Method and system for evaluating WSSV (white spot syndrome virus) resistance of procambarus clarkii
By pretreating and extracting target tissue samples from *Procambarus clarkii*, and combining TRIM23 gene quantification and a relative quantitative mathematical model, the safety and accuracy issues of traditional assessment methods were resolved, enabling rapid, safe, and accurate assessment of WSSV resistance.
Patent Information
- Application Number
- CN202511545353.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-28
- Publication Date
- 2026-01-23
AI Technical Summary
Traditional methods for assessing the resistance of red swamp crayfish to white spot syndrome virus (WSSV) are characterized by high operational risks, long processing times, and high costs, and it is difficult to guarantee the safety and accuracy of the assessment.
By pretreating the red swamp crayfish, collecting target tissue samples and extracting total RNA, performing cDNA synthesis and TRIM23 gene quantification, and combining a relative quantitative mathematical model to evaluate its anti-WSSV ability, the evaluation was automated using a high-performance computing architecture and data storage module.
This method enables a safe, rapid, and accurate assessment of WSSV resistance in Procambarus clarkii, avoiding highly pathogenic procedures, shortening the monitoring process, and improving the accuracy of the assessment.
Smart Images
Figure CN121380355A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crayfish evaluation, and in particular to a method and system for evaluating the resistance of red swamp crayfish to WSSV. Background Technology
[0002] Crayfish, also known as red swamp crayfish, is a freshwater crustacean and an extremely important aquatic product, forming a vast industrial chain from farming and fishing to catering, creating enormous economic value. WSSV, or white spot syndrome virus, is one of the most devastating pathogens affecting shrimp and red swamp crayfish. It is highly contagious and has a very high mortality rate; an outbreak can kill all the shrimp in an entire pond within just a few days. WSSV resistance refers to the inherent potential of the red swamp crayfish's immune system to resist and clear WSSV infection, thus surviving viral attacks. This difference is determined by the genetic and molecular regulatory pathways of the innate immune system, and the TRIM23-TBK1 pathway, which this invention focuses on, is a key component of this immune system.
[0003] Due to the significant threat WSSV poses to crayfish farming, traditional assessment methods have serious drawbacks. For example, traditional methods involve infecting healthy crayfish with a quantitative amount of live virus, then observing and recording mortality and survival time. The disadvantages include the risk of virus leakage and environmental contamination during handling, as well as the long testing time and high costs. Therefore, there is a need to develop alternative methods for assessing the WSSV resistance of Procambarus clarkii, avoiding the handling of highly pathogenic viruses, improving the safety of the process, and significantly shortening the monitoring procedure while increasing accuracy. Summary of the Invention
[0004] This invention overcomes the shortcomings of the prior art and provides a method and system for evaluating the resistance of red swamp crayfish to WSSV.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for evaluating the resistance of red swamp crayfish to WSSV, comprising the following steps: The *Procambarus gracilis* samples to be evaluated were pretreated, and target tissue samples were selected and collected after pretreatment, while the target tissue samples were preserved. Total RNA was extracted from the target tissue sample, and the concentration of the extracted total RNA was precisely quantified and cDNA was synthesized. The TRIM23 gene in the cDNA product was quantitatively detected using a probe method, and the Ct value of the TRIM23 gene was obtained. By combining the target Ct value of the TRIM23 gene, a relative quantitative mathematical model was constructed to evaluate the anti-WSSV ability of the target crayfish sample.
[0006] Furthermore, in a preferred embodiment of the present invention, the pretreatment of the *Procambarus clarkii* to be evaluated, followed by the selection and collection of target tissue samples after pretreatment, and the preservation of the target tissue samples, specifically involves: The culture ponds for raising Procambarus clarkii were identified and designated as target culture ponds. Procambarus clarkii samples were collected from the target culture ponds and placed in the laboratory. The caught Procambarus grahami samples were screened by weight. Samples that were not within the standard weight range were removed. Among the remaining Procambarus grahami samples, those with physical defects were screened again to obtain the target Procambarus grahami samples. The target Procambarus gracilis sample was temporarily pre-treated in the laboratory to obtain the target pre-treated Procambarus gracilis sample. The target tissues of the pretreated Procambarus cristatus sample were selected and collected. Specifically, the hemolymphocyte tissue and gill tissue of the pretreated Procambarus cristatus sample were selected as target tissues. At the same time, the target tissues of the pretreated Procambarus cristatus sample were collected to obtain target tissue samples. Finally, the target tissue samples were preserved in liquid nitrogen.
[0007] Furthermore, in a preferred embodiment of the present invention, the extraction of total RNA from the target tissue sample, and the precise quantification of the extracted total RNA concentration and cDNA synthesis processing, specifically include: The target tissue samples were classified to obtain hemolymphocyte tissue samples for gill tissue sampling; The blood lymphocyte tissue sample was thawed and placed in a centrifuge tube with lysis buffer for centrifugation. Centrifugation was stopped when no precipitate of blood lymphocyte tissue sample appeared in the centrifuge tube. The gill tissue sample was ground in liquid nitrogen, and the ground gill tissue sample was transferred to a centrifuge tube and mixed with the hemolymphocyte tissue sample to obtain the target lysis mixture; The target lysis mixture was subjected to secondary lysis in a centrifuge tube, and the supernatant was extracted after lysis and transferred to a new centrifuge tube. Ethanol was added and mixed to obtain the secondary lysis mixture. An RNA adsorption column was obtained, and protein buffer and washing solution were injected into the RNA adsorption column in sequence. The secondary lysis mixture was then washed with the RNA adsorption column to obtain the total RNA sample of the target tissue sample, i.e., the total RNA eluent. A sample of total RNA elution buffer was taken, and a micro spectrophotometer was used to detect the concentration of total RNA in the sample. The sample of total RNA elution buffer was then subjected to electrophoresis using agarose gel. After electrophoresis, the integrity of the total RNA elution buffer was assessed using an ultraviolet imaging system, and cDNA products were synthesized using total RNA elution buffer with acceptable sample integrity.
[0008] Furthermore, in a preferred embodiment of the present invention, the quantitative detection of the TRIM23 gene in the cDNA product using a probe method to obtain the Ct value of the TRIM23 gene specifically involves: By introducing a big data network, and based on the big data network, the most frequently used amplification primer type for the TRIM23 gene in Procambarus clarkii was retrieved and identified as the target amplification primer type. Based on the target amplification primer type, amplification primers corresponding to the target amplification primer type are added to the cDNA product. The amplification primers corresponding to the target amplification primer type are used to locate the TRIM23 gene in the cDNA product to obtain the located TRIM23 gene. The amplification primers corresponding to the target amplification primer type are primer pairs and are labeled as target amplification primer pairs. TaqMan hydrolysis probe technology was introduced and applied to the TRIM23 gene after localization in cDNA products to generate nucleic acid probes for target amplification primer pairs. The TRIM23 gene was amplified by PCR, and the nucleic acid probe of the target amplification primer pair was detected by real-time fluorescence signal detection. The real-time fluorescence signal was detected by a real-time fluorescence quantitative PCR instrument, and the fluorescence value of the real-time fluorescence signal was read. A standard fluorescence value was preset. When the fluorescence value of the real-time fluorescence signal was equal to the standard fluorescence value, the PCR amplification was stopped, and the PCR amplified TRIM23 gene was obtained. The number of PCR amplification cycles required for the real-time fluorescence signal to equal the standard fluorescence value is calculated and denoted as the Ct value. The target Ct value was obtained by screening through experimental control during the Ct value calculation process.
[0009] Furthermore, in a preferred embodiment of the present invention, the step of obtaining the target Ct value through an experimental control method during the Ct value calculation process specifically involves: Meanwhile, PCR amplification of the TRIM23 gene was performed in different test tubes, and the Ct values in different test tubes were calculated. During the PCR amplification of the TRIM23 gene, an experimental control group was set up. In the experimental control group, pure water was used to replace the cDNA product to detect whether contamination occurred during the PCR amplification of the TRIM23 gene. If contamination occurs, the TRIM23 gene PCR amplification process is repeated. If no contamination occurs, the Ct values in different test tubes are analyzed, and the test tube with the smallest Ct value is selected as the target test tube, and the corresponding Ct value is labeled as the target Ct value.
[0010] Furthermore, in a preferred embodiment of the present invention, the step of constructing a relative quantitative mathematical model based on the target Ct value of the TRIM23 gene to assess the anti-WSSV ability of the target Procambarus clarkii sample specifically involves: Based on the target Ct value, the Ct values of different test tubes are standardized and corrected. That is, the target Ct value is used as the benchmark value, the difference between the Ct value of different test tubes and the target Ct value is calculated, and the sample difference status of TRIM23 gene in different test tubes and the target test tube is calculated based on the difference. The difference is designated as the first difference. Using big data networks, the Ct values of the TRIM23 gene in a population of *Procambarus clarkii* that has been verified and has qualified resistance to WSSV were obtained, and compared with the target Ct value. The difference was calculated and labeled as the second difference. The difference between the first difference and the second difference is obtained, and the relative expression level is calculated based on the difference between the first difference and the second difference, wherein the relative expression level is an indicator of the anti-WSSV ability of the target crayfish sample; The relative expression levels are analyzed, and the classification threshold of the relative expression levels is retrieved through a big data network. Based on the classification threshold of the relative expression levels, the resistance to WSSV in *Procambarus swampus* in the target culture pond is evaluated and output.
[0011] A second aspect of this invention also provides a system for evaluating the WSSV resistance of the red swamp crayfish. The 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 method program for evaluating the WSSV resistance of the red swamp crayfish, equipped with an evaluation engine. When the program is executed in parallel through a superscalar pipeline execution unit within the processor, the following steps are implemented: The *Procambarus gracilis* samples to be evaluated were pretreated, and target tissue samples were selected and collected after pretreatment, while the target tissue samples were preserved. Total RNA was extracted from the target tissue sample, and the concentration of the extracted total RNA was precisely quantified and cDNA was synthesized. The TRIM23 gene in the cDNA product was quantitatively detected using a probe method, and the Ct value of the TRIM23 gene was obtained. By combining the target Ct value of the TRIM23 gene, a relative quantitative mathematical model was constructed to evaluate the anti-WSSV ability of the target crayfish sample.
[0012] This invention addresses the technical deficiencies in the prior art and offers the following advantages: It involves sampling and processing *Procambarus clarkii* (crawfish), extracting target tissue from the samples for total RNA extraction, and then using a probe method to quantitatively detect the TRIM23 gene in *Procambarus clarkii* to calculate the Ct value. Finally, a relative quantitative mathematical model is used to evaluate the anti-WSSV ability of *Procambarus clarkii*. This invention avoids handling highly pathogenic or viral substances, improving operational safety, and significantly shortening the monitoring process while increasing accuracy. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained from these drawings without creative effort.
[0014] Figure 1 A flowchart of a method for evaluating the resistance of red swamp crayfish to WSSV is shown; Figure 2 A flowchart illustrating the method for calculating the Ct value of the TRIM23 gene is shown. Figure 3 A program view of a system for evaluating the resistance of red swamp crayfish to WSSV is shown. Detailed Implementation
[0015] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.
[0016] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.
[0017] Figure 1 A flowchart of a method for evaluating the resistance of red swamp crayfish to WSSV is shown, including the following steps: S102: Pre-treat the swamp crayfish to be evaluated, and select and collect target tissue samples after pre-treatment, while preserving the target tissue samples. S104: Total RNA was extracted from the target tissue sample, and the extracted total RNA was precisely quantified and processed for cDNA synthesis. S106: The TRIM23 gene in the cDNA product was quantitatively detected by probe method to obtain the Ct value of the TRIM23 gene; S108: Based on the target Ct value of the TRIM23 gene, a relative quantitative mathematical model was constructed to evaluate the anti-WSSV ability of the target crayfish sample.
[0018] Furthermore, in a preferred embodiment of the present invention, the pretreatment of the *Procambarus clarkii* to be evaluated, followed by the selection and collection of target tissue samples after pretreatment, and the preservation of the target tissue samples, specifically involves: The culture ponds for raising Procambarus clarkii were identified and designated as target culture ponds. Procambarus clarkii samples were collected from the target culture ponds and placed in the laboratory. The caught Procambarus grahami samples were screened by weight. Samples that were not within the standard weight range were removed. Among the remaining Procambarus grahami samples, those with physical defects were screened again to obtain the target Procambarus grahami samples. The target Procambarus gracilis sample was temporarily pre-treated in the laboratory to obtain the target pre-treated Procambarus gracilis sample. The target tissues of the pretreated Procambarus cristatus sample were selected and collected. Specifically, the hemolymphocyte tissue and gill tissue of the pretreated Procambarus cristatus sample were selected as target tissues. At the same time, the target tissues of the pretreated Procambarus cristatus sample were collected to obtain target tissue samples. Finally, the target tissue samples were preserved in liquid nitrogen.
[0019] It should be noted that *Procambarus clarkii* is the same as crayfish, usually farmed crayfish. However, *Procambarus clarkii* is susceptible to WSSV virus, also known as White Spot Syndrome Virus, which is highly contagious and has a very high mortality rate, commonly referred to as "shrimp cancer." Sampling and testing of *Procambarus clarkii* in the farming pond is necessary to assess their resistance to WSSV, thereby evaluating the WSSV resistance of all *Procambarus clarkii* in the pond. First, samples with substandard weight or physical defects, such as broken legs or tails, are removed to ensure a more accurate and convincing assessment. Second, the *Procambarus clarkii* undergo temporary pre-treatment by feeding them a basic diet to allow them to recover from transport stress and empty their intestinal contents. Feeding is stopped 24 hours before sampling to obtain pre-treated *Procambarus clarkii* samples. Finally, target tissues are selected and collected, specifically hemolymphocytes and gill tissue, as these two tissues are key sites of the immune response and are rich in hemocellular and other immune-active cells. Hemolymphocyte collection involved disinfecting the pericardial sinus region of the red swab using 75% ethanol cotton balls, then aspirating hemolymph from the pericardial sinus region using a sterile syringe and rapidly transferring it into centrifuge tubes pre-filled with sterile anticoagulant. The anticoagulant was used to prevent coagulation. Gill tissue was collected by carefully cutting open and lifting the gill cover from one side of the cephalothorax using sterile dissecting scissors to expose the gill filaments. The gill filament tissue was then cut off using sterile forceps for preservation.
[0020] Furthermore, in a preferred embodiment of the present invention, the extraction of total RNA from the target tissue sample, and the precise quantification of the extracted total RNA concentration and cDNA synthesis processing, specifically include: The target tissue samples were classified to obtain hemolymphocyte tissue samples for gill tissue sampling; The blood lymphocyte tissue sample was thawed and placed in a centrifuge tube with lysis buffer for centrifugation. Centrifugation was stopped when no precipitate of blood lymphocyte tissue sample appeared in the centrifuge tube. The gill tissue sample was ground in liquid nitrogen, and the ground gill tissue sample was transferred to a centrifuge tube and mixed with the hemolymphocyte tissue sample to obtain the target lysis mixture; The target lysis mixture was subjected to secondary lysis in a centrifuge tube, and the supernatant was extracted after lysis and transferred to a new centrifuge tube. Ethanol was added and mixed to obtain the secondary lysis mixture. An RNA adsorption column was obtained, and protein buffer and washing solution were injected into the RNA adsorption column in sequence. The secondary lysis mixture was then washed with the RNA adsorption column to obtain the total RNA sample of the target tissue sample, i.e., the total RNA eluent. A sample of total RNA elution buffer was taken, and a micro spectrophotometer was used to detect the concentration of total RNA in the sample. The sample of total RNA elution buffer was then subjected to electrophoresis using agarose gel. After electrophoresis, the integrity of the total RNA elution buffer was assessed using an ultraviolet imaging system, and cDNA products were synthesized using total RNA elution buffer with acceptable sample integrity.
[0021] It should be noted that the hemolymph tissue was processed first, followed by the gill tissue. The purpose of processing the hemolymph tissue and gill tissue was to extract total RNA for the synthesis of cDNA for quantitative PCR. First, total RNA was extracted by lysing cells in a high-sequence-salt buffer to allow RNA to bind specifically and efficiently to the silica membrane. Impurities were then removed using a washing buffer, and finally, the mixture was eluted with RNase-free water to obtain high-purity total RNA. For the hemolymph tissue extraction, the hemolymph cells were lysed until no significant precipitation occurred. The gill tissue was then ground and added to the lysis buffer along with the hemolymph tissue. Finally, the RNA in the mixture was eluted using an RNA adsorption column to obtain the extracted total RNA. Subsequently, the purity was determined using ultraviolet spectrophotometry, i.e., a sample of the lysis buffer was analyzed using a micro-spectrophotometer. Once the purity was within acceptable limits, agarose gel electrophoresis was performed. The purpose of electrophoresis was to determine the integrity of the total RNA. After electrophoresis, observation using a UV imaging system revealed sharp, untailed ribosomal RNA bands in the total RNA, indicating that the RNA was intact and undegraded. At this point, using reverse transcriptase as a template, and guided by Oligo dT and random primers, complementary DNA strands, i.e., cDNA products, were synthesized. Oligo dT and random primers can be obtained using a reverse transcription kit.
[0022] Furthermore, in a preferred embodiment of the present invention, the step of constructing a relative quantitative mathematical model based on the target Ct value of the TRIM23 gene to assess the anti-WSSV ability of the target Procambarus clarkii sample specifically involves: Based on the target Ct value, the Ct values of different test tubes are standardized and corrected. That is, the target Ct value is used as the benchmark value, the difference between the Ct value of different test tubes and the target Ct value is calculated, and the sample difference status of TRIM23 gene in different test tubes and the target test tube is calculated based on the difference. The difference is designated as the first difference. Using big data networks, the Ct values of the TRIM23 gene in a population of *Procambarus clarkii* that has been verified and has qualified resistance to WSSV were obtained, and compared with the target Ct value. The difference was calculated and labeled as the second difference. The difference between the first difference and the second difference is obtained, and the relative expression level is calculated based on the difference between the first difference and the second difference, wherein the relative expression level is an indicator of the anti-WSSV ability of the target crayfish sample; The relative expression levels are analyzed, and the classification threshold of the relative expression levels is retrieved through a big data network. Based on the classification threshold of the relative expression levels, the resistance to WSSV in *Procambarus swampus* in the target culture pond is evaluated and output.
[0023] It's important to note that since different test tubes can yield corresponding Ct values, and the target Ct value serves as a baseline, obtaining the difference is intended to internally standardize the samples within each test tube. This difference eliminates minor differences in RNA input, reverse transcription efficiency, and loading volume among different samples, ensuring all samples have a level playing field for comparison regarding TRIM23 expression. A smaller difference indicates a higher relative expression level of the gene within the test tube. Simultaneously, a validated reference population with a clearly defined resistance background is needed as a baseline. This population is typically a population of *Procambarus clarkii* with moderate resistance levels, validated through traditional challenge experiments. The difference between the Ct value of this population and the standard value is calculated; this is the second difference. The purpose of calculating both the first and second differences is to compare the difference of the tested sample with the average level of the baseline population, outputting the first calculated relative expression level. High resistance, characterized by a relatively high expression level, indicates that the TRIM23-mediated innate immune pathway is highly prepared, leading to a higher expected survival rate upon WSSV infection. Moderate resistance, characterized by a relatively low expression level, increases the risk of death under intense viral attack. The relative expression level was used to assess the WSSV resistance of *Procambarus clarkii*.
[0024] Figure 2 The flowchart of the method for calculating the Ct value of the TRIM23 gene is shown, including the following steps: S202: The TRIM23 gene in the cDNA product was quantitatively detected by probe method to obtain the Ct value of the TRIM23 gene; S204: The target Ct value is obtained by screening through experimental control during the Ct value calculation process.
[0025] Furthermore, in a preferred embodiment of the present invention, the quantitative detection of the TRIM23 gene in the cDNA product using a probe method to obtain the Ct value of the TRIM23 gene specifically involves: By introducing a big data network, and based on the big data network, the most frequently used amplification primer type for the TRIM23 gene in Procambarus clarkii was retrieved and identified as the target amplification primer type. Based on the target amplification primer type, amplification primers corresponding to the target amplification primer type are added to the cDNA product. The amplification primers corresponding to the target amplification primer type are used to locate the TRIM23 gene in the cDNA product to obtain the located TRIM23 gene. The amplification primers corresponding to the target amplification primer type are primer pairs and are labeled as target amplification primer pairs. TaqMan hydrolysis probe technology was introduced and applied to the TRIM23 gene after localization in cDNA products to generate nucleic acid probes for target amplification primer pairs. The TRIM23 gene was amplified by PCR, and the nucleic acid probe of the target amplification primer pair was detected by real-time fluorescence signal detection. The real-time fluorescence signal was detected by a real-time fluorescence quantitative PCR instrument, and the fluorescence value of the real-time fluorescence signal was read. A standard fluorescence value was preset. When the fluorescence value of the real-time fluorescence signal was equal to the standard fluorescence value, the PCR amplification was stopped, and the PCR amplified TRIM23 gene was obtained. The number of PCR amplification cycles required for the real-time fluorescence signal to equal the standard fluorescence value is calculated and denoted as the Ct value. The target Ct value was obtained by screening through experimental control during the Ct value calculation process.
[0026] It should be noted that the TRIM23 gene is the primary gene used in *Procambarus clarkii* for resistance to WSSV. First, the TRIM23 gene needs to be amplified by PCR to achieve precise gene quantification. The purpose of the amplification primers is to accurately identify and label the TRIM23 gene, binding to both ends to obtain the target amplification primer pair. Subsequently, using TaqMan probe hydrolysis technology, a heterologous oligonucleotide probe is generated on the primer pair. This probe is used to label a fluorophore. When the probe is intact, no fluorescence signal is observed; after hydrolysis, a detectable fluorescence signal is generated, indicating that PCR amplification is complete. The fluorescence value of the real-time fluorescence signal needs to be read, and PCR amplification is stopped when the fluorescence value equals the standard fluorescence value, yielding the PCR-amplified TRIM23 gene. Afterward, the Ct value needs to be calculated to provide direct evidence for the next step of computer-based determination of relative expression level and resistance grade. Fluorescence value calculation and analysis are performed using a real-time quantitative PCR instrument suitable for TaqMan detection; the Ct value is the number of PCR amplification cycles.
[0027] Furthermore, in a preferred embodiment of the present invention, the step of obtaining the target Ct value through an experimental control method during the Ct value calculation process specifically involves: Meanwhile, PCR amplification of the TRIM23 gene was performed in different test tubes, and the Ct values in different test tubes were calculated. During the PCR amplification of the TRIM23 gene, an experimental control group was set up. In the experimental control group, pure water was used to replace the cDNA product to detect whether contamination occurred during the PCR amplification of the TRIM23 gene. If contamination occurs, the TRIM23 gene PCR amplification process is repeated. If no contamination occurs, the Ct values in different test tubes are analyzed, and the test tube with the smallest Ct value is selected as the target test tube, and the corresponding Ct value is labeled as the target Ct value.
[0028] It should be noted that PCR amplification of the TRIM23 gene was performed simultaneously in different test tubes, and the Ct values in each tube were calculated. The purpose was to select a more suitable Ct value as a baseline. Because experimental conditions cannot be completely identical in different test tubes, some tubes may have larger and more standard Ct values for the gene. The purpose of setting up an experimental control group was to detect whether contamination occurred during the PCR amplification process. If so, re-amplification was necessary, as contamination can lead to bias in the assessment of WSSV resistance in *Procambarus clarkii*. Finally, the target Ct value was obtained.
[0029] like Figure 3 As shown, a second aspect of the present invention also provides a system for evaluating the WSSV resistance of the red swamp crayfish. The 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 method program for evaluating the WSSV resistance of the red swamp crayfish, which has an evaluation engine. When the program is executed in parallel through a superscalar pipeline execution unit within the processor, the following steps are implemented: The *Procambarus gracilis* samples to be evaluated were pretreated, and target tissue samples were selected and collected after pretreatment, while the target tissue samples were preserved. Total RNA was extracted from the target tissue sample, and the concentration of the extracted total RNA was precisely quantified and cDNA was synthesized. The TRIM23 gene in the cDNA product was quantitatively detected using a probe method, and the Ct value of the TRIM23 gene was obtained. By combining the target Ct value of the TRIM23 gene, a relative quantitative mathematical model was constructed to evaluate the anti-WSSV ability of the target crayfish sample.
[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 evaluating the resistance of *Procambarus clarkii* to WSSV, characterized in that, Includes the following steps: The *Procambarus gracilis* samples to be evaluated were pretreated, and target tissue samples were selected and collected after pretreatment, while the target tissue samples were preserved. Total RNA was extracted from the target tissue sample, and the concentration of the extracted total RNA was precisely quantified and cDNA was synthesized. The TRIM23 gene in the cDNA product was quantitatively detected using a probe method, and the Ct value of the TRIM23 gene was obtained. By combining the target Ct value of the TRIM23 gene, a relative quantitative mathematical model was constructed to evaluate the anti-WSSV ability of the target crayfish sample.
2. The method for evaluating the resistance of *Procambarus clarkii* to WSSV according to claim 1, characterized in that, The process involves pre-treating the *Procambarus clarkii* to be evaluated, selecting and collecting target tissue samples after pre-treatment, and simultaneously preserving the target tissue samples. Specifically: The culture ponds for raising Procambarus clarkii were identified and designated as target culture ponds. Procambarus clarkii samples were collected from the target culture ponds and placed in the laboratory. The caught Procambarus grahami samples were screened by weight. Samples that were not within the standard weight range were removed. Among the remaining Procambarus grahami samples, those with physical defects were screened again to obtain the target Procambarus grahami samples. The target Procambarus gracilis sample was temporarily pre-treated in the laboratory to obtain the target pre-treated Procambarus gracilis sample. The target tissues of the pretreated Procambarus cristatus sample were selected and collected. Specifically, the hemolymphocyte tissue and gill tissue of the pretreated Procambarus cristatus sample were selected as target tissues. At the same time, the target tissues of the pretreated Procambarus cristatus sample were collected to obtain target tissue samples. Finally, the target tissue samples were preserved in liquid nitrogen.
3. The method for evaluating the resistance of *Procambarus clarkii* to WSSV as described in claim 1, characterized in that, The process of extracting total RNA from the target tissue sample, accurately quantifying the concentration of the extracted total RNA, and synthesizing cDNA specifically involves: The target tissue samples were classified to obtain hemolymphocyte tissue samples for gill tissue sampling; The blood lymphocyte tissue sample was thawed and placed in a centrifuge tube with lysis buffer for centrifugation. Centrifugation was stopped when no precipitate of blood lymphocyte tissue sample appeared in the centrifuge tube. The gill tissue sample was ground in liquid nitrogen, and the ground gill tissue sample was transferred to a centrifuge tube and mixed with the hemolymphocyte tissue sample to obtain the target lysis mixture; The target lysis mixture was subjected to secondary lysis in a centrifuge tube, and the supernatant was extracted after lysis and transferred to a new centrifuge tube. Ethanol was added and mixed to obtain the secondary lysis mixture. An RNA adsorption column was obtained, and protein buffer and washing solution were injected into the RNA adsorption column in sequence. The secondary lysis mixture was then washed with the RNA adsorption column to obtain the total RNA sample of the target tissue sample, i.e., the total RNA eluent. A sample of total RNA elution buffer was taken, and a micro spectrophotometer was used to detect the concentration of total RNA in the sample. The sample of total RNA elution buffer was then subjected to electrophoresis using agarose gel. After electrophoresis, the integrity of the total RNA elution buffer was assessed using an ultraviolet imaging system, and cDNA products were synthesized using total RNA elution buffer with acceptable sample integrity.
4. The method for evaluating the resistance of *Procambarus clarkii* to WSSV as described in claim 1, characterized in that, The method involves quantitatively detecting the TRIM23 gene in the cDNA product using a probe method to obtain the Ct value of the TRIM23 gene. Specifically: By introducing a big data network, and based on the big data network, the most frequently used amplification primer type for the TRIM23 gene in Procambarus clarkii was retrieved and identified as the target amplification primer type. Based on the target amplification primer type, amplification primers corresponding to the target amplification primer type are added to the cDNA product. The amplification primers corresponding to the target amplification primer type are used to locate the TRIM23 gene in the cDNA product to obtain the located TRIM23 gene. The amplification primers corresponding to the target amplification primer type are primer pairs and are labeled as target amplification primer pairs. TaqMan hydrolysis probe technology was introduced and applied to the TRIM23 gene after localization in cDNA products to generate nucleic acid probes for target amplification primer pairs. The TRIM23 gene was amplified by PCR, and the nucleic acid probe of the target amplification primer pair was detected by real-time fluorescence signal detection. The real-time fluorescence signal was detected by a real-time fluorescence quantitative PCR instrument, and the fluorescence value of the real-time fluorescence signal was read. A standard fluorescence value was preset. When the fluorescence value of the real-time fluorescence signal was equal to the standard fluorescence value, the PCR amplification was stopped, and the PCR amplified TRIM23 gene was obtained. The number of PCR amplification cycles required for the real-time fluorescence signal to equal the standard fluorescence value is calculated and denoted as the Ct value. The target Ct value was obtained by screening through experimental control during the Ct value calculation process.
5. The method for evaluating the resistance of *Procambarus clarkii* to WSSV according to claim 4, characterized in that, The process of calculating Ct values involves using an experimental control method to screen and obtain the target Ct value, specifically as follows: Meanwhile, PCR amplification of the TRIM23 gene was performed in different test tubes, and the Ct values in different test tubes were calculated. During the PCR amplification of the TRIM23 gene, an experimental control group was set up. In the experimental control group, pure water was used to replace the cDNA product to detect whether contamination occurred during the PCR amplification of the TRIM23 gene. If contamination occurs, the TRIM23 gene PCR amplification process is repeated. If no contamination occurs, the Ct values in different test tubes are analyzed, and the test tube with the smallest Ct value is selected as the target test tube, and the corresponding Ct value is labeled as the target Ct value.
6. The method for evaluating the resistance of *Procambarus clarkii* to WSSV according to claim 1, characterized in that, The target Ct value of the TRIM23 gene was used to construct a relative quantitative mathematical model to evaluate the anti-WSSV ability of the target Procambarus clarkii samples. Specifically: Based on the target Ct value, the Ct values of different test tubes are standardized and corrected. That is, the target Ct value is used as the benchmark value, the difference between the Ct value of different test tubes and the target Ct value is calculated, and the sample difference status of TRIM23 gene in different test tubes and the target test tube is calculated based on the difference. The difference is designated as the first difference. Using big data networks, the Ct values of the TRIM23 gene in a population of *Procambarus clarkii* that has been verified and has qualified resistance to WSSV were obtained, and compared with the target Ct value. The difference was calculated and labeled as the second difference. The difference between the first difference and the second difference is obtained, and the relative expression level is calculated based on the difference between the first difference and the second difference, wherein the relative expression level is an indicator of the anti-WSSV ability of the target crayfish sample; The relative expression levels are analyzed, and the classification threshold of the relative expression levels is retrieved through a big data network. Based on the classification threshold of the relative expression levels, the resistance to WSSV in *Procambarus swampus* in the target culture pond is evaluated and output.
7. A system for evaluating the resistance of *Procambarus clarkii* to WSSV, characterized in that, The 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 method program for evaluating the WSSV resistance of the red swamp crayfish with an evaluation engine, and when the program is executed in parallel through the superscalar pipeline execution unit in the processor, it implements the steps of evaluating the WSSV resistance of the red swamp crayfish as described in any one of claims 1-6.