Method for monitoring concentration of rare fishes in circulating water culture system based on eDNA quantitative detection

Abstract

The invention relates to the field of eDNA concentration detection, and discloses an eDNA quantitative detection-based method for monitoring the concentration of valuable and rare fishes in a circulating water culture system, which comprises the following steps of: collecting and analyzing a water sample in a closed circulating water culture system, automatically extracting and purifying eDNA, and simultaneously combining a qPCR (quantitative polymerase chain reaction) amplification mode to calculate the concentration of the valuable and rare fishes. According to the method, zero-interference and zero-stress treatment can be performed on the fish only by taking a water sample, the aim of protecting the fish is fulfilled, the operation is simple and convenient, the sensitivity is extremely high, and the concentration monitoring efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of eDNA detection concentration, and particularly to a rare fish concentration monitoring method based on eDNA quantitative detection in a recirculating aquaculture system. BACKGROUND

[0002] eDNA, namely environmental DNA, refers to genetic material fragments released into the surrounding environment (water, soil, air) by organisms through skin mucosa, excrement, reproductive cells, blood, etc. eDNA quantitative detection is a molecular biology technology, which not only detects whether the DNA of a certain organism exists in water, but also accurately measures the concentration of the DNA of the organism in the water. The core process includes sampling filtration, DNA extraction and purification, and quantitative PCR amplification calculation. In this way, the approximate number concentration of the fish species to be detected in the water area can be roughly estimated.

[0003] The traditional method for monitoring the concentration of rare fish has great limitations. For example, the traditional method needs to be fished, weighed, and counted, which may cause the rare fish to be frightened, injured, or even dead. The deviation of the traditional method for fishing in a large-scale aquaculture pond is large. The advantage of the eDNA quantitative detection method is that only water samples need to be taken, which causes zero interference and zero stress to the fish, meets the purpose of protecting the fish, is simple to operate, has high sensitivity, and can realize accurate estimation from "DNA content in water" to "how many fish in the pond" by establishing a mathematical model between eDNA concentration and fish biomass, which is more scientific and reliable than visual observation and sampling and weighing. Finally, the result is obtained quickly, which improves the concentration monitoring efficiency. For rare fish in a recirculating aquaculture system, eDNA quantitative detection is not only a new monitoring tool, but also an innovation of the breeding concept. It upgrades the breeding management from the "fuzzy art" relying on experience to the "precise science" based on data, greatly improves the efficiency and reliability of breeding while respecting and protecting rare organisms to the maximum extent. SUMMARY

[0004] The present application overcomes the shortcomings of the prior art and provides a rare fish concentration monitoring method based on eDNA quantitative detection in a recirculating aquaculture system.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides a rare fish concentration monitoring method based on eDNA quantitative detection in a recirculating aquaculture system, comprising the following steps: Integrated sampling of water samples is performed in a closed recirculating aquaculture system, and the sampled water samples are subjected to in-situ enrichment treatment; The filter membrane obtained after the in-situ enrichment treatment is subjected to automatic eDNA extraction and purification treatment; introducing and detecting the eluate of the dissolved eDNA by real-time fluorescent quantitative amplification detection of the microfluidic chip, and calculating the initial copy number of the eDNA of the target fish; Combined with the initial copy number of the eDNA of the target fish, the concentration of the target fish in the target recirculating aquaculture system is calculated.

[0006] Further, in a preferred embodiment of the present application, the water sample integration sampling in the closed recirculating aquaculture system and the in-situ enrichment treatment of the sampled water sample are specifically as follows: The recirculating aquaculture system to be monitored for the concentration of rare fish is determined as the target recirculating aquaculture system, and a water sample collection device is arranged in the main pipeline of the target recirculating aquaculture system, and the rare fish to be monitored for the concentration is determined as the target fish; The water sample collection device divides the main pipeline into a main line and a sampling branch through a three-way electromagnetic valve, and the water flow in the main line is shunted to the sampling branch, and the nucleic acid removal cleaning treatment is performed on the sampling branch through the cleaning liquid electromagnetic valve of the water sample collection device, and finally the water sample with a fixed volume is extracted in the sampling branch through the quantitative pump of the water sample collection device, which is determined as the target water sample, and the collection concentration of the target water sample is determined; An eDNA enrichment unit is obtained, the target water sample is transported into the eDNA enrichment unit through the quantitative pump, and the water sample state of the target water sample is detected and recorded in real time during the transportation, wherein the water sample state of the target water sample includes temperature, pH value and conductivity; The target water sample is subjected to filter membrane enrichment in the eDNA enrichment unit, wherein the filter membrane enrichment is to enrich the particulate matter larger than the pore size of the filter membrane in the target water sample, and the filter membrane after enrichment is cleaned by the eDNA enrichment unit, wherein the cleaning treatment is to remove the residual PCR inhibitors on the aluminum membrane; The cleaned filter membrane is stored at low temperature, and the sampling state of the target water sample during in-situ enrichment is retained.

[0007] Further, in a preferred embodiment of the present application, the filter membrane obtained after the in-situ enrichment treatment is subjected to automatic eDNA extraction and purification treatment, specifically as follows: The filter membrane stored at low temperature and cleaned is determined as the target filter membrane, and the target filter membrane is placed on the sample processing platform of the nucleic acid extractor; The target filter membrane is subjected to lysate addition on the sample processing platform of the nucleic acid extractor, and the processing temperature of the sample processing platform of the nucleic acid extractor is controlled to maintain equal to the preset temperature, wherein the preset temperature of the target filter membrane during the lysis process is determined according to the target fish; Wherein, before adding the lysis solution, the adding concentration of the lysis solution needs to be determined, a big data network is introduced, and the adding concentration of the lysis solution corresponding to the collection concentration of the target water sample is determined; After adding the lysis solution, the sample processing platform of the nucleic acid extractor is subjected to vortex oscillation treatment, and after a predetermined vortex oscillation treatment time, the magnetic bead suspension liquid is continuously added in the sample processing platform of the nucleic acid extractor, wherein the magnetic bead suspension liquid is a binding liquid for adsorbing all eDNA in the target filter membrane, and after adding the magnetic bead suspension liquid, the binding liquid of the target filter membrane is obtained; Wherein, before adding the magnetic bead suspension liquid, the adding concentration of the lysis solution corresponding to the adding concentration of the magnetic bead suspension liquid needs to be determined in the big data network; The negative pressure pump and the silica gel membrane adsorption column sealed reaction tube are introduced, the binding liquid of the target filter membrane is introduced into the silica gel membrane adsorption column sealed reaction tube, and the binding liquid of the target filter membrane is subjected to negative pressure filtration treatment through the negative pressure pump, wherein the negative pressure filtration treatment obtains waste liquid, and at the same time, the silica gel membrane adsorption column sealed reaction tube adsorbed with eDNA is obtained, which is calibrated as a target reaction tube; The target reaction tube is subjected to negative pressure exhaust waste liquid treatment and drying treatment, and the silica gel membrane adsorption column with only eDNA is obtained, and the silica gel membrane adsorption column with only eDNA is subjected to drying treatment, and at the same time, an eluent is added to obtain an eDNA-dissolved eluent, and finally the eDNA-dissolved eluent is subjected to low-temperature temporary storage treatment; Wherein, before adding the eluent, the standard concentration of the eluent is determined through the big data network.

[0008] Further, in a preferred embodiment of the present application, the qPCR microfluidic chip is introduced and subjected to real-time fluorescent quantitative amplification detection treatment on the eDNA-dissolved eluent, and the initial copy number of eDNA of the target fish is calculated, specifically: The qPCR microfluidic chip and the fluorescence optical detection module are introduced, and the chip information pre-setting is performed on the qPCR microfluidic chip, wherein the chip information pre-setting is to input the type of the target fish and the actual number of the eDNA-dissolved eluent in the qPCR microfluidic chip; The reaction equipment of the eDNA-dissolved eluent is determined, the freeze-dried qPCR reaction reagent is introduced, the qPCR microfluidic chip and the fluorescence optical detection module are integrated in the reaction equipment of the eDNA-dissolved eluent, and at the same time, the eDNA-dissolved eluent and the freeze-dried qPCR reaction reagent are introduced for mixing treatment, so as to construct a PCR reaction system to be amplified, which is calibrated as a target reaction system; Wherein, the mixed liquid in the target reaction system is in contact with the qPCR microfluidic chip and the fluorescence optical detection module; The fluorescence optical detection module is associated with the qPCR microfluidic chip, that is, the qPCR microfluidic chip calculates the initial copy number of eDNA of the target fish by analyzing the fluorescence data state output by the fluorescence optical detection module; The standard temperature for qPCR amplification of the eDNA of the target fish is retrieved through the big data network, and is calibrated as a standard amplification temperature. During the temperature control, the qPCR microfluidic chip and the fluorescence optical detection module are combined to calculate the initial copy number of eDNA of the target fish.

[0009] Further, in a preferred embodiment of the present application, during the temperature control, the qPCR microfluidic chip and the fluorescence optical detection module are combined to calculate the initial copy number of eDNA of the target fish, specifically: During the temperature control, the qPCR microfluidic chip is used to collect the fluorescence data state output by the fluorescence optical detection module in real time, wherein the fluorescence data state output by the fluorescence optical detection module is the wavelength value of the fluorescence signal. In the qPCR microfluidic chip, the eDNA standard curve of the target fish is preset, and the wavelength value of the fluorescence signal is analyzed in real time by the qPCR microfluidic chip according to the collected fluorescence data state output by the fluorescence optical detection module, so as to obtain the real-time amplification curve of eDNA of the target fish. During the temperature control, the qPCR microfluidic chip executes a temperature cycle program in the reaction device of the eDNA-dissolving eluent, wherein the temperature cycle program is that one temperature control process is one cycle, a preset fluorescence baseline threshold is used to analyze the real-time amplification curve of eDNA of the target fish, and when the real-time amplification curve of eDNA of the target fish reaches the fluorescence baseline threshold, the temperature control process is stopped, and the cycle number is recorded and calibrated as a real-time Ct value. The real-time Ct value is imported into the eDNA standard curve of the target fish for regression calculation, and the initial copy number of eDNA of the target fish is output.

[0010] Further, in a preferred embodiment of the present application, the initial copy number of eDNA of the target fish is combined to calculate the concentration of the target fish in the target recirculating aquaculture system, specifically: A concentration conversion formula is retrieved through the big data network, and a biomass-eDNA concentration correction model is constructed based on the concentration conversion formula. Input the water sample state of the target water sample, the water sample concentration of the target water sample, the lysate addition concentration, the magnetic bead suspension addition concentration, the eluent standard concentration, and the eDNA initial copy number of the target fish into the biomass-eDNA concentration correction model; Run the biomass-eDNA concentration correction model, and output the real-time concentration of the target fish in the target recirculating aquaculture system.

[0011] The second aspect of the present application also provides a recirculating aquaculture system rare fish concentration monitoring system based on eDNA quantitative detection. The recirculating aquaculture system rare fish concentration monitoring system integrates a high-performance computing architecture and a data storage module, including a non-volatile memory composed of an ECC-verified DDR4 RDIMM memory module and an NVMe solid-state storage array using 3D NAND flash, and a multi-core processor based on Zen4 microarchitecture. The memory is solidified and deployed with a recirculating aquaculture system rare fish concentration monitoring method program with a concentration monitoring engine. When the program is executed in parallel by the superscalar pipeline execution unit in the processor, the following steps are implemented: Integrated water sampling is performed in a closed recirculating aquaculture system, and the sampled water sample is subjected to in-situ enrichment treatment; The filter membrane obtained after in-situ enrichment treatment is subjected to automatic eDNA extraction and purification treatment; The eluent of the dissolved eDNA is introduced and subjected to real-time fluorescent quantitative amplification detection treatment by a qPCR microfluidic chip, and the eDNA initial copy number of the target fish is calculated; The concentration of the target fish in the target recirculating aquaculture system is calculated in combination with the eDNA initial copy number of the target fish.

[0012] The present application solves the technical defects in the background art. The present application has the following advantages: by collecting and analyzing water samples in a closed recirculating aquaculture system and automatically extracting and purifying eDNA, the concentration of rare fish can be calculated by combining the qPCR amplification method. The present application only needs to take water samples, which causes zero interference and zero stress to the fish, meets the purpose of protecting the fish, is simple to operate, has high sensitivity, and greatly improves the concentration monitoring efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings of embodiments according to these drawings without creative labor.

[0014] Figure 1 A flow chart of a rare fish concentration monitoring method based on eDNA quantitative detection in a recirculating aquaculture system is shown. Figure 2 A flow chart of a method for calculating the initial copy number of eDNA of target fish is shown. Figure 3 A program view of a rare fish concentration monitoring system based on eDNA quantitative detection in a recirculating aquaculture system is shown. DETAILED DESCRIPTION

[0015] In order to enable a clearer understanding of the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0016] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can also be practiced in other ways different from those described herein, and therefore, the scope of protection of the present application is not limited by the specific embodiments disclosed below.

[0017] Figure 1 A flow chart of a rare fish concentration monitoring method based on eDNA quantitative detection in a recirculating aquaculture system is shown, comprising the following steps: S102: Integrated sampling of water samples in a closed recirculating aquaculture system, and in-situ enrichment treatment of the sampled water samples; S104: Automatic eDNA extraction and purification treatment of the filter membrane obtained after in-situ enrichment treatment; S106: Introducing and detecting the eluate of dissolved eDNA by real-time fluorescent quantitative amplification through a qPCR microfluidic chip, and calculating the initial copy number of eDNA of target fish; S108: Combined with the initial copy number of eDNA of target fish, the concentration of target fish in the target recirculating aquaculture system is calculated.

[0018] Further, in a preferred embodiment of the present application, the integrated sampling of water samples in a closed recirculating aquaculture system and the in-situ enrichment treatment of the sampled water samples are specifically: The recirculating aquaculture system to be monitored for rare fish concentration is determined, and is designated as the target recirculating aquaculture system, and water sample collection equipment is arranged in the main pipeline of the target recirculating aquaculture system, and the rare fish to be monitored for concentration is designated as the target fish; The water sampling device uses a three-way solenoid valve to divide the main pipeline into a main path and a sampling branch path. The water flow in the main path is diverted to the sampling branch path within the main pipeline. At the same time, the cleaning fluid solenoid valve of the water sampling device is used to clean the sampling branch path for nucleic acid removal. Finally, the quantitative pump of the water sampling device extracts a fixed volume of water sample from the sampling branch path, which is then labeled as the target water sample, and the collection concentration of the target water sample is determined. An eDNA enrichment unit is obtained, and the target water sample is delivered into the eDNA enrichment unit by a quantitative pump. During the delivery process, the water sample status of the target water sample is detected and recorded in real time, wherein the water sample status of the target water sample includes temperature, pH value and conductivity. Within the eDNA enrichment unit, the target water sample is enriched using a filter membrane. Filter membrane enrichment involves enriching particles larger than the pore size of the filter membrane in the target water sample. The filter membrane after enrichment is then cleaned using the eDNA enrichment unit. The cleaning process removes residual PCR inhibitors from the aluminum membrane. The cleaned filter membrane was stored at low temperature, and the sampling state of the target water sample during in-situ enrichment was preserved.

[0019] It's important to note that in closed-loop recirculating aquaculture systems, fish concentration, i.e., biomass, is determined by analyzing the eDNA in water samples. Quantitative eDNA detection is a molecular biology technique that goes beyond simply detecting the presence of an organism's DNA in the water; it precisely measures the concentration of that organism's DNA in the water. The core process includes sampling and filtration, DNA extraction and purification, and quantitative PCR amplification and calculation to determine the fish concentration. During water sample collection, the sampling path undergoes nucleic acid removal and cleaning to remove any remaining impurities and prevent cross-contamination. The target water sample's concentration is determined for final concentration calculation. The eDNA enrichment unit contains a replaceable filter membrane, typically a polyethersulfone membrane with a pore size of 0.22 μm. Driven by a pump, the water sample passes through the filter membrane, and all particles larger than the pore size, including cell debris containing eDNA and organic matter, are trapped on the membrane surface. Recording the target water sample's conditions, including temperature, pH, and conductivity, is also used for final concentration calculation. The filter membrane needs to be cleaned, including rinsing the filter membrane with a certain volume of sterile phosphate buffer and a second cleaning with a certain volume of nucleic acid remover or ethanol solution to remove PCR inhibitors on the aluminum membrane. Finally, the filter membrane is stored at low temperature, which helps to slow down the enzymatic degradation of eDNA.

[0020] Furthermore, in a preferred embodiment of the present invention, the filter membrane obtained after the in-situ enrichment treatment is subjected to automated eDNA extraction and purification, specifically as follows: The filter membrane after low-temperature preservation and cleaning treatment is calibrated as a target filter membrane, and the target filter membrane is placed on a sample processing platform of a nucleic acid extractor; The target filter membrane is subjected to lysate addition on the sample processing platform of the nucleic acid extractor, and the processing temperature of the sample processing platform of the nucleic acid extractor is controlled to maintain equal to a preset temperature, wherein the preset temperature of the target filter membrane in the lysis process is determined according to the target fish; Wherein, the addition concentration of the lysate needs to be determined before the lysate is added, a big data network is introduced, and the lysate addition concentration corresponding to the collection concentration of the target water sample is determined; After adding the lysate, the sample processing platform of the nucleic acid extractor is subjected to vortex oscillation treatment, and after a predetermined vortex oscillation treatment time, magnetic bead suspension is continuously added in the sample processing platform of the nucleic acid extractor, wherein the magnetic bead suspension is a binding liquid for adsorbing all eDNA in the target filter membrane, and after adding the magnetic bead suspension, a binding liquid of the target filter membrane is obtained; Wherein, the lysate addition concentration corresponding to the magnetic bead suspension addition concentration needs to be determined in the big data network before the magnetic bead suspension is added; A negative pressure pump and a silica gel membrane adsorption column sealed reaction tube are introduced, the binding liquid of the target filter membrane is introduced into the silica gel membrane adsorption column sealed reaction tube, and the binding liquid of the target filter membrane is subjected to negative pressure filtration treatment through the negative pressure pump, wherein the negative pressure filtration treatment obtains a waste liquid, and at the same time, a silica gel membrane adsorption column sealed reaction tube adsorbing eDNA is obtained, which is calibrated as a target reaction tube; The target reaction tube is subjected to negative pressure exhaust waste liquid treatment and drying treatment to obtain a silica gel membrane adsorption column containing only eDNA, and the silica gel membrane adsorption column containing only eDNA is subjected to drying treatment, and at the same time, an eluent is added to obtain an eDNA-dissolved eluent, and finally the eDNA-dissolved eluent is subjected to low-temperature temporary storage treatment; Wherein, the standard concentration of the eluent is determined through the big data network before the eluent is added.

[0021] It should be noted that the sample processing platform of the nucleic acid extractor can automatically and efficiently complete the entire process of cell lysis, eDNA binding, purification, and elution without human intervention, yielding a high-purity eDNA solution suitable for qPCR detection. The purpose of the lysis buffer is to lyse the eDNA; heating and vortexing ensure complete lysis of all biological material on the aluminum membrane, allowing the eDNA to be fully released into the lysis buffer. After lysis, the system is cooled, and a certain volume of binding buffer (magnetic bead suspension) is added to the lysis buffer. This allows the eDNA molecules to adsorb onto the silica gel adsorption column, facilitating subsequent quantification and analysis. The system then activates a negative pressure pump to filter the liquid in the binding buffer through the adsorption column to a waste collection tank, while the eDNA is retained and bound to the silica gel membrane of the adsorption column. Further washing removes protein residues and lipid impurities, creating the necessary conditions for subsequent qPCR. Thorough drying is performed to improve the removal rate of ethanol residues, and finally, elution buffer is added.

[0022] Furthermore, in a preferred embodiment of the present invention, the step of calculating the concentration of the target fish in the target recirculating aquaculture system based on the initial copy number of the target fish's eDNA specifically involves: The concentration conversion formula was retrieved through big data network, and a biomass-eDNA concentration correction model was constructed based on the concentration conversion formula. Input the target water sample status, target water sample concentration, lysis buffer concentration, magnetic bead suspension concentration, elution buffer standard concentration, and initial eDNA copy number of the target fish into the biomass-eDNA concentration correction model. Run the biomass-eDNA concentration correction model to output the real-time concentration of the target fish species within the target recirculating aquaculture system.

[0023] It should be noted that once the initial copy number of the target fish's eDNA is known after sampling, the eDNA concentration in the target water sample at the sampling point is known. By combining the water sample state, water sample concentration, lysis buffer concentration, magnetic bead suspension concentration, elution buffer standard concentration, and the initial copy number of the target fish's eDNA, the concentration can be converted and the real-time concentration of the target fish, i.e., biomass, can be output.

[0024] Figure 2 A flowchart illustrating a method for calculating the initial copy number of eDNA in a target fish species is shown, including the following steps: S202: Introduced and processed using qPCR microfluidic chips for real-time fluorescence quantitative amplification and detection of elution buffer containing dissolved eDNA, and calculated the initial copy number of eDNA in the target fish. S204: During temperature control, the initial copy number of eDNA in the target fish is calculated by combining a qPCR microfluidic chip and a fluorescence optical detection module.

[0025] Furthermore, in a preferred embodiment of the present invention, the process of introducing and performing real-time fluorescence quantitative amplification and detection of the elution buffer containing dissolved eDNA using a qPCR microfluidic chip, and calculating the initial copy number of eDNA in the target fish, specifically involves: A qPCR microfluidic chip and a fluorescence optical detection module are introduced, and the chip information of the qPCR microfluidic chip is pre-set. The chip information is pre-set by inputting the target fish species and the actual number of the elution buffer for dissolving eDNA into the qPCR microfluidic chip. The reaction equipment for dissolving eDNA elution buffer was determined, and lyophilized qPCR reaction reagents were introduced. A qPCR microfluidic chip and a fluorescence optical detection module were integrated into the reaction equipment for dissolving eDNA elution buffer. At the same time, the elution buffer for dissolving eDNA elution buffer and lyophilized qPCR reaction reagents were introduced and mixed to construct the PCR reaction system to be amplified, and labeled as the target reaction system. The mixture in the target reaction system is in contact with the qPCR microfluidic chip and the fluorescence optical detection module. The fluorescence optical detection module is correlated with the qPCR microfluidic chip, that is, the qPCR microfluidic chip calculates the initial copy number of eDNA of the target fish by analyzing the fluorescence data output by the fluorescence optical detection module. By introducing a big data network, the standard temperature for qPCR amplification of the target fish's eDNA is retrieved and calibrated as the standard amplification temperature. In the reaction equipment containing the eluent of dissolved eDNA, the temperature of the target reaction system is controlled to reach the standard amplification temperature within a predetermined time. During temperature control, the initial copy number of eDNA in the target fish was calculated by combining a qPCR microfluidic chip and a fluorescence optical detection module.

[0026] It's important to note that traditional eDNA quantification requires a specialized PCR laboratory and dozens of manual steps performed by trained operators. In contrast, qPCR microfluidic chips highly integrate all these steps onto a postage stamp-sized chip, automating the process and directly outputting digital results. Therefore, real-time quantitative amplification (qPCR) detection is performed using qPCR microfluidic chips. This qPCR process constructs a curve based on fluorescence waveforms to analyze copy numbers. The qPCR microfluidic chip contacts the reaction system liquid, controlling the fluorescence data output from the fluorescence optical detection module to calculate the initial eDNA copy number of the target fish. Amplification is accelerated by increasing the temperature; the entire process is conducted in a closed system, eliminating aerosol contamination, and the temperature control rate is extremely fast.

[0027] Furthermore, in a preferred embodiment of the present invention, the calculation of the initial copy number of eDNA of the target fish during temperature control, in conjunction with the qPCR microfluidic chip and the fluorescence optical detection module, specifically involves: During temperature control, the fluorescence data status output by the fluorescence optical detection module is collected in real time through the qPCR microfluidic chip, wherein the fluorescence data status output by the fluorescence optical detection module is the wavelength value of the fluorescence signal. In the qPCR microfluidic chip, a standard curve of the target fish's eDNA is preset, and the wavelength value of the fluorescence signal is analyzed in real time by the qPCR microfluidic chip based on the fluorescence data output by the acquired fluorescence optical detection module to obtain the real-time amplification curve of the target fish's eDNA. During temperature control, the qPCR microfluidic chip executes a temperature cycling program in the reaction device containing the eluent of dissolved eDNA. The temperature cycling program consists of one temperature control cycle, a preset fluorescence baseline threshold, and analysis of the real-time amplification curve of the target fish's eDNA. When the real-time amplification curve of the target fish's eDNA reaches the fluorescence baseline threshold, the temperature control process is stopped, and the number of cycles is recorded and calibrated as the real-time Ct value. The real-time Ct value is imported into the standard curve of the target fish's eDNA for regression calculation, and the initial copy number of the target fish's eDNA is output.

[0028] It should be noted that the temperature control process involves cyclically raising and lowering the temperature. One cycle consists of one temperature increase and decrease. During these continuous temperature increases and decreases, the elution buffer dissolving the eDNA undergoes a chemical reaction, resulting in qPCR amplification of the eDNA. The cycle is stopped by analyzing the fluorescence data output from the fluorescence optical detection module to determine whether the curve reaches a threshold. When the curve reaches the threshold, the reaction ends, the cycle concludes, and the cycle number is recorded (i.e., the real-time Ct value). This value is then imported into the target fish's eDNA standard curve for regression calculation, outputting the initial eDNA copy number of the target fish. The real-time biomass of the fish is then calculated based on this initial eDNA copy number.

[0029] like Figure 3 As shown, the second aspect of the present invention also provides a rare fish concentration monitoring system for recirculating aquaculture systems based on eDNA quantitative detection. This system integrates a high-performance computing architecture and a data storage module, including a non-volatile memory consisting of a DDR4 RDIMM memory module with ECC verification and an NVMe solid-state storage array using 3D NAND flash memory, and a multi-core processor based on the Zen4 microarchitecture. The memory contains a program for monitoring the concentration of rare fish in a recirculating aquaculture systems, which has a concentration monitoring engine. When the program is executed in parallel through a superscalar pipeline execution unit within the processor, the following steps are implemented: Integrated water sampling was conducted in a closed recirculating aquaculture system, and the sampled water samples were then enriched in situ. The filter membrane obtained after in-situ enrichment was subjected to automated eDNA extraction and purification. We introduced and used a qPCR microfluidic chip to perform real-time fluorescence quantitative amplification and detection of the elution buffer containing dissolved eDNA, and calculated the initial copy number of eDNA in the target fish. Based on the initial copy number of the target fish's eDNA, the concentration of the target fish in the target recirculating aquaculture system is calculated.

[0030] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for monitoring the concentration of rare fish species in recirculating aquaculture systems based on eDNA quantitative detection, characterized in that, Includes the following steps: Integrated water sampling was conducted in a closed recirculating aquaculture system, and the sampled water samples were then enriched in situ. The filter membrane obtained after in-situ enrichment was subjected to automated eDNA extraction and purification. We introduced and used a qPCR microfluidic chip to perform real-time fluorescence quantitative amplification and detection of the elution buffer containing dissolved eDNA, and calculated the initial copy number of eDNA in the target fish. Based on the initial copy number of the target fish's eDNA, the concentration of the target fish in the target recirculating aquaculture system is calculated.

2. The method for monitoring the concentration of rare fish species in a recirculating aquaculture system based on eDNA quantitative detection as described in claim 1, characterized in that, The integrated sampling of water samples in the closed recirculating aquaculture system, followed by in-situ enrichment treatment of the sampled water samples, specifically involves: The recirculating aquaculture system for which the concentration of rare fish species needs to be monitored is identified as the target recirculating aquaculture system. Water sampling equipment is installed in the main pipeline of the target recirculating aquaculture system, and the rare fish species for which the concentration monitoring needs to be monitored are identified as the target fish species. The water sampling device uses a three-way solenoid valve to divide the main pipeline into a main path and a sampling branch path. The water flow in the main path is diverted to the sampling branch path within the main pipeline. At the same time, the sampling branch path is cleaned by the cleaning fluid solenoid valve of the water sampling device to remove nucleic acid. Finally, a fixed volume of water sample is extracted from the sampling branch path by the quantitative pump of the water sampling device, which is then labeled as the target water sample, and the collection concentration of the target water sample is determined. An eDNA enrichment unit is obtained, and the target water sample is delivered into the eDNA enrichment unit by a quantitative pump. During the delivery process, the water sample status of the target water sample is detected and recorded in real time, wherein the water sample status of the target water sample includes temperature, pH value and conductivity. Within the eDNA enrichment unit, the target water sample is enriched using a filter membrane. Filter membrane enrichment involves enriching particles larger than the pore size of the filter membrane in the target water sample. The filter membrane after enrichment is then cleaned using the eDNA enrichment unit. The cleaning process removes residual PCR inhibitors from the aluminum membrane. The cleaned filter membrane was stored at low temperature, and the sampling state of the target water sample during in-situ enrichment was preserved.

3. The method for monitoring the concentration of rare fish species in a recirculating aquaculture system based on eDNA quantitative detection as described in claim 1, characterized in that, The automated eDNA extraction and purification process for the filter membrane obtained after in-situ enrichment treatment is specifically as follows: The filter membrane that has been stored at low temperature and cleaned is labeled as the target filter membrane, and the target filter membrane is placed on the sample processing platform of the nucleic acid extractor; Lysis buffer was added to the target filter membrane on the sample processing platform of the nucleic acid extractor, and the processing temperature of the sample processing platform of the nucleic acid extractor was controlled to be equal to the preset temperature. The preset temperature of the target filter membrane during the lysis process was determined according to the target fish. Before adding the lysis buffer, it is necessary to determine the concentration of the lysis buffer to be added. A big data network is introduced to determine the concentration of the lysis buffer to be added corresponding to the collection concentration of the target water sample. After adding lysis buffer, the sample processing platform of the nucleic acid extractor is subjected to vortex oscillation. After a predetermined vortex oscillation time, magnetic bead suspension is added to the sample processing platform of the nucleic acid extractor. The magnetic bead suspension is a binding solution that adsorbs all eDNA in the target filter membrane. After adding the magnetic bead suspension, the binding solution of the target filter membrane is obtained. Before adding the magnetic bead suspension, it is necessary to determine the magnetic bead suspension concentration corresponding to the lysis solution addition concentration in the big data network. A negative pressure pump and a silica membrane adsorption column closed reaction tube are introduced. The binding solution of the target filter membrane is introduced into the silica membrane adsorption column closed reaction tube, and the binding solution of the target filter membrane is subjected to negative pressure filtration treatment by the negative pressure pump. The negative pressure filtration treatment yields waste liquid and a silica membrane adsorption column closed reaction tube adsorbed with eDNA, which is labeled as the target reaction tube. The target reaction tube was subjected to negative pressure evacuation and drying to obtain a silica membrane adsorption column containing only eDNA. The silica membrane adsorption column containing only eDNA was then dried, and elution buffer was added to obtain an elution buffer containing dissolved eDNA. Finally, the elution buffer containing dissolved eDNA was temporarily stored at low temperature. Before adding the eluent, the standard concentration of the eluent is determined through a big data network.

4. The method for monitoring the concentration of rare fish species in a recirculating aquaculture system based on eDNA quantitative detection as described in claim 1, characterized in that, The process involves introducing and using a qPCR microfluidic chip to perform real-time quantitative amplification and detection of the elution buffer containing dissolved eDNA, and calculating the initial copy number of eDNA in the target fish. Specifically: A qPCR microfluidic chip and a fluorescence optical detection module are introduced, and the chip information of the qPCR microfluidic chip is pre-set. The chip information is pre-set by inputting the target fish species and the actual number of the elution buffer for dissolving eDNA into the qPCR microfluidic chip. The reaction equipment for dissolving eDNA elution buffer was determined, and lyophilized qPCR reaction reagents were introduced. A qPCR microfluidic chip and a fluorescence optical detection module were integrated into the reaction equipment for dissolving eDNA elution buffer. At the same time, the elution buffer for dissolving eDNA elution buffer and lyophilized qPCR reaction reagents were introduced and mixed to construct the PCR reaction system to be amplified, and labeled as the target reaction system. The mixture in the target reaction system is in contact with the qPCR microfluidic chip and the fluorescence optical detection module. The fluorescence optical detection module is correlated with the qPCR microfluidic chip, that is, the qPCR microfluidic chip calculates the initial copy number of eDNA of the target fish by analyzing the fluorescence data output by the fluorescence optical detection module. By introducing a big data network, the standard temperature for qPCR amplification of the target fish's eDNA is retrieved and calibrated as the standard amplification temperature. In the reaction equipment containing the eluent of dissolved eDNA, the temperature of the target reaction system is controlled to reach the standard amplification temperature within a predetermined time. During temperature control, the initial copy number of eDNA in the target fish was calculated by combining a qPCR microfluidic chip and a fluorescence optical detection module.

5. The method for monitoring the concentration of rare fish species in a recirculating aquaculture system based on eDNA quantitative detection as described in claim 4, characterized in that, During temperature control, the initial copy number of eDNA in the target fish is calculated using a qPCR microfluidic chip and a fluorescence optical detection module, specifically as follows: During temperature control, the fluorescence data status output by the fluorescence optical detection module is collected in real time through the qPCR microfluidic chip, wherein the fluorescence data status output by the fluorescence optical detection module is the wavelength value of the fluorescence signal. In the qPCR microfluidic chip, a standard curve of the target fish's eDNA is preset, and the wavelength value of the fluorescence signal is analyzed in real time by the qPCR microfluidic chip based on the fluorescence data output by the acquired fluorescence optical detection module to obtain the real-time amplification curve of the target fish's eDNA. During temperature control, the qPCR microfluidic chip executes a temperature cycling program in the reaction device containing the eluent of dissolved eDNA. The temperature cycling program consists of one temperature control cycle, a preset fluorescence baseline threshold, and analysis of the real-time amplification curve of the target fish's eDNA. When the real-time amplification curve of the target fish's eDNA reaches the fluorescence baseline threshold, the temperature control process is stopped, and the number of cycles is recorded and calibrated as the real-time Ct value. The real-time Ct value is imported into the standard curve of the target fish's eDNA for regression calculation, and the initial copy number of the target fish's eDNA is output.

6. The method for monitoring the concentration of rare fish species in a recirculating aquaculture system based on eDNA quantitative detection as described in claim 1, characterized in that, The concentration of the target fish in the target recirculating aquaculture system is calculated by combining the initial copy number of the target fish's eDNA. Specifically: The concentration conversion formula was retrieved through big data network, and a biomass-eDNA concentration correction model was constructed based on the concentration conversion formula. Input the target water sample status, target water sample concentration, lysis buffer concentration, magnetic bead suspension concentration, elution buffer standard concentration, and initial eDNA copy number of the target fish into the biomass-eDNA concentration correction model. Run the biomass-eDNA concentration correction model to output the real-time concentration of the target fish species within the target recirculating aquaculture system.

7. A system for monitoring the concentration of rare fish species in recirculating aquaculture systems based on eDNA quantitative detection, characterized in that, The recirculating aquaculture system rare fish concentration monitoring system integrates a high-performance computing architecture and a data storage module, including a non-volatile memory consisting of a DDR4 RDIMM memory module with ECC verification and an NVMe solid-state storage array using 3D NAND flash memory, and a multi-core processor based on the Zen4 microarchitecture; the memory contains a recirculating aquaculture system rare fish concentration monitoring method program with a concentration monitoring engine, and when the program is executed in parallel through the superscalar pipeline execution unit in the processor, it realizes the rare fish concentration monitoring steps of the recirculating aquaculture system as described in any one of claims 1-6.

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