CN110055343B - Method for monitoring rapid-acting phosphorus load pollution of sediment by using specific primers - Google Patents
Method for monitoring rapid-acting phosphorus load pollution of sediment by using specific primers Download PDFInfo
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- 239000013049 sediment Substances 0.000 title claims abstract description 46
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 40
- 239000011574 phosphorus Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000012544 monitoring process Methods 0.000 title claims abstract description 12
- 239000012634 fragment Substances 0.000 claims abstract description 16
- 241000233614 Phytophthora Species 0.000 claims abstract description 8
- 241000894007 species Species 0.000 claims abstract description 8
- 238000011109 contamination Methods 0.000 claims 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 19
- 238000012851 eutrophication Methods 0.000 abstract description 11
- 238000009825 accumulation Methods 0.000 abstract description 8
- 238000011156 evaluation Methods 0.000 abstract description 3
- 238000010170 biological method Methods 0.000 abstract description 2
- 238000007619 statistical method Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 101100352374 Oryza sativa subsp. japonica PLA3 gene Proteins 0.000 description 2
- 241000589952 Planctomyces Species 0.000 description 2
- 241000222383 Polyporales Species 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 241000516663 uncultured planctomycete Species 0.000 description 2
- 241000468339 Candidatus Brocadia Species 0.000 description 1
- 241000480585 Candidatus Kuenenia Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 241000589956 Pirellula Species 0.000 description 1
- 241001180199 Planctomycetes Species 0.000 description 1
- 241000233626 Plasmopara Species 0.000 description 1
- 241000635201 Pumilus Species 0.000 description 1
- 241000231139 Pyricularia Species 0.000 description 1
- 241000233639 Pythium Species 0.000 description 1
- 101100029566 Rattus norvegicus Rabggta gene Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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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
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Abstract
The invention relates to a specific primer for monitoring quick-acting phosphorus load pollution of sediments, which is mainly used for carrying out risk prediction on water eutrophication caused by accumulation of phosphorus in sediments such as rivers, lakes and the like. The method comprises the following steps: specific primers are designed according to the species of the phylum Furomyces Pla3, which is sensitive to the change of rapid-acting phosphorus in bottom mud of rivers and lakes: 5'-GCTCACCAAGCCGAAGATG-3', and the 16s universal primer 806R 5'-GGACTACHVGGGTWTCTAAT-3' is used for directly amplifying a fragment with the length of about 534bp and the purpose of non-culturable phytophthora Plan 3 order from the DNA of various sediment samples, and the risk of eutrophication of water bodies caused by sediment phosphorus accumulation is evaluated according to the relative abundance of the copy number of the fragment of the environment sample order. The invention is based on an ecological statistical method and a molecular biological method, overcomes the limitations of traditional index evaluation and biological culture, and can directly amplify a quick-acting phosphorus sensitive indicator target sequence from a mixed DNA sample for comprehensive evaluation.
Description
Technical Field
The invention belongs to the technical field of environmental pollution monitoring, and particularly relates to a method for monitoring quick-acting phosphorus load pollution of sediments by using a specific primer, which can be used for amplifying quick-acting phosphorus sensitive indicator bacteria target sequences of sediment samples.
Background
The endogenous pollution of river and lake sediments is a main factor influencing the water quality of a water body, and when the concentration of the pollutants on the water body changes or the lake bottom sediment is disturbed, the pollutants accumulated in the bottom sediment can be released to the water body again to cause secondary pollution. The quick-acting phosphorus content in the bottom mud often responds to the phosphorus content in the water body, and indirectly represents the eutrophication degree of the water body. Phylum pumilus (planctomyces) is ubiquitous in seawater, brackish water, fresh water, etc., and their growth needs often mutually benefit algae in water. At present, isolated phytophthora (Planctomyces) and pyricularia (Pirellula) and the like are obligate aerobic bacteria, and a plurality of bacteria which are far away from the phytophthora and the like, such as Candidatus Brocadia and Candidatus Kuenenia and the like exist in the phylum of the phytophthora, so far, the bacteria cannot be successfully separated to obtain pure strains, formal naming and classification are not obtained, wherein more non-culturable bacteria exist under the mesh of Pla3 and commonly exist in sediment samples, and the result of Illumina sequencing on different sediment samples is proved that the relative abundance of the bacteria has positive correlation with the content of the available phosphorus. The water eutrophication is generally evaluated by observing the rapid propagation condition of algae and other plankton based on the content of nutrient substances such as phosphorus and the like in the water, but the accumulation trend of phosphorus cannot be predicted by the means, so that the prediction capability of improving the water eutrophication evaluation can be realized by mining the sensitive phytophthora Pla3 genus indicator of the phosphorus change.
The traditional biological indicator bacteria are mainly separated by a classical biological method relying on selective culture medium separation culture, but the method has long separation period and large workload and is not easy to obtain target functional bacteria, so that the separation and abundance measurement of the Nocardia Pla3 order Nossaccus of the Phytophthora are greatly improved, and the probability of excavating the strain resource of the specific species is also reduced. With the rapid development of molecular biology, PCR-based molecular detection technology has become more and more popular for environmental microbial research. The gene sequence of the strain can be obtained by DNA extraction of the strain and amplification of a target fragment by using a bacteria 16s rDNA universal primer 338F/806R, but the specificity of the method is not strong, particularly, a mixed DNA sample with various bacteria coexists, and the partial sequence of a variable region of the species of Aphyllophorales Pla3 cannot be directly amplified from the mixed DNA sample by using the existing primer pair, so that the quick-acting phosphorus accumulation of substrate sludge and water eutrophication are difficult to be judged by using a biological indicator.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for monitoring the rapid-acting phosphorus load pollution of sediments by using a specific primer, the specific primer is designed according to the species of the phylum Furomycetes Pla3 which is sensitive to the rapid-acting phosphorus change in bottom mud of rivers and lakes, the target fragment of the genus Pla3 of the phylum Furomycetes, which is about 534bp and can not be cultured, is directly amplified from the DNA of various sediment samples, the risk of water eutrophication caused by sediment phosphorus accumulation is evaluated according to the relative abundance of the copy number of the target fragment of the environmental sample, and the risk pre-judgment is carried out on the water eutrophication caused by sediment phosphorus accumulation of rivers, lakes and the like.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for monitoring quick-acting phosphorus load pollution of sediment by using specific primers is characterized in that the specific primers are designed according to the species of Furomycetes Pla3 meshes which are sensitive to quick-acting phosphorus change in bottom mud of rivers and lakes: 5'-GCTCACCAAGCCGAAGATG-3', directly amplifying a target fragment of the genus of Planta 3 of the Unculturable Phytophthora with the length of 534bp from DNA of various sediments by matching with a 16s universal primer 806R 5'-GGACTACHVGGGTWTCTAAT-3', indicating the rapid-acting phosphorus load of the sediments by utilizing the characteristic that the relative abundance of the target fragment amplified by the primer is in linear positive correlation with the rapid-acting phosphorus load, and evaluating the risk of water eutrophication caused by sediment phosphorus accumulation.
The sediment is one or more of river and lake sediment.
The 5'-GCTCACCAAGCCGAAGATG-3' is a forward primer, and the 5'-GGACTACHVGGGTWTCTAAT-3' is a reverse primer.
The specific primer is used for amplifying a target fragment of a bacterium of the order of Pla3 in Noncubaceae.
The 5'-GCTCACCAAGCCGAAGATG-3' of the invention was designed by synthesis using Primer5 software based on sequence information in NCBI database of the phylum Furomyces Pla3 mesh.
Compared with the prior art, the invention has the beneficial effects that:
the method can solve the problem that the existing primer is lacked to directly amplify the partial sequence of the 16s rDNA variable region of the Plasmopara 3 genus of the Aphyllophorales with quick-acting phosphorus change sensitivity from a mixed DNA sample, and can accurately predict the water eutrophication caused by sediment sources by using high-sensitivity indicating microorganisms.
Drawings
FIG. 1 is a gel electrophoresis chart of DNA amplification products of 3 sediment samples of the Pla3F/806R primer pair.
FIG. 2 is a regression analysis of the available phosphorus content of 3 sediment samples and the relative abundance of PCR products.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings and embodiments, and it is to be understood that the embodiments described below are some, not all embodiments of the present invention, and do not limit the content and scope of the present invention, and any other embodiments obtained by combining the present invention with other features of the prior art or by the teaching of the present invention will fall within the scope of the present invention.
Example (b): the specific primer 5'-GCTCACCAAGCCGAAGATG-3' (named as Pla3F) is used as a forward primer, 5'-GGACTACHVGGGTWTCTAAT-3' is used as a reverse primer (806R in the universal primer) for pairing, and the gene fragment of the Pythium species of Pla3 order, which is sensitive to the change of the fast-acting phosphorus and has the length of about 534bp, can be directly amplified from the DNA of a sediment sample, so that the accumulation of the fast-acting phosphorus in the sample is predicted.
The specific operation steps are as follows:
and (3) selecting bottom sediment of sampling points in different rivers for sampling, and extracting DNA for carrying out PCR amplification test. The PCR amplification system was 25. mu.L, and included 0.5. mu.L of DNA template (about 10ng of total DNA sample extracted from the sediment sample), 2.5. mu.L of forward primer Pla3F (10pM), 2.5. mu.L of reverse primer 806R (10pM), 2.5. mu.L of 10 XPCR buffer (Mg 2+), 2.0. mu.L of dNTPs (2.5mmol/L), 0.1. mu.L of Taq DNA polymerase (5U/. mu.L), and sterilized double distilled water to make up 25. mu.L.
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 50s, extension at 72 deg.C for 1min, 30 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C. The sequence amplified in this embodiment is detected by agarose gel electrophoresis, and the detection result is shown in FIG. 1, wherein lane M in FIG. 1 is standard DL2000, lanes 1-3 are the amplification results of DNA of the sediment sample, and lane 0 is the amplification result of the blank control. It can be seen that the specific primer Pla3F/806R adopted in the embodiment can accurately amplify the target sequence fragment of the species Pla3 in Aphanomyceta from all the DNA of the sediment samples, and no interference band is generated in the blank control. Randomly selecting corresponding sequence fragments, respectively connecting the sequence fragments with a T vector, then transforming escherichia coli DH5 alpha competent cells for sequencing, wherein the sequencing result is as follows: the sequence length is 534bp, and the sequence is Uncultured planctomycete PLA3 sequence through blast analysis.
The relative abundances of the target bands are converted numerically by QuantityOne software, the relative abundances (strength) of the target bands of the sediment samples 1-3 are respectively 180, 247 and 253, and the rapid-acting phosphorus contents of the samples are directly measured, so that the rapid-acting phosphorus contents of the sediment samples 1-3 are respectively 35.4mg/kg, 48.6mg/kg and 51.1 mg/kg. The result of sexual fitting is shown in FIG. 2, the content of available phosphorus in the sediment sample is well correlated with the relative abundance of PCR products of the Pla3F/806R specific primers, and R is2Greater than 0.99.
The embodiment shows that the specific primer Pla3F/806R designed by the invention can accurately amplify a target sequence fragment of the species of the Planctomycete Pla3 from the mixed DNA of the sediment sample, indicate the change of the rapid-acting phosphorus in the sediment according to the relative abundance, and comprehensively predict the eutrophication risk of the water body.
Sequence listing
<110> Qinghua university
<120> specific primer for monitoring rapid-acting phosphorus load pollution of sediment
<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 method for monitoring quick-acting phosphorus load pollution of sediment by using a specific primer is characterized in that the specific primer is designed according to the species of Apptomycete Plan 3 order which is sensitive to quick-acting phosphorus change in bottom mud of rivers and lakes: 5'-GCTCACCAAGCCGAAGATG-3', directly amplifying the target fragment of the genus of Planta 3 of Nonculturable Phytophthora with the length of 534bp from various sediment DNAs by matching with 16s universal primers 806R 5'-GGACTACHVGGGTWTCTAAT-3', and indicating the quick-acting phosphorus load of the sediment by utilizing the characteristic that the relative abundance of the target fragment amplified by the primers is in linear positive correlation with the quick-acting phosphorus load.
2. The method for monitoring rapid phosphorus burden contamination of sediments using specific primers as claimed in claim 1 wherein said sediments are one or more of river, river and lake bottom sediment sediments.
3. The method for monitoring rapid phosphorus burden contamination of sediments using specific primers as claimed in claim 1, wherein said 5'-GCTCACCAAGCCGAAGATG-3' is forward primer and 5'-GGACTACHVGGGTWTCTAAT-3' is reverse primer.
4. The method for monitoring rapid-acting phosphorus-loading pollution of sediments by using the specific primer as claimed in claim 1, wherein the specific primer is used for amplifying a fragment of the purpose of the genus Pla3 of the phylum Furomyces which can not be cultured.
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