7506015
MONOAZIDE
FIELD OF TECHNOLOGY
[0001] The present invention relates to the technical field of microorganisms, and in particular to a method for screening active microorganisms in soil using propidium bromide azide.
[0002] Active microorganisms refer to microorganisms that have or potentially have growth metabolic characteristics and the ability to adapt to changes in a given environment (Emerson et al., 2017). They are also the main target of microbial isolation, culture or functional identification in soil microbial ecology. However, although high-throughput sequencing technology has become an important tool for studying soil microbial diversity and its community structure (Rinke et al., 2013), soil total DNA amplification sequencing based on non-selective identification of active microorganisms cannot reflect the soil microbial diversity and community structure. The true number of active microorganisms in the soil is likely to be overestimated (Dlott et al., 2015). This is because the total DNA of soil microorganisms mainly includes: 1) living cell DNA, intact cells with metabolic activity and cultivable (Nocker et al., 2009); 2) dormant cell DNA, living cells with low metabolic activity and uncultivable and antibiotic persister cells, but intact cells that can restore their metabolic activity and culturability under certain conditions (Ayrapetyan et al., 2018); 3) Dead cell DNA, including damaged cell membranes that are lysing and dead cells Non-intact cells (Nebe-von-Caron et al, 2000); 4) Extracellular DNA, including free DNA and DNA adsorbed on soil particles (Levy-Booth et al, 2007). Therefore, how to accurately identify and quantify active microorganisms in soil is a key issue that needs to be solved urgently in current soil microbial ecology research.
[0003] Cell membrane integrity is accepted by many researchers as a criterion for microbial activity (Grégori et al, 2001; Emerson et al, 2017). Among the methods for screening viable microorganisms based on cell membrane integrity, propidium iodide (PI) is the earliest nucleic acid dye widely used for screening viable bacteria (Williams et al., 1998). HUS06015
It can only penetrate damaged cells. cell membrane and releases red fluorescence upon embedding in double-stranded DNA, usually bound toFluorescence microscopy, confocal laser scanning microscopy or flow cytometryPerform viable cell assays (Nicoletti et al, 1991;
Pietkiewicz et al., 2015). However, the application of PI dyes also has some limitations and disadvantages. It will stain some growing active cells, such asSphingomonas sp. (Shi et al., 2007). In addition, samples treated with PI dyes cannot be used in subsequent DNA amplification sequencing, that is, PI dyes are not suitable for studying the structure of active microbial communities in complex environmental samples. Nogva et al. (2003) and Nocker et al. (2006) successively proposed that EMA (Ethidium Monoazide Bromide)/PMA (Propidium
Monoazide) nucleic acid dyes can be used to screen active microorganisms. Similar to PI,
EMA/PMA dyes are blocked in the complete cell membrane of viable bacteria. In addition, it irreversibly covalently binds to extracellular DNA and membrane-damaged inactive cellular
DNA, ultimately blocking the PCR amplification reaction of its DNA molecules. Since the
DNA molecules bound to the EMA/PMA dye are permanently modified, the selected active microorganisms can be used for subsequent DNA sequencing and other multi-omics studies, making this method widely used (Elizaquivel et al., 2013; Emerson et al., 2017). However,
EMA dye was found to be somewhat cytotoxic in most subsequent experiments and would bindMycobacterium aviumand other living bacterial DNA (Nocker et al., 2006), because EMA with one positive charge (PMA with two positive charges) is more likely to penetrate the cell membrane of viable bacteria (Fittipaldi et al., 2012), resulting in false negative results in the experiment, making PMA more advantageous in selectively screening active microorganisms.
[0004] Currently, methods for detecting active microorganisms using PMA are mostly used in the inspection and quarantine of water, pure strains or food microorganisms (Nocker et al, 2009; Elizaquivel et al., 2013; Gomez-Alvare et al, 2014). PMA has a high affinity for
DNA and can efficiently penetrate the cell membrane of dead cells during dark culture and produce covalent cross-linking with exposed DNA. Subsequently, under the action of visible light (maximum absorption wavelength of about 460 nm), the photosensitive azide group carried by PMA will be converted into highly reactive nitrene radicals, and react with any hydrocarbons near its binding site to form the stable covalent carbon-nitrogen bond causes the
DNA molecule to be permanently modified, and finally blocks subsequent PCR amplification HUS06015 and other reactions of the DNA molecule. At the same time, the PMA remaining in the solution that is not cross-linked with DNA molecules can react with water molecules to form inactive hydroxylamine under strong light irradiation (Nocker et al., 2006). The above processes are all completed before DNA extraction from environmental samples. Since PMA cannot penetrate the complete cell membrane of active cells, it will not affect the subsequent DNA extraction and PCR amplification of active microorganisms, ultimately allowing relic DNA to be selectively eliminated during nucleic acid purification.
[0005] The use of PMA nucleic acid dye methods to screen active microorganisms in complex soil samples is relatively rare and lacks specific experimental procedures. At the same time, since the optimal excitation wavelength of PMA dye is 464 nm, in previous experiments, the sample was usually placed on ice 20 cm away from the light source and continuously irradiated with a 500~650 W halogen lamp or light and dark cycles for 2~5 minutes. , to stimulate the covalent binding of PMA to DNA molecules. However, the light intensity and spectral characteristics of halogen lamps are difficult to control, and excessive irradiation temperature may damage the cell membrane of viable bacteria or cause the plastic centrifuge tube to melt, increasing the uncertainty of the experimental results.
[0006] In order to solve the above problems, the present invention provides a method for screening active microorganisms in soil using propidium bromide azide. Propidium bromide azide is used to screen active microorganisms in soil, and PMA-Lite is used to screen active microorganisms in soil.™ LED photolysis instrument can induce cross-linked PMA-
DNA with thermal stability, making the experimental process simpler, safer and more efficient.
[0007] In order to achieve the above objects, the present invention provides the following technical solutions:
[0008] The invention provides a method for screening active microorganisms in soil using propidium azide bromide, which includes the following steps:
[0009] 1) Mix and shake the soil sample with PBS buffer to obtain a soil bacterial suspension;
[0010] 2) Mix the soil bacterial suspension obtained in step 1) with the propidium HUS06015 bromide azide solution and incubate it in the dark to obtain a dark incubation product;
[0011] 3) Place the dark incubation obtained in step 2) into PMA-Lite™ Expose in the
LED photolysis instrument to obtain the exposed object;
[0012] 4) Extract the DNA of the exposure obtained in step 3), and determine the active microorganisms in the soil sample through biological experiments.
[0013] Preferably, the volume ratio of the mass of the soil sample to the PBS buffer in step 1) is 0.5g:49.5mL.
[0014] Preferably, the preparation method of the PBS buffer includes: mixing 0.1 mol/L dipotassium hydrogen phosphate solution and 0.1 mol/L disodium hydrogen phosphate solution at a volume ratio of 2:3 to obtain;
[0015] The pH value of the PBS buffer is 6.98.
[0016] Preferably, the oscillation conditions in step 1) include: the rotation speed is 150 rpm and the time is 30 minutes.
[0017] Preferably, the volume ratio of the soil bacteria suspension to the propidium bromide azide solution is 49:1;
[0018] The concentration of the propidium azide bromide solution is 2 mmol/L.
[0019] Preferably, the conditions for dark incubation in step 2) include: the dark incubation time is 10 minutes, and the mixture is shaken every 2 minutes.
[0020] Preferably, the exposure conditions in step 3) include: the exposure time is 20 minutes, and the exposure time is 1 time every 5 minutes.
[0021] Preferably, the exposure temperature is no higher than 37°C.
[0022] Beneficial effects:
[0023] 1. For the first time, the experimental steps for applying PMA nucleic acid dye tothe screening of active microorganisms in complex soil samples were concrete;
[0024] 2. Refine the operating steps, especially fully shake the PMA-soil solution during dark incubation and exposure reactions to avoid local soil adhesion, which may cause
PMA to fail to fully contact dead cell DNA or excessive local dye to damage active microbial cells;
[0025] 3. PMA-Lite™ LED photolysis instrument is equipped with an automatic cooling cycle system. The temperature during exposure is always maintained below 37°C, HUS06015 which avoids damage to active cells caused by excessive temperature during photolysis. A simple and convenient system is established based on a specific PMA photolysis instrument. A safe and efficient method for screening active soil microbiota. 5
[0026] This application provides a method for screening active microorganisms in soil using propidium bromide azide, which includes the following steps:
[0027] 1) Mix and shake the soil sample with PBS buffer to obtain a soil bacterial suspension;
[0028] 2) Mix the soil bacterial suspension obtained in step 1) with the propidium bromide azide solution and incubate it in the dark to obtain a dark incubation product;
[0029] 3) Place the dark incubation obtained in step 2) into PMA-Lite™ Expose in the
LED photolysis instrument to obtain the exposed object;
[0030] 4) Extract the DNA of the exposure obtained in step 3), and determine the active microorganisms in the soil sample through biological experiments.
[0031] In the present invention, soil samples and PBS buffer are mixed and shaken to obtain soil bacterial suspension.
[0032] The present invention has no special limitation on the source of the soil sample, and conventional methods can be used, such as fresh soil, naturally air-dried soil or freeze- dried soil.
[0033] In the present invention, the volume ratio of the mass of the soil sample to the
PBS buffer is preferably 0.5g:49.5mL. In the present invention, the preparation method of the
PBS buffer preferably includes: mixing 0.1 mol/L dipotassium hydrogen phosphate solution and 0.1 mol/L disodium hydrogen phosphate solution at a volume ratio of 2:3 to obtain; The pH value of the PBS buffer is preferably 6.98. In the present invention, the oscillation conditions preferably include: the rotation speed is 150 rpm and the time is 30 minutes.
[0034] In the present invention, the obtained soil bacterial suspension is mixed with a propidium azide bromide solution and incubated in the dark to obtain a dark incubation product.
[0035] In the present invention, the volume ratio of the soil bacterial suspension to the propidium bromide azide solution is preferably 49:1; the concentration of the propidium HUS06015 bromide azide solution is preferably 2 mmol/L. The present invention has no special restrictions on the source of the propidium bromide azide (ie, PMA), and conventional commercially available products can be used. In the present invention, the conditions of the dark incubation preferably include: the dark incubation time is 10 minutes, and shaking is performed every 2 minutes. In the present invention, the dark incubation allows PMA to fully penetrate the damaged cell membrane of dead cells and enter the cells.
[0036] In the present invention, the obtained dark incubation material is placed in
PMA-Lite™ Expose in the LED photolysis instrument to obtain the exposed object.
[0037] In the present invention, the exposure conditions preferably include: the exposure time is 20 minutes, and the exposure time is oscillated every 5 minutes. In the present invention, the temperature of the exposure is preferably not higher than 37°C. In the present invention, the PMA-Lite™ LED photolysis instrument is preferably an instrument sold by
Biotium and the United States. In the present invention, the irreversible covalent bonding between PMA and DNA is promoted, and the excess PMA reacts with water under strong light to generate inactive hydroxylamine, thereby completely passivating the free PMA without affecting the subsequent lysis of viable bacteria. and its DNA amplification reaction to avoid "false negative" results. The present invention samples the PMA-Lite™ LED photolysis instrument (output wavelength 465-475 nm) developed by Biotium Company, which can set up an automatic cooling cycle system on the premise of maintaining the optimal photolysis wavelength required for PMA to bind DNA. The temperature around the sample is always maintained below 37°C, which greatly avoids damage to living cells caused by excessive temperature during photolysis.
[0038] The present invention extracts the DNA of the exposed object and determines the active microorganisms in the soil sample through biological experiments. The method of extracting DNA from exposed objects is not particularly limited in the present invention, and conventional methods for extracting soil DNA can be used. The present invention also has no special limitations on biological experiments. Those skilled in the art can follow routine.
[0039] In order to further illustrate the present invention, the present invention is described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention. HUS060T5
[0040] Example 1
[0041] A method for screening active microorganisms in soil using propidium bromide azide, including the following steps:
[0042] 1. Preparation of phosphate buffered saline solution (PBS buffer)
[0043] Liquid A: 0.1mol/L dipotassium hydrogen phosphate, weigh dipotassium hydrogen phosphate (KH2PO4) 1.36g in a 100mL volumetric flask, add sterile water to dissolve thoroughly;
[0044] Liquid B: 0.1mol/L disodium hydrogen phosphate, weigh disodium hydrogen acetate (Na2HPO4-2H20) 1.78g in a 100 mL volumetric flask, add sterile water and dissolve completely;
[0045] Mix solution A and solution B in a ratio of 2:3 by volume, prepare a PBS buffer with a pH of 6.98, and store at room temperature.
[0046] 2. Preparation of soil bacterial suspension
[0047] Accurately weigh 0.5g of the target soil and place it into a sterile 100mL centrifuge tube. Add 49.5mL of the above PBS buffer into the centrifuge tube to make the soil bacterial suspension concentration 1% (w/v). Then place the centrifuge tube in a constant temperature shaker. Shake culture in the bed (shake at 150 rpm for 30 minutes) to fully disperse the microbial cells in the soil. The prepared soil bacterial suspension can be placed at 4°C Store in refrigerator for short term (within one week).
[0048] 3. PMA solution preparation
[0049] Dissolve PMA dye (Biotium, USA, specification 1 mg, 40013) powder in 98 uL of 20% dimethyl sulfoxide to prepare a 20 mmol/L stock solution, or purchase 20 mmol/L
PMAxx (an improved version of PMA, 100 pL, 40019) solution, when using, dilute both to a 2 mmol/L use solution, and both the stock solution and the use solution need to be wrapped in tin foil and placed at -20 °C Store away from light.
[0050] 4. PMA dark incubation
[0051] Take 490 uL of soil bacteria suspension in a 1.5 mL centrifuge tube, add 10 uL of 2 mmol/L PMA or PMAxx into the centrifuge tube to make a PM A-soil solution with a final concentration of 40 umol/L, and then shake the centrifuge tube manually and fully Immediately incubate in the dark for 10 minutes (the centrifuge tube can be placed in a dark box or fully HUS06015 wrapped in tin foil). During the incubation period, shake the centrifuge tube fully again every 2 minutes. This step allows PMA to fully penetrate the damaged cell membrane of dead cells and enter the cells.
[0052] 5. PMA exposure reaction
[0053] After the dark incubation of PMA, place the centrifuge tube containing the
PMA-soil solution in the PMA-LiteTM LED photolysis instrument (Biotium, USA), turn on the light source (wavelength 470nm), the total exposure time is 20min, and the temperature is lower than 37°C, shake the centrifuge tube every 5 minutes during the exposure period. This step promotes the completion of irreversible covalent binding of PMA and DNA. The excess
PMA reacts with water under strong light to generate inactive hydroxylamine. This completely inactivates the free PMA and will not affect the subsequent lysis of viable bacteria and its DNA amplification. reaction to avoid “false negative” results.
[0054] 6. Identification of active soil microorganisms
[0055] The PMA-soil solution that has undergone the PMA exposure reaction only contains active microorganisms with intact cell membranes. The soil DNA extraction kit can be directly used to extract the DNA of the active microorganisms. The extracted DNA can be used in molecular biology experiments such as fluorescence quantitative PCR. To determine the abundance of active microorganisms in soil samples, or for multi-omics analysis such as high-throughput sequencing to determine the diversity and metabolic functions of active microbial communities in soil samples.
[0056] Example 2
[0057] Using the method of screening active microorganisms in soil using propidium azide bromide in Example 1 (dead cell DNA and extracellular DNA are collectively referred to asrelic DNA), sampling fluorescence quantitative PCR was performed on fresh, naturally air- dried and frozen samples before and after adding PMA dye. The bacterial abundance of dry soil (both natural air-drying and freeze-drying soil stored for 100 days) was measured. Soil samples were collected from typical rice field soil in Xiaoji Town, Jiangdu District, Yangzhou
City, Jiangsu Province (32°35'N, 119°42'E). The soil samples collected were non-flooded.
Paddy soil during the water period. The abundance of total bacteria in fresh, naturally air-dried and freeze-dried soil was 2.29 x 10 respectively.",0.95x10"and 1.85 x 107, after PMA treatment, HUS060T5 the abundance of active bacteria in fresh, naturally air-dried and freeze-dried soil was 1.46 x 10 respectively.”,0.82x10"and 1.34 x 10”, therefore, the proportions of relic DNA in fresh, naturally air-dried and freeze-dried soil were 36.2%, 13.7% and 27.6% respectively. The proportion of relic DNA in fresh soil detected in this example is close to the 33% proportion of relic DNA in fresh soil detected by Lennon et al. (mBio, 2018, e00637-18) using DNase. This example also found that relic DNA accounted for the smallest proportion in naturally air-dried soil, and there was no significant difference between total bacterial abundance and active bacterial abundance. This also shows that during the slow natural air-drying process, relic DNA is degraded to the maximum extent. Or be used as nutrients by other active microorganisms.
[0058] Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.
Claims (8)
1. A method for screening active microbiota in soil using propidium monoazide, comprising the following steps: 1) mixing a soil sample with a phosphate buffered saline (PBS) solution and performing shaking to obtain a soil bacterial suspension; 2) mixing the soil bacterial suspension obtained in step 1) with a propidium monoazide solution and performing dark incubation to obtain a dark incubated product; 3) placing the dark incubated product obtained in step 2) into a PMA-Lite™ LED photolysis device for exposure to obtain an exposed product; 4) extracting the DNA of the exposed product obtained in step 3), and determining active microorganisms in the soil sample through biological experiments.
2. The method according to claim 1, wherein in step 1), the ratio of the mass of the soil sample to the volume of the PBS solution is 0.5g : 49.5mL.
3. The method according to claim 1 or 2, wherein the PBS solution is prepared by mixing 0.1 mol/L of a dipotassium hydrogen phosphate solution and 0.1 mol/L of a disodium hydrogen phosphate solution with the volume ratio of 2:3; and the pH value of the PBS solution is 6.98.
4. The method according to claim 1, wherein a shaking condition in step 1) comprises a rotation speed of 150 rpm and the time of 30 minutes.
5. The method according to claim 1, wherein the volume ratio of the soil bacterial suspension to the propidium monoazide solution is 49:1; and the propidium monoazide solution has a concentration of 2 mmol/L.
6. The method according to claim 1, wherein a dark incubation condition in step 2) comprises: the dark incubation time is 10 minutes, and shaking is performed every 2 minutes.
7. The method according to claim 1, wherein an exposure condition in step 3) comprises: the exposure time is 20 minutes, and shaking is performed every 5 minutes.
8. The method according to claim 7, wherein the exposure temperature is not higher than 37°C.
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