Macrobrachium rosenbergii spf scale-up breeding method and breeding system

Abstract

The present application relates to the technical field of aquaculture and seed breeding, and particularly relates to a method and system for SPF scale breeding of Macrobrachium rosenbergii. The method sets online continuous enrichment units at the entry disinfection pool of the breeding field, the source water main pipe, the water distribution pipe of the breeding workshop, and the batch water pipe of the breeding pond, etc. The method continuously enriches nucleic acid in the water environment, and injects DNA / RNA synchronous preservation liquid before the filter membrane is exposed. The upper and lower filter membranes are detected for environmental DNA / environmental RNA, respectively. According to the positive combination of upstream and downstream nodes, the results of continuous monitoring time periods, and the filter membrane hierarchical distribution, the path of pathogen input and the risk level are determined. According to the determination results, the source water switching, water inflow blocking, disinfection strengthening, fry isolation, removal and batch release are linked, and the semi-saline water breeding time sequence is embedded, so that the stable breeding of SPF fry of Macrobrachium rosenbergii is realized.

Description

Technical Field

[0001] This invention relates to the field of aquatic breeding and seedling propagation technology, and particularly to a method and system for large-scale SPF (Special Purity Flood Freshwater Prawn) propagation. It is applicable to the continuous enrichment of specific pathogens in the water used for seedling production during the processes of introducing broodstock, releasing larvae, larval rearing, acclimatization, and seedling release of Macrobrachium rosenbergii. This includes environmental DNA / RNA detection, risk classification, influent water control, and batch release management of seedlings. Background Technology

[0002] The giant freshwater prawn (Macrobrachium rosenbergii) is one of my country's important freshwater economic shrimp species. Its seedling production process typically includes broodstock selection, overwintering and intensive rearing of broodstock, mating and egg-carrying, larval release, larval rearing, freshwater acclimatization, and hatching. Because giant freshwater prawn larvae and postlarvae are highly sensitive to changes in the aquatic environment and pathogen invasion, under high-density seedling production conditions, once pathogens enter the seedling system through broodstock, source water, water used in the seedling production workshop, operating tools, feed organisms, or during the transfer of production materials or seedlings, it can lead to decreased larval survival rates, unstable batch hatching, or even the complete culling of entire batches. Therefore, how to detect and promptly treat specific pathogens during large-scale seedling production, and establish a traceable seedling release mechanism, is a crucial issue that urgently needs to be addressed in the SPF (Special Priority Farming) seedling propagation of giant freshwater prawns.

[0003] Existing SPF or virus-free seedling breeding technologies for giant freshwater prawns mainly focus on broodstock selection, enhanced broodstock rearing, water pretreatment, pond and equipment disinfection, regular virus testing, and disease prevention. For example, Chinese patent document CN106922583A discloses a method for breeding SPF virus-free seedlings of giant freshwater prawns, which involves steps such as seedling selection, temporary rearing of prawns, pond culture, disinfection and isolation measures during prawn rearing, broodstock selection, overwintering pond pretreatment, environmental condition control, feed feeding, regular virus testing of broodstocks, water pretreatment, and disease prevention to prevent muscle turbidity disease in giant freshwater prawns and improve seedling survival rate. The technical solution emphasizes disinfection, isolation, and regular testing during the breeding and management of broodstock and seedlings. However, its testing methods mainly focus on broodstock, seedlings, or phased production management. It does not establish a continuous monitoring system for environmental input checkpoints such as inlet disinfection pools, main source water pipes, distribution water pipes in seedling workshops, and batch water pipes in seedling ponds. It also does not publicly utilize environmental nucleic acid test results to coordinate the control of source water switching, water inlet blocking, seedling isolation, and batch release.

[0004] For example, Chinese patent document CN107295990A discloses a method for breeding virus-free SPF (Special Pressure Flood) seedlings of Macrobrachium rosenbergii, which includes the selection of virus-free broodstock, enhanced overwintering cultivation of broodstock, and breeding of virus-free seedlings. It proposes measures such as regular virus testing of broodstock, water pretreatment, disinfection of nursery ponds and tools, increased water exchange volume and frequency, and regular virus testing of larvae, especially those after desalination. This method can reduce the pathogen risk in broodstock and during the seedling process to some extent, but its focus remains on broodstock nutritional enhancement, routine water treatment, artificial management, and periodic virus testing. It does not address the continuous detection of low-load pathogen input from environmental pathways, short-term pulse input, or cross-contamination of water in workshop distribution. Furthermore, it does not involve technical solutions for parallel eDNA / eRNA detection, RNA activity risk assessment, double-layer filter membrane stratified detection, or determining pathogen input pathways based on positive combinations of upstream and downstream water flow nodes.

[0005] Therefore, although the existing SPF breeding method for giant freshwater prawns has proposed measures such as virus-free screening of broodstock, disinfection of pond equipment, water pretreatment, and regular virus testing, it still has the following shortcomings: First, the testing targets are mostly concentrated on broodstock, larvae, or postlarvae, and the pathogen monitoring point is relatively lagging, making it difficult to detect pathogen risks at environmental input points such as source water, workshop distribution water, and batch water in a timely manner; Second, existing testing is mostly staged sampling or manual sampling, with limited sampling volume and time coverage, which easily leads to missed detection of pathogens with low load, intermittent or pulsed input; Third, a simple positive nucleic acid result is difficult to distinguish between residual environmental nucleic acid and recent active input risk, which can easily lead to insufficient treatment or accidental production stoppage; Fourth, the test results are usually reported for manual judgment and are not linked to source water switching, water inlet blocking, enhanced disinfection, seedling isolation, seedling removal, and batch release in an automatic or semi-automatic manner. Summary of the Invention

[0006] The technical objective of this invention is to provide a method and system for large-scale breeding of Giant freshwater prawns (SPF). By setting up an online continuous enrichment and detection linkage mechanism for environmental nucleic acids at key water flow checkpoints such as the entrance to the hatchery, source water, workshop distribution water, and batch water use in the hatchery ponds, the method combines environmental DNA / RNA detection results with pathogen input path identification, risk classification, biosafety disposal, and batch release of seedlings. This solves the problems in existing Giant freshwater prawn SPF breeding, such as delayed detection of environmental pathogen input, insufficient sampling and testing coverage, easy misjudgment due to residual nucleic acids, and difficulty in directly guiding production and disposal with monitoring results.

[0007] Firstly, in order to achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] A method for large-scale breeding of giant freshwater prawns (SPF), comprising the following steps within one seedling production cycle:

[0009] S1. According to the water flow entry path and seedling transfer path of the seedling nursery, set up online continuous enrichment units at at least three types of checkpoints in the inlet disinfection pool, the main source water pipe, the distribution water pipe of the seedling workshop and the batch water pipe of the seedling pool. Filter the water at each node continuously according to the preset monitoring period and record the cumulative filtered water volume.

[0010] S2. After the enrichment membrane is isolated from the water flow and before the membrane is exposed, inject DNA / RNA synchronous preservation solution into the enrichment membrane and number and preserve the upper large-pore membrane and the lower small-pore membrane respectively.

[0011] S3. Environmental DNA and environmental RNA were detected in the upper large-pore filter membrane and the lower small-pore filter membrane at the same monitoring time and the same checkpoint node, and the detected amounts were normalized.

[0012] S4. Based on the positive combinations of the same pathogen at upstream and downstream checkpoints, the positive results of environmental RNA during continuous monitoring periods, and the detection distribution of the upper and lower filter membranes, determine the pathogen input pathway and activity risk level.

[0013] S5. When the preset risk level is reached, the corresponding water intake shall be blocked or the backup water source shall be switched, and the associated batch of seedlings shall be isolated or removed; when the environmental nucleic acid test results are continuously met, the associated batch shall be allowed to enter the next seedling stage.

[0014] S6. Insert S1 to S5 into a parent stock with a salinity of 12‰ to 14‰, and follow the sequence of stocking, releasing, larval rearing, acclimatization, and hatching to obtain giant freshwater prawn larvae that meet the release conditions of negative nucleic acid in a specific pathogen environment.

[0015] As a further improvement, in step S1, the checkpoint nodes are divided into external source input checkpoints, source water input checkpoints, workshop distribution checkpoints, and batch water use checkpoints according to the water flow topology. The external source input checkpoints are set at the entrance disinfection pool or personnel and vehicle disinfection channel. The source water input checkpoints are set at the water intake, the main pipe after source water treatment, or the backup source water pipe. The workshop distribution checkpoints are set at the main water inlet pipe or distribution branch pipe of the seedling workshop. The batch water use checkpoints are set at the water inlet pipes of the seedling pond, the larval discharge pond, the desalination pond, or the parent stock temporary rearing pond.

[0016] As a further improvement, in step S1, the online continuous enrichment unit includes a pre-filter, a flow meter, a metering pump or peristaltic pump, an enrichment filter membrane assembly, and a bypass parallel valve group; the bypass parallel valve group includes at least two parallel enrichment branches, so that when one enrichment branch is undergoing filter membrane replacement, the other enrichment branch remains in a continuous enrichment state.

[0017] As a further improvement, in step S2, the upper large-pore filter membrane is used to retain cells, tissue debris, fecal particles, and large-particle nucleic acid carriers, while the lower small-pore filter membrane is used to retain virus particles, free nucleic acids, and small-particle nucleic acid carriers; the upper large-pore filter membrane and the lower small-pore filter membrane are used to preserve, extract nucleic acids, or generate detection data, respectively.

[0018] Preferably, the upper large-pore filter membrane is a glass fiber membrane with a pore size of 1.0 μm to 3.0 μm, and the lower small-pore filter membrane is a mixed cellulose ester membrane, polyethersulfone membrane, or polyvinylidene fluoride membrane with a pore size of 0.22 μm to 0.80 μm; the cumulative filtration water volume for each preset monitoring period is 50 L to 1000 L.

[0019] As a further improvement, in step S2, the DNA / RNA synchronous preservation solution is added by a preservation solution injection module located in the filter membrane quick-change chamber; the filter membrane quick-change chamber remains sealed after the inlet valve and outlet valve are closed, and the preservation solution injection is completed before the chamber is opened, so as to reduce the degradation of environmental RNA during the membrane removal process.

[0020] As a further improvement, in step S3, the environmental DNA detection uses qPCR or digital PCR, and the environmental RNA detection uses RT-qPCR or RT digital PCR; the normalization of the detection amount is determined based on the cumulative filtered water volume, blank control, historical negative baseline of the current site, spiked recovery rate, and detection limit of the detection method.

[0021] As a further improvement, in step S4, when the same pathogen only shows positive environmental DNA and negative environmental RNA at the upstream checkpoint node, it is determined to be a level 1 residual nucleic acid risk; when the same pathogen shows positive environmental RNA for two consecutive preset monitoring periods at the same checkpoint node, or shows positive environmental RNA successively at the upstream and downstream checkpoint nodes along the water flow direction, it is determined to be a level 2 active input risk.

[0022] Preferably, for the primary risk of residual nucleic acid, measures such as enhanced disinfection, extended disinfection contact time, increased retesting frequency, increased water change frequency, or suspension of seedling transfer should be implemented; for the secondary risk of active input, measures such as blocking the corresponding water intake, switching to backup water source, isolating related batches of seedlings, removing related batches of seedlings, disinfecting the related ponds, or initiating upstream pollution tracing should be implemented.

[0023] As a further improvement, in step S5, the environmental nucleic acid negative release condition is as follows: within at least two consecutive preset monitoring periods corresponding to the current seedling node, the inlet disinfection pool, the main source water pipe, the seedling workshop distribution water pipe, and the corresponding batch water pipe of the seedling pool have not reached the level of primary residual nucleic acid risk or secondary active input risk; after the environmental nucleic acid negative release condition is met, a release result bound to the seedling identification of that batch is generated.

[0024] As a further improvement, in step S6, the sequence of parent stock entry into the pool, juvenile discharge, juvenile release, juvenile cultivation, desalination, and seedling emergence is respectively bound to the preset monitoring period of the corresponding checkpoint node; if any seedling node fails to meet the environmental nucleic acid negative release condition, the transfer of that batch of seedlings to the next seedling node is stopped.

[0025] As a further improvement, in step S6, the specific pathogens include at least three of the following: giant freshwater prawn nodavirus, giant freshwater prawn Taihu virus, giant freshwater prawn flavivirus, decapod iridovirus 1, and hepatocellular carcinoma; for RNA viruses, the positive result of environmental RNA is used as the basis for determining the activity risk; for DNA viruses or eukaryotic parasites, the positive result of environmental DNA combined with the positive result of its transcript is used as the basis for determining the activity risk.

[0026] Secondly, the present invention also provides a breeding system for implementing the method, comprising a checkpoint node deployment unit, an online continuous enrichment unit, a filter membrane quick-change and storage unit, a detection data access unit, and a data-threshold-response linkage control unit; the checkpoint node deployment unit is set at least three types of checkpoint nodes in the inlet disinfection pool, the main source water pipe, the distribution water pipe of the seedling workshop, and the batch water pipe of the seedling pool according to the water flow entry path of the seedling farm and the seedling transfer path; the data-threshold-response linkage control unit is communicatively connected to the source water switching valve, the seedling workshop inlet valve, the disinfection dosing device, and the seedling batch release identification device.

[0027] As a further improvement, the online continuous enrichment unit includes a pre-filter, a flow meter, a metering pump or a peristaltic pump, at least two parallel enrichment branches and an enrichment filter membrane assembly; each parallel enrichment branch is equipped with an inlet control valve, an outlet control valve and a filter membrane quick-change compartment, so as to complete the filter membrane replacement without interrupting the continuous enrichment of the corresponding gate node.

[0028] Preferably, the enrichment filter membrane assembly includes an upper large-pore filter membrane and a lower small-pore filter membrane arranged sequentially along the water flow direction; the filter membrane quick-change chamber is provided with a filter membrane clamping structure for fixing the upper large-pore filter membrane and the lower small-pore filter membrane respectively, and a sample numbering structure for generating filter membrane numbers respectively;

[0029] Preferably, the filter membrane quick-change and preservation unit includes a chamber sealing detection module, a preservation solution injection module, and a low-temperature temporary storage module; the preservation solution injection module is configured to inject DNA / RNA synchronous preservation solution into the enrichment filter membrane assembly after the inlet control valve and outlet control valve of the filter membrane quick-change chamber are closed and before the filter membrane quick-change chamber is opened.

[0030] As a further improvement, the detection data access unit is used to receive environmental DNA detection data and environmental RNA detection data corresponding to the same monitoring period, the same checkpoint node, the upper large-pore filter membrane and the lower small-pore filter membrane respectively, and to associate and store the detection data with the cumulative filtered water volume, blank control, historical negative baseline of the current field and spiked recovery rate.

[0031] As a further improvement, the data-threshold-response linkage control unit includes a filtered water volume normalization module, an upstream and downstream positive combination judgment module, a continuous time period positive judgment module, a filter membrane stratification distribution judgment module, a risk level judgment module, and a production and disposal output module; the production and disposal output module is used to output at least one control result among source water switching, inlet water blocking, disinfection dosing, re-inspection, seedling isolation, seedling removal, and seedling release.

[0032] As a further improvement, the seedling batch release identification device is used to bind the seedling batch number, the current seedling node, the monitoring result of the corresponding checkpoint node, the risk level, and the release result; when the data-threshold-response linkage control unit determines that the environmental nucleic acid negative release condition is not met, the seedling batch release identification device generates an identification that prohibits transfer to the next seedling node.

[0033] As a further improvement, the data-threshold-response linkage control unit is also communicatively connected to the tailwater discharge valve and the tailwater disinfection device; when the tailwater node reaches the secondary active input risk, it controls the tailwater discharge valve to close or controls the tailwater disinfection device to increase the disinfection intensity.

[0034] This invention has the following technical effects: By setting up multi-level checkpoint nodes according to the water flow topology of the hatchery, pathogen risk monitoring can be shifted from single shrimp sampling to environmental input links such as the inlet disinfection pool, the main source water pipe, the distribution water pipe in the hatchery workshop, and the batch water pipe in the hatchery pond, thereby increasing the probability of detecting low-load, intermittent, or pulsed pathogen input; by replacing single manual bottle sampling with online continuous enrichment, the effective sampling volume per unit monitoring period can be significantly increased, reducing the risk of missed detection due to sampling time deviation; by injecting DNA / RNA synchronous preservation solution after the filter membrane is separated from the water flow and before the filter membrane is exposed, the degradation of environmental RNA during membrane removal can be reduced, improving the reliability of RNA pathogen and transcript detection results; by numbering and detecting the upper large-pore filter membrane and the lower small-pore filter membrane separately, the distribution differences of nucleic acid carriers of different particle sizes can be combined to assist in judging the source and transmission mode of pathogens; by combining environmental DNA and environmental RNA... Parallel detection can identify recent or active pathogen risks while detecting residual environmental nucleic acids, reducing the risk of erroneous production stoppages or removals due to environmental DNA positivity alone. By combining positive results from upstream and downstream checkpoints, environmental RNA positivity during continuous monitoring periods, and filter membrane stratification results for risk classification, it is possible to locate pathogen input pathways and make precise judgments on risk levels. By linking risk levels with source water switching, influent blockage, enhanced disinfection, seedling isolation, seedling removal, and batch release, environmental nucleic acid monitoring results can directly affect the seedling production control process, reducing the chance of pathogen spread within the seedling system. By embedding the above monitoring and treatment mechanisms into the timing of broodstock introduction, larval discharge, larval rearing, larval desalination, and seedling release at a salinity of 12‰–14‰, a biosafety closed loop covering the entire process of giant freshwater prawn seedling production can be formed, improving the stability, traceability, and batch consistency of large-scale SPF shrimp seedling production. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the process for the large-scale SPF breeding method of giant freshwater prawns of the present invention.

[0036] Figure 2 This is a schematic diagram of the online enrichment and detection linkage system for SPF breeding farms of giant freshwater prawns according to the present invention.

[0037] Figure 3 This is a schematic diagram showing the correspondence between the checkpoint nodes of this invention and the semi-brackish water seedling sequence of giant freshwater prawns.

[0038] Figure 4 This is a schematic diagram of the online continuous enrichment unit and the double-layer enrichment filter membrane assembly of the present invention.

[0039] Figure 5 This is a schematic diagram illustrating the linkage logic between risk level determination and production disposal in this invention.

[0040] Figure 6 This is a comparison chart of the detection rates of low-load pathogen nucleic acid using different sampling methods.

[0041] Figure 7 A comparative graph showing the impact of different preservation methods on the recovery rate of environmental RNA.

[0042] Explanation of reference numerals in the attached diagram: 111. Inlet disinfection tank; 112. Main source water pipe; 113. Distribution water pipe for seedling workshop; 114. Batch water pipe for seedling pond; 115. Tailwater outlet; 200. Online continuous enrichment unit; 210. Pre-filter; 220. Flow meter; 230. Peristaltic pump; 240. Bypass parallel valve assembly; 241. First enrichment branch; 242. Second enrichment branch; 250. Filter membrane quick-change chamber; 251. Inlet control valve; 252. Outlet control valve; 253. Chamber body ; 260, Double-layer enrichment filter membrane assembly; 261, Upper layer large-pore filter membrane; 262, Lower layer small-pore filter membrane; 270, Preservative injection module; 280, Low-temperature temporary storage module; 300, Detection data access unit; 400, Data-threshold-response linkage control unit; 510, Source water switching valve; 520, Seedling workshop inlet valve; 530, Disinfection dosing device; 540, Seedling batch release identification device; 550, Tailwater discharge valve; 560, Tailwater disinfection device. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the following specific embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Without departing from the concept of the present invention, those skilled in the art can make adaptive adjustments to the number of checkpoints, monitoring periods, filtration water volume, filter membrane material, pathogen combination for detection, risk threshold, and production and disposal methods according to the scale of the hatchery, source water conditions, giant freshwater prawn species, pathogen prevalence, detection platform configuration, and production management system.

[0044] The "large-scale SPF breeding of giant freshwater prawns" as referred to in this invention refers to a large-scale breeding process that establishes continuous environmental nucleic acid monitoring, risk-based grading, and batch release conditions for specific pathogen combinations at various stages of the giant freshwater prawn breeding and production process, including stocking broodstock, intensive cultivation, mating and brooding, larval release, larval rearing, freshwater acclimatization, and hatching. This ensures that each hatching batch meets the release condition of negative environmental nucleic acid for the specific pathogen. Here, "SPF prawn larvae" does not mean that the larvae are absolutely free of all pathogens under any conditions, but rather that, under the checkpoints, detection targets, detection methods, monitoring periods, and release rules set by this invention, the relevant breeding batches meet the release condition of negative environmental nucleic acid for the specific pathogen.

[0045] The term "environmental nucleic acid" in this invention includes environmental DNA and environmental RNA present in the inlet disinfection tank, source water, distribution water in the seedling workshop, batch water in the seedling pond, and tailings water. Environmental DNA is abbreviated as eDNA, and environmental RNA is abbreviated as eRNA. eDNA is mainly used to indicate the presence of target pathogen nucleic acid, while eRNA or target pathogen transcripts are used to assist in judging the risk of recent input or activity. Because environmental nucleic acid detection results can be affected by factors such as cumulative filtration water volume, nucleic acid recovery rate, detection limit of the detection method, nucleic acid storage conditions, and water body inhibitors, this invention does not directly equate a single positive environmental nucleic acid result with a conclusion of shrimp infection, but rather uses it as a basis for biosafety control of the seedling production environment.

[0046] The "DNA / RNA synchronous preservation solution" referred to in this invention is a nucleic acid preservation solution containing nuclease inhibitors, chelating agents and denaturing protective components, or a commercially available DNA / RNA Shield or RNAlater synchronous preservation solution; the amount added is 1.0-5.0 times the liquid holding capacity of the filter membrane covering the effective filtration surface of the double-layer filter membrane assembly.

[0047] The "checkpoint node" referred to in this invention refers to a monitoring node set up according to the water flow entry path of the hatchery, the distribution path of the workshop, and the seedling transfer path. The checkpoint node includes an inlet disinfection pool 111, a main source water pipe 112, a distribution water pipe for the hatchery workshop 113, and a batch water pipe for the hatchery pond 114. Depending on the actual situation of the hatchery, additional nodes may be added, such as an exhaust water node 115, a broodstock holding pond inlet node, a larvae discharge pond inlet node, a desalination pond inlet node, or a backup source water node. The technical significance of the checkpoint node lies in: shifting the traditional staged monitoring of broodstock or shrimp larvae to the environmental input end and the batch water supply end; identifying pathogen input paths through water flow topology; and directly using the monitoring results for water inlet blocking, source water switching, enhanced disinfection, seedling isolation, seedling removal, and batch release.

[0048] The "online continuous enrichment" referred to in this invention refers to the continuous passage of the water body to be monitored through an enrichment filter membrane assembly during a preset monitoring period, thereby trapping cells, tissue debris, fecal particles, viral particles, free nucleic acids, and small nucleic acid carriers in a larger volume of water on the filter membrane. Here, "online" primarily means that the enrichment sampling process is connected to the water system of the seedling nursery and operates continuously according to the monitoring period. Subsequent qPCR, digital PCR, RT-qPCR, or RT digital PCR testing can be completed in the seedling nursery's testing laboratory or sent to a qualified testing platform.

[0049] like Figure 1As shown, the SPF large-scale breeding method for giant freshwater prawns of the present invention includes S1 to S6. S1 involves setting up checkpoints according to the water flow entry path and seedling transfer path of the nursery and performing online continuous enrichment; S2 involves injecting DNA / RNA synchronous preservation solution after the enrichment filter membrane is isolated from the water flow and before the filter membrane is exposed; S3 involves detecting eDNA and eRNA on the upper large-pore filter membrane and the lower small-pore filter membrane, respectively; S4 involves determining the pathogen input path and activity risk level based on the positive combination of upstream and downstream checkpoints, the positive results of eRNA during continuous monitoring, and the detection distribution of the double-layer filter membrane; S5 involves implementing water inflow blocking, source water switching, enhanced disinfection, seedling isolation, seedling removal, or batch release according to the risk level; S6 involves embedding the above monitoring and treatment mechanisms into the semi-brackish water nursery sequence of giant freshwater prawns with a salinity of 12‰ to 14‰.

[0050] like Figure 2 As shown, the online enrichment and detection linkage system for implementing the method of the present invention includes a checkpoint node deployment unit, an online continuous enrichment unit 200, a filter membrane quick-change and storage unit, a detection data access unit 300, and a data-threshold-response linkage control unit 400. The checkpoint node deployment unit is arranged according to the water flow topology of the seedling nursery at at least three types of nodes: the inlet disinfection pool 111, the source water main pipe 112, the seedling workshop distribution water pipe 113, and the batch water pipe of the seedling pool 114. For seedling nurseries with centralized drainage or tailwater disinfection facilities, an online continuous enrichment unit 200 can also be set at the tailwater node 115 to monitor the risk of tailwater discharge.

[0051] The online continuous enrichment unit 200 includes a pre-filter 210, a flow meter 220, a peristaltic pump 230, a bypass parallel valve assembly 240, a membrane quick-change chamber 250, and a dual-layer enrichment membrane assembly 260. The pre-filter 210 removes large suspended solids, uneaten feed, algae flocs, and larger organic debris from the seedling water, preventing rapid clogging of the dual-layer enrichment membrane assembly 260. The flow meter 220 records the cumulative filtered water volume for each preset monitoring period. The peristaltic pump 230 drives the water to pass through the dual-layer enrichment membrane assembly 260 at a stable flow rate. The peristaltic pump 230 can also be replaced with a diaphragm pump, metering pump, or other pump capable of stably controlling the filtration flow rate.

[0052] like Figure 4As shown, the bypass parallel valve group 240 includes a first enrichment branch 241 and a second enrichment branch 242. Both the first enrichment branch 241 and the second enrichment branch 242 are equipped with an inlet control valve 251, an outlet control valve 252, and a filter membrane quick-change chamber 250. When the first enrichment branch 241 reaches a preset monitoring period and membrane removal is required, the second enrichment branch 242 opens and continues enrichment; when the second enrichment branch 242 needs membrane removal, the first enrichment branch 241 takes over. Through the above-mentioned bypass parallel structure, the present invention can maintain the continuous enrichment state of the corresponding key nodes during membrane removal and replacement, avoiding monitoring gaps.

[0053] A dual-layer enrichment filter membrane assembly 260 is disposed within the filter membrane quick-change chamber 250, and sequentially comprises an upper large-pore filter membrane 261 and a lower small-pore filter membrane 262 along the water flow direction. The upper large-pore filter membrane 261 is mainly used to retain cells, tissue debris, fecal particles, and large-particle nucleic acid carriers; the lower small-pore filter membrane 262 is mainly used to retain virus particles, free nucleic acids, and small-particle nucleic acid carriers. In one specific embodiment, the upper large-pore filter membrane 261 is a glass fiber membrane with a pore size of 2.0 μm, and the lower small-pore filter membrane 262 is a mixed cellulose ester membrane with a pore size of 0.45 μm. Alternatively, depending on the water turbidity and the target being detected, the upper large-pore filter membrane 261 can be set to a glass fiber membrane with a pore size of 1.0 μm to 3.0 μm, and the lower small-pore filter membrane 262 can be set to a mixed cellulose ester membrane, polyethersulfone membrane, or polyvinylidene fluoride membrane with a pore size of 0.22 μm to 0.80 μm.

[0054] The membrane quick-change and preservation unit includes a preservation solution injection module 270 and a low-temperature temporary storage module 280. The preservation solution injection module 270 is connected to the membrane quick-change chamber 250. When a certain enrichment branch reaches a preset monitoring period, the data-threshold-response linkage control unit 400 first closes the inlet control valve 251 and the outlet control valve 252 of that enrichment branch, isolating the double-layer enrichment membrane assembly 260 from the water flow. At this time, the membrane quick-change chamber 250 remains sealed, and the chamber has not yet been opened. Subsequently, the preservation solution injection module 270 injects DNA / RNA synchronous preservation solution into the membrane quick-change chamber 250, so that the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 are wetted by the preservation solution before being exposed to the outside air. After injecting the preservation solution, the membrane quick-change chamber 250 is kept for 1 to 5 minutes before being opened to remove the filter membrane. The removed upper large-pore filter membrane 261 and lower small-pore filter membrane 262 are numbered and temporarily stored in the low-temperature storage module 280. This structure can reduce eRNA degradation during membrane removal and improve the reliability of RNA virus or transcript detection results.

[0055] The detection data access unit 300 is used to receive eDNA and eRNA detection data corresponding to each checkpoint node, each preset monitoring period, and each filter membrane layer. The detection data may include the target pathogen name, Ct value, copy number, positive or negative determination, standard curve information, blank control results, positive control results, spiked recovery results, detection limit of the detection method, sample inhibition status, cumulative filtration water volume, and detection time. The detection data access unit 300 associates and stores the above data with the seedling batch number, checkpoint node number, monitoring period number, and filter membrane number.

[0056] The data-threshold-response linkage control unit 400 includes a filtered water volume normalization module, an upstream and downstream positive combination judgment module, a continuous time period positive judgment module, a filter membrane stratification distribution judgment module, a risk level judgment module, and a production and disposal output module. The filtered water volume normalization module converts the test results into the detection amount per unit volume of water. This conversion must at least consider the cumulative filtered water volume, and, where possible, further consider the spiked recovery rate, the historical negative baseline of the site, and the detection limit of the detection method. The following normalization methods can be used:

[0057] ;

[0058] in, This represents the normalized target nucleic acid detection volume; This indicates the target nucleic acid copy number obtained from the detection and conversion. Indicates the cumulative amount of water filtered; This indicates the spiked recovery rate or the method recovery correction factor. If recovery rate correction has not yet been performed in the actual testing phase, preliminary normalization can be performed based on the cumulative filtered water volume, and recovery rate correction can be supplemented after the methodology validation is completed in this field.

[0059] The upstream-downstream positive combination judgment module is used to determine whether the same pathogen exhibits a positive transmission combination from upstream to downstream along the water flow direction between the inlet disinfection pool 111, the source water main pipe 112, the seedling workshop distribution water pipe 113, and the seedling pool batch water pipe 114. The continuous time period positive judgment module is used to determine whether the same checkpoint node repeatedly shows eRNA positivity within two or more consecutive preset monitoring periods. The filter membrane layer distribution judgment module is used to determine whether the target pathogen is mainly distributed in the upper large-pore filter membrane 261 or the lower small-pore filter membrane 262. The risk level judgment module forms a level 1 residual nucleic acid risk or a level 2 active input risk based on the above judgment results. The production disposal output module is communicatively connected to the source water switching valve 510, the seedling workshop inlet valve 520, the disinfection dosing device 530, the seedling batch release marking device 540, the tailwater discharge valve 550, and the tailwater disinfection device 560, and is used to output the source water switching, inlet water blocking, enhanced disinfection, re-inspection, seedling isolation, seedling removal, or seedling release control results.

[0060] The following is combined with Figures 1 to 5 The specific implementation process of the method of the present invention will be described in detail.

[0061] In step S1, a water flow topology diagram is first drawn based on the actual waterway of the hatchery. For a conventional giant freshwater prawn hatchery, external personnel, vehicles, and production materials enter the core breeding area through the inlet disinfection pool 111; external water sources, after pretreatment, enter the main source water pipe 112; the main source water pipe 112 distributes water to each hatchery workshop, forming the hatchery workshop distribution water pipe 113; the water distributed from the hatchery workshops further enters the parent stocking pond, larvae discharge pond, hatchery pond, and desalination pond, forming the hatchery pond batch water supply pipe 114. Based on this water flow topology, online continuous enrichment units 200 are set at at least three types of checkpoint nodes in the inlet disinfection pool 111, the main source water pipe 112, the hatchery workshop distribution water pipe 113, and the hatchery pond batch water supply pipe 114. Preferably, online continuous enrichment units 200 are set at all four types of checkpoint nodes to form a continuous monitoring chain from external source input to batch water supply.

[0062] Each online continuous enrichment unit 200 operates according to a preset monitoring period. The preset monitoring period can be set to 6 hours, 8 hours, 12 hours, or 24 hours, preferably 12 hours. At the beginning of each preset monitoring period, the system records the checkpoint node number, seedling batch number, enrichment branch number, filter membrane number, start time, and initial flow rate. The peristaltic pump 230 drives the water to be monitored to pass sequentially through the pre-filter 210, flow meter 220, and double-layer enrichment filter membrane assembly 260. The cumulative filtration volume for each preset monitoring period can be 50L to 1000L; in the demonstration application in the giant freshwater prawn seedling workshop, the preferred cumulative filtration volume is 300L to 800L. For batch water in seedling ponds with high turbidity, residual feed, and organic particles, the filtration flow rate can be appropriately reduced or the monitoring period shortened; for nodes with relatively clear water quality, such as the main source water pipe 112, the filtration volume can be appropriately increased.

[0063] Step S1 transforms traditional single-stage manual bottle sampling into continuous enrichment sampling deployed at checkpoint nodes, enabling pathogen risk monitoring to cover key environmental input links such as inlet, source water, workshop distribution, and batch water use. Since pathogen input in seedling farms may manifest as low-load, short-duration, or pulsed input, online continuous enrichment can increase the effective sampling volume per unit monitoring period and reduce the risk of missed detection due to sampling time deviation.

[0064] In step S2, when a certain enrichment branch reaches a preset monitoring period, the system enters the membrane extraction and storage procedure. Taking the first enrichment branch 241 as an example, the data-threshold-response linkage control unit 400 first opens the second enrichment branch 242, allowing the second enrichment branch 242 to take over from the first enrichment branch 241 to continue online continuous enrichment. Subsequently, the system closes the inlet control valve 251 and the outlet control valve 252 of the first enrichment branch 241, isolating the double-layer enrichment filter membrane assembly 260 in the filter membrane quick-change chamber 250 from the water flow. At this time, the filter membrane quick-change chamber 250 remains sealed.

[0065] Before opening the filter membrane quick-change compartment 250, the preservation solution injection module 270 injects DNA / RNA synchronous preservation solution into the filter membrane quick-change compartment 250, ensuring that the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 are wetted with the preservation solution. The amount of preservation solution injected is sufficient to fully cover the double-layer enrichment filter membrane assembly 260. After the preservation solution is injected, the system is maintained for 1 to 5 minutes to allow the preservation solution to fully contact the nucleic acid carriers on the filter membrane surface. Subsequently, the operator opens the filter membrane quick-change compartment 250, removes the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 respectively, labels them, places them in sample preservation tubes, and places them in the low-temperature temporary storage module 280. The low-temperature temporary storage conditions can be short-term storage at 4°C, or transferred to a lower temperature for storage according to the detection cycle.

[0066] Step S2 completes the simultaneous preservation of DNA / RNA before the filter membrane is exposed to air, reducing eRNA degradation during membrane removal, transfer, and temporary storage. For target pathogens involving RNA detection, such as Macrobrachium rosenbergii nodavirus, Macrobrachium rosenbergii Taihu virus, and Macrobrachium rosenbergii flavivirus, the quality of eRNA preservation directly affects the reliability of the risk assessment for active input. This invention, through the structural cooperation of the filter membrane quick-change compartment 250 and the preservation solution injection module 270, moves the nucleic acid preservation action forward to before membrane removal. Compared with the ordinary method of adding preservation solution after membrane removal, it is more suitable for parallel detection of eDNA / eRNA.

[0067] In step S3, nucleic acid extraction is performed on the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 at the same monitoring time period and the same checkpoint node. For eDNA detection, qPCR or digital PCR can be used; for eRNA detection, RNA extraction and reverse transcription can be performed first, followed by RT-qPCR or RT digital PCR. The detection targets may include at least three of the following: Macrobrachium rosenbergii nodavirus, Macrobrachium rosenbergii Taihu virus, Macrobrachium rosenbergii flavivirus, Decapoda iridovirus 1, and Enterocytozoon hepatocellular carcinoma. For RNA viruses, a positive eRNA result is used as the basis for determining activity risk; for DNA viruses or eukaryotic parasites, a positive eDNA result combined with a positive transcript result is used as the basis for determining activity risk.

[0068] During testing, template-free controls, negative extraction controls, positive standards, and spiked recovery controls should be included. If the template-free control is positive, the batch of test results is invalid and should be retested. If the spiked recovery rate is significantly lower than the method validation range, it indicates the presence of inhibitors in the water sample or abnormal nucleic acid extraction efficiency, and template dilution, purification, or re-extraction should be performed. For each test result, the data access unit 300 binds the pathogen name, checkpoint node, monitoring period, filter membrane level, Ct value or copy number, cumulative filtered water volume, blank control, spiked recovery rate, and testing personnel records. After normalization, the system generates the detection quantity per unit water volume for subsequent risk stratification.

[0069] This invention does not simply involve mixing and detecting the two filter layers; instead, it detects the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 separately. A high detection rate of the upper large-pore filter membrane 261 may indicate the introduction of cells, tissue debris, fecal particles, or larger organic particles; a high detection rate of the lower small-pore filter membrane 262 may indicate the introduction of viral particles, small particle carriers, or free nucleic acids. Combining this stratified detection result with the results from upstream and downstream checkpoints can help determine the source and transmission pattern of pathogens.

[0070] In step S4, the data-threshold-response linkage control unit 400 determines the pathogen input path and activity risk level based on the positive combinations of the same pathogen at upstream and downstream checkpoint nodes, the positive results of eRNA during continuous monitoring periods, and the detection distribution of the upper and lower filter membranes. For example... Figure 5 As shown, when the same pathogen is only positive for eDNA and negative for eRNA in the main water pipe 112, and is not detected in the distribution water pipe 113 in the seedling workshop or the batch water pipe 114 in the seedling pond, the system determines it as a primary residual nucleic acid risk at the source water end; when the same pathogen is positive for eRNA in the main water pipe 112, and is positive for eRNA in the distribution water pipe 113 in the seedling workshop or the batch water pipe 114 in the seedling pond during subsequent monitoring periods, the system determines it as a secondary active input risk along the water flow direction; when the same pathogen is positive for eRNA in two consecutive preset monitoring periods at the same checkpoint node, even if the downstream node is not positive, the checkpoint node is determined to have a secondary active input risk.

[0071] In one specific judgment rule, the primary risk of residual nucleic acid refers to the target pathogen showing only eDNA positivity and eRNA negativity, and not recurring during continuous monitoring periods, or appearing only at low levels at upstream nodes and not detected at downstream nodes. The secondary risk of active input refers to the target pathogen showing eRNA positivity at the same checkpoint for two consecutive preset monitoring periods, or showing eRNA positivity sequentially at upstream and downstream checkpoints along the water flow direction. For DNA viruses or eukaryotic parasites, if their target transcripts are positive, the risk can be upgraded based on eRNA positivity.

[0072] Step S4 transforms the single-point detection result into a risk assessment with temporal continuity and spatial directionality. Traditional detection methods typically only provide positive or negative results, making it difficult to distinguish between residual nucleic acid, single-use contamination, continuous input, or diffusion along waterways. This invention, through a comprehensive assessment of "positive combinations at upstream and downstream checkpoints, positive results over consecutive time periods, and stratified distribution of the filter membrane," can improve the ability to identify pathogen input pathways and reduce the risk of erroneous production stoppages or false clearances due to a single positive eDNA test.

[0073] In step S5, the system executes corresponding production procedures based on the risk level. For Level 1 residual nucleic acid risk, the production procedure output module 460 can output instructions such as enhanced disinfection, extended disinfection contact time, increased retesting frequency, increased water change frequency, or suspension of seedling transfer. For example, if Level 1 residual nucleic acid risk occurs in the main water pipe 112, the system controls the disinfection dosing device 530 to increase the disinfection intensity of the source water and increases the retesting frequency at this node from once every 12 hours to once every 6 hours; if consecutive retests are negative, the Level 1 warning is lifted.

[0074] For secondary active input risks, the production disposal output module 460 implements stricter measures based on the location of the positive checkpoint node. If the positive node is located in the main source water pipe 112, the source water switching valve 510 is controlled to switch to the backup source water or the corresponding inlet water is shut off; if the positive node is located in the seedling workshop distribution water pipe 113, the seedling workshop inlet valve 520 is controlled to block the inlet water to the corresponding workshop and the disinfection intensity of the disinfection dosing device 530 is increased; if the positive node is located in the batch water pipe 114 of the seedling pond, the associated batch of seedlings is marked as isolated, and entry into the next seedling node is suspended. If necessary, the batch of seedlings is removed and the associated pond is disinfected; if the positive node is located in the tailwater node 115, the tailwater discharge valve 550 is controlled to close or the tailwater disinfection device 560 is controlled to increase the disinfection intensity.

[0075] If, within at least two consecutive preset monitoring periods corresponding to the current seedling stage, the inlet disinfection pool 111, the main source water pipe 112, the seedling workshop distribution water pipe 113, and the corresponding batch water pipe 114 in the seedling pool all fail to meet the Level 1 residual nucleic acid risk or Level 2 active input risk, the system determines that the batch meets the environmental nucleic acid negative release conditions. The seedling batch release labeling device 540 binds the seedling batch number, the current seedling stage, the monitoring results of the corresponding checkpoint node, the risk level, and the release result to generate a release label that allows entry into the next seedling stage. If the release conditions are not met, a prohibition on transfer label is generated until the release conditions are met through re-inspection or the batch is isolated or removed.

[0076] Step S5 directly transforms the environmental nucleic acid monitoring results into production control actions, so that the test is no longer a standalone experimental report, but becomes a trigger condition for source water switching, water inlet blocking, disinfection control, seedling isolation, seedling removal and batch release, thereby constructing a closed loop of biosafety in the seedling production environment.

[0077] In step S6, steps S1 to S5 are embedded into the brackish water rearing sequence of giant freshwater prawns. For example... Figure 3 As shown, before the broodstock or berried shrimp are introduced into the pond, continuous monitoring is conducted at the inlet disinfection pond 111, the main source water pipe 112, and the inlet water nodes of the broodstock temporary rearing pond. Before the larvae are discharged, continuous monitoring is conducted at the inlet water node of the larvae discharge pond and the distribution water pipe 113 of the nursery workshop. Before the larvae are introduced, continuous monitoring is conducted at the batch water pipe 114 of the nursery pond. During the larval rearing period, continuous monitoring is conducted at the main source water pipe 112, the distribution water pipe 113 of the nursery workshop, and the batch water pipe 114 of the nursery pond according to the preset monitoring period. Before desalination, continuous monitoring is conducted at the inlet water node of the desalination pond. Before hatching, the batch water pipe 114 of the nursery pond corresponding to the hatching batch and the tailwater node 115 are re-monitored. The salinity of the nursery water is controlled at 12‰~14‰, and the salinity is gradually reduced during the desalination stage according to the adaptability of the giant freshwater prawn larvae. At each nursery node, seedlings are only allowed to enter the next node or hatch when the corresponding checkpoint node continuously meets the environmental nucleic acid negative release conditions.

[0078] Through the above steps, this invention forms an environmental biosafety closed loop covering the entire process of giant freshwater prawn (SPF) seedling production. This closed loop covers not only source water and workshop water, but also batch water and seedling transfer points, which can improve the stability, traceability, and batch consistency of large-scale SPF prawn seedling production.

[0079] Example 1: Verification of SPF (Special Price Flooding) Seedling Production of Giant Freshwater Prawns under a Four-Level Checkpoint System

[0080] Four checkpoints were set up at a giant freshwater prawn hatchery: an inlet disinfection pond (111), a main source water pipe (112), a distribution water pipe for the hatchery workshop (113), and a batch water pipe for the hatchery ponds (114). The salinity of the hatchery water was controlled between 12‰ and 14‰. Environmental nucleic acid monitoring was implemented during the stages of broodstock introduction, larval discharge, larval distribution, larval rearing, acclimatization, and hatching. Each checkpoint was equipped with an online continuous enrichment unit (200). The double-layer enrichment filter membrane module (260) used an upper 2.0μm glass fiber membrane and a lower 0.45μm mixed cellulose ester membrane. A preset monitoring period was defined as 12 hours, with the cumulative filtration volume controlled between 600L and 750L per period.

[0081] The target pathogens for detection included macrophage nodavirus, macrophage Taihu virus, macrophage flavivirus, decapod iridovirus 1, and enterocytozoon hepatocellular carcinoma. For macrophage nodavirus, macrophage Taihu virus, and macrophage flavivirus, both eDNA and eRNA were detected simultaneously; for decapod iridovirus 1 and enterocytozoon hepatocellular carcinoma, eDNA was detected and activity risk was determined in conjunction with the target transcript results.

[0082] During a monitoring period on day 5 of larval rearing, a weak positive result for Macrobrachium rosenbergii nodavirus eDNA was detected in the lower small-pore filter membrane 262 of the main source water pipe 112, with a normalized detection limit of 78 copies / L. eRNA was not detected in the same sample, nor in the inlet disinfection tank 111, the nursery distribution water pipe 113, or the batch water pipe 114 in the nursery pond. The system determined this to be a Level 1 residual nucleic acid risk at the source water end, controlling the disinfection dosing device 530 to increase the source water disinfection intensity, and increasing the retesting frequency of the main source water pipe 112 to once every 6 hours. In two consecutive retesting periods, the corresponding eDNA and eRNA were not detected in the main source water pipe 112, the nursery distribution water pipe 113, or the batch water pipe 114 in the nursery pond. The system lifted the Level 1 warning, and the batch of seedlings continued larval rearing.

[0083] The above results indicate that the present invention can distinguish between the residual nucleic acid risk of eDNA-positive but eRNA-negative and the active input risk corresponding to eRNA-positive, avoiding the direct elimination of the entire seedling batch due to a single weak eDNA positive, while also preventing the continued input of potential risks through enhanced disinfection and retesting.

[0084] Example 2: Comparison of low-load detection capabilities between online continuous enrichment and manual bottle sampling

[0085] To verify the online continuous enrichment capability of this invention for detecting low-load pathogen nucleic acids, simulated seedling water samples were prepared using inactivated Macrobrachium rosenbergii nodavirus nucleic acid standards or equivalent nucleic acid standards, with three target concentration levels set at 5 copies / L, 20 copies / L, and 100 copies / L. Twelve replicates were prepared for each concentration.

[0086] The example group employed the online continuous enrichment method of this invention, with each sample having a filtered water volume of 600L. Detection was performed using an upper 2.0μm glass fiber membrane and a lower 0.45μm mixed cellulose ester membrane. Comparative Example 1 used manual bottle sampling, collecting 1L of water for each sample, which was then filtered through a 0.45μm single-layer filter membrane before detection. The detection results are shown in the table below.

[0087] Table 1 Comparison of low-load pathogen nucleic acid detection rates using different sampling methods

[0088]

[0089] As shown in Table 1, under low loading conditions of 5 copies / L and 20 copies / L, the detection rate of the online continuous enrichment method of this invention is significantly higher than that of manual bottle sampling. This is because the water volume processed by this invention within a preset monitoring period is much larger than that of manual bottle sampling, and it can cover a longer time window, reducing the possibility of missing pathogen pulse input. This result corresponds to... Figure 6 The technical effects shown demonstrate that the present invention can improve the detection probability of low-load or intermittent pathogen input.

[0090] Example 3: Effect of pre-membrane preservation on eRNA recovery rate

[0091] To verify the effect of pre-membrane preservation on eRNA stability, simulated seedling water samples containing target RNA standards were used for enrichment. All groups used the same double-layer enrichment filter membrane assembly 260, with a filtration volume of 300 L. In the example group, DNA / RNA simultaneous preservation solution was injected through the preservation solution injection module 270 after the filter membrane was isolated from the water flow and before the filter membrane quick-change chamber 250 was opened; in Comparative Example 2, preservation solution was added 10 minutes after the filter membrane was removed and exposed; in Comparative Example 3, preservation solution was added 30 minutes after the filter membrane was removed and exposed. Six replicates were set up for each group. The eRNA recovery rate was calculated using the standard curve conversion results under immediate preservation conditions as a reference, and the results are shown in the table below.

[0092] Table 2. Effects of different preservation methods on eRNA recovery rate

[0093]

[0094] As shown in Table 2, the eRNA recovery rate in the example group was significantly higher than that in the delayed preservation group after membrane removal. This result indicates that the present invention, through the cooperation of the filter membrane quick-change compartment 250 and the preservation solution injection module 270, completes simultaneous DNA / RNA preservation before the filter membrane is exposed, which can reduce eRNA degradation during membrane removal and improve the stability of eRNA detection. This effect corresponds to... Figure 7 The technical effects shown provide a basis for assessing the risk of active input using eRNA, demonstrating sample stability.

[0095] Example 4: The role of double-layer filter membrane stratification in determining the morphology of pathogen input

[0096] To verify the effectiveness of the dual-layer filter membrane stratified detection, simulated seedling water samples were used. Target nucleic acid standards containing tissue debris carriers were used to simulate large particle input, while small-particle nucleic acid carriers were used to simulate viral particles or free nucleic acid input. Six replicates were prepared for each type of sample, and the dual-layer enrichment filter membrane assembly 260 of this invention was used to detect the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262, respectively. The results are shown in the table below.

[0097] Table 3 Distribution of stratified detection in double-layer filter membranes

[0098]

[0099] As shown in Table 3, this invention, by detecting the upper large-pore filter membrane 261 and the lower small-pore filter membrane 262 separately, can obtain the distribution information of the target nucleic acid in carriers of different particle sizes. This distribution information, combined with positive results from upstream and downstream checkpoint nodes, helps to distinguish between different scenarios such as residual debris input from source water, small particles input from workshop waterways, and batch water contamination in seedling ponds. Compared with mixed detection using two filter membranes, this invention can provide more detailed information on the morphological characteristics of pathogen input.

[0100] Example 5: Verification of the effect of seedling batch release

[0101] Under identical nursery conditions, an example control group and a routine management group were established. The example control group underwent continuous online enrichment, eDNA / eRNA stratified detection, risk classification, and batch release using the method of this invention. The routine management group underwent pre-introduction testing of parent stock, pond disinfection, water treatment, and periodic artificial water sample testing. Both groups were raised in brackish water with a salinity of 12‰–14‰ for giant freshwater prawns. For each group, the hatching pass rate, environmental nucleic acid release results, and number of abnormal handling procedures were recorded for three batches. The results are shown in the table below.

[0102] Table 4. Results of Seedling Batch Production Verification

[0103]

[0104] As shown in Table 4, all three batches in the Example Group met the release condition of negative environmental nucleic acid for the target pathogen before emergence, with an average emergence pass rate of 86.7% and a standard deviation of 1.2%. In the Routine Management Group, one batch showed a weak positive result for target pathogen eDNA in the pre-emergence artificial water sample retest, requiring delayed emergence and retesting, with an average emergence pass rate of 76.5% and a standard deviation of 1.9%. Under the same seedling farm management conditions, this invention helps improve the stability of seedling production and batch traceability by moving environmental input monitoring forward, improving the detection capability of low load, distinguishing between residual nucleic acid risk and active input risk, and linking risk level with batch release.

[0105] It should be noted that the seedling qualification rate is also affected by factors such as parent stock quality, feed quality, salinity changes, temperature control, larval density, water exchange schedule, and daily management level. Table 4 is used to illustrate the control effect of this invention through the identification of environmental pathogen input and the linkage between production and disposal under the same management conditions, and does not mean that this invention can eliminate all seedling risks by relying solely on environmental nucleic acid monitoring.

[0106] Comparative Example 1: Manual Bottle Sampling Monitoring Method

[0107] Comparative Example 1 used conventional manual bottle sampling. At the end of each monitoring period, 1L of water samples were collected from the main water pipe 112 and the inlet of the seedling pond. After being brought back to the testing room, the samples were filtered through a 0.45μm single-layer filter membrane and then analyzed for eDNA and eRNA. This method involves a small sample volume, and the sampling points only represent the instantaneous state. When the pathogen input is low-load or pulsed, it is easy to miss detections because the sampling time point does not cover the pathogen input window. The results are shown in Table 1.

[0108] Comparative Example 2: Online Enrichment Methods Based on eDNA Detection Only

[0109] Comparative Example 2 employed the same online continuous enrichment method as Example 1, but only detected eDNA, not eRNA or target transcripts. During larval rearing, Comparative Example 2 detected a low-level nodavirus eDNA positivity in the main source water pipe 112. Since it was impossible to determine whether this positivity originated from residual nucleic acid or live virus input, conventional positive handling procedures required suspending batch transfer and conducting prolonged retesting. Compared to Example 1, Comparative Example 2 could not effectively distinguish between residual nucleic acid risk and live virus input risk, potentially leading to unwarranted production stoppages or over-treatment.

[0110] Comparative Example 3: Delayed Preservation Method After Membrane Removal

[0111] Comparative Example 3 employed the same online continuous enrichment method as Example 1, but the filter membrane was removed and exposed for 30 minutes before the preservation solution was added. Its eRNA recovery rate was significantly lower than that of the Example group, as detailed in Table 2. Since eRNA is easily degraded by environmental temperature, nucleases, and operation time, Comparative Example 3 was prone to underestimation of eRNA or false negatives. This comparative example illustrates that without the pre-exposure preservation solution injection structure, even with parallel eDNA / eRNA detection, eRNA degradation may affect the assessment of the risk of active input.

[0112] Comparative Example 4: Detection Method for Mixed Double-Layer Filter Membranes

[0113] Comparative Example 4 used an upper large-pore filter membrane and a lower small-pore filter membrane for filtration, but after removing the membranes, the two layers were mixed to extract nucleic acids without separate detection. This method can improve the overall enrichment, but it cannot obtain information on the distribution of nucleic acid carriers on filter membranes of different particle sizes, and cannot help determine the pathogen input morphology. When low-level positive results for eDNA were found in both the main water pipe 112 and the batch water pipe 114 in the seedling pond, Comparative Example 4 could only determine the presence of target nucleic acids, and it was difficult to distinguish between large-particle residual input and small-particle active input by combining the filter membrane layer information. Compared with Example 4, Comparative Example 4 has a weaker ability to determine the source and transmission morphology of pathogens.

[0114] In summary, this invention solves the problems of delayed detection of environmental pathogens, insufficient sampling coverage, easy misjudgment of residual nucleic acids, and disconnect between detection results and production and disposal in existing SPF (Special Purity for Freshwater Prawn) seedling cultivation by employing specific checkpoint node layout, continuous enrichment structure, pre-exposure preservation structure of filter membrane, stratified detection of double-layer filter membranes, and coordinated control of production and disposal. Those skilled in the art can implement this invention without creative effort based on the system structure, checkpoint node settings, enrichment sampling method, nucleic acid preservation method, detection method, risk assessment rules, and production and disposal logic disclosed in this specification.

Claims

1. A method for large-scale SPF (Special Purity Flood) breeding of giant freshwater prawns, characterized in that, This method includes the following within a seedling production cycle: S1. According to the water flow entry path and seedling transfer path of the seedling nursery, set up online continuous enrichment units at at least three types of checkpoints in the inlet disinfection pool, the main source water pipe, the distribution water pipe of the seedling workshop and the batch water pipe of the seedling pool. Filter the water at each node continuously according to the preset monitoring period and record the cumulative filtered water volume. S2. After the enrichment membrane is isolated from the water flow and before the membrane is exposed, inject DNA / RNA synchronous preservation solution into the enrichment membrane and number and preserve the upper large-pore membrane and the lower small-pore membrane respectively. S3. Environmental DNA and environmental RNA were detected in the upper large-pore filter membrane and the lower small-pore filter membrane at the same monitoring time and the same checkpoint node, and the detected amounts were normalized. S4. Based on the positive combinations of the same pathogen at upstream and downstream checkpoints, the positive results of environmental RNA during continuous monitoring periods, and the detection distribution of the upper and lower filter membranes, determine the pathogen input pathway and activity risk level. S5. When the preset risk level is reached, the corresponding water intake shall be blocked or the backup water source shall be switched, and the associated batch of seedlings shall be isolated or removed; when the environmental nucleic acid test results are continuously met, the associated batch shall be allowed to enter the next seedling stage. S6. Insert S1 to S5 into a parent stock with a salinity of 12‰ to 14‰, and follow the sequence of stocking, releasing, larval rearing, acclimatization, and hatching to obtain giant freshwater prawn larvae that meet the release conditions of negative nucleic acid in a specific pathogen environment.

2. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S1, the checkpoint nodes are divided into external source input checkpoints, source water input checkpoints, workshop distribution checkpoints, and batch water use checkpoints according to the water flow topology. The external source input checkpoints are set at the entrance disinfection pool or personnel and vehicle disinfection channel. The source water input checkpoints are set at the water intake, the main pipe after source water treatment, or the backup source water pipe. The workshop distribution checkpoints are set at the main water inlet pipe or distribution branch pipe of the seedling workshop. The batch water use checkpoints are set at the water inlet pipes of the seedling pond, the larval discharge pond, the desalination pond, or the parent stock temporary rearing pond. And / or, the online continuous enrichment unit includes a pre-filter, a flow meter, a metering pump or peristaltic pump, an enrichment filter membrane assembly, and a bypass parallel valve group; the bypass parallel valve group includes at least two parallel enrichment branches, such that when one enrichment branch is undergoing filter membrane replacement, the other enrichment branch remains in a continuous enrichment state.

3. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S2, the upper large-pore filter membrane is used to retain cells, tissue debris, fecal particles, and large-particle nucleic acid carriers, while the lower small-pore filter membrane is used to retain virus particles, free nucleic acids, and small-particle nucleic acid carriers; the upper large-pore filter membrane and the lower small-pore filter membrane are used to preserve, extract nucleic acids, or generate detection data, respectively. The upper large-pore filter membrane is a glass fiber membrane with a pore size of 1.0μm to 3.0μm, and the lower small-pore filter membrane is a mixed cellulose ester membrane, polyethersulfone membrane, or polyvinylidene fluoride membrane with a pore size of 0.22μm to 0.80μm; the cumulative filtration water volume for each preset monitoring period is 50L to 1000L. And / or, in step S2, the DNA / RNA synchronous preservation solution is added by a preservation solution injection mechanism located in the filter membrane quick-change chamber; the filter membrane quick-change chamber remains sealed after the inlet valve and outlet valve are closed, and the preservation solution injection is completed before the chamber is opened, so as to reduce the degradation of environmental RNA during the membrane removal process.

4. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S3, the environmental DNA detection uses qPCR or digital PCR, and the environmental RNA detection uses RT-qPCR or RT digital PCR; the normalization of the detection amount is determined based on the cumulative filtered water volume, blank control, historical negative baseline of the current site, spiked recovery rate, and detection limit of the detection method.

5. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S4, when the same pathogen is positive for environmental DNA but negative for environmental RNA only at the upstream checkpoint node, it is determined to be a level 1 residual nucleic acid risk; when the same pathogen is positive for environmental RNA for two consecutive preset monitoring periods at the same checkpoint node, or when it is positive for environmental RNA successively at the upstream and downstream checkpoint nodes along the water flow direction, it is determined to be a level 2 active input risk. For Level 1 residual nucleic acid risk, implement enhanced disinfection, extend disinfection contact time, increase retesting frequency, increase water change frequency, or suspend seedling transfer; for Level 2 active input risk, implement measures such as blocking corresponding influent, switching to backup water source, isolating related batches of seedlings, removing related batches of seedlings, disinfecting empty pools of related pools, or initiating upstream pollution tracing.

6. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S5, the environmental nucleic acid negative release condition is as follows: within at least two consecutive preset monitoring periods corresponding to the current seedling node, the inlet disinfection pool, the main source water pipe, the distribution water pipe of the seedling workshop, and the batch water pipe of the corresponding seedling pool have not reached the level of primary residual nucleic acid risk or secondary active input risk; after the environmental nucleic acid negative release condition is met, a release result bound to the identifier of the batch of seedlings is generated.

7. The method for large-scale SPF breeding of giant freshwater prawns according to claim 1, characterized in that, In step S6, the sequence of parent stock entry into the pool, juvenile discharge, juvenile distribution, juvenile cultivation, desalination, and seedling emergence is respectively bound to the preset monitoring period of the corresponding checkpoint node; if any seedling node fails to meet the environmental nucleic acid negative release condition, the transfer of that batch of seedlings to the next seedling node is stopped. And / or, in step S6, the specific pathogen includes at least three of the following: giant freshwater prawn nodavirus, giant freshwater prawn Taihu virus, giant freshwater prawn flavivirus, decapod iridovirus 1, and hepatocellular carcinoma; for RNA viruses, a positive result for environmental RNA is used as the basis for determining the activity risk; for DNA viruses or eukaryotic parasites, a positive result for environmental DNA combined with a positive result for its transcript is used as the basis for determining the activity risk.

8. A linked system for online enrichment and detection of SPF (Special Purity Fiber) in a Macrobrachium rosenbergii hatchery for implementing the method according to any one of claims 1 to 7, characterized in that, It includes a checkpoint node deployment unit, an online continuous enrichment unit, a filter membrane quick-change and storage unit, a detection data access unit, and a data-threshold-response linkage control unit; the checkpoint node deployment unit is set at least three types of checkpoint nodes in the inlet disinfection pool, the main source water pipe, the distribution water pipe of the seedling workshop, and the batch water pipe of the seedling pool according to the water flow entry path and seedling transfer path of the seedling farm; the data-threshold-response linkage control unit is communicatively connected to the source water switching valve, the seedling workshop inlet valve, the disinfection dosing device, and the seedling batch release identification device.

9. The online enrichment and detection linkage system for Giant Freshwater Prawn SPF hatchery according to claim 8, characterized in that, The online continuous enrichment unit includes a pre-filter, a flow meter, a metering pump or a peristaltic pump, at least two parallel enrichment branches and an enrichment membrane assembly; each parallel enrichment branch is equipped with an inlet control valve, an outlet control valve and a quick-change filter membrane compartment, so as to complete the filter membrane replacement without interrupting the continuous enrichment at the corresponding gate node. The enrichment filter membrane assembly includes an upper large-pore filter membrane and a lower small-pore filter membrane arranged sequentially along the water flow direction; the filter membrane quick-change chamber is provided with a filter membrane clamping structure for fixing the upper large-pore filter membrane and the lower small-pore filter membrane respectively, and a sample numbering structure for generating filter membrane numbers respectively. The filter membrane quick-change and preservation unit includes a chamber sealing detection module, a preservation solution injection module, and a low-temperature temporary storage module. The preservation solution injection module is configured to inject DNA / RNA synchronous preservation solution into the enrichment filter membrane assembly after the inlet and outlet control valves of the filter membrane quick-change chamber are closed and before the filter membrane quick-change chamber is opened.

10. The online enrichment and detection linkage system for Giant freshwater prawn SPF hatchery according to claim 8, characterized in that, The detection data access unit is used to receive environmental DNA detection data and environmental RNA detection data corresponding to the same monitoring period, the same checkpoint node, the upper large-pore filter membrane and the lower small-pore filter membrane respectively, and to associate and store the detection data with the cumulative filtered water volume, blank control, the historical negative baseline of this field and the spiked recovery rate. And / or, the data-threshold-response linkage control unit includes a filtered water volume normalization module, an upstream and downstream positive combination judgment module, a continuous time period positive judgment module, a filter membrane stratification distribution judgment module, a risk level judgment module, and a production and disposal output module; the production and disposal output module is used to output at least one control result among source water switching, inlet water blocking, disinfection dosing, re-inspection, seedling isolation, seedling removal, and seedling release; And / or, the seedling batch release identification device is used to bind the seedling batch number, the current seedling node, the monitoring result of the corresponding checkpoint node, the risk level, and the release result; When the data-threshold-response linkage control unit determines that the environmental nucleic acid negative release condition is not met, the seedling batch release identification device generates an identification that prohibits transfer to the next seedling stage; And / or, the data-threshold-response linkage control unit is also communicatively connected to the tailwater discharge valve and the tailwater disinfection device; when the drainage node or tailwater treatment node reaches the secondary active input risk, it controls the tailwater discharge valve to close or controls the tailwater disinfection device to increase the disinfection intensity.

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