CN111471750A - Detection method of bacterial strain for rapidly degrading nitrogen in sewage and application thereof - Google Patents
Detection method of bacterial strain for rapidly degrading nitrogen in sewage and application thereof Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 166
- 239000010865 sewage Substances 0.000 title claims abstract description 96
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 83
- 230000000593 degrading effect Effects 0.000 title claims abstract description 75
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title abstract description 14
- 238000012408 PCR amplification Methods 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000001962 electrophoresis Methods 0.000 claims abstract description 6
- 238000012790 confirmation Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 33
- 239000013612 plasmid Substances 0.000 claims description 17
- 238000011529 RT qPCR Methods 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000013642 negative control Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- 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 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 239000005549 deoxyribonucleoside Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 239000001226 triphosphate Substances 0.000 claims description 2
- 235000011178 triphosphate Nutrition 0.000 claims description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 claims 1
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 239000003344 environmental pollutant Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 231100000719 pollutant Toxicity 0.000 description 3
- 238000012271 agricultural production Methods 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000003911 water pollution Methods 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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Abstract
The invention belongs to the technical field of environmental engineering, and discloses a detection method of a bacterial strain for rapidly degrading nitrogen in sewage and application thereof, wherein the detection method of the bacterial strain for rapidly degrading nitrogen in sewage comprises the following steps: obtaining a sample of the bacterial strain for rapidly degrading nitrogen in the sewage, and preparing a bacterial strain standard substance; establishing a standard curve corresponding to the concentration of the strain standard substance and the critical cycle number; carrying out PCR amplification and electrophoresis confirmation on a sample of the strain, and establishing a standard curve corresponding to the concentration of the sample of the strain and the critical cycle number; and obtaining the copy number of the strain genes in the sample of the strain according to a standard curve corresponding to the concentration of the sample of the strain and the critical cycle number, namely obtaining the number of the strains in the sample of the strain for rapidly degrading the nitrogen in the sewage. The detection standard substance of the strain provided by the invention has high sensitivity, simple and convenient preparation method, good purity and wide detection range, and can be used for quickly and quantitatively detecting the strain for degrading nitrogen in sewage, thereby realizing the reduction of the denitrification cost of the sewage.
Description
Technical Field
The invention belongs to the technical field of environmental engineering, and particularly relates to a detection method of a strain for rapidly degrading nitrogen in sewage and application thereof.
Background
Currently, the closest prior art: people pay more attention to environmental protection in recent decades, and the national strength is increased to prevent and treat polluted water, but the pollution of rivers, lakes and the like can not be effectively inhibited for many years, the water pollution load is continuously increased, the eutrophication problem is still serious, and the safety of drinking water is still threatened. Along with the sewage receiving range of a sewage treatment plant is enlarged, some industrial sewage is also brought into the sewage treatment plant, the pollutant components are complex and various, the problems of overhigh concentration of nutrient salts such as nitrogen, phosphorus and the like frequently occur, and the prior art is not basically provided with the advanced treatment of nitrogen and phosphorus removal. In addition, with the increase of sewage quantity, sewage treatment plants exceed the original treatment capacity of the sewage treatment plants, the normal operation of sewage treatment facilities is influenced, and the nitrogen, phosphorus and the like in the effluent water are discharged in an overproof manner. Waste in agricultural production activities, livestock and poultry breeding, aquaculture, rural resident life and the like enters water through surface runoff, interflow, farmland drainage and underground leakage, and serious pollution is caused to the water. Especially, in agricultural production, excessive fertilizer, improperly-treated livestock and poultry manure, high-density aquaculture, excessive feed addition, kitchen garbage in residential areas, excrement in toilets and the like cause nitrogen pollution of water. The non-point source pollution has the characteristics of scattered and multiple pollution sources, wide pollution range, large pollutant amount, more complex pollutant components and process and the like, so that the nitrogen pollution is more difficult to control. The traditional denitrification technology has complex denitrification process in sewage; the newly appeared biological denitrification technology has high cost and is difficult to popularize and use.
In summary, the problems of the prior art are as follows: the traditional denitrification technology has complex denitrification process in sewage; the newly appeared biological denitrification technology has high cost and is difficult to popularize and use.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a detection method of a bacterial strain for rapidly degrading nitrogen in sewage and application thereof.
The invention is realized in such a way that a method for detecting a bacterial strain capable of rapidly degrading nitrogen in sewage comprises the following steps:
acquiring a sample of a strain for rapidly degrading nitrogen in sewage to obtain a standard product of the strain for rapidly degrading the nitrogen in the sewage;
establishing a standard curve corresponding to the concentration of a bacterial strain standard substance for rapidly degrading nitrogen in the sewage and the critical cycle number;
performing PCR amplification and electrophoresis confirmation on the sample of the strain for rapidly degrading the nitrogen in the sewage in the step one, and establishing a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number;
and step four, obtaining the copy number of the strain gene in the sample of the strain for rapidly degrading the nitrogen in the sewage according to a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number, and obtaining the number of the strains in the sample of the strain for rapidly degrading the nitrogen in the sewage.
Further, the method for obtaining the sample of the strain capable of rapidly degrading the nitrogen in the sewage comprises the following steps:
(1) collecting sewage of 5m L, adding dropwise into a triangular flask containing 30m L of enrichment culture solution, and culturing at 28 deg.C for 3-5 days;
(2) dripping the cultured liquid onto a flat plate, and separating by adopting a dilution coating flat plate method;
(3) culturing for 2 days at 28 ℃, wherein the strain falling into the inclined plane is a sample of the strain for rapidly degrading nitrogen in the sewage.
Further, the strain standard substance for rapidly degrading nitrogen in sewage comprises recombinant plasmids or recombinant cells of the strain genes.
Further, the preparation method of the bacterial strain standard substance for rapidly degrading nitrogen in sewage comprises the following steps:
(1) designing specific primers aiming at the genes of the bacterial strains capable of degrading nitrogen in sewage;
(2) taking a sample of the strain for rapidly degrading nitrogen in the sewage, extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample, and performing PCR amplification by using the DNA sample as a template;
(3) purifying the PCR amplification product, and linking the purified PCR amplification product with a plasmid vector to obtain a white colony transformed with the plasmid;
(4) and (3) selecting a full colony to inoculate in a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a bacterial strain standard substance for rapidly degrading nitrogen in sewage.
Further, the PCR amplification method comprises the following steps:
extracting DNA of a sample of a strain for rapidly degrading nitrogen in sewage;
constructing a PCR amplification system;
performing PCR reaction at 95 deg.C for 3min, performing denaturation at 93 deg.C for 30s, annealing at 55 deg.C for 30s, and extending at 72 deg.C for 3 min; 45 cycles, and final extension at 70 ℃ for 4 min.
Further, the method for constructing the PCR amplification system comprises the following steps:
1) carrying out gradient dilution on the plasmid standard substance by a plurality of times to be used as a quantitative PCR template DNA, and carrying out real-time quantitative PCR amplification;
2) operating according to the kit instructions, adding reagents required by real-time quantitative PCR, wherein the PCR reaction system is 20 mu L, and adding 2 mu L plasmid standard substance as a reaction template to perform real-time quantitative PCR reaction;
3) and (3) establishing a real-time quantitative PCR standard curve by taking deionized water as a negative control, taking the logarithm value of the dilution gradient of the standard substance as an abscissa and taking the critical cycle number as an ordinate.
Further, the PCR reaction system is as follows:
50% of glycerol with the mass ratio of 3 mu L, 1 mu L dimethyl sulfoxide, deoxyribonucleoside triphosphate with the concentration of 1 mu L of 2.5 mol/L, Tris-HCl with the concentration of 2 mu L of 25 mmol/L, MgCl with the concentration of 2 mu L of 25 mmol/L2Taq enzyme with concentration of 0.5 mu L of 2U/L and two primers.
Further, the PCR amplification product was confirmed by electrophoresis on an agarose gel electrophoresis apparatus.
The invention also aims to provide an application method of the strain for rapidly degrading nitrogen in sewage, which comprises the following steps:
culturing a strain capable of rapidly degrading nitrogen in sewage to obtain thalli, collecting the thalli, and inoculating the thalli into the sewage;
fermenting at 35 deg.C under 150rpm shaking for 40 h.
Further, the operating conditions applied to the sewage treatment are as follows:
the dissolved oxygen is 0.5 mg/L-5.0 mg/L, the mass ratio of carbon to nitrogen is 15-25, the temperature is 35 ℃, and the pH is 7.0.
In summary, the advantages and positive effects of the invention are: the detection standard substance of the strain for rapidly degrading the nitrogen in the sewage provided by the invention has the characteristics of combination of spectral recognition and specificity, and is high in sensitivity; the detection method is simple and convenient, and the detection result can be obtained only by analyzing a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number; the linear detection range is wide, the bacterial strain for degrading nitrogen in the surface sewage can be rapidly and quantitatively detected, and the sewage denitrification cost is reduced.
Drawings
FIG. 1 is a flow chart of a method for detecting strains capable of rapidly degrading nitrogen in sewage according to an embodiment of the present invention.
FIG. 2 is a flow chart of a method for obtaining a sample of a strain that rapidly degrades nitrogen in wastewater according to an embodiment of the present invention.
FIG. 3 is a flow chart of a method for preparing a standard bacterial strain for rapidly degrading nitrogen in sewage according to an embodiment of the present invention.
FIG. 4 is a flowchart of a method for constructing a PCR amplification system according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a detection method of a bacterial strain for rapidly degrading nitrogen in sewage and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in FIG. 1, the method for detecting the bacterial strain capable of rapidly degrading nitrogen in sewage provided by the embodiment of the invention comprises the following steps:
s101, obtaining a sample of a strain for rapidly degrading nitrogen in sewage to obtain a standard product of the strain for rapidly degrading nitrogen in sewage;
s102, establishing a standard curve corresponding to the concentration of a strain standard substance for rapidly degrading nitrogen in the sewage and the critical cycle number;
s103, carrying out PCR amplification and electrophoresis confirmation on the sample of the strain for rapidly degrading the nitrogen in the sewage in the S101, and establishing a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number;
s104, obtaining the copy number of the strain gene in the sample of the strain for rapidly degrading the nitrogen in the sewage according to a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number, and obtaining the number of the strains in the sample of the strain for rapidly degrading the nitrogen in the sewage.
As shown in fig. 2, the method for obtaining a sample of a strain that rapidly degrades nitrogen in sewage provided by the embodiment of the present invention comprises:
s201, collecting 5m L of sewage, dropwise adding the sewage into a triangular flask containing 30m L of enrichment culture solution, and culturing for 3-5 days at 28 ℃;
s202, dripping the cultured liquid onto a flat plate, and separating by adopting a dilution coating flat plate method;
s203, culturing for 2 days at the temperature of 28 ℃, wherein the strain falling into the inclined plane is a sample of the strain for rapidly degrading nitrogen in the sewage.
As shown in fig. 3, the preparation method of the standard strain for rapidly degrading nitrogen in sewage provided by the embodiment of the invention is as follows:
s301, designing a specific primer aiming at the gene of the strain capable of degrading the nitrogen in the sewage;
s302, taking a sample of the strain for rapidly degrading nitrogen in the sewage, extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample, and performing PCR amplification by using the DNA sample as a template;
s303, purifying the PCR amplification product, and linking the purified PCR amplification product with a plasmid vector to obtain a white colony converted with the plasmid;
s304, selecting full colonies, inoculating the colonies into a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a bacterial strain standard substance for rapidly degrading nitrogen in sewage.
As shown in fig. 4, the method for constructing a PCR amplification system provided in the embodiment of the present invention includes:
s401, carrying out gradient dilution on the plasmid standard substance by multiple times to serve as a quantitative PCR template DNA, and carrying out real-time quantitative PCR amplification;
s402, operating according to the kit instructions, adding reagents required by real-time quantitative PCR, wherein the PCR reaction system is 20 mu L, adding 2 mu L plasmid standard substance as a reaction template, and carrying out real-time quantitative PCR reaction;
and S403, establishing a real-time quantitative PCR standard curve by taking deionized water as a negative control, taking the logarithm value of the dilution gradient of the standard substance as an abscissa and taking the critical cycle number as an ordinate.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
<110> Taizhou institute of occupational and technology
<120> detection method of bacterial strain for rapidly degrading nitrogen in sewage and application thereof
<160>1
<210>1
<211>1091
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<213> Artificial Sequence (Artificial Sequence)
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cgtatacgcc ctttggggga aagatttatc ggcaaaggat tagcccgcgt tggattaggt 180
agttggtggg gtaatggcct accaagccga cgatccatag ctggtttgag aggatgatca 240
gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatctta 300
gacaatgggg gcaaccctga tctagccatg ccgcgtgagt gatgaaggcc ctagggttgt 360
aaagctcttt cagctgggaa gataatgacg gtaccagcag aagaagcccc ggctaactcc 420
gtgccagcag ccgcggtaat acggaggggg ctagcgttgt tcggaattac tgggcgtaaa 480
gcgcacgtag gcggaccgga aagttggggg tgaaatcccg gggctcaacc tcggaactgc 540
cttcaaaact atcggtctgg agttcgagag aggtgagtgg aattccgagt gtagaggtga 600
aattcgtaga tattcggagg aacaccagtg gcgaaggcgg ctcactggct cgatactgac 660
gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta 720
aacgatgaat gccagtcgtc gggcagcatg ctgttcggtg acacacctaa cggattaagc 780
attccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca 840
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc aacccttgac 900
atcgcaggac cgctccagag atggagtttt ctcgtaagag acctgtggac aggtgctgca 960
tggctgtcgt cagctcgtgt cgtgagatgt tcgggttaag tcccggcaac gagcgcaacc 1020
cacactctta gttgccagca tttggttggg gcactctaag agaactgccg atgatagtcg 1080
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Claims (11)
1. A method for detecting a bacterial strain capable of rapidly degrading nitrogen in sewage is characterized by comprising the following steps:
acquiring a sample of a strain for rapidly degrading nitrogen in sewage to obtain a standard product of the strain for rapidly degrading the nitrogen in the sewage;
establishing a standard curve corresponding to the concentration of a bacterial strain standard substance for rapidly degrading nitrogen in the sewage and the critical cycle number;
performing PCR amplification and electrophoresis confirmation on the sample of the strain for rapidly degrading the nitrogen in the sewage in the step one, and establishing a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number;
and step four, obtaining the copy number of the strain gene in the sample of the strain for rapidly degrading the nitrogen in the sewage according to a standard curve corresponding to the concentration of the sample of the strain for rapidly degrading the nitrogen in the sewage and the critical cycle number, and obtaining the number of the strains in the sample of the strain for rapidly degrading the nitrogen in the sewage.
2. The method for detecting the bacterial strain capable of rapidly degrading the nitrogen in the sewage according to claim 1, wherein the method for obtaining the sample of the bacterial strain capable of rapidly degrading the nitrogen in the sewage comprises the following steps:
(1) collecting sewage of 5m L, adding dropwise into a triangular flask containing 30m L of enrichment culture solution, and culturing at 28 deg.C for 3-5 days;
(2) dripping the cultured liquid onto a flat plate, and separating by adopting a dilution coating flat plate method;
(3) culturing for 2 days at 28 ℃, wherein the strain falling into the inclined plane is a sample of the strain for rapidly degrading nitrogen in the sewage.
3. The method for detecting a strain capable of rapidly degrading nitrogen in sewage according to claim 1, wherein the standard substance of the strain capable of rapidly degrading nitrogen in sewage comprises a recombinant plasmid or a recombinant cell of a gene of the strain.
4. The method for detecting the strain capable of rapidly degrading the nitrogen in the sewage according to claim 1, wherein the preparation method of the standard strain capable of rapidly degrading the nitrogen in the sewage comprises the following steps:
(1) designing specific primers aiming at the genes of the bacterial strains capable of degrading nitrogen in sewage;
(2) taking a sample of the strain for rapidly degrading nitrogen in the sewage, extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample, and performing PCR amplification by using the DNA sample as a template;
(3) purifying the PCR amplification product, and linking the purified PCR amplification product with a plasmid vector to obtain a white colony transformed with the plasmid;
(4) and (3) selecting a full colony to inoculate in a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a bacterial strain standard substance for rapidly degrading nitrogen in sewage.
5. The method for detecting the bacterial strain for rapidly degrading the nitrogen in the sewage according to claim 1, wherein the PCR amplification method comprises the following steps:
extracting DNA of a sample of a strain for rapidly degrading nitrogen in sewage;
constructing a PCR amplification system;
PCR reaction was performed.
6. The method according to claim 4, wherein the PCR reaction is performed at 95 ℃ for 3min, followed by denaturation at 93 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 3 min; 45 cycles, and final extension at 70 ℃ for 4 min.
7. The method for detecting the bacterial strain capable of rapidly degrading the nitrogen in the sewage according to claim 4, wherein the method for constructing the PCR amplification system comprises the following steps:
1) carrying out gradient dilution on the plasmid standard substance by a plurality of times to be used as a quantitative PCR template DNA, and carrying out real-time quantitative PCR amplification;
2) operating according to the kit instructions, adding reagents required by real-time quantitative PCR, wherein the PCR reaction system is 20 mu L, and adding 2 mu L plasmid standard substance as a reaction template to perform real-time quantitative PCR reaction;
3) and (3) establishing a real-time quantitative PCR standard curve by taking deionized water as a negative control, taking the logarithm value of the dilution gradient of the standard substance as an abscissa and taking the critical cycle number as an ordinate.
8. The method according to claim 6, wherein the PCR reaction system comprises 50% by mass of glycerol 3 μ L, dimethyl sulfoxide 1 μ L, deoxyribonucleoside triphosphate 1 μ L at 2.5 mol/L, Tris-HCl 2 μ L at 25 mmol/L, MgCl 2 μ L at 25 mmol/L2Taq enzyme with concentration of 0.5 mu L of 2U/L and two primers.
9. The method according to claim 1, wherein the PCR amplification product is confirmed by electrophoresis on an agarose gel electrophoresis apparatus.
10. An application method of the strain for rapidly degrading nitrogen in sewage according to any one of claims 1 to 8 is characterized in that the application method of the strain for rapidly degrading nitrogen in sewage comprises the following steps: culturing a strain capable of rapidly degrading nitrogen in sewage to obtain thalli, collecting the thalli, and inoculating the thalli into the sewage; fermenting at 35 deg.C under 150rpm shaking for 40 h.
11. The method for detecting bacterial strains capable of rapidly degrading nitrogen in sewage as claimed in claim 9, wherein the operating conditions for sewage treatment are that the dissolved oxygen is 0.5 mg/L-5.0 mg/L, the carbon-nitrogen mass ratio is 15-25, the temperature is 35 ℃, and the pH is 7.0.
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| CN102230009A (en) * | 2011-06-14 | 2011-11-02 | 河海大学 | Method for quantifying ammonia oxidizing archaea in lacustrine deposit |
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