Target gene and detection reagent for identifying carp edema virus and application of target gene and detection reagent in breeding of carp fry without carp edema virus

Abstract

The invention belongs to the field of disease prevention and control in aquaculture, and particularly relates to a target gene and a detection reagent for identifying a carp edema virus and application of the target gene and the detection reagent in breeding of carp fries without the carp edema virus. The invention discovers that DNA-binding protein can be used as a specific target gene for identifying the carp edema virus for the first time. According to the present invention, the RAA amplification technology and the CRISPR / Cas12a system are combined to establish the system capable of rapidly detecting the CEV virus on site, and the method has advantages of high detection sensitivity, convenient operation and rapid reaction, and can be used for monitoring the CEV pathogen retention condition on the breeding site. Furthermore, a method suitable for rapid detection of the carp edema virus on a culture site is established. CEV pathogen purification and purification maintenance and parent artificial breeding in a pathogen-free environment are realized. According to the method, CEV pathogen purification and purification maintenance of the cultured carp are realized, the culture rate of the cultured carp is increased, and the use of fish medicine is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of aquaculture disease prevention and control, and particularly relates to a target gene for identifying carp edema virus, a detection reagent and application of the target gene and the detection reagent in breeding of carp fry free of carp edema virus. BACKGROUND

[0002] Carp and koi carp play an important role in aquaculture in China. With the continuous expansion of the scale of aquaculture, various fish diseases have emerged, causing serious harm to aquaculture. Carp edema virus disease (CEVD), also known as koi sleepy disease (KSD), is a viral infectious disease caused by carp edema virus (CEV) that leads to the death of cyprinid fish. It mainly infects carp and koi carp. The main pathological changes of the diseased fish are pale necrosis of the gills, sunken eyes, erosive or hemorrhagic skin lesions, systemic edema, hyperemia and ulceration around the mouth and tail fin, and excessive mucus secretion on the skin and gills. The disease was first discovered and reported in koi carp in Japan in 1970. The high incidence period of the disease is from June to July each year, and the disease can occur at water temperatures of 7-27℃. The diseased fish moves slowly and usually gathers on the water surface or in the corner of the pond, showing a sleepy state. When touched, the diseased fish will swim for a while and then return to a sleepy state. The disease course is generally 7-10 days, and the mortality rate of the diseased fish can be as high as 70-100% within two weeks. Therefore, the establishment of a carp edema virus-free breeding farm is of great significance for reducing the harm of viral infection to carp (koi carp) and improving the quality and efficiency of aquaculture.

[0003] Carp edema virus (CEV) is the pathogen of CEVD. CEV belongs to the family of poxviridae and is a linear DNA double-stranded virus. The CEV virion is between 250-400 nm and appears like a mulberry under an electron microscope, has a capsule, and is one of the largest viruses. The currently reported CEV detection methods all use the gene as the detection target gene. Although it has been reported that the gene has good conservation and heterogeneity, the gene fragment also has sufficient variation for distinguishing different genotypes of CEV isolates such as gI and gII. However, it is precisely because the gene is highly conserved that it is difficult to distinguish different genotypes of CEV isolates, and the detection method is not sensitive enough. P4a As the gene is highly conserved, it is difficult to distinguish different genotypes of CEV isolates, and the detection method is not sensitive enough. P4aThe differences in genes among different isolates lead to certain clinical false negatives in existing CEV detection methods. Therefore, the current high carrier rate of carp (koi) seedlings makes pathogen eradication impossible. In conventional carp (koi) seedling production, CEVD can infect offspring through vertical transmission from pathogen-carrying parents, or through horizontal transmission between seedlings due to environmental viral contamination. These infected seedlings are highly susceptible to hemorrhagic disease outbreaks when subjected to stress during cultivation. Selecting appropriate target genes to improve the detection rate of pathogen-carrying carp (koi) and enhance the accuracy of CEV pathogen detection is one of the most effective ways to cut off the CEV transmission chain at its source, ensuring successful cultivation and industry benefits. Simultaneously, establishing accurate, reliable, rapid, and convenient on-site CEV rapid detection technology can provide technical support for establishing a stable, efficient, and scalable CEV-SPF seedling breeding system, addressing key technical aspects such as parent purification, environmental isolation, and pathogen blocking.

[0004] RAA technology enables rapid, isothermal amplification of DNA in vitro, and the Cas12a protein can be used to edit target DNA. A system consisting of CRISPR-Cas12a (Cpf1), guide RNA, fluorescent reporter molecules, and RPA (recombinase polymerase amplification) reagents allows for rapid, convenient, and immediate detection of small amounts of DNA in clinical samples using a single-tube reaction. The combination of RAA technology and CRISPR / Cas12a technology improves detection sensitivity and simplifies result interpretation.

[0005] This invention analyzes the whole genomes of CEV strains isolated from Asia, Europe, and the Americas published on NCBI, screens genes encoding proteins related to viral genome replication and transcription as candidate genes, and systematically analyzes the homology of these candidate genes among different prevalent CEV strains. It identifies highly conserved gene sequences among prevalent strains in different regions as target genes for detection, establishes a RAA-CRISPR / Cas12a method for rapid on-site diagnosis of CEVD, and provides a method for establishing virus-free carp (koi) farms. Virus monitoring begins from the introduction of carp (koi) fry, enabling the purification and maintenance of CEV pathogens in carp (koi) farms, thereby improving the yield and quality of carp (koi). Summary of the Invention

[0006] The first aspect of the present invention aims to provide a detection method. DNA-binding protein Application of gene-based reagents in the preparation of products for identifying carp edema virus.

[0007] A second aspect of the present invention is to provide a set of reagents for detecting carp edema virus.

[0008] A third aspect of the present invention is to provide a reagent kit.

[0009] The fourth aspect of this invention aims to provide the use of the reagents and kits of the second and third aspects of this invention in detecting carp edema virus for non-diagnostic and therapeutic purposes and / or in preparing products for identifying carp edema virus.

[0010] The fifth aspect of this invention aims to provide a method for detecting carp edema virus for non-diagnostic purposes.

[0011] The sixth aspect of the present invention is to provide a method for breeding carp fry free of carp edema virus pathogen.

[0012] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: The first aspect of the present invention provides a detection DNA-binding protein Application of gene-based reagents in the preparation of products for identifying carp edema virus.

[0013] The DNA-binding protein (BCT22629.1) gene shows good homology in different genotypes of CEV isolates and can be used as a specific target gene for CEV.

[0014] A second aspect of the invention provides a set of reagents comprising primer pairs for detecting crRNA of carp edema virus (CEV) and / or amplifying carp edema virus (CEV).

[0015] In some embodiments of the present invention, the primer pair sequences are shown in SEQ ID NO.3 and SEQ ID NO.4; the crRNA sequence is shown in SEQ ID NO.8.

[0016] A third aspect of the present invention provides a reagent kit comprising the reagents described in the second aspect of the present invention.

[0017] In some embodiments of the present invention, the kit includes cas12a protein and / or signal reporter probe and / or positive standard.

[0018] A fourth aspect of the present invention provides the use of the reagents and kits of the second and third aspects of the present invention in detecting carp edema virus for non-diagnostic and therapeutic purposes and / or in preparing products for identifying carp edema virus.

[0019] A fifth aspect of the present invention provides a method for detecting carp edema virus for non-diagnostic purposes, comprising the step of detecting a sample to be tested using the kit described in the third aspect of the present invention.

[0020] The steps include: (1) Extract DNA from the sample to be tested; (2) Using the DNA extracted in step (1) as a template, amplification was performed using primer pairs to obtain the amplification product; (3) Take the amplification product obtained in step (2), add cas12a protein and crRNA, perform CRISPR reaction detection, read the detection signal, and determine the detection result based on the detection signal.

[0021] In some embodiments of the present invention, the amplification conditions in step (2) are 39 °C for 30 minutes.

[0022] In some embodiments of the present invention, the amplification system in step (2) includes: A Buffer, primer pair, template DNA, water, B Buffer, reaction powder, and phenol:chloroform:isoamyl alcohol (25:24:1) extraction solution.

[0023] In some embodiments of the present invention, the amplification system in step (2) includes: 25 μL of A Buffer, 2 μL each of upstream and downstream primers (primer concentration is 10 µM), 5 μL of DNA, 13.5 μL of water, 2.5 μL of B Buffer, 1 tube of reaction powder, and 50 μL of phenol:chloroform:isoamyl alcohol (25:24:1) extraction solution.

[0024] In some embodiments of the present invention, the CRISPR reaction detection conditions in step (3) are 37 °C for 30 to 60 minutes.

[0025] In some embodiments of the present invention, the CRISPR reaction detection system in step (3) includes Cleavage Buffer, signal reporter probe, Cas12a protein, crRNA, and amplification product.

[0026] In some embodiments of the present invention, the CRISPR reaction detection system in step (3) consists of 2 μL Leavage Buffer (10 ×), 0.6 μL Reporter probe (4 μM), 1 μL Cas12a protein (1 μM), 1 μL crRNA (Cas12a) (1 μM), 13.4 μL Nuclease-free H2O, and 2 μL amplification product from step (2).

[0027] A sixth aspect of the present invention provides a method for breeding carp fry free of carp edema virus pathogen, comprising the following steps: 1) Using the kit described in the third aspect of this invention, the parent plants were tested for carp edema virus, and the parent plants carrying the virus were eliminated to obtain the parent population; 2) Artificial egg collection and insemination were performed on the parent population to obtain fertilized eggs; 3) Place the fertilized eggs in the fish nest and disinfect them with povidone-iodine; 4) Use the kit described in the third aspect of this invention to test the fertilized eggs, discard the fertilized eggs carrying the virus, and hatch the virus-free fertilized eggs into fry. 5) Using the kit described in the third aspect of this invention, the fry are tested, and the fry carrying the virus are eliminated to obtain carp seedlings free of carp edema virus pathogen.

[0028] In some embodiments of the present invention, the disinfection concentration of povidone-iodine is 18~22 mg / L; In some embodiments of the present invention, the disinfection time of the povidone-iodine is 24-48 hours.

[0029] In some embodiments of the present invention, reserve parent lines that do not carry the CEV pathogen are selected and introduced into the breeding farm for rearing. They are disinfected with povidone-iodine and the detection method of the fifth aspect of the present invention is used to regularly detect the virus. All seedlings carrying the virus are eliminated to obtain reserve parent lines that do not carry the virus.

[0030] In some embodiments of the present invention, healthy, mature, and virus-free reserve parent stock are selected and then nutritionally fortified to cultivate specific pathogen-free koi (carp) parent stock.

[0031] In some embodiments of the present invention, cultivating a virus-free parent population includes performing virus testing on the reserve parents every 7 days using the detection method of the fifth aspect of the present invention, eliminating individuals carrying the virus and all seedlings in the cultivation space, and conducting at least 3 batches of virus testing. By adopting the above scheme, during the reserve parent cultivation stage, virus transmission in the reserve parents is prevented through layer-by-layer screening, thus avoiding the transmission of the virus to the next generation of seedlings.

[0032] In some embodiments of the present invention, prenatal nutritional fortification is performed on the parent plants. Since the parent plants are in their reproductive physiological cycle before birth, their energy consumption and demands are high. Malnutrition at this time can provide an opportunity for viral invasion. Prenatal nutritional fortification with nutritional fortifiers can improve the parent plants' own immunity and simultaneously improve the health of offspring, resulting in high-quality seedlings.

[0033] The beneficial effects of this invention are: This invention is the first discovery DNA-binding protein(BCT22629.1) can serve as a specific target gene for identifying carp edema virus. Furthermore, by combining RAA amplification technology and the CRISPR / Cas12a system, a system for rapid on-site detection of CEV virus was established. This method has the advantages of high detection sensitivity, convenient operation, and rapid response, and can be used to monitor the persistence of CEV pathogens in aquaculture sites.

[0034] This invention further analyzes the safety of povidone-iodine as a CEV inactivating agent, evaluating its effectiveness in inactivating pathogens in production equipment, aquaculture water, and fertilized eggs, and determining the appropriate concentration and treatment time. A rapid CEV detection method and virus inactivating agent are prepared, and CEV-free koi carp (breeding carp) are screened and bred as replacement broodstock. CEV-SPF koi carp (breeding carp) seedlings are produced using the bred CEV-free broodstock. The embodiments of this invention are simple and easy to learn, can be scaled up, and the obtained CEV-SPF koi carp (breeding carp) seedlings have a high survival rate. Attached Figure Description

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 For different genotypes of CEV DNA-binding protein Systematic Evolutionary Analysis of Genes Figure 2 The results of CEV-RAA primer screening were obtained by using CEV-positive DNA as a template and performing PCR amplification with three pre-designed primers; M: DL2000 DNA marker; 1, 2, and 3 are the screening primer numbers, and N is the negative control.

[0036] Figure 3 The results are for crRNA screening, where (a) is the fluorescence color development result under UV light source, and (b) is the fluorescence intensity quantification result.

[0037] Figure 4 The results show the sensitivity assays for CEV RAA-Cas12a and PCR methods, where: (a) 1.23 × 10⁻⁶ under 470 nm blue light and UV light source. 6 ~1.23×10 0 (a) Fluorescence intensity of RAA-CRISPR / Cas12a using 7 concentration standard plasmids as templates (copies / μL); (b) Fluorescence curve of RAA-CRISPR / Cas12a; (c) Results of sensitivity test of nested PCR method for primary amplification; NC: negative control; (d) Results of sensitivity test of nested PCR method for secondary amplification; NC: negative control.

[0038] Figure 5The results are CEV RAA-Cas12a specificity test results, where: (a) is the fluorescence color development result under UV light source, and (b) is the fluorescence intensity quantification result.

[0039] Figure 6 The results of CEV detection in different samples using RAA-Cas12a and PCR methods are shown, where: (a) is the fluorescence color development result under UV light source, and (b) is the PCR amplification result. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0041] Example 1: Screening of specific conserved gene fragments of carp edema virus Using the genome sequence of the Asian isolate FTI2020 of CEV as a reference sequence (GenBank: LC613089.1), the full-length genome of CEV FTI2020 is 456,821 bp, with a G+C content of 28.8%, and is predicted to contain 392 genes (some of which have no annotated proteins). CEV FTI2020 is classified as an isolate of CEV genome group IIa.

[0042] Based on the published CEV genome sequence, multiple sequence homology analysis was performed using bioinformatics software: BLAST, ClustalW, and Geneious. Fourteen genes related to viral genome structure, replication, and transcription were preliminarily screened as candidate conserved target sequences. These are: DNA-binding protein (BCT22629.1), NTPase (BCT22635.1), putative membrane protein (BCT22636.1), deoxynucleoside monophosphate kinase (BCT22638.1), late transcription factor VLTF-2 (BCT22643.1), late genetranscription factor VLTF-3 (BCT22644.1), virion core protein P4b (BCT22646.1), RNA polymerase subunit RPO19 (BCT22648.1), VITF-3 subunit protein (BCT22651.1), 4a protein (BCT22653.1), and viral membrane formation. protein (BCT22654.1), divergentFlap Endonuclease (BCT22659.1), DNA helicase (BCT22664.1) and putative apurinic-apyrimidinic endonuclease 1 (BCT22665.1) genes.

[0043] The conserved genes obtained from the initial screening were sequence aligned using MEGA to construct a phylogenetic tree, and the genetic relationships among different strains were analyzed. Through sequence alignment and phylogenetic analysis, the conservation and representativeness of the selected fragments were further confirmed.

[0044] Analysis results as follows Figure 1 As shown, among all candidate genes, the DNA-binding protein (BCT22629.1) gene showed good homology in different genotype CEV isolates and can be used as a specific target gene for CEV to establish nucleic acid diagnostic methods.

[0045] Example 2: Establishment of a rapid on-site detection method for carp edema virus 1. Reagents and Instruments RAA Nucleic Acid Amplification Reagent (Basic Type) (S001ZC), Hangzhou Zhongce Biotechnology Co., Ltd.; CRISPR / Cas12a DNA Detection Kit (DF-CAS12-2S), Shenzhen Yizhi Biotechnology Co., Ltd.; PCR Master Mix, Shandong Sikejie Biotechnology Co., Ltd.; pEASY-T1 Cloning Kit T1 Gene Cloning Kit (Double Antibiotic), Beijing TransGen Biotechnology Co., Ltd.; LB Agar Plate (Ampicillin Antibiotic), Sangon Biotech (Shanghai) Co., Ltd.; Gel Extraction / PCR Product Purification Kit, Shandong Sikejie Biotechnology Co., Ltd.; SPARKeasy Plasmid Mini-Range Rapid Extraction Kit, SPARKeasy Viral Genomic DNA Extraction Kit, Shandong Sikejie Biotechnology Co., Ltd.; Phenol, Chloroform, Isoamyl Alcohol, Wuhan Chucheng Zhengmao Technology Engineering Co., Ltd.

[0046] 2. Preparation of standard templates Using DNA from CEV-positive samples as templates, the target gene sequence was amplified by conventional PCR to obtain the target product. The PCR product was then recovered from the gel using an agarose gel extraction kit. Following the pEASY-T1 Cloning Kit instructions, 1 μL of pEASY-T1 Cloning Vector and 4 μL of the recovered PCR product were added to a 200 μL centrifuge tube. The mixture was gently stirred and incubated at 25 °C (controlled by the PCR instrument) for 5 minutes. The ligation product was then transformed into 50 μL of Trans1-T1 competent cells. After shaking, the product was plated onto a solid medium containing ampicillin and incubated at 37 °C to form single colonies. White single colonies were selected for PCR identification of positive clones. The PCR amplification products were detected by gel electrophoresis, and the PCR results were used to determine whether a clone was positive. A single colony grown on the plate from the previous step was picked and inoculated into 5 mL of LB broth containing 2.5 μL of ampicillin for shaking culture. The positive bacterial culture was cultured in a large system and plasmids were extracted. The concentration of recombinant plasmid DNA was determined to be 59.305 ng / μL using an ultra-micro spectrophotometer. The plasmid concentration was calculated using the formula (6.02 × 10⁻⁶). 23 ) × plasmid concentration (ng / μL) × 10 -9 / (DNA length × 660) = copies / μL. The initial concentration of the plasmid standard for determination is 1.23 × 10⁻⁶. 10 (copies / μL) The recombinant plasmid was used as the stock solution of the standard in this experiment and stored at –20 ℃.

[0047] 3. Establishment of CEV RAA--CRISPR / Cas12a detection method Primer and crRNA design. The industrial synthesis of oligonucleotides (primers and crRNAs) was carried out by Sangon Biotech (Shanghai) Co., Ltd. Specific information is shown in Tables 1 and 2.

[0048] Table 1 CEV-RAA primers

[0049] Table 2 CEV RAA crRNA

[0050] Primer and crRNA screening. Using CEV-positive DNA as a template, RAA-AGE detection was performed. 25 μL of A Buffer, 13.5 μL of Nuclease-free H2O, and 2 μL each of the RAA forward and reverse primers (both at 10 µM) were mixed thoroughly and added to a detection unit tube containing the reaction powder. Then, 5 μL of the DNA sample to be tested was added. Finally, 2.5 μL of B Buffer was added to the cap of the detection unit tube. The tube was capped, and the mixture was gently shaken up and down 5-6 times to mix thoroughly. The tube was then centrifuged at low speed for 10 seconds. The detection unit tube was incubated at 39℃ for 30 min. After the reaction, 50 μL of phenol:chloroform:isoamyl alcohol (25:24:1) extraction buffer was added for purification. After thorough mixing, the mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was collected for electrophoresis detection.

[0051] See results Figure 2 , 3 Based on the brightness of the bands in agarose gel electrophoresis, a pair of primers F2 / R2 with good performance was selected. Using CEV-positive DNA as a template, the optimal primer combination was used to screen three groups of crRNA reactions using the RAA-Cas12a detection method. The optimal crRNA primer set was selected based on the fluorescence value and peak time of the amplification curve. According to the time and fluorescence intensity of the RAA-CRISPR / Cas12a fluorescence curve, the optimal crRNA was determined to be CEV-crRNA-2.

[0052] Determination of optimal reaction conditions for RAA-CRISPR / Cas12a. The first step amplification reaction system for RAA-CRISPR / Cas12a was as follows: 25 μL A Buffer, 13.5 μL water, 2 μL each of RAA forward and reverse primers (primer concentration 10 µM), mixed thoroughly and added to a detection unit tube containing reaction powder. 5 μL of DNA sample was added to the detection unit tube, followed by 2.5 μL of B Buffer to the tube cap, and mixed thoroughly. The detection unit was incubated in a constant temperature water bath at 37 ℃, 38 ℃, 39 ℃, and 40 ℃ for 30 min, respectively. The detection unit was then incubated at 38 ℃ for 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min, respectively. After the reaction, electrophoresis analysis was performed to determine that the optimal reaction conditions for the first step were 39 ℃ for 30 min.

[0053] The second-step detection reaction system of RAA-CRISPR / Cas12a is as follows: 2 μL Cleavage Buffer (10 ×), 0.6 μL Reporter (4 μM), 1 μL Cas12a protein (1 μM), 1 μL crRNA (Cas12a) (1 μM), 13.4 μL Nuclease-free H2O, and finally 2 μL of the first-step amplification product of RAA-CRISPR / Cas12a. The reaction tubes were incubated at 37 ℃, 38 ℃, 39 ℃, and 40 ℃ for 30 min, respectively. The detection unit was incubated at 38 ℃ for 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min, respectively. The optimal reaction conditions (37 ℃ for 30 min) were determined by the time and fluorescence intensity of the fluorescence curves.

[0054] 4. Evaluation of the CEV RAA--CRISPR / Cas12a detection method Sensitivity analysis. The positive plasmid was diluted to 1.23 × 10⁻⁶. 6 ~1.23×10 0 Seven concentration gradients were established using copies / μL. The detection sensitivity of this method was determined by amplification curves using the diluted positive plasmids and RNase-free water as templates, following the method established in step 3.

[0055] The results are as follows Figure 4 As shown, the minimum detection limit is 1.23 × 10⁻⁶. 0The detection sensitivity was improved by 1000 times compared with the CEV detection method recommended by the current aquatic industry standard. The blank control showed no obvious fluorescence signal.

[0056] Specificity analysis. Clinical samples containing CEV-positive DNA, SVCV-positive DNA, GCRV-positive DNA, KHV-positive DNA, and LMBV-positive DNA were selected as amplification templates. Nuclease-free water was used as a negative control. The detection specificity of this invention was evaluated using the detection method established in step 3.

[0057] The results are as follows Figure 5 As shown, only CEV samples showed amplification curves, while blank control (water without nuclease), SVCV positive DNA, GCRV positive DNA, KHV positive DNA, and LMBV positive DNA samples did not show amplification. This indicates that the CEV RAA-CRISPR / Cas12a detection method established in this invention does not cross-react with other pathogens and has good specificity.

[0058] Analysis of clinical sample detection. Nineteen carp (koi) samples preserved in the laboratory were tested using the method established in step 3 and the current recommended testing method in the aquaculture industry standard. The consistency of the test results was analyzed, and the results are as follows: Figure 6 As shown, out of 19 samples, 5 were positive, with a positive rate of 26.32%. The concordance rate between the two methods was 100%.

[0059] Example 3: Preparation and Evaluation of a CEV Disinfectant for Koi (Carp) Fertilized Eggs and Larvae Povidone-iodine is an effective disinfectant for inactivating CEVs and is also a disinfectant for aquatic use as stipulated by the Ministry of Agriculture and Rural Affairs of my country. However, the disinfection effect and toxic side effects on fertilized eggs and larvae vary considerably depending on the concentration and disinfection time. This invention uses a static aquatic organism acute toxicity test method to determine the appropriate concentration of povidone-iodine for inactivating the virus in larvae and fertilized eggs.

[0060] During the evaluation process, no feeding or water changes were performed, and the oxygen content of the water was maintained using an aerator. Based on the effective inactivation dose of povidone-iodine for CEVs, five different concentration groups were set up, with three replicates for each concentration group, and one blank control group. After preparing the disinfectant, randomly selected fertilized eggs and larvae were placed in a plastic box containing the disinfectant. The hatching rate of fertilized eggs and the mortality rate of larvae were statistically analyzed after treatment with different concentrations of disinfectant. The results are shown in Tables 4 and 5. Soaking larvae or fertilized eggs in a 20 mg / L povidone-iodine solution for 36 hours was found to be the optimal disinfection dose for koi (carp) fertilized eggs and larvae.

[0061] Table 4. Hatching rates of fertilized eggs soaked in povidone-iodine at different concentrations for different times.

[0062] Table 5. Survival rate of fry after soaking in different concentrations of povidone-iodine for different times

[0063] Example 4: A method for breeding CEV-free koi carp (koi) parents 1. Preparations before breeding CEV backup parents The farm has pathogen isolation facilities. Multiple systems are in place for diagnosis, isolation, and harmless disposal of dead animals. Harmless disposal measures comply with the relevant provisions of the "Technical Specifications for Harmless Disposal of Dead and Diseased Animals." Manure and waste in the production area are stored in a designated area and cleaned regularly. Facilities or measures are in place to prevent rodents, insect vectors, dogs, cats, and birds from entering. The farm entrance should have a vehicle disinfection pool, full-vehicle disinfection facilities, and personnel disinfection facilities. Vehicles and personnel entering and exiting the farm should be disinfected according to regulations. Personnel disinfection, shower, and changing facilities should be provided at the production area entrance.

[0064] Disinfection measures for production tools and aquaculture water. The disinfectant determined in Example 3 shall be used to disinfect the production tools and aquaculture water. After disinfection, the aquaculture water shall be tested for pathogens using the CEV detection technology established in Example 2, and the test result should be negative. Furthermore, the water quality of the aquaculture water should meet the requirements of "Fishery Water Quality Standard GB 11607-89".

[0065] 2. Screening and breeding of CEV-free parent stock Koi carp with no history of carp edema disease, robust physique, and no external injuries were selected and isolated in an isolation pond for 4 weeks. During this period, the feeding and swimming behaviors of the koi carp were observed daily, and individuals exhibiting abnormal feeding, swimming alone, or surfacing were culled. Weekly gill mucus samples were collected from the isolated koi carp, and pathogen detection was performed using the CEV detection technology established in Example 2. All individuals from ponds with positive CEV test results were discarded as backup broodstock. Individuals that showed no abnormal behavior after continuous observation and had all four rounds of negative pathogen test results were selected as CEV-free backup broodstock and introduced into the farm for further cultivation.

[0066] Without CEV-free broodstock, 1% of individuals were randomly selected from each pond every 4 weeks, and gill mucus was scraped for pathogen monitoring using the CEV detection technology established in Example 2. Broodstock with negative monitoring results continued to be cultured; all individuals from ponds with positive monitoring results were disposed of according to the "SC / T 7015-2022 Standard for Harmless Treatment of Dead and Diseased Aquatic Animals and Products," and all tools, water, and feed were thoroughly disinfected. After disinfection, individuals with negative CEV detection results using the technology established in Example 2 continued to be cultured and used.

[0067] Example 5: A method for producing CEV-free koi carp seedlings Containers for storing fish eggs, broodstock holding tanks, fish nests, and other facilities were disinfected using the disinfectant determined in Example 3. Production tools were then tested for pathogens using the CEV detection technology established in Example 2 to ensure they were free of pathogens. In the cultivated CEV-free broodstock population, selectively mature carp (koi) were chosen, injected with oxytocin, and isolated for observation. When male and female fish began chasing each other, they were removed and artificially inseminated for egg collection and fertilization. After artificial insemination, the fertilized eggs were quickly and evenly spread on the disinfected fish nests.

[0068] Fish nests with fertilized eggs attached to them in the spawning pond were disinfected and purified by soaking in the disinfectant determined in Example 3, and then rinsed with clean water three times for 5 minutes each time. 0.01% of the fertilized eggs were randomly collected and pathogen detection was performed using the CEV detection technology established in Example 2. For fertilized eggs with positive test results, they were disposed of according to the "SC / T 7015-2022 Standard for Harmless Treatment of Dead and Diseased Aquatic Animals and Aquatic Animal Products," and the tools, water, and feed used were thoroughly disinfected. Fertilized eggs with negative test results were transferred to the hatching pond for incubation. The incubation water temperature was 18-25 ℃, and the incubation time was 72-120 hours. After hatching, the fry attached themselves to the nests and began to swim slowly after about 24 hours, with a hatching rate of 80%-90%.

[0069] Forty-eight hours after hatching, 0.01% of the larvae were randomly collected and pathogens were detected using the CEV detection technology established in Example 2. For fertilized eggs with positive test results, they were disposed of in accordance with the provisions of "SC / T 7015-2022 Standard for Harmless Treatment of Dead and Diseased Aquatic Animals and Aquatic Animal Products", and the tools, water and feed used were thoroughly disinfected.

[0070] In this program, the fertilized eggs and fry were tested for viruses and found to be 100% non-viral, meaning that carp (koi) seedlings free of CEV pathogens were obtained.

[0071] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. detecting DNA-binding protein Use of the reagent of the gene in the preparation of the product for identifying the carp edema virus.

2. A set of reagents for detecting the carp edema virus, characterized in that: the reagents comprise a crRNA for detecting the carp edema virus and / or a primer pair for amplifying the carp edema virus; The crRNA and / or primer target DNA-binding protein a gene.

3. The reagents of claim 2, characterized in that: the sequence of the crRNA is shown in SEQ ID NO: 8; the sequences of the primer pair are shown in SEQ ID NO: 3 and 4.

4. A kit, characterized in that: the kit comprises the reagents of claim 2 or 3.

5. The kit of claim 4, characterized in that: the kit further comprises a Cas12a protein.

6. Use of the reagents of claim 2 or 3, or the kit of claim 4 or 5, for detecting the carp edema virus and / or preparing a product for identifying the carp edema virus for non-diagnostic therapeutic purposes.

7. A method for detecting the carp edema virus for non-diagnostic purposes, comprising the step of detecting a sample to be tested using the kit of claim 4 or 5.

8. The method of claim 7, characterized in that: the step comprises: (1) extracting DNA from the sample to be tested; (2) using the primer pair to amplify the DNA extracted in step (1) as a template to obtain an amplification product; (3) taking the amplification product obtained in step (2), adding a cas12a protein and a crRNA, and performing a CRISPR reaction detection, reading the detection signal, and determining the detection result according to the detection signal.

9. A method for breeding carp fry without the carp edema virus pathogen, comprising the following steps: 1) detecting the carp edema virus in the parents using the kit of claim 4 or 5, eliminating the parents carrying the virus, and obtaining a parent population; 2) artificially inseminating and fertilizing the parent population to obtain fertilized eggs; 3) placing the fertilized eggs in a fish nest and disinfecting them with povidone-iodine; 4) detecting the fertilized eggs using the kit of claim 4 or 5, eliminating the fertilized eggs carrying the virus, and hatching the fertilized eggs without the virus into fry; 5) detecting the fry using the kit of claim 4 or 5, eliminating the fry carrying the virus, and obtaining carp fry without the carp edema virus pathogen.

10. The method for breeding carp fry according to claim 9, characterized in that: the disinfecting concentration of povidone-iodine is 18-22 mg / L; the disinfecting time of povidone-iodine is 24-48 h.

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