Water pollution source identification and tracing method based on environment DNA macro bar code

Abstract

The invention discloses a water pollution source identification and source tracing method based on an environmental DNA macro bar code, which comprises the following steps: S1: eDNA sample collection: collecting a water sample through an alcohol preservation sample method, and collecting genetic materials in an aquatic environment by collecting the water sample and filtering instead of sampling an organism; s2, eDNA extraction: extracting eDNA by a CTAB (cetyltrimethyl ammonium bromide) method or a kit. According to the water pollution source identification and traceability method based on the environmental DNA macro bar code, through an eDNA macro bar code detection method, the specialty level requirement for species classification of researchers is not high; especially for plankton, part of benthic organisms, larvae and other objects which are difficult to identify by non-professionals, the eDNA macro bar code detection method has relatively high operability; for rare and endangered organisms with a small number of individuals, the eDNA macro bar code detection method does not need to catch target organisms, so that the operation process is greatly simplified.

Description

Technical Field

[0001] The present invention relates to the technical field of water pollution detection, and in particular to a method for identifying and tracing water pollution sources based on environmental DNA macrobarcodes. Background Art

[0002] DNA barcode technology is a new biological identification system created by using standard, sufficiently variable, easily amplified and relatively short DNA fragments to identify species within species and between species. It can quickly and automatically identify species. The principle of DNA barcode: DNA is the genetic information carrier of organisms. The difference in genetic material determines the diversity of organisms. Since the DNA sequence of each biological species is unique, it provides a material basis for DNA barcodes. Due to the conservatism of some bases, the length of dozens of bases cannot provide enough coding information. Therefore, the current DNA barcode analysis is based on DNA sequences of hundreds of bases. After searching, it is found that the water pollution detection method in the prior art is typical such as the publication number CN112083132A, a sewage pollution tracing method, which includes obtaining sewage sampling data of the main pipeline; when the sewage sampling data of the main pipeline is detected to reach the preset pollution value, the sewage sampling data is traced back step by step to obtain the upper pipeline where the sewage sampling data is abnormal; according to the recording time of the sewage sampling data of the main pipeline, and the recording time of the sewage sampling data of the pipelines at all levels where the sewage sampling data is abnormal, the primary pipeline where the polluted sewage is located is traced back step by step. Its main feature is that due to the highest accuracy of the sensor on the main pipeline, it can detect whether there is sewage discharge in the pipelines at all levels, and when sewage discharge is detected, it is traced back step by step along the pipelines at all levels according to the time correlation relationship to determine the primary pipeline that produces pollution, which solves the problem of inconvenient tracing of existing sewage pollution.

[0003] In summary, existing water pollution detection can only trace the source of industrial pollution, but cannot trace the source of biological pollution in the water flow. To address the above problems, it is necessary to establish and improve the sampling sample and statistical algorithm system of the existing methods. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding, so as to solve the problem that the existing water pollution detection proposed in the above background technology can only trace the source of industrial pollution but cannot trace the source of biological pollution in the water flow.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding, comprising:

[0006] S1: eDNA sample collection: water samples are collected through the alcohol preservation method, and genetic materials in the aquatic environment are collected by filtering the collected water samples instead of sampling the organisms themselves;

[0007] S2: eDNA extraction: CTAB method or kit to extract eDNA;

[0008] S3: PCR amplification: Select appropriate molecular markers and design universal primers to perform PCR amplification on eDNA;

[0009] S4: High-throughput sequencing: Sequencing data are processed through software platforms such as qiime and usearch;

[0010] S5: Bioinformatics analysis: Compare the detected sequences with the sequence information in the existing database to obtain the operational taxonomic units (OTU) information corresponding to each sample, and use the NCBI database to annotate the OTU species to complete the identification and tracing of pollutants.

[0011] Preferably, the eDNA in the S1 stage includes the sum of free DNA fragments released by organisms directly extracted from environmental samples such as soil, water and atmosphere without any separation, including secretions, saliva, skin, hair, mirrors and feces.

[0012] Preferably, the S1 stage needs to avoid contamination and preserve the specimens in cold storage, which is mainly reflected in the collection of sediments at the bottom of the water body, cleaning, and freezing; environmental DNA extraction includes thawing, ethanol extraction, centrifugation, and then DNA extraction.

[0013] Preferably, the sample collection in the S1 stage includes: collecting sediments at the bottom of the water body using tools such as a Peterson mud sampler and a triangular trawl, screening, filtering, and rinsing, substantially draining the mixture of water, and freezing it at -40°C to -80°C for 1-3 days.

[0014] Preferably, the S2 stage eDNA extraction process includes: weighing after thawing, converting 1g of the mixture into 1mL, adding 3 to 5 times the volume of anhydrous ethanol, and extracting with ethanol for 3 to 30 days, shaking well, extracting 2mL of the upper liquid after standing, and obtaining dry tissue residues after vacuum centrifugation, and extracting environmental DNA using a kit. Furthermore, the centrifugation conditions are 8000 rpm and the centrifugation time is 5 minutes.

[0015] Preferably, the S3 stage uses COI primers for the amplification step, and the primers for the S3 stage also include the mitochondrial cytochrome oxidase subunit I gene, 658 bp at the 5' end.

[0016] Preferably, the sequencing process in the S4 stage is library construction, cluster generation, cluster amplification and sequencing in sequence.

[0017] Preferably, the library construction is to add universal adapters with known sequences to both ends of the DNA fragments to construct the library, and the library is loaded into the sequencing chip Flowcell.

[0018] Preferably, the cluster generation is that the known sequences at both ends of the library are complementary to the Oligo sequences on the flow cell substrate, and each library fragment is amplified by bridge PCR to form a cluster.

[0019] Preferably, the cluster amplification and sequencing are performed simultaneously with cluster generation. That is, during the base extension process, each SBS cycle reaction can only extend one correct complementary base, and the base type is confirmed according to four different fluorescent signals to ensure the quality of the final nucleic acid sequence. After multiple cycles, the nucleic acid sequence is completely read.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the water pollution source identification and tracing method based on environmental DNA macrobarcodes can

[0021] The present invention can effectively solve the problem that the existing water pollution detection can only trace the source of industrial pollution, but cannot trace the source of biological pollution in the water flow through the PCR amplification of eDNA macrobarcodes and the coordinated use of high-throughput sequencing. The eDNA macrobarcode detection method does not require high professional level of species classification for researchers, especially for objects that are difficult for non-professionals to identify, such as plankton, some benthic organisms and fry. Secondly, the eDNA macrobarcode detection method has relatively high operability; for rare, endangered species with a small number of individuals or species with hidden actions, the eDNA macrobarcode detection method does not need to catch the target organism, which greatly simplifies the operation process; for some areas where fishing is difficult to implement, it saves high fishing costs; in addition, the eDNA macrobarcode detection method will not affect populations and habitats, especially for rare species with a limited number. In summary, compared with traditional methods, the eDNA macrobarcode detection method has high efficiency and sensitivity, and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the identification and tracing process of the present invention;

[0023] Figure 2 This is the website address map of the database of the present invention;

[0024] Figure 3 This is a diagram of the DNA macrobarcoding technology of the present invention;

[0025] Figure 4 The bacterial species composition diagram of Yongding River water samples of the present invention (phylum level);

[0026] Figure 5 The bacterial species composition diagram of Yongding River water samples of the present invention (genus level);

[0027] Figure 6 The bacterial species composition diagram of the Yongding River sediment of the present invention (phylum level);

[0028] Figure 7 The bacterial species composition diagram of the Yongding River sediment of the present invention (genus level);

[0029] Figure 8 The bacterial species composition diagram of the Chaobai River water sample of the present invention (phylum level);

[0030] Fig. 9 The bacterial species composition diagram of the Chaobai River water sample of the present invention (genus level);

[0031] Fig.10 The bacterial species composition diagram of the Chaobai River sediment of the present invention (phylum level);

[0032] Fig.11 This is the species composition diagram of Chaobai River sediment bacteria (genus level) of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] See also Figure 1-11 ,

[0035] Example 1

[0036] The present invention provides a technical solution: a method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding, comprising:

[0037] S1: eDNA sample collection: water samples are collected through the alcohol preservation method, and genetic materials in the aquatic environment are collected by filtering the collected water samples instead of sampling the organisms themselves;

[0038] S2: eDNA extraction: CTAB method or kit to extract eDNA;

[0039] S3: PCR amplification: Select appropriate molecular markers and design universal primers to perform PCR amplification on eDNA;

[0040] S4: High-throughput sequencing: Sequencing data are processed through software platforms such as qiime and usearch;

[0041] S5: Bioinformatics analysis: Compare the detected sequences with the sequence information in the existing database to obtain the operational taxonomic units (OTU) information corresponding to each sample, and use the NCBI database to annotate the OTU species to complete the identification and tracing of pollutants.

[0042] S1 stage eDNA includes the sum of free DNA fragments released by organisms directly extracted from environmental samples such as soil, water and atmosphere without any separation, including secretions, saliva, skin, hair, mirrors and feces.

[0043] In the S1 stage, contamination must be avoided and specimens must be kept refrigerated, which mainly involves collecting sediments from the bottom of the water body, cleaning them, and freezing them. Environmental DNA extraction includes thawing, ethanol extraction, centrifugation, and then DNA extraction.

[0044] Sample collection in the S1 stage includes: collecting sediments at the bottom of the water body using tools such as Peterson mud samplers and triangular trawls, screening, filtering, and rinsing, basically draining the mixture of water, and freezing it at -40℃ to -80℃ for 1-3 days.

[0045] The eDNA extraction process in the S2 stage includes: weighing after thawing, converting 1 g of the mixture into 1 mL, adding 3 to 5 times the volume of anhydrous ethanol, and ethanol extraction for 3 to 30 days, shaking well, extracting 2 mL of the upper liquid after standing, and obtaining dry tissue residues after vacuum centrifugation, and extracting environmental DNA using a kit. Furthermore, the centrifugation conditions were 8000 rpm and the centrifugation time was 5 min.

[0046] The S3 stage uses COI primers for the amplification step, and the primers in the S3 stage also include the mitochondrial cytochrome oxidase subunit I gene, 658 bp at the 5' end.

[0047] The sequencing process in the S4 stage is library construction, cluster generation, cluster amplification and sequencing.

[0048] Library construction is to add universal adapters with known sequences at both ends of the DNA fragments to construct the library, and the library is loaded into the sequencing chip Flowcell.

[0049] Cluster generation means that the known sequences at both ends of the library are complementary to the oligo sequences on the flow cell substrate, and each library fragment is amplified by bridge PCR to form a cluster.

[0050] Cluster amplification and sequencing are performed simultaneously with cluster generation. That is, during the base extension process, each SBS cycle reaction can only extend one correct complementary base, and the base type is confirmed based on four different fluorescent signals to ensure the quality of the final nucleic acid sequence. After multiple cycles, the nucleic acid sequence is completely read.

[0051] Example 2

[0052] Yongding River Sample Analysis (YD)

[0053] Species composition

[0054] Analysis of bacterial composition of water samples

[0055] The bacterial composition of Yongding River water samples is as follows Figure 4 and Figure 5 As shown, DB_YD1~8 represent dry season samples, DB_YD1_2~DB_YD8_2 represent wet season samples, and the phylum level microorganisms mainly include Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, and Cyanobacteria. The total proportion of the five bacterial phyla at each point in the Yongding River water samples during the dry season was 74.72%-97.92%, and the total proportion of the five bacterial phyla at each point in the flood season was 81.47%-97.25%. The genus-level microorganisms mainly included Exiguobacterium, hgcl_clade, Cyanobium_PCC6307, Planococcus, norank_f_norank_o_Chloroplast, Flavobacterium, etc., among which the change of cyanobacteria (Cyanobium_PCC6307) was particularly significant. The content of cyanobacteria (Cyanobium_PCC6307) was relatively high in points 1, 2, and 3 in the front section of the Yongding River, and the abundance in the dry season was higher than that in the flood season.

[0056] Analysis of sediment bacterial composition

[0057] The composition of sediment bacteria is as follows: phylum, class, order, family, and genus Figure 6 and Figure 7As shown. Microorganisms at the phylum level mainly include Proteobacteria, Actinobacteriota, Chloroflexi, Firmicutes, Acidobacteriota, etc. The total proportion of the five phyla at each point in the Yongding River sediment during the dry season was 76.82%-79.64%, and the total proportion of the five phyla at each point in the Yongding River sediment during the flood season was 75.79%-86.11%. The proportion of the five main phyla was higher during the flood season. The proportion of Cyanobacteria in the sediment at Point 7 of the Yongding River was relatively high, accounting for about 10%.

[0058] Microorganisms at the genus level mainly include Pseudarthrobacter, Flavobacterium, Pseudomonas, norank_f_norank_o_norank_c_KD4-96, norank_f_Steroidobacteraceanorank_f_norank_o_Vicinamibacterales.

[0059] Example 3

[0060] Chaobai River Sample Analysis

[0061] Species composition

[0062] Analysis of bacterial composition of water samples

[0063] The bacterial composition of Chaobai River water samples is as follows Figure 8 and Fig. 9 As shown, DB_CB1~8 represent dry season samples, DB_CB1_2~DB_YD8_2 represent wet season samples, and the phylum-level microorganisms mainly include Proteobacteria, Actinobacteriota, Bacteroidota, Cyanobacteria, and Verrucomicrobia. The total abundance of the five phyla at each point of the Chaobai River water sample during the dry season accounted for 94.06%-99.12%, and the total abundance of the five phyla at each point of the water sample during the wet season accounted for 56.97%-97.04%. The total abundance of the five phyla in the dry season was generally higher than that in the wet season. The abundance of Cyanobacteria was relatively high at sites 3 and 4 in the middle section of Chaobai River, and the genus-level microorganisms mainly included hgcl_clade, norank_f__norank_o__Chloroplast, Limnohabitans, Flavobacterium, and Brevundimonasunclassified_f_Sporichthyaceae.

[0064] Analysis of sediment bacterial composition

[0065] The bacterial composition of the Chaobai River sediment is as follows Fig.10 and Fig.11 As shown. Microorganisms at the phylum level mainly include Proteobacteria, Actinobacteriota, Chloroflexi, Firmicutes, Acidobacteriota, etc. The total abundance of the five phyla at each point of the Chaobai River water sample during the dry season accounted for 75.72%-81.49%, and the total abundance of the five phyla at each point of the water sample during the wet season accounted for 74.52%-85.78%. The total abundance of the five phyla in the wet season and the dry season was not much different.

[0066] Microorganisms at the genus level mainly include Bacillus, Pseudarthrobacter, norank_f_Anaerolineaceae, and norank_f_Steroidobacteraceae.

[0067] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding, characterized in that: include: S1: eDNA sample collection: water samples are collected through the alcohol preservation method, and genetic materials in the aquatic environment are collected by filtering the collected water samples instead of sampling the organisms themselves; S2: eDNA extraction: CTAB method or kit to extract eDNA; S3: PCR amplification: Select appropriate molecular markers and design universal primers to perform PCR amplification on eDNA; S4: High-throughput sequencing: Sequencing data were processed using qiime and usearch software platforms; S5: Bioinformatics analysis: Compare the detected sequences with the sequence information in the existing database to obtain the operational taxonomic unit information corresponding to each sample, and use the NCBI database to annotate the OTU species to complete the identification and tracing of pollutants.

2. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The eDNA in the S1 stage includes the sum of free DNA fragments released by organisms that are directly extracted from environmental samples such as soil, water and atmosphere without any separation.

3. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The S1 stage also includes collecting bottom sediments of the water body, cleaning and freezing them for storage.

4. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The sample collection in the S1 stage includes: collecting sediments at the bottom of the water body using a Peterson mud sampler and a triangular trawl net tool, screening, filtering, and washing, draining the mixture to substantially dry the mixture, and freezing it at -40°C to -80°C for 1-3 days.

5. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The S2 stage eDNA extraction process includes: weighing after thawing, converting 1g of the mixture into 1mL, adding 3 to 5 times the volume of anhydrous ethanol, and ethanol extraction for 3 to 30 days, shaking well, extracting 2mL of the upper liquid after standing, and obtaining dry tissue residues after vacuum centrifugation, and extracting environmental DNA using a kit. Furthermore, the centrifugation conditions are 8000 rpm and the centrifugation time is 5 minutes.

6. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The S3 stage uses COI primers for amplification, and the primers in the S3 stage also include mitochondrial cytochrome oxidase subunit I gene, 658 bp at the 5' end.

7. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 1, characterized in that: The sequencing process in the S4 stage is library construction, cluster generation, cluster amplification and sequencing.

8. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 7, characterized in that: The library construction is to add universal adapters with known sequences to both ends of the DNA fragments to construct the library, and the library is loaded into the sequencing chip Flowcell.

9. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 8, characterized in that: The cluster generation is that the known sequences at both ends of the library are complementary to the Oligo sequence on the flow cell substrate, and each library fragment is amplified by bridge PCR to form a cluster.

10. The method for identifying and tracing water pollution sources based on environmental DNA macrobarcoding according to claim 9, characterized in that: The cluster amplification and sequencing are performed simultaneously with cluster generation, that is, during the base extension process, each SBS cycle reaction can only extend one correct complementary base, and the base type is confirmed according to four different fluorescent signals to ensure the quality of the final nucleic acid sequence. After multiple cycles, the nucleic acid sequence is completely read.

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