CN110055343A - A kind of specific primer polluted for monitoring the quick-acting phosphorus loads of deposit - Google Patents
A kind of specific primer polluted for monitoring the quick-acting phosphorus loads of deposit Download PDFInfo
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- CN110055343A CN110055343A CN201910363294.7A CN201910363294A CN110055343A CN 110055343 A CN110055343 A CN 110055343A CN 201910363294 A CN201910363294 A CN 201910363294A CN 110055343 A CN110055343 A CN 110055343A
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- 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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Abstract
The present invention relates to a kind of for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit, causes water eutrophication to carry out risk anticipation mainly for the accumulation of the deposits phosphorus element such as river, lake.Step of the invention is as follows: designing specific primer: 5'-GCTCACCAAGCCGAAGATG-3' according to the floating mould door Pla3 mesh kind sensitive to rapid available phosphorus variation in riverbed sludge, the use of can directly amplify length from all kinds of sediment sample DNA is about that 534bp can not cultivate floating mould door Pla3 mesh Pseudomonas target fragment with 16s universal primer 806R 5'-GGACTACHVGGGTWTCTAAT-3' pairing, the accumulation of deposit phosphorus element is evaluated according to the relative abundance of environmental sample target fragment copy number causes water eutrophication risk.The present invention is to overcome the limitation of traditional index evaluation and biological culture based on ecological statistical method and molecular biology method, and rapid available phosphorus sensitive indicator target sequence can be directly amplified from hybrid dna sample and carries out overall merit.
Description
Technical field
It is the invention belongs to environmental pollution monitoring technical field, in particular to a kind of dirty for monitoring the quick-acting phosphorus loads of deposit
The specific primer of dye can be used for the amplification of sediment sample rapid available phosphorus sensitive indicator target sequence.
Background technique
The endogenous pollution of fluviolacustrine deposit object is the principal element for influencing water quality, when overlying water pollutant concentration occurs
When variation or bottom mud in lake are disturbed, the pollutant being accumulated in bed mud can be discharged to water body again again, cause " secondary dirt
Dye ".Available phosphorus contents often respond phosphorus content in water body in bed mud, characterize water body eutrophication degree indirectly.Floating mould door
(Planctomycetes) Pseudomonas is prevalent in seawater, brackish water, fresh water etc., their growth demand often with algae in water body
Class is mutually beneficial.Currently, separated floating mould category (Planctomyces) and small pyriform Pseudomonas (Pirellula) etc. are all obligate good
Oxygen bacterium, in floating mould door there are many more and floating mould the farther away bacterium of relationships such as belong to, as Candidatus Brocadia,
Candidatus Kuenenia belongs to etc., they fail isolated pure bacterial strain so far, and not yet obtain definite designation and point
Class, wherein Pla3 exist now it is more can not cultivate Pseudomonas, and be prevalent in bed mud sample, count different bed mud samples
The discovery of Illumina sequencing result, there are positive correlations with available phosphorus contents for the relative abundance of these Pseudomonas.Water body richness is sought
Feedingization is generally bred rapidly situation and is commented based on nutrition contents such as water systems'phosphoruses, by observation algae and other planktonic organisms
Estimate, but these means tend not to anticipation phosphorus element accumulation tendency, therefore, excavates the sensitive floating mould door Pla3 changed to phosphorus element
Mesh kind biological indicator can play the ability to predict for improving water eutrophication assessment.
The separation of traditional biological indicator bacteria relies primarily on the classical biological method that selective medium is separately cultured, but
The method separation cycle is long, heavy workload and is not easy to obtain purpose function bacterium, this makes floating mould door Pla3 mesh that can not cultivate Pseudomonas
Separation and abundance metering greatly promote, and also reduce the probability for excavating this specific kind strain resource.With molecular biology
Rapid development, the molecular detection technology of based on PCR is using more and more common in environmental microorganism research.By to bacterial strain
DNA extraction is carried out, target fragment is expanded using bacterium 16s rDNA universal primer 338F/806R, is sequenced it can be seen that its gene sequence
Column, but specific not strong, the hybrid dna sample that especially a variety of bacterium coexist of the method are not possible to existing primer pair from mixing
It closes and directly amplifies floating mould door Pla3 mesh kind Variable Area partial sequence in DNA sample, and then microbial administration is difficult to be utilized
Agent prejudges the accumulation of bed mud rapid available phosphorus and water eutrophication.
Summary of the invention
In order to overcome the disadvantages of the above prior art, it is quick-acting for monitoring deposit that the purpose of the present invention is to provide one kind
The specific primer of phosphorus load pollution is designed according to the floating mould door Pla3 mesh kind sensitive to rapid available phosphorus variation in riverbed sludge
It is about that 534bp can not cultivate floating mould door that specific primer directly amplifies length from all kinds of sediment sample DNA
Pla3 mesh Pseudomonas target fragment is evaluated the accumulation of deposit phosphorus element according to the relative abundance of environmental sample target fragment copy number and is caused
Water eutrophication risk causes water eutrophication to carry out risk anticipation the accumulation of the deposits phosphorus element such as river, lake.
To achieve the goals above, the technical solution adopted by the present invention is that:
A kind of specific primer polluted for monitoring the quick-acting phosphorus loads of deposit, becomes according to rapid available phosphorus in riverbed sludge
Change sensitive floating mould door Pla3 mesh kind and designs specific primer are as follows: 5'-GCTCACCAAGCCGAAGATG-3' is logical with 16s
Length is directly amplified from all kinds of deposit DNA with primer 806R 5'-GGACTACHVGGGTWTCTAAT-3' pairing use
Floating mould door Pla3 mesh Pseudomonas target fragment can not be cultivated for 534bp, utilizes primer amplification target fragment relative abundance and speed
Phosphorus load linearly positively related feature is imitated, indicates the quick-acting phosphorus loads of deposit, and evaluates the accumulation of deposit phosphorus element and causes water body
Outrophication risk.
The deposit is one or more of river, river and lake Sediments.
The 5'-GCTCACCAAGCCGAAGATG-3' is forward primer, and 5'-GGACTACHVGGGTWTCTAAT-3' is
Reverse primer.
The specific primer is expanded for that can not cultivate floating mould door Pla3 mesh Pseudomonas target fragment.
5'-GCTCACCAAGCCGAAGATG-3' of the present invention is to use Primer5 software according to floating mould door
Sequence information comprehensive design in Pla3 mesh ncbi database.
Compared with prior art, the beneficial effects of the present invention are:
Method of the invention can solve shortage primer at present and directly amplify from hybrid dna sample to be become with rapid available phosphorus
Change the problem of sensitive floating mould door Pla3 mesh kind 16s rDNA can be changed region partial sequence, it is micro- using high sensitive instruction
Biology accurately prejudges water eutrophication caused by bed mud source.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure of 3 kinds of bed mud sample DNA amplified productions of Pla3F/806R primer pair.
Fig. 2 is 3 kinds of bed mud sample available phosphorus contents and the regression analysis of PCR product relative abundance.
Specific embodiment
Clear, complete description is carried out to technical solution of the present invention with reference to the accompanying drawings and examples, it is clear that retouch below
The embodiment stated is a part of the embodiments of the present invention, instead of all the embodiments, not to the contents of the present invention and protection model
It encloses and is construed as limiting, anyone is combined and is not having under the inspiration of the present invention or by the feature of the present invention and other prior arts
Other embodiments obtained under the premise of creative work are made, protection scope of the present invention is belonged to.
Embodiment: Pla3F is named as with specific primer 5'-GCTCACCAAGCCGAAGATG-3'() it is forward primer,
5'-GGACTACHVGGGTWTCTAAT-3' is that reverse primer (806R in universal primer) is that reverse primer pairing uses, can be from
The floating mould door Pla3 mesh kind mesh that length is about the rapid available phosphorus variation sensitivity of 534bp is directly amplified in bed mud sample DNA
Genetic fragment, prejudge sample in rapid available phosphorus accumulation.
Specific steps are as follows:
It chooses sampled point bed mud at 3 in different rivers to be sampled, extracts DNA and carry out PCR amplification test.PCR amplification body
System is 25 μ L, positive including 0.5 μ L DNA profiling of following component (from the total DNA sample about 10ng of bed mud sample extraction), 2.5 μ L
Primer Pla3F (10pM), 2.5 μ L reverse primer 806R (10pM), 2.5 μ 10 × PCR of L buffer (containing Mg2+), 2.0 μ L
DNTP s (2.5mmol/L), 0.1 μ L Taq archaeal dna polymerase (5U/ μ L), sterilizing distilled water supply 25 μ L.
Pcr amplification reaction condition are as follows: 95 DEG C of 5min of initial denaturation are denaturalized 94 DEG C of 1min, and anneal 56 DEG C of 50s, extend 72 DEG C
1min, totally 30 recycle, 72 DEG C of extension 10min, 4 DEG C of preservations.The sequence that present embodiment is amplified carries out Ago-Gel
Electrophoresis detection, for testing result as shown in Figure 1, M swimming lane is standard DL2000 in Fig. 1,1-3 swimming lane is respectively bed mud sample DNA
Amplification, No. 0 swimming lane is the amplification of blank control.It can be seen that the specific primer that present embodiment uses
Pla3F/806R can accurately amplify the target sequence piece of floating mould door Pla3 mesh kind from all bed mud sample DNAs
Section, and interference stripes will not be generated in blank control.Corresponding sequence fragment is selected at random to be ligated and transformed into carrier T respectively greatly
The sequencing of enterobacteria DH5 α competent cell, sequencing result are as follows: sequence length 534bp, and analyzed through blast, it is
Uncultured planctomycete PLA3 sequence.
Numerical value conversion, bed mud sample 1-3 target stripe are carried out to target stripe relative abundance by QuantityOne software
Relative abundance (intensity) is respectively 180,247,253, by directly measuring sample available phosphorus contents, as a result, it has been found that bed mud sample 1-
3 available phosphorus contents are respectively 35.4mg/kg, 48.6mg/kg and 51.1mg/kg.Property fitting result is as shown in Fig. 2, bed mud sample
Preferable correlation, R is presented with Pla3F/806R specific primer PCR product relative abundance in available phosphorus contents2Greater than 0.99.
The specific primer Pla3F/806R designed through the invention it can be seen from the embodiment can be accurately the bottom of from
The target sequence segment of floating mould door Pla3 mesh kind is amplified in mud sample product hybrid dna and bed mud speed is indicated according to relative abundance
Imitate phosphorus variation, comprehensive anticipation water eutrophication risk.
Sequence table
<110>Tsinghua University
<120>a kind of for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 534
<212> DNA
<213> Uncultured planctomycete clone PLA3
<400> 1
gctcaccaag ccgaagatgg gtaccgggtg tgagagcatg gtccggctca ctgggactga 60
gacactgtcc agacgtctac ggatggctgc agtcgagaat cttccgcaat gggcgaaagc 120
ctgacggagc gacgccgcgt gcgggatgaa ggccttcggg ttgtaaaccg ctgtcagttg 180
ggaggaagtg ctatggggtt ctcttcatag cttgaccgat cttcagagga agtacgggct 240
aagtacgtgc cagcagccgc ggtaatacgt accgtacgaa cgttattcgg aattactggg 300
cttaaagagt ccgtaggcgg ctttaaaggt gaggtgtgaa atcccacggc ttaaccgtgg 360
aattgcgctt caaaccataa ggcttgaggg agatagagga aagcggaact gatggtggag 420
cggtgaaatg cgttgatatc atcaggaacg ccggtggcga aagcggctca ctggatcttt 480
tctgacgctg aggaacgaaa gctagggtag cgaacgggat tagatacccc ggta 534
Claims (4)
1. a kind of for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit, which is characterized in that according to riverbed sludge
The sensitive floating mould door Pla3 mesh kind of middle rapid available phosphorus variation designs specific primer are as follows: 5'-GCTCACCAAGCCGAAGATG-
3' directly expands from all kinds of deposit DNA with 16s universal primer 806R5'-GGACTACHVGGGTWTCTAAT-3' pairing use
Increase the floating mould door Pla3 mesh Pseudomonas target fragment that can not cultivate that length out is 534bp, it is opposite using primer amplification target fragment
The linear positively related feature of abundance and quick-acting phosphorus loads, indicates the quick-acting phosphorus loads of deposit.
2. according to claim 1 for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit, which is characterized in that institute
Stating deposit is one or more of river, river and lake Sediments.
3. according to claim 1 for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit, which is characterized in that institute
Stating 5'-GCTCACCAAGCCGAAGATG-3' is forward primer, and 5'-GGACTACHVGGGTWTCTAAT-3' is reverse primer.
4. according to claim 1 for monitoring the specific primer of the quick-acting phosphorus load pollutions of deposit, which is characterized in that institute
It states specific primer and is expanded for floating mould door Pla3 mesh Pseudomonas target fragment can not be cultivated.
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| CN201910363294.7A CN110055343B (en) | 2019-04-30 | 2019-04-30 | Method for monitoring rapid-acting phosphorus load pollution of sediment by using specific primers |
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| CN201910363294.7A CN110055343B (en) | 2019-04-30 | 2019-04-30 | Method for monitoring rapid-acting phosphorus load pollution of sediment by using specific primers |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398539A (en) * | 2020-03-09 | 2020-07-10 | 上海交通大学 | Water quality microorganism indication method based on big data and molecular biotechnology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807593A (en) * | 2006-02-14 | 2006-07-26 | 浙江大学 | Separation and authentication method for Planctomyces with anaerobic ammoxidation activity |
| CN104899475A (en) * | 2015-05-21 | 2015-09-09 | 上海大学 | Method for evaluating water quality by using microbial diversity indicators in water sediments |
| CN107460240A (en) * | 2017-08-03 | 2017-12-12 | 广东省实验动物监测所 | A kind of method using changes in gene expression quick detection seawater or Polycyclic Aromatic Hydrocarbons |
-
2019
- 2019-04-30 CN CN201910363294.7A patent/CN110055343B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807593A (en) * | 2006-02-14 | 2006-07-26 | 浙江大学 | Separation and authentication method for Planctomyces with anaerobic ammoxidation activity |
| CN104899475A (en) * | 2015-05-21 | 2015-09-09 | 上海大学 | Method for evaluating water quality by using microbial diversity indicators in water sediments |
| CN107460240A (en) * | 2017-08-03 | 2017-12-12 | 广东省实验动物监测所 | A kind of method using changes in gene expression quick detection seawater or Polycyclic Aromatic Hydrocarbons |
Non-Patent Citations (3)
| Title |
|---|
| ANASTASIA A. IVANOVA等: "Distinct diversity patterns of Planctomycetes associated with the freshwater macrophyte Nuphar lutea (L.) Smith", 《ANTONIE VAN LEEUWENHOEK》 * |
| 舒青龙: "海洋浮霉状菌分子生态学研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
| 黄佩蓓等: "海洋浮霉状菌多样性与生态学功能研究进展", 《微生物学通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398539A (en) * | 2020-03-09 | 2020-07-10 | 上海交通大学 | Water quality microorganism indication method based on big data and molecular biotechnology |
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