CN106048058A - Primers and method for identifying diversity of eukaryotic algae in activated sludge of epoxypropane saponified wastewater - Google Patents
Primers and method for identifying diversity of eukaryotic algae in activated sludge of epoxypropane saponified wastewater Download PDFInfo
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Abstract
The invention relates to primers and a method for identifying the diversity of eukaryotic algae in activated sludge of epoxypropane saponified wastewater. There is a pair of the primers. The upstream primer nucleotide sequence is as shown in SEQ ID NO. 1, and the downstream primer nucleotide sequence is as shown in SEQ ID NO. 2. The invention also relates to a method for identifying the diversity of eukaryotic algae in activated sludge of epoxypropane saponified wastewater. By the design of the eukaryotic algae-specific primers, metagenome DNA of the activated sludge of epoxypropane saponified wastewater can undergo specific amplification to obtain different eukaryotic algae. Then, eukaryotic algae species in the activated sludge can be identified.
Description
Technical field
The present invention relates to the multifarious primer of Eukaryotic Algae and side in a kind of qualification epoxy propane saponified wastewater activated sludge
Method, belongs to biological technical field.
Background technology
Epoxy propane saponified wastewater is a kind of large amount of sewage being produced the generation of expoxy propane process by chlorohydrination.This waste water has
Have that pH value is high, calcium chloride content high, feature high for COD, wherein contain the chloride being difficult to biochemical degradation in a large number, as chloropropyl alcohol,
Nemamort, dichloropropane etc..This high salt, environment can be caused severe contamination rich in organic waste water, use rationally
Effective manner processes this waste water and can not be ignored.
Biological activated sludge method is the major way that current epoxy propane saponified wastewater processes.Activated sludge is sewage disposal
The main body that in plant aeration tank, organic pollution materials converts, its bioactive height fundamentally determines a sewage treatment plant
Sewage treatment capacity, and the biological activity of activated sludge depends primarily on microorganism species 26S Proteasome Structure and Function therein, bag
Include the microorganisms such as antibacterial, ancient bacterium, virus and Eukaryotic Algae.
Chinese patent literature CN103849678A (application number 201210517032.X) discloses a kind of fast micro algae kind
The method identified.Mainly comprise the following steps: the picking that fallen by single algae is in the PCR pipe adding 10～20 μ L water in advance, or algae-containing water body
Centrifugal collection frustule 104～106Add in the PCR pipe of 10～20 μ L water after individual, after frustule piping and druming is scattered, directly by PCR
Pipe is placed in PCR instrument, heats 20～30min in 99 DEG C, and piping and druming makes DNA discharge as template, with the general 18S of eukaryotic cell again
RDNA or ITS primer carries out PCR reaction.By product is checked order and sequence analysis, the kind information of algae strain to be measured can be obtained.
Although above-mentioned authentication method can identify the kind information of algae strain to be measured, but due to Microbial Communities in Activated Sludge group
Structure is complicated, and said method cannot be carried out precise Identification, and most of microorganism is can not to be separated training by traditional method
Support, by traditional enrichment, separate, cultivate be difficult to sludge microbe group is accurately analyzed.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that eukaryotic algae in a kind of qualification epoxy propane saponified wastewater activated sludge
The multifarious primer of class and method.
Technical scheme is as follows:
The multifarious primer of Eukaryotic Algae in a kind of qualification epoxy propane saponified wastewater activated sludge, this primer is a pair,
Forward primer nucleotide sequence is as shown in SEQ ID NO.1, and downstream primer nucleotide sequence is as shown in SEQ ID NO.2.
Forward primer: 5 '-AAAGTTAGGTGAGCG-3 '；SEQ ID NO.1
Downstream primer: 5 '-CAGTCAATCGGTATG-3 '.SEQ ID NO.2
A kind of multifarious method of Eukaryotic Algae in qualification epoxy propane saponified wastewater activated sludge, comprises the steps:
(1) take epoxy propane saponified wastewater activated sludge sample, extract STb gene；
(2) with step (1) prepare STb gene as template, utilize in above-mentioned qualification epoxy propane saponified wastewater activated sludge
The multifarious primer of Eukaryotic Algae carries out PCR amplification, prepares amplified production；The clip size of amplified production is at about 700bp；
(3) amplified production preparing step (2) checks order, and sequencing result carries out blastn comparison and surely plants；
(4) sequencing result of step (3) is inputted GenBank, in GenBank, carry out similarity by blast program
Search for, and the sequence finding similarity-rough set high the result of gained after comparison compares, apply MAGA4.0 software
Use N-J method constructing system cladogram, click on Bootstrap Test of Phylogeny and select Neighbor-Joining to enter
Row bootstrap value is analyzed, repeated sampling, obtain that credibility is the highest one.
According to currently preferred, in described step (1), the step extracting STb gene is as follows:
Taking epoxy propane saponified wastewater activated sludge sample, under the conditions of 4 DEG C, 4000r/min is centrifuged 10min, abandons supernatant, makes
Extract test kit with soil DNA and extract the macro genome DNA in activated sludge.
According to currently preferred, in described step (2), PCR amplification system is as follows, total system 50 μ L:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 54 DEG C of annealing 45s, 72 DEG C extend 45s, carry out 35 circulations；72℃
Extend 7min.
According to currently preferred, in described step (3), the step checking order amplified production is as follows:
The amplified production that recycling step (2) prepares, connects into PGM-T carrier, prepares and connects product, then converts large intestine bar
Bacterium JM109 competent cell, then checks order.
It is further preferred that the linked system of the above-mentioned PGM-T of connecting into carrier is 10 μ L:
Condition of contact is: 16 DEG C connect overnight.
It is further preferred that above-mentioned escherichia coli jm109 competent cell is adopted and is prepared with the following method:
(I) cultivation of recipient bacterium
Take the mono-bacterium colony of escherichia coli (E.coli) JM109 of activation, be inoculated in 3～5ml LB fluid mediums, 37 DEG C
Lower shaken cultivation 10～14 hours are to the logarithmic growth later stage, by this bacteria suspension with 1:(50～100) volume ratio be inoculated in
In 100mlLB fluid medium, 37 DEG C of shaken cultivation 2～3 hours to OD600=0.4～0.6, prepare culture fluid；
(II)CaCl2Method prepares competent cell
1. proceeding in centrifuge tube by culture fluid, ice is put 10 minutes, and then at 4 DEG C, 3000g is centrifuged 10 minutes；
2. supernatant is abandoned, with the CaCl of the 0.05mol/L of pre-cooling2Solution 10ml suspension cell gently, ice puts 15～30 minutes
After, at 4 DEG C, 3000g is centrifuged 10 minutes；
3. abandon supernatant, add the 4ml pre-cooling CaCl containing the 0.05mol/L that mass concentration is 15% glycerol2Solution, hangs gently
Floating cell, ice is put 3 minutes, prepares competent cell suspension；
It is further preferred that the step of above-mentioned conversion escherichia coli jm109 competent cell is as follows:
1. escherichia coli jm109 competent cell 200 μ L add connection product 1 μ L mixture in centrifuge tube, place on ice
30min, period shake prevents recipient bacterium from sinking to the bottom 3 times；
2. 42 DEG C of heating in water bath 90 seconds, take out and put into 2min on ice；
3. sterilizing LB fluid medium 800 μ L is added, mixing；
4. 37 DEG C, 180rpm shaking table cultivation 45min, make thalline recover；
5. 3000rpm is centrifuged 2min, removes supernatant 800 μ L, remaining 200 μ L, resuspended；
6. re-suspension liquid is applied on solid medium, and room temperature stands 30 minutes until liquid is absorbed；
7. 37 DEG C of calorstats, are inverted and cultivate 12～20h, prepare.
It is further preferred that above-mentioned LB fluid medium, every liter of component is as follows:
Tryptone 10g, yeast powder 5g, sodium chloride 10g.
Above-mentioned solid medium on the basis of LB fluid medium, every liter add following component:
50mg/ml ampicillin (Amp) 200ml, the IPTG 350 μ L of mass concentration 20%, the X-of mass concentration 2%
Gal 2ml, agar 20g.
According to currently preferred, in described step (3), sequencing result is carried out blastn (http: //
Blast.ncbi.nlm.nih.gov) step that comparison is planted surely is as follows:
Website login http://www.ncbi.nlm.nih.gov, clicks on BLAST；Subsequently into tblatn, at Enter
Sequence replicating after order-checking is pasted by Query Sequence dialog box, clicks on BLAST, Gen Bank automatically the sequence of input
Row compare with all correlated serieses in sequence resources storehouse；In comparative result, find out the highest one of homology and determine not
Know the kind of algae.
According to currently preferred, in described step (4), repeated sampling number of times is 1000 times.
Beneficial effect
1, the present invention is by design Eukaryotic Algae specific primer, can be from the grand base of epoxy propane saponified wastewater activated sludge
Because in group DNA, specific amplification obtains different Eukaryotic Algae such that it is able to the Eukaryotic Algae kind in identified activity mud；
2, the multifarious method of Eukaryotic Algae in qualification epoxy propane saponified wastewater activated sludge of the present invention, step
Simply, it is possible to efficiently separate the Eukaryotic Algae identifying in activated sludge, for research Eukaryotic Algae further in activated sludge
Effect and the effect of whole mud flora lay a good foundation.
Accompanying drawing explanation
Fig. 1 is PCR electrophoresis result photo described in embodiment 2 and comparative example；
In figure: A, be to use Standard PCR procedure result in comparative example 1；B, be for specific primer improve after PCR journey
Sequence result；C, it is to use 18S rDNA universal primer PCR amplification described in comparative example 2；
Fig. 2 is the evolution that embodiment 2 is set up when analyzing Eukaryotic Algae multiformity in epoxy propane saponified wastewater activated sludge
Tree.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is described further, but institute's protection domain of the present invention not only
It is limited to this.
Biological material source
Escherichia coli (E.coli) JM109 is purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd.
PGM-T carrier is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
Embodiment 1
1.NCBI searches nucleotide sequence.Log in www.ncbi.nlm.nih.gov/Gen-Bank, can be from Gen Bank
Obtain gene order as template.
The target amplification monoid of the present invention is Eukaryotic Algae, according to Burki (2008) to photosynthesis eukaryote, Adl
(2012) to Eukaryotic molecular system classification method, target amplification monoid is refined as chlorella (Chlorophyceae), red algae
(Porphyridium), Euglena (Euglena), charophyta (Charophyta), dinoflagellate (Pyrrophyta), hidden algae
(Cryptophyceae) whip algae (Haptophyta), Chlorarachniophyta, chrysophyceae (Chrysophyceae), silicon, are determined
12 Eukaryotic Algae doors such as algae (Diatom), Brown algae (Phaeophyta), xanthophyta (Xanthophyta).Come according to test requirements document
Determining the DNA sequence needing amplification, and know its coding structure gene region array, concrete grammar is as follows:
In Search dialog box, select Nucleotide, dialog box is inserted Nucleotide name to be searched
Claim, such as input Chlorophyceae (chlorella), click on search and i.e. obtain the many sequences letter relevant to Chlorophyceae
Breath, therefrom selects close 1 of species, then its sequence is compared with correlated series in Gen Bank.
2. sequence homology analysis with compare.
Use the related softwares such as DNAMAN to carry out gene comparision, concretely comprise the following steps and open DNAMAN, click on Sequences,
Multiple sequence alignment is selected, by the mesh of above-mentioned acquisition in seeking scope dialog box in File menu
Sequence insert one by one, click on Done, software the most automatically input all sequences compare, determine homology region.Look for
Go out the region that homology is higher, in this region, select the template of design of primers, the most permissible by above computer operation
Complete sequence editor and process and the determination of design of primers position.
3. use Primer Premier 5.0 to design primer.
Primer Premier 5.0 is used to design the application software being best suitable for primer, utilizes its senior primer function to enter
The design of row primer database search, nested primer, primer editor and analysis etc., can be designed that the ideal of efficient amplification ability
Primer, it is also possible to design the primer sequence of the PCR primer being up to more than 50kb for amplification.This software is mainly by Gene
Bank sequence editor, Primer design of primers, Align gene comparision, Enzyme restriction analysis and Motif motif analysis etc. are several
Major function plate forms.
As a example by Algae, design primer, can directly open Primer Premier 5.0, click in File menu New
DNA Sequence, then blank space inputs Algae gene order below, new interface occurs, clicks on Primer, then clicks on
Search, pop-up dialogue box, click on OK button, the new window containing various possible primer information occurs.Draw accordingly by clicking on
Thing searches each pair of primer in interface, will show the various information of primer accordingly.Certainly at primer editor to program finally
Dynamic and the manual primer sequence found is edited, and the type, research purpose and the meaning that design according to concrete gene primer are entered
Row selects.
4.BLAST comparison.
First log into http://www.ncbi.nlm.nih.gov, then click on BLAST；Next enters Nucleotide
Blast, pastes the sequence replicating of above-mentioned acquisition in Search dialog box, and clicking on BLAST, Gen Bank will be automatically input
Sequence compare with all correlated serieses in sequence resources storehouse.In comparative result, list what all of correlated series compared
Situation, the most also include same species different length and different plant species in Gen Bank registration related gene sequence combine
Composition and division in a proportion is relatively.
If the specificity analyses to special primer, it is also possible to log in primer before and after www.Gramene.org
Blastn position analysis the most only has in the close positions of genome mates, then completely it is believed that have preferable specificity.
Then can select to use corresponding gene comparision data according to research purpose, it is also possible to utilize the information acquisition provided in result
A kind of gene order oneself needed most, the PCR primer design for next step lays the first stone.
5. the overall merit of primer after screening.
After screening, first primer can carry out assay on software, and the software primer to be possessed of design primer divides
Analysis Function of Evaluation.May then pass through the gel imaging system after PCR amplification and carry out verification experimental verification: one be PCR primer amount with
And the specificity and efficiency of PCR amplification；Two are whether to form primer dimer band；Three is with DNA as template design primer
Time, pcr amplification product whether with expection PCR primer sizableness.
Fig. 2 is the PCR primer electrophoretogram of the Algae mentioned by the present invention, it can be seen that this PCR primer not only yield is high,
And specific band is clearly, without hairpin structure and primer dimer, product is identical with expection PCR primer size.
6. the detection again of gene after design of primers
It is to be appropriate to drawing of special, High Efficiency PC R amplification to find out in the multipair primer of design that primer repeatedly screens
Thing.Main it should be noted that following 2 points: one is to carry out back a series of primers obtained respectively examining, i.e. every in Gen Bank
Primer carries out homology search in the blastnr of comparison instrument (http://www.ncbi.nlm.nih.gov/blast), abandons
Fall and compare high primer with other Homoeologies of genome, be namely likely to form the primer of mispairing.General continuous 10bp
Above homology is likely to form 3 ' ends of more stable mispairing, particularly primer should avoid the homology of continuous 5～6bp.Two
When being with mRNA for template design primer, to substantially judge the montage of exon and intron first with the knowledge of bioinformatics
Site, then discards the primer being placed exactly in splice site.
The most finally determine and can the primer sequence of specific amplification Eukaryotic Algae be:
Forward primer: 5 '-AAAGTTAGGTGAGCG-3 '；SEQ ID NO.1
Downstream primer: 5 '-CAGTCAATCGGTATG-3 '.SEQ ID NO.2
Embodiment 2
Epoxy propane saponified wastewater activated sludge described in the present embodiment takes from BinHua Group Co., Ltd's epoxy
Activated sludge in propane saponification waste-water processing procedure.
A kind of multifarious method of Eukaryotic Algae in qualification epoxy propane saponified wastewater activated sludge, comprises the steps:
(1) take epoxy propane saponified wastewater activated sludge sample, extract STb gene；
Activated sludge takes from the activated sludge of Shandong sewage treatment plant of Bin Hua group, and this mud is that process is epoxy propane saponified
The activated sludge of waste water.The fresh sludge fetched from sewage treatment plant, 4 DEG C of 4000r/min are centrifuged 10min, abandon supernatant and be placed on 4
DEG C refrigerator preserves.Remembered according to embodiment 1 in Chinese patent literature CN103789300A (application number 201410067311.X)
The method carried extracts the macro genome DNA in activated sludge；
(2) with step (1) prepare STb gene as template, utilize in above-mentioned qualification epoxy propane saponified wastewater activated sludge
The multifarious primer of Eukaryotic Algae carries out PCR amplification, extracts the main bar Han wanted PCR primer with clean scalpel under uviol lamp
The residing agar block of band, purge process subsequently reclaims test kit explanation according to TIANGEN company agarose gel DNA and carries out, system
Obtain amplified production；The clip size of amplified production is at about 700bp；
Pcr amplification primer thing sequence is as follows:
Forward primer: 5 '-AAAGTTAGGTGAGCG-3 '；SEQ ID NO.1
Downstream primer: 5 '-CAGTCAATCGGTATG-3 '.SEQ ID NO.2
PCR amplification system is as follows, total system 50 μ L:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 53.5 DEG C of annealing 45s, 72 DEG C extend 45s, carry out 35 circulations；72
DEG C extend 7min.
Above-mentioned escherichia coli jm109 competent cell is adopted and is prepared with the following method:
(I) cultivation of recipient bacterium
Take the mono-bacterium colony of escherichia coli (E.coli) JM109 of activation, be inoculated in 3～5ml LB fluid mediums, 37 DEG C
Lower shaken cultivation 10～14 hours are to the logarithmic growth later stage, by this bacteria suspension with 1:(50～100) volume ratio be inoculated in
In 100mlLB fluid medium, 37 DEG C of shaken cultivation 2～3 hours to OD600=0.4～0.6, prepare culture fluid；
(II)CaCl2Method prepares competent cell
1. proceeding in centrifuge tube by culture fluid, ice is put 10 minutes, and then at 4 DEG C, 3000g is centrifuged 10 minutes；
2. supernatant is abandoned, with the CaCl of the 0.05mol/L of pre-cooling2Solution 10ml suspension cell gently, ice puts 15～30 minutes
After, at 4 DEG C, 3000g is centrifuged 10 minutes；
3. abandon supernatant, add the CaCl of the 4ml pre-cooling 0.05mol/L containing 15% glycerol2Solution, gently suspension cell, ice
Put 3 minutes, prepare competent cell suspension；
The linked system of the above-mentioned PGM-T of connecting into carrier is 10 μ L:
Condition of contact is: 16 DEG C connect overnight.
The step of above-mentioned conversion escherichia coli jm109 competent cell is as follows:
1. escherichia coli jm109 competent cell 200 μ L add connection product 1 μ L mixture in centrifuge tube, place on ice
30min, period shake prevents recipient bacterium from sinking to the bottom 3 times；
2. 42 DEG C of heating in water bath 90 seconds, take out and put into ice chest 2min on ice；
3. sterilizing LB fluid medium 800 μ L is added, mixing；
4. 37 DEG C, 180rpm shaking table cultivation 45min, make bacterium recover；
5. 3000rpm is centrifuged 2min, removes supernatant 800 μ L, remaining 200 μ L, resuspended；
6. re-suspension liquid is applied on solid medium, and room temperature stands 30 minutes until liquid is absorbed；
7. 37 DEG C of calorstats, are inverted and cultivate 12～20h, prepare.
Above-mentioned LB fluid medium, every liter of component is as follows:
Tryptone 10g, yeast powder 5g, sodium chloride 10g
Above-mentioned solid medium on the basis of LB fluid medium, every liter add following component:
50mg/ml ampicillin (Amp) 200ml, the IPTG 350 μ L of mass concentration 20%, the X-of mass concentration 2%
Gal 2ml, agar 20g.
(3) to step (2) prepare amplified production check order, sequencing result carry out blastn (http: //
Blast.ncbi.nlm.nih.gov) comparison is planted surely；
Website login http://www.ncbi.nlm.nih.gov, clicks on BLAST；Subsequently into tblatn, at Enter
Sequence replicating after order-checking is pasted by Query Sequence dialog box, clicks on BLAST, Gen Bank automatically the sequence of input
Row compare with all correlated serieses in sequence resources storehouse；In comparative result, find out the highest one of homology and determine not
Know the kind of algae.
The PCR primer of purebred Eukaryotic Algae DNA and bacterium solution PCR primer submit to Bo Shang biotechnology (Shanghai) Co., Ltd. to enter
Row order-checking, sequencing result carries out blastn (http://blast.ncbi.nlm.nih.gov) comparison and surely plants.
(4) sequencing result of step (3) is inputted GenBank, by blast program at GenBank
(www.ncbi.nlm.nih.gov) carry out similarity searching in, and the result of gained finds similarity-rough set high after comparison
Sequence compare, application MAGA4.0 software use N-J method constructing system cladogram, click on Bootstrap Test
Of Phylogeny selects Neighbor-Joining to carry out the analysis of bootstrap value, repeated sampling 1000 times, obtains credible
Spending the highest one, result is as shown in Figure 2.
Comparative example 1
Epoxy propane saponified wastewater activated sludge described in the present embodiment takes from BinHua Group Co., Ltd's epoxy
Activated sludge in propane saponification waste-water processing procedure.
Standard PCR reaction system and program comprise the steps:
(1) take epoxy propane saponified wastewater activated sludge sample, extract STb gene；
Activated sludge takes from the activated sludge of Shandong sewage treatment plant of Bin Hua group, and this mud is that process is epoxy propane saponified
The activated sludge of waste water.The fresh sludge fetched from sewage treatment plant, 4 DEG C of 4000r/min are centrifuged 10min, abandon supernatant and be placed on 4
DEG C refrigerator preserves.Remembered according to embodiment 1 in Chinese patent literature CN103789300A (application number 201410067311.X)
The method carried extracts the macro genome DNA in activated sludge；
(2) with step (1) prepare STb gene as template, utilize in above-mentioned qualification epoxy propane saponified wastewater activated sludge
The multifarious primer of Eukaryotic Algae carries out PCR amplification, and the clip size of amplified production is at about 700bp；
Pcr amplification primer thing sequence is as follows:
Forward primer: 5 '-AAAGTTAGGTGAGCG-3 '；SEQ ID NO.1
Downstream primer: 5 '-CAGTCAATCGGTATG-3 '.SEQ ID NO.2
PCR amplification system is as follows, total system 50 μ L:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 50 DEG C of annealing 45s, 72 DEG C extend 45s, carry out 30 circulations；72℃
Extending 10min, agarose gel electrophoresis result is as shown in Figure 1A.
Comparative example 2
Epoxy propane saponified wastewater activated sludge described in this comparative example is again taken from BinHua Group Co., Ltd
Activated sludge in epoxy propane saponified wastewater processing procedure.
This comparative example uses described in Chinese patent literature CN103849678A (application number 201210517032.X) description
Eukaryotic cell general 18s rDNA primer carries out PCR amplification.
Eukaryote general 18s rDNA primer, sequence is:
18s-F 5’-CCGAATTCGTCGACAACCTGGTTGATCCTGCCAGT-3’；
18s-R 5’-CCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTAC-3’.
In this example, other operating process are with embodiment 2.
Test example
Take the PCR primer 5 μ L point sample obtained by the method for comparative example and embodiment 2 respectively in containing 1 μ g/mL ethidium bromide
0.8% agarose gel carry out electrophoresis detection.Electrophoretic buffer is 1 × TAE, and voltage is 100V, and electrophoresis time is 20min,
Gel imaging system is observed and records accordingly result, as shown in Figure 1.
By Figure 1A it can be seen that use specific primer to use conventional method to carry out PCR amplification, effect is poor, although have
Specific band is amplified out, and size is at about 700bp, and band is not it is obvious that there is several sample not to be amplified out
Come.
By Figure 1B it can be seen that use the PCR program after adjusting can amplify clearly for specific primer
Specific band, size is at about 700bp, and occurs without other non-specific bands, and amplification efficiency is high.Can by this primer
With the simple and effective Eukaryotic Algae amplified in activated sludge, and its multiformity can be studied.
By Fig. 1 C it can be seen that use general 18S rDNA primer can amplify specific band, size is at 1800bp
Left and right, the band amplified relatively disperse.Follow-up to its be analyzed finding this primer amplification out do not only have Eukaryotic Algae,
The most also including the gene of part protozoacide etc., therefore it can not by specific for Eukaryotic Algae amplification out.Through surveying
This is a kind of to find only have chrysophyceae through its Eukaryotic Algae amplifying activated sludge after sequence, it is impossible to expand out by other algae,
Thus do not reach the multifarious purpose of Eukaryotic Algae in analysis activated sludge.
Claims (9)
1. identify the multifarious primer of Eukaryotic Algae in epoxy propane saponified wastewater activated sludge for one kind, it is characterised in that this draws
Thing is a pair, forward primer nucleotide sequence as shown in SEQ ID NO.1, downstream primer nucleotide sequence such as SEQ ID NO.2
Shown in.
2. identify the multifarious method of Eukaryotic Algae in epoxy propane saponified wastewater activated sludge for one kind, it is characterised in that include
Following steps:
(1) take epoxy propane saponified wastewater activated sludge sample, extract STb gene；
(2) STb gene prepared with step (1) is as template, utilizes and identifies described in claim 1 that epoxy propane saponified wastewater activity is dirty
In mud, the multifarious primer of Eukaryotic Algae carries out PCR amplification, prepares amplified production；The clip size of amplified production is left at 700bp
Right；
(3) amplified production preparing step (2) checks order, and sequencing result carries out blastn comparison and surely plants；
(4) sequencing result of step (3) is inputted GenBank, in GenBank, carries out similarity searching by blast program,
And the sequence finding similarity-rough set high the result of gained after comparison compares, application MAGA4.0 software uses
N-J method constructing system cladogram, clicks on Bootstrap Test of Phylogeny and selects Neighbor-Joining to carry out
Bootstrap value is analyzed, repeated sampling, obtain that credibility is the highest one.
3. method as claimed in claim 2, it is characterised in that in described step (1), the step extracting STb gene is as follows:
Taking epoxy propane saponified wastewater activated sludge sample, under the conditions of 4 DEG C, 4000r/min is centrifuged 10min, abandons supernatant, uses soil
Earth DNA extraction kit extracts the macro genome DNA in activated sludge.
4. method as claimed in claim 2, it is characterised in that in described step (2), PCR amplification system is as follows, total system 50
μ L:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 54 DEG C of annealing 45s, 72 DEG C extend 45s, carry out 35 circulations；72 DEG C of extensions
7min。
5. method as claimed in claim 2, it is characterised in that in described step (3), the step that amplified production is checked order
As follows:
The amplified production that recycling step (2) prepares, connects into PGM-T carrier, prepares and connects product, then converts escherichia coli
JM109 competent cell, then checks order.
6. method as claimed in claim 5, it is characterised in that described in connect into the linked system of PGM-T carrier be 10 μ L:
Condition of contact is: 16 DEG C connect overnight.
7. method as claimed in claim 5, it is characterised in that described escherichia coli jm109 competent cell uses such as lower section
Prepared by method:
(I) cultivation of recipient bacterium
Take the mono-bacterium colony of escherichia coli (E.coli) JM109 of activation, be inoculated in 3～5ml LB fluid mediums, shake at 37 DEG C
Swing cultivation 10～14 hours to the logarithmic growth later stage, by this bacteria suspension with 1:(50～100) volume ratio be inoculated in 100ml LB
In fluid medium, 37 DEG C of shaken cultivation 2～3 hours to OD600=0.4～0.6, prepare culture fluid；
(II)CaCl2Method prepares competent cell
1. proceeding in centrifuge tube by culture fluid, ice is put 10 minutes, and then at 4 DEG C, 3000g is centrifuged 10 minutes；
2. supernatant is abandoned, with the CaCl of the 0.05mol/L of pre-cooling2Solution 10ml suspension cell gently, after ice puts 15～30 minutes, 4 DEG C
Lower 3000g is centrifuged 10 minutes；
3. abandon supernatant, add the 4ml pre-cooling CaCl containing the 0.05mol/L that mass concentration is 15% glycerol2Solution, suspends thin gently
Born of the same parents, ice is put 3 minutes, prepares competent cell suspension；
It is further preferred that the step of above-mentioned conversion escherichia coli jm109 competent cell is as follows:
1. escherichia coli jm109 competent cell 200 μ L add connection product 1 μ L mixture in centrifuge tube, place on ice
30min, period shake prevents recipient bacterium from sinking to the bottom 3 times；
2. 42 DEG C of heating in water bath 90 seconds, take out and put into 2min on ice；
3. sterilizing LB fluid medium 800 μ L is added, mixing；
4. 37 DEG C, 180rpm shaking table cultivation 45min, make thalline recover；
5. 3000rpm is centrifuged 2min, removes supernatant 800 μ L, remaining 200 μ L, resuspended；
6. re-suspension liquid is applied on solid medium, and room temperature stands 30 minutes until liquid is absorbed；
7. 37 DEG C of calorstats, are inverted and cultivate 12～20h, prepare.
8. method as claimed in claim 7, it is characterised in that described LB fluid medium, every liter of component is as follows:
Tryptone 10g, yeast powder 5g, sodium chloride 10g；
Described solid medium on the basis of LB fluid medium, every liter add following component:
50mg/ml ampicillin (Amp) 200ml, the IPTG 350 μ L of mass concentration 20%, the X-gal of mass concentration 2%
2ml, agar 20g.
9. method as claimed in claim 2, it is characterised in that in described step (3), sequencing result is carried out blastn comparison
The step of fixed kind is as follows:
Website login http://www.ncbi.nlm.nih.gov, clicks on BLAST；Subsequently into tblatn, at Enter
Sequence replicating after order-checking is pasted by Query Sequence dialog box, clicks on BLAST, Gen Bank automatically the sequence of input
Row compare with all correlated serieses in sequence resources storehouse；In comparative result, find out the highest one of homology and determine not
Know the kind of algae；
Preferably, in described step (4), repeated sampling number of times is 1000 times.
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