A method for quantitatively detecting an indicator species based on environmental DNA

Abstract

A kind of environmental DNA-based indicator species quantitative detection method, first get indicator species list, then design and verify primer and probe combination to obtain optimal combination, then environmental DNA is enriched in the sampling point of target water area, while, in-situ biomass data of indicator species is obtained by sampling point measurement, then environmental DNA is extracted, mixed with optimal combination to obtain reaction solution, then amplification procedure is carried out to carry out quantitative analysis, then environmental DNA quantitative data of indicator species is obtained, then environmental DNA, in-situ biomass is constructed to obtain quantitative relationship model;The environmental DNA quantitative data of indicator species of the water area to be detected is obtained, and the in-situ biomass data of the indicator species is obtained after being substituted into the quantitative relationship model;Optimal combination does not amplify non-indicator species, reduces the interference of background DNA, target signal is concentrated, which can significantly improve the detection ability and quantitative accuracy of indicator species.Therefore, the design detects indicator species DNA and is not easy to be disturbed, and the quantitative accuracy of indicator species is better.

Description

Technical Field

[0001] This invention relates to a method for quantitative detection of indicator species, belonging to the technical field of quantitative detection of indicator species in aquatic ecological monitoring, and particularly to a method for quantitative detection of indicator species based on environmental DNA. Background Technology

[0002] In recent years, environmental DNA technology has developed rapidly and has been gradually applied to the field of aquatic organism monitoring. During their survival, activities, and metabolism, organisms continuously release cell-free DNA into water bodies. By detecting cell-free DNA, the in-situ biomass of indicator species can be retrieved, thereby enabling aquatic ecological monitoring.

[0003] Chinese patent application number 202510033016.0, filed on January 9, 2025, discloses a method for assessing and identifying aquatic ecological damage based on high-throughput environmental DNA sequencing. The method includes compiling a species list of the target area, searching and downloading species sequence information, constructing a local database, and supplementing the local database information through sample sequencing. It also involves collecting benthic animal samples from multiple target area sampling points, selecting test samples, extracting sample DNA, amplifying and performing high-throughput sequencing, comparing species based on sequencing results, setting multiple read count thresholds, determining whether species are detected in random mixed species samples and quantitatively matched mixed species samples, constructing a mapping relationship model between environmental DNA sequencing and morphology using a generalized linear model, optimizing the mapping relationship model, calculating the population resource quantity of specific species in natural samples, and assessing and identifying the resource quantity of indicator species for aquatic ecological damage. Although this design can calculate the in-situ biomass of indicator species, it still has the following shortcomings:

[0004] This design uses high-throughput sequencing for broad-spectrum screening. Its principle is to simultaneously detect the DNA information of a large number of species in the water. However, the target indicator species are easily interfered with by background DNA under low abundance conditions, resulting in poor quantitative accuracy of this design.

[0005] The information disclosed in this background section is intended only to enhance understanding of the overall background of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects and problems of existing technologies, such as the susceptibility of indicator species DNA to interference and poor quantitative accuracy of indicator species, and to provide a method for quantitative detection of indicator species based on environmental DNA that is less susceptible to interference and has better quantitative accuracy.

[0007] To achieve the above objectives, the technical solution of the present invention is:

[0008] A method for quantitative detection of indicator species based on environmental DNA, the method comprising the following steps:

[0009] Step 1: First, screen according to the screening method to obtain a candidate indicator species library for the target water area, and then screen the candidate indicator species library according to the specified criteria to obtain a list of indicator species;

[0010] The second step is to design multiple primer and probe combinations based on the indicator species in the indicator species list, then verify the multiple primer and probe combinations, and finally obtain the optimal primer and probe combination.

[0011] The third step is to first set up multiple sampling points in the target water area, then collect and filter water samples at the sampling points to obtain environmental DNA; at the same time, measure the in-situ biomass data of indicator species at the sampling points.

[0012] Step 4: First, extract environmental DNA to obtain template DNA. Then, mix the template DNA with the optimal primer and probe combination to obtain a reaction solution. Next, perform an amplification program on the reaction solution for quantitative analysis to obtain quantitative data of environmental DNA of the indicator species.

[0013] Step 5: Using the in-situ biomass data of indicator species obtained from actual sampling points as the independent variable and the environmental DNA quantitative data of indicator species as the dependent variable, construct a quantitative relationship model;

[0014] Step 6: Perform steps 1 through 4 sequentially on the water area to be tested to obtain quantitative environmental DNA data of the indicator species. Then, input the quantitative environmental DNA data of the indicator species into the quantitative relationship model to obtain in-situ biomass data of the indicator species.

[0015] In the fifth step, constructing a quantitative relationship model refers to: In the formula, To obtain in-situ biomass data of indicator species by sampling points, These are the model calibration parameters obtained by fitting in-situ field data. Quantitative data on the environmental DNA of species, These are the model calibration parameters obtained by fitting in-situ field data;

[0016] The coefficient of determination in constructing a quantitative relationship model The construction of the quantitative relationship model .

[0017] In the first step, the list of indicator species includes 1 to 5 indicator species.

[0018] In the second step, the verification refers to: specificity verification, sensitivity and amplification efficiency verification; obtaining the optimal primer-probe combination refers to: obtaining the optimal primer-probe combination by comprehensively considering the specificity verification results, sensitivity and amplification efficiency verification results.

[0019] In the second step, the specificity verification refers to: using the DNA of the indicator species, DNA of closely related species, DNA of common aquatic organisms, and DNA of aquatic microorganisms as templates to perform amplification programs respectively, while recording the changes in fluorescence signal intensity of each template to generate multiple amplification curves that correspond one-to-one; then screening the multiple amplification curves and retaining primer-probe combinations that only show specific amplification curves with the target indicator species, have no cross-amplification signals, and have no non-specific products.

[0020] In the second step, the verification of sensitivity and amplification efficiency refers to: firstly, extracting the DNA of the indicator species, then serially diluting the DNA of the indicator species by 10-fold to obtain standard solutions of multiple concentration gradients, then performing an amplification program on the standard solutions of multiple concentration gradients, then plotting multiple standard curves corresponding one-to-one with multiple combinations of reaction solutions of multiple gradient concentrations, then calculating the amplification efficiency, linear correlation coefficient, and detection limit based on the multiple standard curves, and finally screening primer and probe combinations based on the calculated amplification efficiency, linear correlation coefficient, and detection limit.

[0021] In the second step, the design of multiple primer and probe combinations based on indicator species in the indicator species list refers to: selecting gene fragments of cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA from the indicator species to design primer and probe combinations; the primers are 18–25 bp in length, have a Tm value of 57–63 °C, a GC content of 40%–60%, and are free of hairpin structures, dimers, and cross-complementary sequences; the probes are 20–28 bp in length, have a Tm value 5–8 °C higher than the primers, are labeled with FAM or a corresponding fluorescent reporter group at the 5' end, and are labeled with BHQ1 or a corresponding quencher group at the 3' end.

[0022] In the first step, the process of obtaining a candidate indicator species library for the target water area by screening according to the screening method refers to: firstly, integrating multi-source data based on the target water area to obtain an indicator species database; then, designing an evaluation system based on the molecular biological characteristics and macro-ecological characteristics of the target water area; then, using machine learning algorithms to comprehensively score and rank the indicator species in the indicator species database based on their contribution; and finally, screening to obtain a candidate indicator species library.

[0023] In the first step, the process of screening the candidate indicator species library according to specified standards to obtain the indicator species list refers to the following: screening the candidate indicator species library according to national ecological and environmental standards, long-term ecological monitoring results of the target watershed, and recognized indicator taxa, and including indicator species that meet the following criteria: native species, sensitive to heavy metal / organic matter / low dissolved oxygen / eutrophication stress, widely distributed in nature, easily identifiable and sampled in the field, with an eDNA amplification efficiency of 90% to 110%, no cross-amplification interference, quantifiable biomass, and listed in the national / industry recognized indicator list.

[0024] In the first step, the evaluation system includes ecological sensitivity, regional distribution breadth, feasibility of field monitoring, eDNA amplification adaptability, and species stability.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The present invention provides a method for quantitative detection of indicator species based on environmental DNA, comprising the following steps: Step 1: Obtaining a list of indicator species according to screening methods and specified standards; Step 2: Designing and validating primer and probe combinations based on indicator species, and obtaining the optimal primer and probe combination; Step 3: Deploying multiple sampling points in the target water area, and obtaining environmental DNA and in-situ biomass data of indicator species at the sampling points; Step 4: Extracting environmental DNA, mixing it with the optimal primer and probe combination, and performing an amplification procedure for quantitative analysis, thereby obtaining quantitative data of environmental DNA of indicator species; Step 5: Constructing a quantitative relationship model using the in-situ biomass data of indicator species obtained from the sampling points as the independent variable and the quantitative data of environmental DNA of indicator species as the dependent variable; Step 6: Performing steps 1 to 4 sequentially in the water area to be tested to obtain quantitative data of environmental DNA of indicator species, and then substituting the quantitative data of environmental DNA of indicator species into the quantitative relationship model to obtain in-situ biomass data of indicator species. The advantages of the present invention also include:

[0027] Firstly, the quantitative relationship model constructed in this invention corresponds the environmental DNA quantitative data of indicator species with the in-situ biomass data of indicator species. Therefore, after substituting the environmental DNA quantitative data of indicator species in the water area to be tested into the quantitative relationship model, the in-situ biomass data of indicator species can be obtained.

[0028] Secondly, this invention screens primer and probe combinations, retaining only the optimal primer and probe combination. The optimal primer and probe combination only amplifies the DNA of the indicator species and does not amplify non-indicator species, reducing interference from background DNA. Therefore, the target signal is more concentrated, which can significantly improve the detection ability and quantitative accuracy of indicator species.

[0029] Thirdly, in existing technologies, various simulation models are almost all constructed under controlled and idealized laboratory conditions, resulting in a huge gap with the field environment and their results being largely unusable. In constructing the quantitative relationship model, this invention directly uses real, complex, and variable water environment data from the field, fully considering the impact of field interference factors such as water temperature, turbidity, microorganisms, wind and waves, and sediment resuspension on environmental DNA. The model closely matches the actual field conditions, has strong applicability, and achieves a breakthrough improvement in inversion accuracy, fundamentally solving the industry pain points of existing technology models having large deviations and being unusable in the field.

[0030] Fourthly, the direct, accurate, and standardized quantitative relationship established by this invention between environmental DNA quantitative data and in-situ biomass data in the field breaks through the limitations of existing technologies, which are qualitative, semi-quantitative, and lack quantitative standards. The results are standardized in g / L, making them intuitive, comparable, quantifiable, and calculable. They can be directly used for quantitative assessment of water ecological damage, ecological restoration acceptance, environmental law enforcement evidence collection, judicial compensation calculation, and judicial judgment acceptance, filling the gap in the field of environmental DNA technology for quantitative assessment of water ecology.

[0031] Fifthly, the process of this invention is highly standardized, with clear parameters, standardized operation, and repeatability; the third, fourth, and sixth steps are executed until biomass data is obtained, and it only takes 2 hours, which is more than 80% more efficient than traditional methods; it can realize large-scale, high-frequency, and dynamic monitoring, meeting the needs of refined and intelligent water environment management;

[0032] Sixth point: This invention does not require destructive operations such as fishing, digging, or electrofishing. It is non-damaging, low-interference, and eco-friendly. Even in heavily polluted water bodies with extremely low abundance of indicator species, it can still accurately detect and quantify, avoiding missed detections and misjudgments, and improving the accuracy and reliability of monitoring.

[0033] Therefore, the DNA of indicator species detected by this invention is not easily interfered with, and the quantitative accuracy of indicator species is good.

[0034] 2. In the quantitative detection method for indicator species based on environmental DNA of the present invention, the fifth step, namely, constructing a quantitative relationship model, refers to: ; To obtain in-situ biomass data of indicator species by sampling points, , These are the model calibration parameters obtained by fitting in-situ field data. Quantitative data on environmental DNA of species, coefficient of determination The construction of the quantitative relationship model When applied, the environmental DNA quantitative data of the species ( Substituting these values ​​into the quantitative relationship model, the in-situ biomass data of the indicator species at the sampling points were calculated. This linear fitting model is more tolerant of a small number of outliers, and can obtain stable results even with small sample datasets. Furthermore, the model's meaning is intuitive, and the results are easy to interpret. The formula is mature, and parameter calculation for different species is fast without complex iterations. Statistical tests have shown that the model's coefficient of determination is high. And significance level This indicates that the model possesses high statistical significance and excellent predictive accuracy, and can accurately and objectively reflect the intrinsic transformation relationship between indicator species biomass data and environmental DNA quantitative data. Therefore, the model of this invention has strong scientific validity.

[0035] 3. In the quantitative detection method for indicator species based on environmental DNA of the present invention, the first step includes a list of 1 to 5 indicator species. In application, selecting 1 to 5 indicator species is an optimized range that balances ecological representativeness, detection efficiency, and engineering feasibility. Too few indicator species may fail to fully reflect the ecological state, while too many indicator species will increase primer design complexity, cross-amplification risk, and detection cost. Therefore, 1 to 5 indicator species achieve a good balance between accuracy, stability, and economy. Thus, the present invention combines data accuracy with ecosystem representativeness.

[0036] 4. In the quantitative detection method for indicator species based on environmental DNA of the present invention, the second step of the verification refers to: specificity verification, sensitivity and amplification efficiency verification; the specificity verification refers to: using the DNA of the indicator species, DNA of closely related species, DNA of common aquatic organisms, and DNA of aquatic microorganisms as templates to perform amplification programs respectively, recording and screening amplification curves, and then retaining primer-probe combinations that only show specific amplification curves with the target indicator species; the sensitivity and amplification efficiency verification refers to: serially diluting the DNA of the indicator species to obtain multiple standard solutions, then performing amplification programs to obtain multiple standard curves, and then calculating the standard curves. The amplification efficiency, linear correlation coefficient, and detection limit are evaluated, and primer-probe combinations are then screened. During application, the primer-probe combinations screened and validated for specificity ensure that the detection signal originates solely from the target indicator species, fundamentally eliminating erroneous results caused by interference from closely related species, common aquatic organisms, and aquatic microorganisms. Combinations screened and validated for sensitivity and amplification efficiency guarantee stable exponential amplification capabilities and extremely high detection sensitivity across a wide concentration range. This completely resolves the technical pain points of existing technologies, such as non-specific amplification, cross-contamination, insufficient sensitivity, large quantitative deviations, and missed detection of low-abundance samples, ensuring accurate, reliable, and interference-free absolute quantification results of environmental DNA. Therefore, the detection results of this invention are accurate.

[0037] 5. In the quantitative detection method for indicator species based on environmental DNA of the present invention, in the second step, the design of multiple primer and probe combinations based on indicator species in the indicator species list refers to: selecting gene fragments of cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA of the indicator species to design primer and probe combinations; the primers are 18-25 bp in length, have a Tm value of 57-63℃, a GC content of 40%-60%, and are free of hairpin structures, dimers, and cross-complementary sequences; the probes are 20-28 bp in length, have a Tm value 5-8℃ higher than the primers, are labeled with FAM or a corresponding fluorescent reporter group at the 5' end, and are labeled with BHQ1 or a corresponding quencher group at the 3' end. In application, multiple primer and probe combinations are designed for specific gene fragments of the indicator species, which can cover the molecular detection needs of major indicator groups such as fish, benthic animals, and algae; by setting the primer length to 18-25 bp... The primers and probes, with a Tm value of 57–63℃ and a GC content of 40%–60%, combined with screening criteria of no hairpin structures, dimers, and cross-complementary sequences, effectively ensured the specificity of primer-template binding and the consistency of annealing temperature, reducing the risk of non-specific amplification. Simultaneously, limiting the probe length to 20–28 bp and the Tm value to be 5–8℃ higher than the primers ensured that the release of fluorescence signals occurred only during specific amplification. The pairing of fluorescent reporter groups such as FAM with quencher groups such as BHQ1 significantly improved the sensitivity and signal-to-noise ratio of quantitative detection, laying a molecular biological foundation for obtaining high-quality quantitative data. Therefore, the primer and probe design principles of this invention are superior.

[0038] 6. In the quantitative detection method for indicator species based on environmental DNA of the present invention, the first step of obtaining a candidate indicator species library for the target water area by screening according to the screening method refers to: firstly, integrating multi-source data based on the target water area to obtain an indicator species database; then, designing a multi-dimensional evaluation system based on the molecular biological characteristics and macro-ecological characteristics of the target water area; then, using machine learning algorithms to comprehensively score and rank the indicator species in the indicator species database based on their contribution; and finally, screening to obtain a candidate indicator species library. In application, by integrating multi-source heterogeneous data such as bibliometrics, monitoring reports, and gene databases, the one-sidedness and lag caused by a single data source are effectively avoided, ensuring the completeness and timeliness of the indicator species database. On this basis, a multi-dimensional evaluation system including molecular biological characteristics (such as eDNA amplification adaptability) and macro-ecological characteristics (such as ecological sensitivity and distribution breadth) is constructed, which can comprehensively measure the potential of species as indicator species. Furthermore, using machine learning algorithms (such as random forests or gradient boosting trees) to comprehensively score and rank the species based on their contribution not only greatly improves the screening efficiency, but more importantly, quantifies the contribution weight of each evaluation indicator to the screening results, eliminating the subjective bias present in the traditional expert scoring method. Therefore, the present invention is effective in screening indicator species. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the process of the present invention.

[0040] Figure 2 This is a flowchart illustrating the second step in this invention.

[0041] Figure 3 This is a schematic diagram of in-situ synchronous sampling in Example 7.

[0042] Figure 4 The standard curve for the small cyclophosphamide plasmid experiment in Example 7 is shown.

[0043] Figure 5 This is a schematic diagram of the linear regression fitting between the concentration of Cyclocarya paliurus eDNA and in situ biomass in Example 7.

[0044] Figure 6 This is a schematic diagram of in-situ synchronous sampling in Example 8.

[0045] Figure 7 The standard curve for the plasmid experiment of *Rotaria spp.* in Example 8 is shown.

[0046] Figure 8 This is a schematic diagram of the linear regression fitting between the eDNA concentration and in situ biomass of *Rotaria spp.* in Example 8. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] Please see Figure 1 — Figure 8 A method for quantitative detection of indicator species based on environmental DNA, the method comprising the following steps:

[0049] Step 1: First, screen according to the screening method to obtain a candidate indicator species library for the target water area, and then screen the candidate indicator species library according to the specified criteria to obtain a list of indicator species;

[0050] The second step is to design multiple primer and probe combinations based on the indicator species in the indicator species list, then verify the multiple primer and probe combinations, and finally obtain the optimal primer and probe combination.

[0051] The third step is to first set up multiple sampling points in the target water area, then collect and filter water samples at the sampling points to obtain environmental DNA; at the same time, measure the in-situ biomass data of indicator species at the sampling points.

[0052] Step 4: First, extract environmental DNA to obtain template DNA. Then, mix the template DNA with the optimal primer and probe combination to obtain a reaction solution. Next, perform an amplification program on the reaction solution for quantitative analysis to obtain quantitative data of environmental DNA of the indicator species.

[0053] Step 5: Using the in-situ biomass data of indicator species obtained from actual sampling points as the independent variable and the environmental DNA quantitative data of indicator species as the dependent variable, construct a quantitative relationship model;

[0054] Step 6: Perform steps 1 through 4 sequentially on the water area to be tested to obtain quantitative environmental DNA data of the indicator species. Then, input the quantitative environmental DNA data of the indicator species into the quantitative relationship model to obtain in-situ biomass data of the indicator species.

[0055] In the fifth step, constructing a quantitative relationship model refers to: In the formula, To obtain in-situ biomass data of indicator species by sampling points, These are the model calibration parameters obtained by fitting in-situ field data. Quantitative data on the environmental DNA of species, These are the model calibration parameters obtained by fitting in-situ field data;

[0056] The coefficient of determination in constructing a quantitative relationship model The construction of the quantitative relationship model .

[0057] In the first step, the list of indicator species includes 1 to 5 indicator species.

[0058] In the second step, the verification refers to: specificity verification, sensitivity and amplification efficiency verification; obtaining the optimal primer-probe combination refers to: obtaining the optimal primer-probe combination by comprehensively considering the specificity verification results, sensitivity and amplification efficiency verification results.

[0059] In the second step, the specificity verification refers to: using the DNA of the indicator species, DNA of closely related species, DNA of common aquatic organisms, and DNA of aquatic microorganisms as templates to perform amplification programs respectively, while recording the changes in fluorescence signal intensity of each template to generate multiple amplification curves that correspond one-to-one; then screening the multiple amplification curves and retaining primer-probe combinations that only show specific amplification curves with the target indicator species, have no cross-amplification signals, and have no non-specific products.

[0060] In the second step, the verification of sensitivity and amplification efficiency refers to: firstly, extracting the DNA of the indicator species, then serially diluting the DNA of the indicator species by 10-fold to obtain standard solutions of multiple concentration gradients, then performing an amplification program on the standard solutions of multiple concentration gradients, then plotting multiple standard curves corresponding one-to-one with multiple combinations of reaction solutions of multiple gradient concentrations, then calculating the amplification efficiency, linear correlation coefficient, and detection limit based on the multiple standard curves, and finally screening primer and probe combinations based on the calculated amplification efficiency, linear correlation coefficient, and detection limit.

[0061] In the second step, the design of multiple primer and probe combinations based on indicator species in the indicator species list refers to: selecting gene fragments of cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA from the indicator species to design primer and probe combinations; the primers are 18–25 bp in length, have a Tm value of 57–63 °C, a GC content of 40%–60%, and are free of hairpin structures, dimers, and cross-complementary sequences; the probes are 20–28 bp in length, have a Tm value 5–8 °C higher than the primers, are labeled with FAM or a corresponding fluorescent reporter group at the 5' end, and are labeled with BHQ1 or a corresponding quencher group at the 3' end.

[0062] In the first step, the process of obtaining a candidate indicator species library for the target water area by screening according to the screening method refers to: firstly, integrating multi-source data based on the target water area to obtain an indicator species database; then, designing an evaluation system based on the molecular biological characteristics and macro-ecological characteristics of the target water area; then, using machine learning algorithms to comprehensively score and rank the indicator species in the indicator species database based on their contribution; and finally, screening to obtain a candidate indicator species library.

[0063] In the first step, the process of screening the candidate indicator species library according to specified standards to obtain the indicator species list refers to the following: screening the candidate indicator species library according to national ecological and environmental standards, long-term ecological monitoring results of the target watershed, and recognized indicator taxa, and including indicator species that meet the following criteria: native species, sensitive to heavy metal / organic matter / low dissolved oxygen / eutrophication stress, widely distributed in nature, easily identifiable and sampled in the field, with an eDNA amplification efficiency of 90% to 110%, no cross-amplification interference, quantifiable biomass, and listed in the national / industry recognized indicator list.

[0064] In the first step, the evaluation system includes ecological sensitivity, regional distribution breadth, feasibility of field monitoring, eDNA amplification adaptability, and species stability.

[0065] The following are supplementary descriptions of the present invention:

[0066] This invention can be widely applied to: routine monitoring at ecological and environmental monitoring stations, on-site evidence collection for ecological and environmental law enforcement, emergency response to pollution accidents, judicial appraisal of ecological damage, ecological research in scientific research institutes, and refined management and control by watershed management departments. It provides solid technical support for ecological civilization construction, water ecological protection, and environmental judicial fairness, and has enormous social, ecological, and judicial value.

[0067] The environmental DNA mentioned in this invention refers to eDNA, which is released into the aquatic environment by organisms during their survival, activity, and metabolism through shedding cells, mucus, excrement, and decomposition of corpses. This DNA can remain stable in the aquatic environment for a certain period of time and degrades dynamically within a certain time scale. Therefore, it can still reflect the presence and abundance of the target species within a certain time range. This invention employs a third step (multi-site deployment, in-situ synchronous sampling in the field, on-site real-time filtration and enrichment, and low-temperature preservation), a fourth step (qPCR absolute quantification), and a fifth step (constructing a quantitative relationship model) to minimize the error caused by eDNA degradation and improve the stability and reliability of detection.

[0068] Example 1:

[0069] Please see Figure 1 — Figure 8 A method for quantitative detection of indicator species based on environmental DNA, the method comprising the following steps:

[0070] Step 1: First, screen according to the screening method to obtain a candidate indicator species library for the target water area, and then screen the candidate indicator species library according to the specified criteria to obtain a list of indicator species;

[0071] The second step is to design multiple primer and probe combinations based on the indicator species in the indicator species list, then verify the multiple primer and probe combinations, and finally obtain the optimal primer and probe combination.

[0072] The third step is to first set up multiple sampling points in the target water area, then collect and filter water samples at the sampling points to obtain environmental DNA; at the same time, measure the in-situ biomass data of indicator species at the sampling points.

[0073] Step 4: First, extract environmental DNA to obtain template DNA. Then, mix the template DNA with the optimal primer and probe combination to obtain a reaction solution. Next, perform an amplification program on the reaction solution for quantitative analysis to obtain quantitative data of environmental DNA of the indicator species.

[0074] Step 5: Using the in-situ biomass data of indicator species obtained from actual sampling points as the independent variable and the environmental DNA quantitative data of indicator species as the dependent variable, construct a quantitative relationship model;

[0075] Step 6: Perform steps 1 through 4 sequentially on the water area to be tested to obtain quantitative environmental DNA data of the indicator species. Then, input the quantitative environmental DNA data of the indicator species into the quantitative relationship model to obtain in-situ biomass data of the indicator species.

[0076] Example 2:

[0077] The basic content is the same as in Example 1, except that:

[0078] Please see Figure 1 —8. In the fifth step, constructing a quantitative relationship model refers to: In the formula, To obtain in-situ biomass data of indicator species by sampling points, These are the model calibration parameters obtained by fitting in-situ field data. Quantitative data on the environmental DNA of species, These are the model calibration parameters obtained by fitting in-situ field data; the determination coefficients for constructing the quantitative relationship model. The construction of the quantitative relationship model .

[0079] When applied, the quantitative data of indicator species environmental DNA obtained from sampling points in the water area to be tested will be used. Substitute into the quantitative relationship model In this process, through mathematical transformations, the in-situ biomass data of the indicator species at the corresponding sampling points can be calculated. The quantitative relationship model is a linear fitting model. Compared with complex nonlinear models, linear models are more tolerant of the small number of anomalous samples inevitably mixed in during field monitoring. Even with small field datasets, stable and reliable fitting results can be obtained, and the verification criteria are uniform, greatly reducing the difficulty of implementing the model in engineering projects. Secondly, the model parameters are intuitive, and the output... (Calibration parameters) reflect the eDNA flux released per unit biomass of the indicator species. The calibration parameters correspond to the background noise or baseline values ​​in the environment, making it easy for technicians to understand and interpret. Furthermore, the algorithm formula is mature, the parameter solution process is convenient, and there is no need for complex iterative calculations, which effectively improves data processing efficiency and can meet the timeliness requirements of large-scale environmental DNA monitoring scenarios.

[0080] Example 3:

[0081] The basic content is the same as in Example 1, except that:

[0082] Please see Figure 1 — Figure 2 In the first step, the list of indicator species includes 1 to 5 indicator species.

[0083] When applied, limiting the indicator species list to 1-5 species balances ecological representativeness and detection efficiency. The optimized range of 1-5 species can comprehensively cover the core community structure of the target water area, avoiding the inability to accurately represent the overall ecological state due to too few species, thus ensuring the representativeness and accuracy of the monitoring data. It also effectively controls the cost and complexity of engineering implementation. Compared with combinations of more species, 1-5 species can improve the stability of monitoring results by eliminating random errors caused by a single species through redundancy verification among multiple species, without significantly increasing the complexity of primer design, the risk of cross-amplification, and the cost of subsequent sequencing and analysis, achieving the best balance between accuracy, stability, and economy.

[0084] Example 4:

[0085] The basic content is the same as in Example 1, except that:

[0086] Please see Figure 1 — Figure 2In the second step, the verification refers to: specificity verification, sensitivity and amplification efficiency verification; obtaining the optimal primer-probe combination refers to: obtaining the optimal primer-probe combination by comprehensively considering the specificity verification results, sensitivity and amplification efficiency verification results. In the second step, the specificity verification refers to: using DNA from the indicator species, DNA from closely related species, DNA from common aquatic organisms, and DNA from aquatic microorganisms as templates for amplification programs, while simultaneously recording the changes in fluorescence signal intensity of each template to generate multiple corresponding amplification curves; then screening these multiple amplification curves, retaining primer-probe combinations that only show specific amplification curves with the target indicator species, have no cross-amplification signals, and have no non-specific products. In the second step, the sensitivity and amplification efficiency verification refers to: first extracting DNA from the indicator species, then performing a 10-fold serial dilution of the indicator species DNA to obtain multiple concentration gradient standard solutions, then performing amplification programs on these standard solutions, then plotting multiple standard curves corresponding to the multiple combinations of reaction solutions with different concentration gradients, then calculating the amplification efficiency, linear correlation coefficient, and detection limit based on the multiple standard curves, and finally screening primer-probe combinations based on the calculated amplification efficiency, linear correlation coefficient, and detection limit.

[0087] In application, specificity verification is first performed: using target indicator species DNA, closely related species DNA, common aquatic organism DNA, and aquatic microbial DNA as templates, qPCR amplification is performed simultaneously, and the fluorescence signal intensity changes of each template are recorded to generate multiple corresponding amplification curves. Primer-probe combinations that only show specific amplification curves for the indicator species, have no cross-amplification signals, and produce no non-specific products are then selected, completely eliminating interference from other biological DNA in complex aquatic environments and greatly improving the accuracy of the detection results. Next, sensitivity and amplification efficiency are verified: indicator species DNA is extracted first, and then the indicator species DNA standard is serially diluted to 10-1. 2 10 3 10 4 10 5 10 6 10 7 Multiple gradient concentrations of reaction solutions (copy / μL) were prepared, and qPCR amplification was performed on each solution. Multiple standard curves corresponding to the reaction solutions were then plotted. Amplification efficiency, linear correlation coefficient, and detection limit were calculated based on these standard curves. Primer-probe combinations with amplification efficiency of 90%–110%, linear correlation coefficient R² ≥ 0.99, and detection limit ≤ 10 copies / μL were selected. This ensured that the optimal primer-probe combination possessed high sensitivity and high quantitative accuracy, enabling stable detection and accurate quantification even in aquatic environments with extremely low concentrations of indicator species' environmental DNA. The optimal primer-probe combination was then determined based on a comprehensive evaluation of specificity, sensitivity, and amplification efficiency verification results.

[0088] Example 5:

[0089] The basic content is the same as in Example 1, except that:

[0090] Please see Figure 1 — Figure 2 In the second step, the design of multiple primer and probe combinations based on indicator species in the indicator species list refers to: selecting gene fragments of cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA from the indicator species to design primer and probe combinations; the primers are 18–25 bp in length, have a Tm value of 57–63 °C, a GC content of 40%–60%, and are free of hairpin structures, dimers, and cross-complementary sequences; the probes are 20–28 bp in length, have a Tm value 5–8 °C higher than the primers, are labeled with FAM or a corresponding fluorescent reporter group at the 5' end, and are labeled with BHQ1 or a corresponding quencher group at the 3' end.

[0091] When applying this method, target gene screening is performed first: Based on the list of indicator species, the full sequences of mitochondrial DNA (mtDNA) or ribosomal DNA (rDNA) of the target indicator species, their closely related species, common aquatic organisms, and aquatic microorganisms are downloaded from the GenBank database. Priority is given to gene fragments such as cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA, as these genes have moderate evolutionary rates, significant sequence differences between species, and strong evolutionary conservation, making them the optimal choice for designing specific primers. Then, primer design is performed: Primers are used... Specific PCR primers were designed using Premier 6.0 or OligoCalc software. Primer lengths were designed to be 18–25 bp, with a Tm value controlled at 57–63℃ and a GC content of 40%–60%. Software simulations were used to ensure the absence of hairpin structures, dimers, and cross-complementary sequences, thus guaranteeing the stability of primer-template binding and amplification efficiency. Probe design followed: TaqMa fluorescent probes were designed with a length of 20–28 bp, a Tm value 5–8℃ higher than the primers, and the 5' end labeled with FAM or a corresponding fluorescent reporter group, while the 3' end was labeled with BHQ1 or a corresponding quencher group, ensuring high specificity and low background interference. This resulted in multiple primer-probe combinations.

[0092] Example 6:

[0093] The basic content is the same as in Example 1, except that:

[0094] Please see Figure 1 — Figure 2In the first step, the selection of candidate indicator species for the target water area based on the screening method refers to: firstly, integrating multi-source data to obtain an indicator species database based on the target water area; secondly, designing an evaluation system based on the molecular biological and macro-ecological characteristics of the target water area; and thirdly, using machine learning algorithms to comprehensively score and rank the indicator species in the database based on their contribution, thus selecting the candidate indicator species. In the second step, the selection of the indicator species list based on specified standards refers to: selecting the candidate indicator species based on national ecological and environmental standards, long-term ecological monitoring results of the target watershed, and recognized indicator taxa; and including indicator species that meet the following criteria: native species, sensitive to heavy metal / organic matter / low dissolved oxygen / eutrophication stress, widely distributed in nature, easily identifiable in the field, with an eDNA amplification efficiency of 90%–110%, no cross-amplification interference, quantifiable biomass, and listed in the national / industry-recognized indicator species list. In the third step, the evaluation system includes ecological sensitivity, regional distribution breadth, feasibility of field monitoring, eDNA amplification adaptability, and species stability.

[0095] In application, first, multi-source data integration is performed according to the steps disclosed in application number 202510548578.9, invention titled "A Method for Screening Indicator Species of Aquatic Ecological Damage Based on Data Integration": The system searches CNKI, Wanfang, Web of Science, GBIF, and the China Aquatic Ecology Database, integrating multi-source literature data, survey data, and ecological data on aquatic ecological damage, indicator species, aquatic organism distribution, eDNA application, and field monitoring to construct an indicator species database for the target water area; then, based on the molecular biological and macro-ecological characteristics of the target water area, five core quantitative evaluation indicators are designed, including ecological sensitivity, regional distribution breadth, field monitoring feasibility, eDNA amplification adaptability, and species stability; then, a random forest machine learning algorithm is used to comprehensively score and rank the species in the indicator species database, initially screening out candidate indicator species that respond most significantly to aquatic ecological damage; then, based on HJ... Based on the "Technical Guidelines for Water Ecological Monitoring: Monitoring and Evaluation of Aquatic Organisms in Rivers" (1295—2023), the "Technical Guidelines for Establishing Water Quality Standards for Freshwater Aquatic Organisms," and the long-term ecological monitoring results of the target watershed, 1–5 native aquatic organisms will be selected from the candidate indicator species pool for inclusion in the indicator species list. Priority will be given to three recognized indicator groups: sensitive fish, large benthic invertebrates, and large daphnia. At the same time, the following criteria will be strictly followed: native species, sensitive to heavy metal / organic matter / low dissolved oxygen / eutrophication stress, widely distributed in nature, easily identifiable and sampled in the field, with an eDNA amplification efficiency of 90%–110%, no cross-amplification interference, quantifiable biomass, and listed in the national / industry recognized indicator species list.

[0096] Example 7:

[0097] The basic content is the same as in Example 1, except that:

[0098] Please see Figure 1 — Figure 2 In the third step, setting up multiple sampling points in the target water area means: scientifically setting up no fewer than 6 sampling points based on the water morphology, hydrological characteristics, pollution distribution gradient, and pollution sources of the target water area, evenly covering clean areas, lightly polluted areas, moderately polluted areas, and heavily polluted areas, to ensure that the data gradient is complete, representative, and can reflect the ecological status of the entire watershed; collecting water samples means: using a sterile deep-water sampler to collect surface water at a depth of 0.5–1.0 m, collecting 500–1000 mL of water sample from each point, with sterile operation throughout to strictly avoid exogenous DNA contamination; filtering to obtain environmental DNA means: immediately after water sample collection, performing vacuum filtration in the field using sterile mixed fibers with a diameter of 47 mm and a pore size of 0.22–0.8 μm. Polycarbonate or ester filter membranes are used to enrich free environmental DNA in water. After filtration, the filter membrane is aseptically sealed in a centrifuge tube and stored at -20°C to prevent DNA degradation and contamination. The in-situ biomass data of indicator species obtained at the sampling point refers to the following: at the same sampling point, during the same time period, and under the same environmental conditions, standardized survey methods matching the indicator species type are used to measure the in-situ biomass data of the indicator species, and the units are converted to g / L. For example, for fish, electrofishing or net fishing methods are used, and the conversion is based on the sample area, water depth, and catch weight; for large benthic animals, mud samples are collected, sorted, identified, and weighed, and the conversion is based on the sample volume; for large daphnia, planktonic net filtration, concentration, counting, and weighing are used, followed by conversion.

[0099] Example 8:

[0100] The basic content is the same as in Example 1, except that:

[0101] Please see Figure 1 — Figure 5 This embodiment uses a typical river in the middle and lower reaches of the Yangtze River as the target water area (e.g., Figure 3 As shown in the figure, Cyclotella sp., a native sensitive phytoplankton, was selected as an indicator species, and in-situ biomass quantification was performed in the field according to the method of this invention:

[0102] First, based on the steps disclosed in application number 202510548578.9, entitled "A Method for Screening Indicator Species of Aquatic Ecological Damage Based on Data Integration" and the nationally recognized list in HJ1295-2023, *Cyclocarya paliurus* was selected as the indicator species. This species is a native small phytoplankton in the Yangtze River Basin, which is sensitive to heavy metal and organic pollution, widely distributed, easily identifiable in the wild, and has easy eDNA amplification, thus having clear ecological indicator significance.

[0103] Multiple primer and probe combinations were designed using conserved fragments of the *Cyclocarya spp.* rbc gene. After verification, the optimal primer and probe combination was obtained: primers included upstream 5'-TTGCGTACTGGTGTCGTGAA-3' and downstream 5'-CCCAGCCATACGCATCCATT-3'; probes included 5'-CY5-TTACATTTACACCGTGCTGGTAACT-MGB-3'. This optimal primer and probe combination could specifically amplify *Cyclocarya spp.* without cross-amplification, with an amplification efficiency of 90.37% and a correlation coefficient R² = 1.00.

[0104] Seven sampling points were then established in the target water area, covering clean, lightly polluted, moderately polluted, and heavily polluted areas. 1L of surface water was collected from each sampling point, and environmental DNA was enriched by filtration through a 0.22μm sterile filter membrane and stored at -20℃. Simultaneously, the biomass of *Cyclocarya microphylla* was measured using a synchronous method, with in-situ biomass data ranging from 0 to 8.25 × 10⁻⁶. -5 g / L;

[0105] Environmental DNA was then extracted to obtain template DNA. The template DNA and the optimal primer-probe combination were then mixed to prepare a reaction solution. The reaction solution was then subjected to an amplification program for quantitative analysis, yielding quantitative data of environmental DNA for the indicator species ranging from 0 to 3.23 × 10⁻⁶. 6 Copies / L, such as Figure 4 As shown;

[0106] Then, the in-situ biomass data of indicator species obtained from actual sampling points were used as independent variables. Quantitative environmental DNA data of indicator species are used as the dependent variable. , construct as Figure 5 The quantitative relationship model shown:

[0107] ;

[0108] Then, following the aforementioned steps, the environmental DNA quantification data of *Cyclocarya paliurus* in the tested water area was obtained: 1.17 × 10⁻⁶. 6 Copies / L, 2.09×10 6 Copies / L, 2.28×10 6 Copies / L, 2.52×106 Copies / L, 3.24×10 6 Copies / L; then, substituting the aforementioned environmental DNA quantitative data into the quantitative relationship model, the in-situ biomass data of *Cyclocarya paliurus* was obtained: 3.38 × 10⁻⁶. -5 g / L, 6.44×10 -5 g / L, 7.06×10 -5 g / L, 7.88×10 -5 g / L, 1.03×10 -4 g / L indicates the completion of quantitative detection of in-situ biomass of Cyclops microphylla.

[0109] Example 9:

[0110] The basic content is the same as in Example 1, except that:

[0111] Please see Figure 1 — Figure 8 This embodiment uses a medium-sized reservoir in East China as the target water area (e.g., Figure 6 As shown in the figure, the native sensitive zooplankton *Rotaria spp.* was selected as the indicator species, and in-situ biomass quantification was performed in the field according to the method of this invention:

[0112] First, based on the steps disclosed in application number 202510548578.9, entitled "A Method for Screening Indicator Species of Aquatic Ecological Damage Based on Data Integration" and the nationally recognized list in HJ1295-2023, *Rotaria spp.* was selected as the indicator species. This species is a native zooplankton in the target waters, sensitive to heavy metal and organic pollution, widely distributed, easily identifiable in the wild, and has easily amplified eDNA, thus having clear ecological indicator significance.

[0113] The COI gene primer and probe combination was selected as the optimal primer and probe combination. This optimal primer and probe combination can specifically amplify Cyclocarya paliurus without cross-reactivity, and the amplification efficiency is 96.3% with a correlation coefficient R² = 0.995.

[0114] Seven sampling points were then established in the target water area, covering clean, lightly polluted, moderately polluted, and heavily polluted areas. 1L of surface water was collected from each sampling point, and environmental DNA was enriched by filtration through a 0.22μm sterile filter membrane and stored at -20℃. Simultaneously, the biomass of *Rotaria spiraea* was measured using a synchronous method, with in-situ biomass data ranging from 0 to 7.44 × 10⁻⁶. -8 g / L;

[0115] Environmental DNA was then extracted to obtain template DNA. The template DNA and the optimal primer-probe combination were then mixed to prepare a reaction solution. The reaction solution was then subjected to an amplification program for quantitative analysis, yielding quantitative data of environmental DNA for the indicator species ranging from 1075 to 1561 copies / L. Figure 7 As shown;

[0116] Then, the in-situ biomass data of indicator species obtained from actual sampling points were used as independent variables. Quantitative environmental DNA data of indicator species are used as the dependent variable. , construct as Figure 8 The quantitative relationship model shown:

[0117] ;

[0118] Following the aforementioned steps, the environmental DNA quantitative data of *Rotaria spp.* in the tested water area were obtained: 112 copies / L, 265 copies / L, 347 copies / L, 468 copies / L, and 1075 copies / L. Then, these environmental DNA quantitative data were substituted into a quantitative relationship model to obtain the in-situ biomass data of *Rotaria spp.*: 3.38 × 10⁻⁶. -5 g / L, 6.44×10 -5 g / L, 7.06×10 -5 g / L, 7.88×10 -5 g / L, 1.03×10 -4 g / L indicates the completion of quantitative detection of in-situ biomass of *Rotaria spp.*.

[0119] The above description is only a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. Any equivalent modifications or changes made by those skilled in the art based on the content disclosed in the present invention should be included within the scope of protection set forth in the claims.

Claims

1. A method for quantitative detection of indicator species based on environmental DNA, characterized in that: The method includes the following steps: Step 1: First, screen according to the screening method to obtain a candidate indicator species library for the target water area, and then screen the candidate indicator species library according to the specified criteria to obtain a list of indicator species; The second step is to design multiple primer and probe combinations based on the indicator species in the indicator species list, then verify the multiple primer and probe combinations, and finally obtain the optimal primer and probe combination. The third step is to first set up multiple sampling points in the target water area, then collect and filter water samples at the sampling points to obtain environmental DNA; at the same time, measure the in-situ biomass data of indicator species at the sampling points. Step 4: First, extract environmental DNA to obtain template DNA. Then, mix the template DNA with the optimal primer and probe combination to obtain a reaction solution. Next, perform an amplification program on the reaction solution for quantitative analysis to obtain quantitative data of environmental DNA of the indicator species. Step 5: Using the in-situ biomass data of indicator species obtained from actual sampling points as the independent variable and the environmental DNA quantitative data of indicator species as the dependent variable, construct a quantitative relationship model; Step 6: Perform steps 1 through 4 sequentially on the water area to be tested to obtain quantitative environmental DNA data of the indicator species. Then, input the quantitative environmental DNA data of the indicator species into the quantitative relationship model to obtain in-situ biomass data of the indicator species.

2. The method for quantitative detection of indicator species based on environmental DNA according to claim 1, characterized in that: In the fifth step, constructing a quantitative relationship model refers to: In the formula, To obtain in-situ biomass data of indicator species by sampling points, These are the model calibration parameters obtained by fitting in-situ field data. Quantitative data on the environmental DNA of species, These are the model calibration parameters obtained by fitting in-situ field data; The coefficient of determination in constructing a quantitative relationship model The construction of the quantitative relationship model .

3. The method for quantitative detection of indicator species based on environmental DNA according to claim 1 or 2, characterized in that: In the first step, the list of indicator species includes 1 to 5 indicator species.

4. A method for quantitative detection of indicator species based on environmental DNA according to claim 1 or 2, characterized in that: In the second step, the verification refers to: specificity verification, sensitivity and amplification efficiency verification; obtaining the optimal primer-probe combination refers to: obtaining the optimal primer-probe combination by comprehensively considering the specificity verification results, sensitivity and amplification efficiency verification results.

5. The method for quantitative detection of indicator species based on environmental DNA according to claim 4, characterized in that: In the second step, the specificity verification refers to: using the DNA of the indicator species, DNA of closely related species, DNA of common aquatic organisms, and DNA of aquatic microorganisms as templates to perform amplification programs respectively, while recording the changes in fluorescence signal intensity of each template to generate multiple amplification curves that correspond one-to-one; then screening the multiple amplification curves and retaining primer-probe combinations that only show specific amplification curves with the target indicator species, have no cross-amplification signals, and have no non-specific products.

6. The method for quantitative detection of indicator species based on environmental DNA according to claim 5, characterized in that: In the second step, the verification of sensitivity and amplification efficiency refers to: firstly, extracting the DNA of the indicator species, then serially diluting the DNA of the indicator species by 10-fold to obtain standard solutions of multiple concentration gradients, then performing an amplification program on the standard solutions of multiple concentration gradients, then plotting multiple standard curves corresponding one-to-one with multiple combinations of reaction solutions of multiple gradient concentrations, then calculating the amplification efficiency, linear correlation coefficient, and detection limit based on the multiple standard curves, and finally screening primer and probe combinations based on the calculated amplification efficiency, linear correlation coefficient, and detection limit.

7. A method for quantitative detection of indicator species based on environmental DNA according to claim 1 or 2, characterized in that: In the second step, the design of multiple primer and probe combinations based on indicator species in the indicator species list refers to: selecting gene fragments of cytochrome b (Cytb), COI, rbcL, 12S rRNA, and 16S rRNA from the indicator species to design primer and probe combinations; the primers are 18–25 bp in length, have a Tm value of 57–63 °C, a GC content of 40%–60%, and are free of hairpin structures, dimers, and cross-complementary sequences; the probes are 20–28 bp in length, have a Tm value 5–8 °C higher than the primers, are labeled with FAM or a corresponding fluorescent reporter group at the 5' end, and are labeled with BHQ1 or a corresponding quencher group at the 3' end.

8. A method for quantitative detection of indicator species based on environmental DNA according to claim 1 or 2, characterized in that: In the first step, the process of obtaining a candidate indicator species library for the target water area by screening according to the screening method refers to: firstly, integrating multi-source data based on the target water area to obtain an indicator species database; then, designing an evaluation system based on the molecular biological characteristics and macro-ecological characteristics of the target water area; then, using machine learning algorithms to comprehensively score and rank the indicator species in the indicator species database based on their contribution; and finally, screening to obtain a candidate indicator species library.

9. The method for quantitative detection of indicator species based on environmental DNA according to claim 8, characterized in that: In the first step, the process of screening the candidate indicator species library according to specified standards to obtain the indicator species list refers to the following: screening the candidate indicator species library according to national ecological and environmental standards, long-term ecological monitoring results of the target watershed, and recognized indicator taxa, and including indicator species that meet the following criteria: native species, sensitive to heavy metal / organic matter / low dissolved oxygen / eutrophication stress, widely distributed in nature, easily identifiable and sampled in the field, with an eDNA amplification efficiency of 90% to 110%, no cross-amplification interference, quantifiable biomass, and listed in the national / industry recognized indicator list.

10. The method for quantitative detection of indicator species based on environmental DNA according to claim 8, characterized in that: In the first step, the evaluation system includes ecological sensitivity, regional distribution breadth, feasibility of field monitoring, eDNA amplification adaptability, and species stability.

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