Port water eDNA continuous monitoring method

Abstract

The invention provides a port water eDNA continuous monitoring method which comprises the following steps: S1, arranging eDNA sampling points in a port water area, and arranging corresponding automatic acquisition equipment at each sampling point; s2, continuously sampling and enriching eDNA regularly / quantitatively, and sampling regularly; s3, eluting the enriched eDNA substances, removing impurities, recovering all DNA fragment solutions, and extracting DNA; s4, DNA fragment amplification is carried out, and the DNA sequence copy number is increased through copying; s501, DNA sequencing is conducted, continuous data information is obtained in combination with continuous sampling, the continuous data information is compared with sequence information in a database for data analysis, and in-situ species information is obtained; and S502, carrying out QPCR, and carrying out rapid monitoring on invasive species. Through reasonable point location layout and automatic continuous sampling, species classification in the water body can be rapidly and accurately judged, long-term continuous monitoring is achieved, and then invasive species monitoring, species trend change analysis, diversity analysis and the like of the water area can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of environmental protection, and in particular to a method for continuous monitoring of eDNA in port water. Background Art

[0002] With the application of global transportation technology, water transportation has achieved unprecedented development, driving regional coordinated development and strengthening exchanges and trade, but at the same time, it has also inadvertently increased the potential risk of alien species invasion, and its spread has become more diverse and hidden. Alien species invasion is difficult to detect and monitor in the early stages, and once the invasion is successful, it is difficult to control, posing threats or harm to ecosystems, habitats, and species, affecting my country's ecological environment, and even causing the extinction of rare species, with serious consequences.

[0003] At present, the best technical method to prevent biological invasion of water bodies is early protection, early detection and early treatment, and prevention before it happens. Observation is still the mainstream method to determine whether there is biological invasion in the water environment, such as electric shock, fishing, visual or acoustic survey methods, but this method is difficult to effectively monitor the invasion of low-abundance species in water bodies, and it is difficult to judge when the invasive species and local species are similar, and it will also cause certain damage to the local habitat system. In particular, the monitoring of invasive species in dock waters is still a blind spot. Based on the above problems and the characteristics of eDNA (environmental DNA) technology, the present invention proposes a continuous monitoring method of port water eDNA. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention proposes a method for continuous monitoring of port water eDNA, which can quickly and accurately determine the species classification in the water body through reasonable point layout and automatic continuous sampling.

[0005] To achieve the above object, a method for continuous monitoring of eDNA in port water of the present invention comprises the following steps:

[0006] S1: Arrange eDNA sampling points in the port waters and set up corresponding automatic collection equipment at each sampling point;

[0007] S2: Timed / quantitative continuous sampling to enrich eDNA and take samples regularly;

[0008] S3: Elute the enriched eDNA material, remove impurities, recover all DNA fragment solutions, and extract DNA;

[0009] S4: Amplify DNA fragments and increase the number of DNA sequence copies through replication;

[0010] S501: DNA sequencing, combining continuous sampling to obtain continuous data information, comparing it with the sequence information in the database for data analysis, and obtaining in situ species information;

[0011] S502: QPCR, for rapid monitoring of invasive species.

[0012] Further, in step S1, when the port waters are a river basin system, the sampling points are respectively set at 50-100m upstream of the port area, in the port area and 50-100m downstream of the port area; when the port waters are open waters, the sampling points include peripheral sampling points and port area sampling points. The peripheral sampling points are set within 50-100m of the waters outside the port area. At least two peripheral sampling points are set, and the two peripheral sampling points are respectively set at 60° on both sides of the left and right sides of the line connecting the center points of the port shore base.

[0013] Further, in step S1, the sampling points include an upper point at 0.5 m below the water surface, a middle point at 1 / 2 of the water depth, and a lower point at 0.5 m above the water bottom.

[0014] Furthermore, in step S2, eDNA is continuously sampled at a fixed time / quantity, and the process is automatically triggered to run at a set time, the water body is sucked in, filtered and discharged, and the eDNA substances contained in the water are filtered and retained for fixed time / quantity enrichment.

[0015] Furthermore, in step S501, sequencing is achieved by analyzing the base sequence of a specific DNA fragment based on high-throughput sequencing technology. The database refers to a collection of nucleotide sequences, single nucleotide polymorphisms, structures, properties and related descriptions of all known nucleic acids. The comparison process includes simulating the barcode sequence annotation process based on sequence similarity, and statistically annotating the species of the sequence obtained by sequencing in a public database or a self-built database. When the sequence to be annotated has only one highly similar species to itself in the reference database, the sequence is marked as accurately annotated.

[0016] Furthermore, in step S502, QPCR uses a real-time fluorescent quantitative nucleic acid amplification detection system to measure PCR amplification products based on the amplification cycle data through the fluorescent staining in the DNA molecules, so as to quickly monitor the possible presence of invasive species in the dock waters, and combine continuous sampling for long-term species invasion monitoring.

[0017] The port water eDNA continuous monitoring method of the present invention can realize continuous monitoring of invasive species in port waters, and real-time analysis of species diversity and trend changes; provide data support for continuous early warning and monitoring of relevant invasive species in dock waters; and provide technical support for the later detection department to formulate and establish invasive species monitoring and protection policies for dock waters. It can more accurately and quickly determine the species classification in the relevant water bodies, so that measures can be taken to control the species invasion in the early stage and it is easy to achieve full coverage and continuous monitoring of the watershed; reduce the monitoring requirements for professionals and personnel, and is easy to implement and promote in a standardized manner; and through reasonable point layout and automatic continuous sampling, it can realize continuous early warning monitoring of relevant species and also realize analysis of species diversity and trend changes in the area. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described and explained below in conjunction with the accompanying drawings.

[0019] Figure 1 It is a step diagram of a method for continuous monitoring of port water eDNA according to the present invention.

[0020] Figure 2 It is a sampling point layout map of the watershed system.

[0021] Figure 3 This is a map of the sampling point layout in open waters.

[0022] Figure 4 It is a statistical chart of species census data at each point.

[0023] Figure 5 It is a time trend change analysis chart. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be more clearly and completely explained below through description of preferred embodiments of the present invention in combination with the accompanying drawings.

[0025] like Figure 1 As shown, a method for continuous monitoring of eDNA in port water according to a preferred embodiment of the present invention comprises the following steps:

[0026] S1: Arrange eDNA sampling points in the port waters, and set corresponding automatic collection equipment at each sampling point. The automatic collection equipment can adopt any collection equipment in the prior art, such as the device mentioned in the Chinese invention patent with publication number CN114608886A previously submitted by the applicant: "A fully automatic collection device and method for eDNA samples of aquatic organisms". The specific structure of the device will not be repeated in this application.

[0027] like Figure 2As shown in Figure 1, when the port water area is a river basin system, the sampling points are set at 50-100m upstream of the port area, between the port area and 50-100m downstream of the port area. Figure 3 As shown in the figure, when the port waters are open waters, the sampling points include peripheral sampling points and port area sampling points. The peripheral sampling points are set within 50-100m of the waters outside the port area. At least two peripheral sampling points are set, and the two peripheral sampling points are set at 60° on both sides of the line connecting the center points of the port shore base; each sampling point includes an upper point at 0.5m below the water surface, a middle point at 1 / 2 of the water depth, and a lower point at 0.5m above the water bottom.

[0028] S2: Timed / quantitative continuous sampling and enrichment of eDNA, set the time to automatically trigger the process flow, absorb water, filter and discharge, filter and intercept the eDNA contained in the water through the filter membrane or filter in the integrated filter, regularly sample and replace the standardized enricher.

[0029] The sampling period performed by the automatic collection device is shown in Table 1. In Table 1, X day X time means X time on X day of a certain month, for example, 10:00 on the 10th day.

[0030] Table 1

[0031]

[0032]

[0033] S3: Elute the enriched eDNA material to remove impurities such as proteins, ions, and small primer fragments, recover all DNA fragment solutions, and extract DNA.

[0034] S4: Perform DNA fragment amplification. Based on a specific reaction system and reaction conditions, the extracted DNA is replicated through relevant equipment and methods to increase the number of copies of a specific DNA sequence.

[0035] S501: DNA sequencing, combining continuous sampling to obtain continuous data information, comparing it with the sequence information in the database for data analysis, obtaining in situ species information, and analyzing the base sequence of specific DNA fragments based on high-throughput sequencing technology to achieve sequencing. The database refers to the nucleotide sequence, single nucleotide polymorphism, structure, properties and related descriptions of all known nucleic acids. You can choose public bioinformatics databases such as GenBank in the United States, EMBL in Europe, and DDBJ in Japan, or collect samples to build your own database. The comparison process includes simulating the barcode sequence annotation process based on sequence similarity, and counting the species annotation of the sequence obtained by sequencing in the public database or the self-built database. When the highly similar species to the sequence to be annotated is only one of itself in the reference database, the sequence is marked as accurately annotated.

[0036] S502: QPCR, QPCR uses a real-time fluorescent quantitative nucleic acid amplification detection system. According to the amplification cycle data, the PCR amplification product is determined through the fluorescent staining in the DNA molecule, so as to achieve rapid monitoring of invasive species that may appear in the dock waters, such as invasive fish, pathogenic bacteria, etc. It can also be combined with continuous sampling to conduct long-term invasive species monitoring.

[0037] In step S501, the specific data analysis project may be a species census at each point, such as Figure 4 As shown in the figure, statistical analysis shows that the total number of relevant fish, benthic, pelagic, viral microorganisms and other species and the family, order, genus and species statistics of each species are regularly measured in this area; based on this data, a census of the entire species and diversity analysis such as species point distribution and the presence or absence of invasive species can be achieved.

[0038] Specific data analysis projects can also be time trend change analysis, such as Figure 5 As shown, long-term data analysis and statistics can realize the time trend change analysis of related species, and then understand the continuous monitoring and early warning, trend change analysis of each species; it can also realize correlation analysis by combining water temperature, season, sampling depth, etc.

[0039] Through the above report analysis, it is possible to achieve continuous monitoring of invasive species in port waters, species diversity census, trend change analysis, etc.; provide data support for continuous early warning and monitoring of relevant invasive species in terminal waters; and provide technical method support for the subsequent detection department for port water environment monitoring, management and protection policy formulation.

[0040] The above specific implementations are only descriptions of the preferred implementations of the present invention, and do not limit the protection scope of the present invention. Without departing from the design concept and spirit of the present invention, various modifications, substitutions and improvements made by ordinary technicians in this field to the technical solution of the present invention based on the text description and drawings provided by the present invention should all fall within the protection scope of the present invention. The protection scope of the present invention is determined by the claims.

Claims

1. A method for continuous monitoring of eDNA in port water, characterized in that: The following steps are involved: S1: Arrange eDNA sampling points in the port waters and set up corresponding automatic collection equipment at each sampling point; S2: Timed / quantitative continuous sampling to enrich eDNA and take samples regularly; S3: Elute the enriched eDNA material, remove impurities, recover all DNA fragment solutions, and extract DNA; S4: Amplify DNA fragments and increase the number of DNA sequence copies through replication; S501: DNA sequencing, combining continuous sampling to obtain continuous data information, comparing it with the sequence information in the database for data analysis, and obtaining in situ species information; S502: QPCR, for rapid monitoring of invasive species.

2. A method for continuous monitoring of port water eDNA according to claim 1, characterized in that: In step S1, when the port water area is a river basin system, the sampling points are respectively set at 50-100m upstream of the port area, 50-100m between the port area and 50-100m downstream of the port area; when the port water area is an open water area, the sampling points include peripheral sampling points and port area sampling points, and the peripheral sampling points are set within 50-100m water area outside the port area. At least two peripheral sampling points are set, and the two peripheral sampling points are respectively set at 60° on the left and right sides of the line connecting the center points of the port shore base.

3. A method for continuous monitoring of port water eDNA according to claim 1, characterized in that: In step S1, the sampling points include an upper point at 0.5 m below the water surface, a middle point at 1 / 2 of the water depth, and a lower point at 0.5 m above the water bottom.

4. A method for continuous monitoring of port water eDNA according to claim 1, characterized in that: In step S2, the eDNA is sampled continuously and regularly / quantitatively, and the process is automatically triggered to run at a set time, the water body is sucked in, filtered and discharged, and the eDNA substances contained in the water are filtered and intercepted for regular / quantitative enrichment.

5. The method for continuous monitoring of port water eDNA according to claim 1, characterized in that: In step S501, sequencing is achieved by analyzing the base sequence of a specific DNA fragment based on high-throughput sequencing technology. The database refers to a collection of nucleotide sequences, single nucleotide polymorphisms, structures, properties and related descriptions of all known nucleic acids. The comparison process includes simulating the barcode sequence annotation process based on sequence similarity, and statistically annotating the species of the sequence obtained by sequencing in a public database or a self-built database. When the sequence to be annotated has only one highly similar species to itself in the reference database, the sequence is marked as accurately annotated.

6. A method for continuous monitoring of port water eDNA according to claim 1, characterized in that: In step S502, the QPCR uses a real-time fluorescent quantitative nucleic acid amplification detection system to measure the PCR amplification product based on the amplification cycle data through the fluorescent staining in the DNA molecules, quickly monitor the possible invasive species in the dock waters, and combine continuous sampling for long-term species invasion monitoring.

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