CN101519643A - Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby - Google Patents
Method for rapidly separating anaerobic denitrifying bacteria and particular primer thereby Download PDFInfo
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- CN101519643A CN101519643A CN200910071694A CN200910071694A CN101519643A CN 101519643 A CN101519643 A CN 101519643A CN 200910071694 A CN200910071694 A CN 200910071694A CN 200910071694 A CN200910071694 A CN 200910071694A CN 101519643 A CN101519643 A CN 101519643A
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Abstract
The invention relates to a method for rapidly separating bacteria and a particular primer thereby, in particular to a method for rapidly separating anaerobic denitrifying bacteria and a particular primer thereby, solving the problems that the prior anaerobic denitrifying bacteria have long separation period and great workload and are difficult to obtain bacterial strains and pure bacterial strains with the destination functions. The method comprises the following steps: 1, extracting microorganism DNA from a sample for PCR augmentation and separation culture and prescreening the anaerobic denitrifying bacteria; 2, preparing particular culture media to be purified until a colony grows up; 3, carrying out the enrichment culture of the colony for the PCR augmentation and re-screening the anaerobic denitrifying bacteria; 4, repeating the separation culture, the purification culture and the enrichment culture, identifying and carrying out the PCR augmentation for removing repetitive bacterial strains and then, repeating the separation culture and the purification culture to obtain the pure bacterial strains. An upstream primer is SEQ ID of NO.1, and a downstream primer is SEQ ID NO.2.The invention shortens the separation period by 40 percent to 50 percent, has little workload and is easy to obtain destination bacterial strains and pure bacterial strains.
Description
Technical field
The present invention relates to fast separating process and the used primer of a kind of bacterium.
Background technology
Anaerobic denitrifying bacteria is mainly used in the processing of nitrate sewage, and is more with the research of anaerobic denitrifying bacteria control sulfate reduction in recent years simultaneously, and obtained good effect.
At present, the separation cycle of anaerobic denitrifying bacteria is longer, and workload is big in the sepn process, and anaerobic condition is difficult to control, is difficult for obtaining pure bacterial strain simultaneously; Having obtained bacterial strain even also exist, but be not the purpose function stem, and non-purpose bacterial strain is bad to the degradation effect of nitrate and nitrous acid.The separation difficulty of anaerobic denitrifying bacteria causes the research of many basic theories to carry out, and along with the aggravation of going from bad to worse of environment, particularly groundwater azotate pollution, the separation of anaerobic denitrifying bacteria highlights importance just with relevant research.
Summary of the invention
The present invention seeks to long for the separation cycle that solves existing anaerobic denitrifying bacteria, workload is big and be difficult for obtaining the problem of purpose function stem and pure bacterial strain, and provides a kind of fast separating process and used Auele Specific Primer of anaerobic denitrifying bacteria.
The fast separating process of anaerobic denitrifying bacteria is realized according to the following steps: one, adopt DNA extraction agent box to extract the DNA of microorganism in the sample, adopt Auele Specific Primer that DNA is carried out pcr amplification then, selection contains the sample of anaerobic denitrifying bacteria, be inoculated in then in the liquid substratum of anerobe, adopt Hungate anaerobic operation technology (Heng Gaite anaerobic operation technology) to carry out the separation and Culture of anaerobic denitrifying bacteria, the bacterium that separation and Culture is obtained is tested then, just sifts out anaerobic denitrifying bacteria; Two, the anaerobic denitrifying bacteria that primary dcreening operation is obtained is inoculated in the anerobe solid medium, makes to roll purifying that pipe carries out anaerobic denitrifying bacteria and be cultured to and grow bacterium colony; Three, in the anaerobism work box picking list colony inoculation in the anerobe liquid nutrient medium, carry out enrichment culture 7～14 days bacterium liquid, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification then, sift out anaerobic denitrifying bacteria again; Four, to the multiple bacterium liquid that sieves the anaerobic denitrifying bacteria that obtains, carry out separation and Culture, purifying cultivation and enrichment culture successively, bacterium liquid to enrichment culture carries out mirror mirror and gramstaining then, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification again, remove and repeat bacterial strain, repetitive operation separation and Culture then and purifying are cultured to and obtain pure bacterial strain, promptly finish the sharp separation of anaerobic denitrifying bacteria; Wherein the pcr amplification program is that 94 ℃ of preheating 5min, 94 ℃ of sex change 30s, 55.9 ℃ of renaturation 45s, 72 ℃ of extension 90s, circulations are extended 10min 30 times and 72 ℃ in the step 1; Auele Specific Primer is upstream Auele Specific Primer 5 '-GC[T/C in the step 1] [C/T] ACCAAG[G/C] CGACGATC-3 ', downstream Auele Specific Primer 5 '-ACCAGGGTATCTAATCCTG-3 '; The reaction system of pcr amplification is as follows in the step 1:
10×PCR?Buffer 2μL
dNTPs(0.3mmol/L) 2μL
Upstream primer (0.1 μ mol/L) 1 μ L
Downstream primer (0.1 μ mol/L) 1 μ L
rTaq?E(0.3U/μL) 0.3μL
DNA sample (2 μ g/L) 2 μ L
Supply dH
2O to 20 μ L;
The liquid substratum of anerobe is by the KNO of 2.0g in the step 1
3, 2.0g NaNO
3, the MgSO of 0.03g
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O and 1750mL is formed, pH=7.5; The anerobe solid medium is by the KNO of 2.0g in the step 2
3, 2.0g NaNO
3, 0.03g MgSO
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O, 12g agar powder and 1750mL is formed, pH=7.5; Step 1 is identical with the liquid medium component of anerobe in the step 3; Adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification in step 3 and the step 4, identical in the method for employing and the step 1.
The used Auele Specific Primer of the fast separating process of anaerobic denitrifying bacteria, upstream Auele Specific Primer are 5 '-GC[T/C] [C/T] ACCAAG[G/C] CGACGATC-3 ', the downstream Auele Specific Primer is 5 '-ACCAGGGTATCTAATCCTG-3 '.
The sharp separation of anaerobic denitrifying bacteria is compared with the separation method of existing anaerobic denitrifying bacteria among the present invention, and the cycle has shortened 40%～50%, and workload is little; By the pcr amplification of Auele Specific Primer, avoided of the interference of non-anaerobic denitrifying bacteria to detecting, make that whole experimental result error is little, accurately and reliably, obtain purpose bacterial strain and pure bacterial strain easily, and to the good degrading effect of nitrate and nitrous acid.
Description of drawings
Fig. 1 is for separating the sem photograph of the anaerobic denitrifying bacteria that obtains in the embodiment six, Fig. 2 is for separating the atomic force microscope observation figure of the anaerobic denitrifying bacteria that obtains in the embodiment six, Fig. 3 is for separating the Phylogenetic Analysis figure of the anaerobic denitrifying bacteria that obtains in the embodiment six.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the fast separating process of present embodiment anaerobic denitrifying bacteria is realized according to the following steps: one, adopt DNA extraction agent box to extract the DNA of microorganism in the sample, adopt Auele Specific Primer that DNA is carried out pcr amplification then, selection contains the sample of anaerobic denitrifying bacteria, be inoculated in then in the liquid substratum of anerobe, adopt Hungate anaerobic operation technology to carry out the separation and Culture of anaerobic denitrifying bacteria, the bacterium that separation and Culture is obtained is tested then, just sifts out anaerobic denitrifying bacteria; Two, the anaerobic denitrifying bacteria that primary dcreening operation is obtained is inoculated in the anerobe solid medium, makes to roll purifying that pipe carries out anaerobic denitrifying bacteria and be cultured to and grow bacterium colony; Three, in the anaerobism work box picking list colony inoculation in the anerobe liquid nutrient medium, carry out enrichment culture 7～14 days bacterium liquid, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification then, sift out anaerobic denitrifying bacteria again; Four, to the multiple bacterium liquid that sieves the anaerobic denitrifying bacteria that obtains, carry out separation and Culture, purifying cultivation and enrichment culture successively, bacterium liquid to enrichment culture carries out mirror mirror and gramstaining then, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification again, remove and repeat bacterial strain, repetitive operation separation and Culture then and purifying are cultured to and obtain pure bacterial strain, promptly finish the sharp separation of anaerobic denitrifying bacteria; Wherein the pcr amplification program is that 94 ℃ of preheating 5min, 94 ℃ of sex change 30s, 55.9 ℃ of renaturation 45s, 72 ℃ of extension 90s, circulations are extended 10min 30 times and 72 ℃ in the step 1; Auele Specific Primer is upstream Auele Specific Primer 5 '-GC[T/C in the step 1] [C/T] ACCAAG[G/C] CGACGATC-3 ', downstream Auele Specific Primer 5 '-ACCAGGGTATCTAATCCTG-3 '; The reaction system of pcr amplification is as follows in the step 1:
10×PCR?Buffer 2μL
dNTPs(0.3mmol/L) 2μL
Upstream primer (0.1 μ mol/L) 1 μ L
Downstream primer (0.1 μ mol/L) 1 μ L
rTaq?E(0.3U/μL) 0.3μL
DNA sample (2 μ g/L) 2 μ L
Supply dH
2O to 20 μ L;
The liquid substratum of anerobe is by the KNO of 2.0g in the step 1
3, 2.0g NaNO
3, the MgSO of 0.03g
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O and 1750mL is formed, pH=7.5; The anerobe solid medium is by the KNO of 2.0g in the step 2
3, 2.0g NaNO
3, 0.03g MgSO
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O, 12g agar powder and 1750mL is formed, pH=7.5; Step 1 is identical with the liquid medium component of anerobe in the step 3; Adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification in step 3 and the step 4, identical in the method for employing and the step 1.
DNA extraction agent box test kit by specification operation in the present embodiment.
Drive away the oxygen in the substratum in the present embodiment step 1 in the liquid substratum process for preparation of anerobe, be in the substratum boiling part, feed bulk purity and be 99.99% nitrogen to drive away oxygen, in substratum, add simultaneously reductive agent (L-halfcystine), utilize its reductive action to remove oxygen, reducing redox potential (is lower than-100mV), obtain anaerobic condition.
The separation and Culture of anaerobic denitrifying bacteria in the present embodiment step 1 is to cultivate under 35～38 ℃ anaerobic condition 7～12 days.
The bacterium that in the present embodiment step 1 separation and Culture is obtained is tested, by " simple and clear the 8th edition uncle Jie Shi Bacteria Identification handbook tested.
The purifying of anaerobic denitrifying bacteria is cultivated in the present embodiment step 2, is to be cultured to grow bacterium colony under 35～38 ℃ anaerobic condition.
Bacterium liquid to enrichment culture in the present embodiment step 3 carries out mirror mirror and gramstaining, by " simple and clear the 8th edition uncle Jie Shi Bacteria Identification handbook tested.
The pure bacterial strain that obtains in the present embodiment step 4 carries out functional verification, and anaerobic condition is removed nitrate and nitrous nitrate.
The pure bacterial strain that obtains in the present embodiment step 4 carries out Physiology and biochemistry evaluation, fatty acid analysis and 16s rDNA Phylogenetic Analysis, proves that further pure bacterial strain is an anaerobic denitrifying bacteria.
Embodiment two: present embodiment and embodiment one are different is to put in the electromagnetism pot after the composition of anerobe solid medium in the step 2 configures, boiling the back, to add L-halfcystine 0.5g and mass concentration be that 0.2% resazurin solution (indicator) has oxygen to exist this moment, the color of substratum is a pink, continuous heated and boiled, cover pot cover, the feeding quality purity is nitrogen flooding oxygen 30～40min of 99.99%, become colourless by pink until substratum, anaerobism pipe packing then, 20min sterilizes under 121 ℃ condition.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment and embodiment two are different is enrichment culture 8～12 days in the step 3.Other step and parameter are identical with embodiment two.
Embodiment four: present embodiment and embodiment two are different is enrichment culture 10 days in the step 3.Other step and parameter are identical with embodiment two.
Embodiment five: the used Auele Specific Primer of the fast separating process of present embodiment anaerobic denitrifying bacteria, the upstream Auele Specific Primer is 5 '-GC[T/C] [C/T] ACCAAG[G/C] CGACGATC-3 ', the downstream Auele Specific Primer is 5 '-ACCAGGGTATCTAATCCTG-3 '.
Embodiment six: the fast separating process of present embodiment anaerobic denitrifying bacteria is realized according to the following steps: one, adopt DNA extraction agent box to extract the DNA of microorganism in the sample, adopt Auele Specific Primer that DNA is carried out pcr amplification then, selection contains the sample of anaerobic denitrifying bacteria, be inoculated in then in the liquid substratum of anerobe, adopt Hungate anaerobic operation technology to carry out the separation and Culture of anaerobic denitrifying bacteria, the bacterium that separation and Culture is obtained is tested then, just sifts out anaerobic denitrifying bacteria; Two, the anaerobic denitrifying bacteria that primary dcreening operation is obtained is inoculated in the anerobe solid medium, makes to roll purifying that pipe carries out anaerobic denitrifying bacteria and be cultured to and grow bacterium colony; Three, in the anaerobism work box picking list colony inoculation in the anerobe liquid nutrient medium, carry out enrichment culture 10 days bacterium liquid, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification then, sift out anaerobic denitrifying bacteria again; Four, to the multiple bacterium liquid that sieves the anaerobic denitrifying bacteria that obtains, carry out separation and Culture, purifying cultivation and enrichment culture successively, bacterium liquid to enrichment culture carries out mirror mirror and gramstaining then, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification again, remove and repeat bacterial strain, repetitive operation separation and Culture then and purifying are cultured to and obtain pure bacterial strain, promptly finish the sharp separation of anaerobic denitrifying bacteria; Wherein the pcr amplification program is that 94 ℃ of preheating 5min, 94 ℃ of sex change 30s, 55.9 ℃ of renaturation 45s, 72 ℃ of extension 90s, circulations are extended 10min 30 times and 72 ℃ in the step 1; Auele Specific Primer is upstream Auele Specific Primer 5 '-GC[T/C in the step 1] [C/T] ACCAAG[G/C] CGACGATC-3 ', downstream Auele Specific Primer 5 '-ACCAGGGTATCTAATCCTG-3 '; The reaction system of pcr amplification is as follows in the step 1:
10×PCR?Buffer 2μL
dNTPs(0.3mmol/L) 2μL
Upstream primer (0.1 μ mol/L) 1 μ L
Downstream primer (0.1 μ mol/L) 1 μ L
rTaq?E(0.3U/μL) 0.3μL
DNA sample (2 μ g/L) 2 μ L
Supply dH
2O to 20 μ L;
The liquid substratum of anerobe is by the KNO of 2.0g in the step 1
3, 2.0g NaNO
3, the MgSO of 0.03g
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O and 1750mL is formed, pH=7.5; The anerobe solid medium is by the KNO of 2.0g in the step 2
3, 2.0g NaNO
3, 0.03g MgSO
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O, 12g agar powder and 1750mL is formed, pH=7.5; Step 1 is identical with the liquid medium component of anerobe in the step 3; Adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification in step 3 and the step 4, identical in the method for employing and the step 1.
Sample picks up from Daqing oil field ground waterflood system in the present embodiment, according to the on-the-spot oil recovery situation of Daqing oil field present situation, chooses Daqing oil field four factory's apricots nine associating sewage workss.
The sharp separation of anaerobic denitrifying bacteria is compared with existing separation method in the present embodiment, and the cycle has shortened 50%, and has obtained the pure bacterial strain of purpose bacterial strain.
Separate the anaerobic denitrifying bacteria that obtains in the present embodiment and carry out the physiology evaluation, as depicted in figs. 1 and 2, bacterium is that tyrothricin, thalline size are wide 0.4～0.8 μ m * length 1.0～2.5 μ m; The anaerobic denitrifying bacteria that separation obtains in the present embodiment carries out biochemical identification, and the bacterium gramstaining is negative, catalase test is positive, methyl red test is negative, the hydrolyzed starch test is positive, the hydrolysate oil test is positive, the pure test of acetylmethyl is negative, the product indole test is positive, Citrate trianion utilization test is positive, litmus milk reduces and acid cure is solid, nitrate reduction test is positive, the liquefy gelatin test is negative, produce H
2S test is negative, produce that ammonia test is positive, the hydrolysis of urea test is positive, the glucose oxidase fermentation and acid, the glucose fermentation test is negative, the fructose fermentation test is negative, lactose-fermentation test is negative, the sucrose fermentation test is negative, the ethanol fermentation test is negative, growth temperature is that 20～40 ℃, growth pH value are 7.5, flat-plate bacterial colony is rounded, surface elevation, white.
Separate the anaerobic denitrifying bacteria that obtains in the present embodiment and carry out fatty acid analysis, lipid acid mainly is distributed in C
12:0～C
18:0, main lipid acid is C
16:1-CIS-9-FAME, C
16:0-FAME, C
18:1-CIS-11And C
18:0-FAME, content is respectively 9.05%, 35.65%, 27.78% and 6.00% and accounts for 78.48% of lipid acid sum.
Separate the anaerobic denitrifying bacteria that obtains obtains 1446bp after checking order 16SrDNA sequence in the present embodiment, the GenBank accession number is DQ450463, adopt adjacent method (NJ) constructing system evolutionary tree, as shown in Figure 3, the average genetic of 15 bacterial strains of phylogenetic tree is 0.065, phyletic evolution shows, the similarity of the 16S rDNA sequence of bacterial strain and Thauera sp. (AM231040) is 99%, in conjunction with the morphology and the physiology characteristic of bacterial strain, preliminary evaluation belongs to anaerobic denitrifying bacteria for Thauera sp..
Sequence table
＜110〉Harbin Teachers' Univ.
＜120〉a kind of fast separating process of anaerobic denitrifying bacteria and used Auele Specific Primer
<160>2
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉upstream Auele Specific Primer
<220>
<221>misc_feature
<222>(3，4，11)
＜223〉y=t or c, s=g or c
<400>1
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉downstream Auele Specific Primer
<400>2
Claims (5)
1, a kind of fast separating process of anaerobic denitrifying bacteria, the fast separating process that it is characterized in that anaerobic denitrifying bacteria is realized according to the following steps: one, adopt DNA extraction agent box to extract the DNA of microorganism in the sample, adopt Auele Specific Primer that DNA is carried out pcr amplification then, selection contains the sample of anaerobic denitrifying bacteria, be inoculated in then in the liquid substratum of anerobe, adopt Hungate anaerobic operation technology to carry out the separation and Culture of anaerobic denitrifying bacteria, the bacterium that separation and Culture is obtained is tested then, just sifts out anaerobic denitrifying bacteria; Two, the anaerobic denitrifying bacteria that primary dcreening operation is obtained is inoculated in the anerobe solid medium, makes to roll purifying that pipe carries out anaerobic denitrifying bacteria and be cultured to and grow bacterium colony; Three, in the anaerobism work box picking list colony inoculation in the anerobe liquid nutrient medium, carry out enrichment culture 7～14 days bacterium liquid, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification then, sift out anaerobic denitrifying bacteria again; Four, to the multiple bacterium liquid that sieves the anaerobic denitrifying bacteria that obtains, carry out separation and Culture, purifying cultivation and enrichment culture successively, bacterium liquid to enrichment culture carries out mirror mirror and gramstaining then, adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification again, remove and repeat bacterial strain, repetitive operation separation and Culture then and purifying are cultured to and obtain pure bacterial strain, promptly finish the sharp separation of anaerobic denitrifying bacteria; Wherein the pcr amplification program is that 94 ℃ of preheating 5min, 94 ℃ of sex change 30s, 55.9 ℃ of renaturation 45s, 72 ℃ of extension 90s, circulations are extended 10min 30 times and 72 ℃ in the step 1; Auele Specific Primer is upstream Auele Specific Primer 5 '-GC[T/C in the step 1] [C/T] ACCAAG[G/C] CGACGATC-3 ', downstream Auele Specific Primer 5 '-ACCAGGGTATCTAATCCTG-3 '; The reaction system of pcr amplification is as follows in the step 1:
10×PCR?Buffer 2μL
dNTPs(0.3mmol/L) 2μL
Upstream primer (0.1 μ mol/L) 1 μ L
Downstream primer (0.1 μ mol/L) 1 μ L
rTaq?E(0.3U/μL) 0.3μL
DNA sample (2 μ g/L) 2 μ L
Supply dH
2O to 20 μ L;
The liquid substratum of anerobe is by the KNO of 2.0g in the step 1
3, 2.0g NaNO
3, the MgSO of 0.03g
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O and 1750mL is formed, pH=7.5; The anerobe solid medium is by the KNO of 2.0g in the step 2
3, 2.0g NaNO
3, 0.03g MgSO
47H
2The K of O, 0.5g
2HPO
4, the Seignette salt of 10g, the KH of 1.0g
2PO
4, 0.5g FeCl
26H
2The CaCl of O, 0.2g
22H
2The distilled water of O, 12g agar powder and 1750mL is formed, pH=7.5; Step 1 is identical with the liquid medium component of anerobe in the step 3; Adopt Auele Specific Primer that the DNA of bacterium in the bacterium liquid is carried out pcr amplification in step 3 and the step 4, identical in the method for employing and the step 1.
2, the fast separating process of a kind of anaerobic denitrifying bacteria according to claim 1, after configuring, puts in the electromagnetism pot composition that it is characterized in that anerobe solid medium in the step 2, boiling the back, to add L-halfcystine 0.5g and mass concentration be that 0.2% resazurin solution (indicator) has oxygen to exist this moment, the color of substratum is a pink, continuous heated and boiled, cover pot cover, the feeding quality purity is nitrogen flooding oxygen 30～40min of 99.99%, become colourless by pink until substratum, anaerobism pipe packing then, 20min sterilizes under 121 ℃ condition.
3, the fast separating process of a kind of anaerobic denitrifying bacteria according to claim 2 is characterized in that in the step 3 enrichment culture 8～12 days.
4, the fast separating process of a kind of anaerobic denitrifying bacteria according to claim 2 is characterized in that in the step 3 enrichment culture 10 days.
5, the used Auele Specific Primer of a kind of fast separating process of anaerobic denitrifying bacteria, it is characterized in that the upstream Auele Specific Primer is 5 '-GC[T/C] [C/T] ACCAAG[G/C] CGACGATC-3 ', the downstream Auele Specific Primer is 5 '-ACCAGGGTATCTAATCCTG-3 '.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884040A (en) * | 2016-05-11 | 2016-08-24 | 长沙学院 | Preparation method of water body denitrification material |
| CN109593838A (en) * | 2018-12-28 | 2019-04-09 | 江苏大学 | A kind of screening technique of the heterotrophic nitrification of saline-alkali tolerant-anaerobic denitrifying bacterial strain |
| CN109628309A (en) * | 2018-12-28 | 2019-04-16 | 江苏大学 | A kind of separation method of saline-alkali tolerant denitrifying microorganism bacterial strain |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372942C (en) * | 2005-10-21 | 2008-03-05 | 哈尔滨工业大学 | Quick-speed PCR quantitative determination method of denitrifying bacteria direct dilution by multiple proportions |
| CN101086023A (en) * | 2007-06-08 | 2007-12-12 | 哈尔滨工业大学 | Primer and fluorescent probe for detecting anaerobic denitrifying bacteria |
| CN101368200B (en) * | 2008-07-18 | 2011-09-21 | 哈尔滨师范大学 | Culture medium for sifting motion anaerobic coreduction sulfate and inverse nitrification function bacterial strain and sifting motion method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884040A (en) * | 2016-05-11 | 2016-08-24 | 长沙学院 | Preparation method of water body denitrification material |
| CN109593838A (en) * | 2018-12-28 | 2019-04-09 | 江苏大学 | A kind of screening technique of the heterotrophic nitrification of saline-alkali tolerant-anaerobic denitrifying bacterial strain |
| CN109628309A (en) * | 2018-12-28 | 2019-04-16 | 江苏大学 | A kind of separation method of saline-alkali tolerant denitrifying microorganism bacterial strain |
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