Primer-probe set, kit and application for real-time fluorescence quantitative PCR detection of fish rhabdovirus CAPRV2023

Abstract

The present invention discloses a primer-probe set and a kit for detecting fish rhabdovirus CAPRV2023 by real-time fluorescence quantitative PCR, and also discloses a method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes; as well as the application of the above primer-probe set, kit or method in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes. The primer-probe set and the kit of the present invention have strong specificity and high sensitivity, and the method of the present invention has a short detection time, can be monitored in real time, is easy to operate and has less pollution.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus molecular detection, and particularly relates to a primer-probe set, a kit and an application for detecting Carpione rhabdovirus (CAPRV2023) in fish based on real-time fluorescence quantitative PCR, especially a primer-probe set, a kit and an application for detecting Carpione rhabdovirus CAPRV2023 in fish based on TaqMan real-time fluorescence quantitative PCR. Background Art

[0002] Rhabdoviruses are linear negative-strand single-stranded RNA viruses. The virus particles are (100-430) nm × (45-100) nm in size, and are rod-shaped or bullet-shaped in morphology, and usually have 5 main structural proteins (L, G, N, P, M). The family Rhabdoviridae includes more than 318 members and can infect vertebrates, invertebrates and plants. Rhabdoviruses in fish seriously endanger various freshwater and seawater fish due to their wide range of infected hosts, many strain types and strong virulence. So far, more than 20 rhabdoviruses infecting fish have been reported, among which the more harmful ones include Spring viremia of carp virus (SVCV), Viral hemorrhagic septicemia virus (VHSV), Infectious hematopoietic necrosis virus (IHNV), Halichoeres rubescens rhabdovirus (HRV), Siniperca chuatsi rhabdovirus (SCRV), etc. Classified into 5 genera in the family Rhabdoviridae, namely Novirhabdovirus, Perhabdovirus, Siniperhavirus, Sprivivirus and Scophrhavirus, but there are still some that have not been classified.

[0003] Trachinotus ovatus, commonly known as the golden pompano, is one of the nine key fish species developed by the National Marine Fish Industry Technology System of China. It has a fast growth rate and high economic value, and possesses excellent characteristics such as the ability to resist strong ocean currents, make full use of large aquaculture spaces, feed entirely on artificial compound feeds throughout the process, and be suitable for frozen preservation and processing. It has become one of the main species for deep-sea aquaculture in the southern marine ranches of China. The output in 2023 reached 245,000 tons, ranking second in China's marine fish production. In 2023, an acute infectious disease broke out in farmed Trachinotus ovatus, with a fast transmission speed, wide prevalence, high incidence and mortality rates, causing serious economic losses. The diseased fish showed clinical symptoms such as lethargy, anorexia, rotational movement in water, and bleeding at the base of the fins and jaws. During dissection, pale gills, a large amount of red ascites, enlarged liver and spleen, and congestion and bleeding in the liver, spleen, kidneys, and intestines were observed. Through etiological, epidemiological investigations, and whole-genome sequencing, a new virus was isolated and identified, named CAPRV2023, which is a new member of the rhabdovirus Carpione rhabdovirus and was globally discovered for the first time to be able to infect marine cultured fish. As a newly emerging infectious disease, there is no effective drug yet, and at present, it mainly focuses on prevention or cutting off the pathogen transmission route. There is currently no detection and diagnosis technology for this pathogen. Therefore, there is an urgent need to develop a method for accurately and rapidly detecting the rhabdovirus CAPRV2023 in fish, which provides technical support for clinical diagnosis and prevention and control and is of great significance for the healthy and sustainable development of the Trachinotus ovatus aquaculture industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a primer-probe set and a kit for real-time fluorescence quantitative PCR detection of the rhabdovirus CAPRV2023 in fish.

[0005] The present invention also aims to provide a method for detecting the rhabdovirus CAPRV2023 in fish for non-disease diagnosis purposes.

[0006] The last purpose of the present invention is to provide the application of the above primer-probe set or kit in the detection of the rhabdovirus CAPRV2023 in fish for non-disease diagnosis purposes.

[0007] The above first purpose of the present invention can be achieved through the following technical solutions: A primer-probe set for real-time fluorescence quantitative PCR detection of the rhabdovirus CAPRV2023 in fish, the primer-probe set includes primers and a probe, the primers include a forward primer CAPRVFq and a reverse primer CAPRVRq, the nucleotide sequence of the forward primer CAPRVFq is as shown in SEQ ID NO.1, the nucleotide sequence of the reverse primer CAPRVRq is as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3.

[0008] Preferably, the primers and probes are designed based on a specific gene with a nucleotide sequence as shown in SEQ ID NO.4.

[0009] Preferably, the 5′ end of the probe is labeled with a fluorescent group, and the 3′ end is labeled with a quenching group. The fluorescent reporter group is FAM, HEX, VIC, ROX, JOE, Cy3, TxR or Cy5, and the quenching group is BHQ1, BHQ2, BHQ3, TAMRA or MGB.

[0010] More preferably, the 5′ end of the probe is labeled with a fluorescent group, and the 3′ end is labeled with a quenching group. The fluorescent reporter group is FAM, and the quenching group is BHQ1.

[0011] The present invention also provides a kit for real-time fluorescence quantitative PCR detection of fish rhabdovirus CAPRV2023, including the primer-probe group as described above.

[0012] Preferably, the molar ratio of the forward primer, reverse primer and probe in the kit is 4.8:4.8:1.

[0013] Furthermore, the kit further includes a qPCR MIX reaction solution, a positive control and a negative control.

[0014] Preferably, the qPCR MIX reaction solution contains the following components in the following amounts: 5U Taq enzyme, 2.5 mM dNTPs, 25 mM MgCl2 and 2×PCR buffer.

[0015] Preferably, the positive control is a T-vector clone of the G protein gene fragment of rhabdovirus CAPRV2023. Its preparation method is: extracting the RNA of rhabdovirus CAPRV2023 and reverse-transcribing it into cDNA, performing a PCR amplification reaction on the template cDNA using the forward primer CAPRVFq and the reverse primer CAPRVRq to obtain DNA containing the target gene sequence, recovering the amplified fragment, and ligating it into a T-vector by a conventional cloning method, which is the positive control.

[0016] Preferably, the negative control is deionized water.

[0017] The present invention also discloses the application of the primer-probe group in the preparation of a kit for detecting fish rhabdovirus CAPRV2023.

[0018] The above second object of the present invention can be achieved by the following technical solution: A method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes, including the following steps:

[0019] (1) Extracting the RNA of the sample to be detected and reverse-transcribing it into cDNA;

[0020] (2) Using the cDNA as a template, establish a real-time fluorescence quantitative PCR reaction system and reaction conditions including the primer-probe set, and perform fluorescence quantitative PCR detection with the reaction system and reaction conditions to obtain the amplification curve of the sample to be detected.

[0021] (3) Judgment of the detection result: After the reaction ends, judge whether the sample to be detected is positive or negative for fish rhabdovirus CAPRV2023 according to the Ct value and amplification curve of the sample to be detected.

[0022] In the above detection method of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes:

[0023] Preferably, the real-time fluorescence quantitative PCR reaction system in step (2) is: 10 μL of qPCR MIX reaction solution, 1.2 μL each of the upstream and downstream primers with a concentration of 10 μmol / mL, 0.5 μL of the probe with a concentration of 5 μmol / mL, 1.0 μL of cDNA template, and supplemented with ddH2O to a total volume of 20 μL.

[0024] Preferably, the real-time fluorescence quantitative PCR reaction conditions in step (2) are: 95°C for 3 min, 95°C for 10 s, 55°C for 10 s, for 40 cycles.

[0025] Preferably, when judging the detection result in step (3), if the Ct value ≤ 35 and a typical amplification curve appears, the result is positive, that is, the sample to be detected contains the fish rhabdovirus strain CAPRV2023; if there is no C t t value or no amplification curve, the result is negative; when the Ct value > 35, repeat the sample. If the repeated result has no Ct value, it is negative, otherwise it is positive.

[0026] The above third object of the present invention can be achieved by the following technical solution: Application of the above primer-probe set, the above kit or the above method in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes.

[0027] The present invention has the following advantages:

[0028] (1) High-efficiency detection: The present invention can complete the detection of fish rhabdovirus CAPRV2023 within 40 minutes. Compared with the traditional PCR technology (3 - 4 hours), the detection time is greatly shortened, the detection efficiency is improved, enabling relevant institutions to obtain detection results more quickly, and providing strong support for subsequent prevention, control and treatment work;

[0029] (2) Real-time monitoring: Through fluorescence signal monitoring technology, the present invention realizes real-time online monitoring of the detection process, enabling operators to more intuitively observe the increase in products, accurately judge the detection results. At the same time, the detection results can be directly read by computer software without cumbersome subsequent processing, improving the convenience and accuracy of detection;

[0030] (3) High specificity: The present invention introduces fluorescence probe technology, enabling primers and fluorescence probes to specifically bind to the template simultaneously, improving the specificity of detection, ensuring that only the CAPRV2023 virus can be detected, effectively avoiding interference from other viruses, and enhancing the accuracy and reliability of detection;

[0031] (4) Simple operation: The process is simple, only requiring three steps: liquid preparation, instrument loading, and result reading, reducing the operation difficulty and time cost, having low requirements for operators, and being conducive to the wide promotion and application of this method;

[0032] (5) Less pollution: The present invention adopts a closed-tube detection method, effectively avoiding the potential risks brought by opening the lid to handle PCR products, avoiding the generation of false positives in detection results and environmental pollution by aerosols;

[0033] (6) High detection sensitivity: The detection limit can reach 10 copies, and the quantification is accurate within the range of 10 2 copies - 10 9 copies. Description of the Drawings

[0034] The following further describes the present invention with reference to the drawings in conjunction with the embodiments.

[0035] Figure 1 It is the result of optimizing the primer concentration, probe concentration, and reaction temperature for the fluorescence quantitative PCR reaction in Example 2; among them, A is the optimization result of the primer concentration, B is the optimization result of the probe concentration, and C is the optimization result of the reaction annealing temperature;

[0036] Figure 2 It is the determination diagram of the detection results in Example 2;

[0037] Figure 3 It is the detection standard curve of Example 3;

[0038] Figure 4 It is the amplification curve for the sensitivity analysis of Example 3;

[0039] Figure 5Amplification curve for specific detection in Example 4; where 1: positive control; 2: spleen tissue of Trachinotus ovatus infected with fish rhabdovirus CAPRV2023; 3: kidney tissue of Trachinotus ovatus infected with fish rhabdovirus CAPRV2023; 4: SGIV; 5: SCRV; 6: IHNV; 7: NNV; 8: Vibrio harveyi; 9: Streptococcus iniae; 10: Lactococcus garvieae; 11: Streptococcus dysgalactiae; 12: Vibrio alginolyticus; 13: Photobacterium damselae subsp. piscicida; 14: Vibrio vulnificus; 15: cDNA of healthy Trachinotus ovatus; 16: negative control;

[0040] Figure 6 Amplification curve for repeatability detection in Example 5. Detailed implementation manners

[0041] The following further illustrates the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0042] Unless otherwise specified, the materials, reagents and equipment used in the present invention can be obtained from commercial sources without special instructions. The experimental methods used in the following implementation cases are all conventional methods unless otherwise specified. The primer probes are recommended but not limited to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] Example 1

[0044] This example provides the design and synthesis of fluorescence quantitative PCR primers and probes for fish rhabdovirus CAPRV2023:

[0045] For a partial sequence of the G protein gene of rhabdovirus CAPRV2023 (as shown in SEQ ID NO.4), primers are designed:

[0046] Forward primer CAPRVFq: 5’-CCAGAACCTGTTTGCGCTTG-3’ (as shown in SEQ ID NO.1);

[0047] Reverse primer CAPRVRq: 5’-AGTCCCTATTCACCACCCAGA-3’ (as shown in SEQ ID NO.2).

[0048] Specific TaqMan probe: 5’-FAM-CTCACAGATAATGTGGCGGCAAAGAGGGAG-BHQ1-3’ (as shown in SEQ ID NO.3).

[0049] Partial sequence of the G protein gene of rhabdovirus CAPRV2023:

[0050]

[0051] Among them, the straight lines below are the forward and reverse primers, and the wavy lines below are the probes.

[0052] Among them, the 5'-end of the probe is labeled with the fluorescent reporter group FAM, and the 3'-end is labeled with the fluorescent quenching group BHQ1.

[0053] The probe was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0054] The rhabdovirus CAPRV2023 was isolated and preserved by the laboratory where the inventors are located. It is recommended but not limited to the following method to obtain it repeatedly: Take the liver, spleen and kidney tissues of diseased Trachinotus ovatus, add 9 times the volume of sterile PBS, homogenize using a homogenizer under ice bath conditions, then centrifuge at 10000g for 10 min, take the supernatant, and pass it through a 0.22 μm filter membrane to prepare the virus stock solution. After inoculating the virus stock solution on FHM cells for 1 h, discard the supernatant, and add L-15 culture medium containing 10% fetal bovine serum to the FHM cells again and continue to culture at 28°C for 48 h to obtain a solution containing a large amount of rhabdovirus CAPRV2023.

[0055] This primer-probe group can be applied in the preparation of a detection kit for fish rhabdovirus CAPRV2023. For example: This example also provides a kit for real-time fluorescence quantitative PCR detection of fish rhabdovirus CAPRV2023, including the primer-probe group described above.

[0056] The molar ratio of the forward primer, reverse primer and probe in the kit is 4.8:4.8:1.

[0057] The kit also includes a qPCR MIX reaction solution, a positive control and a negative control.

[0058] Among them, the qPCR MIX reaction solution contains the following components in the following contents: 5 U Taq enzyme, 2.5 mM dNTPs, 25 mM MgCl2 and 2×PCR buffer.

[0059] The positive control is a T-vector clone of the rhabdovirus CAPRV2023 G protein gene fragment. Its preparation method is: Extract the RNA of rhabdovirus CAPRV2023 and reverse transcribe it into cDNA. Use the forward primer CAPRVFq and the reverse primer CAPRVRq to perform a PCR amplification reaction on the template cDNA to obtain DNA containing the target gene sequence. Recover this amplified fragment (SEQ ID NO. 4) and ligate it into the T-vector using a conventional cloning method, which is the positive control.

[0060] The negative control is deionized water.

[0061] Example 2

[0062] The method for rapid detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes based on real-time fluorescence quantitative PCR technology provided in this embodiment includes the following steps:

[0063] (1) Take about 10 mg of liver, spleen and kidney tissues of suspected diseased Trachinotus ovatus, extract its RNA using the tissue RNA extraction reagent FastPure Complex Tissue Total RNA Isolation Kit (Nanjing Novoprotein), and reverse transcribe it into cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Nanjing Novoprotein);

[0064] (2) Adopt the TaqMan probe technology, add cDNA, the forward primer CAPRVFq, reverse primer CAPRVRq, probe, and qPCR MIX reaction solution designed in Example 1 to the reaction system, and perform PCR amplification using Lightcycle 96 (ROCHE, Switzerland).

[0065] Set the primer concentrations at 0.2, 0.4, 0.6, 0.8 μmol / L, probe concentrations at 0.0625, 0.125, 0.25 μmol / L, and annealing temperatures at 53, 55, 57, and 59 °C respectively, to obtain the standard of the lowest Ct value and the highest increase in fluorescence intensity, and screen the optimal primer concentration, probe concentration, and annealing temperature.

[0066] The results are as Figure 1 shown. The concentrations of the forward primer, reverse primer, and probe are 0.6:0.6:0.125 ( Figure 1 in Figures A - B), the molar ratio is 4.8:4.8:1, and the optimal annealing temperature is 55 °C ( Figure 1 in Figure C).

[0067] (3) The optimized real-time fluorescence quantitative PCR reaction system is: 10 μL of qPCR MIX reaction solution, 1.2 μL each of the forward and reverse primers (10 μmol / mL), 0.5 μL of the probe (5 μmol / mL), 1.0 μL of cDNA template, and make up to a total volume of 20 μL with ddH2O; set positive and negative controls; mix the prepared PCR tubes and centrifuge, and place them in a fluorescence quantitative PCR instrument for reaction.

[0068] (4) The real-time fluorescence PCR reaction conditions are: 95 °C for 3 min, 95 °C for 10 s, 55 °C for 10 s, for 40 cycles; perform fluorescence quantitative PCR detection using the above reaction system, collect fluorescence signals at the end of each cycle extension, and obtain the amplification curve of the sample;

[0069] (5) Judgment of test results: Observe the Ct value amplified by the fluorescence quantitative PCR instrument to judge the amplification result. If the Ct value ≤ 35 and a typical amplification curve appears, the result is positive (as shown in Figure A of Figure 2 ), that is, the test sample contains the fish rhabdovirus strain CAPRV2023; without Ct value or without amplification curve, the result is negative (as shown in Figure B of Figure 2 ), that is, the test sample does not contain the fish rhabdovirus strain CAPRV2023; when the Ct value > 35, re-test this sample. If the re-test result has no Ct value, it is negative, otherwise it is positive.

[0070] Example 3

[0071] Establishment of quantitative formula and sensitivity test of the kit for preparing fluorescence quantitative PCR primers of fish rhabdovirus CAPRV2023

[0072] (1) Template preparation: Connect the partial sequence of the G protein gene of fish rhabdovirus CAPRV2023 (shown in SEQ ID NO.4) into the T vector (Ultra-Universal TOPO Cloning Kit, Nanjing Novoprotein), then transform the plasmid into Escherichia coli DH5α for proliferation and extract it by alkaline lysis method, and purify it with a DNA purification kit to obtain a positive recombinant plasmid standard.

[0073] (2) Standard curve preparation: Apply the fluorescence quantitative PCR reaction system and reaction conditions described in Example 2 to perform real-time fluorescence quantitative PCR amplification on standard products with different concentrations (1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 copies) to obtain amplification curves of standard products with different concentrations.

[0074] Use the common logarithm (logC) of the starting concentration (C) of the standard product as the abscissa and the cycle threshold (Ct value) at which fluorescence appears as the ordinate to obtain the amplification curve of this amplification method.

[0075] The results show ( Figure 3 ) that there is a good linear relationship in the range of template concentration from 10-10 9 in the reaction system. The linear relationship between the logarithm of the initial copy number (copies / μL) and the C t value is Y = -3.214*X + 37.94, and the correlation coefficient R 2 = 0.9990, meeting the expected effect of the experimental design.

[0076] (4) Figure 4 This is the amplification curve for the sensitivity analysis of the fluorescence quantitative PCR method for fish rhabdovirus CAPRV2023. Real-time fluorescence quantitative PCR detection and analysis were performed on the linearity and sensitivity of standard products with different concentrations. The detection limit of the real-time fluorescence quantitative PCR detection method for fish rhabdovirus CAPRV2023 established in the present invention is 10 copies / μL ( Figure 4 as shown in Figure A), and the sensitivity is more than 100 times higher than that of the PCR established in this laboratory ( Figure 4 as shown in Figure B).

[0077] Example 4 Specificity test of the fluorescence quantitative RT-PCR detection kit for fish rhabdovirus CAPRV2023

[0078] Using the method described in Example 2, specific detections were performed on the cDNA or DNA of healthy Trachinotus ovatus, spleen and kidney tissues of Trachinotus ovatus infected with CAPRV2023, and ten pathogens that often appear in aquaculture, including grouper iridovirus (SGIV), siniperca chuatsi rhabdovirus (SCRV), megalocytivirus, infectious hematopoietic necrosis virus (IHNV), nervous necrosis virus (NNV), Vibrio harveyi, Streptococcus iniae, Lactococcus garvieae, Streptococcus dysgalactiae, and Vibrio alginolyticus. At the same time, a negative control group (double-distilled water) and a positive control were set up. The specific amplification curve is shown in Figure 5 . Only the cDNA of the spleen and kidney tissues of Trachinotus ovatus infected with CAPRV2023 and the positive control showed typical amplification curves, indicating that the RT-PCR detection method for fish rhabdovirus CAPRV2023 has good specificity and extremely high sensitivity.

[0079] Example 5 Repeatability test of the fluorescence quantitative RT-PCR detection kit for fish rhabdovirus CAPRV2023

[0080] Using the method described in Example 2, 40 fluorescence quantitative RT-PCR detections were performed on the same sample. The amplification curve is as shown in Figure 6 . The standard deviation of the Cq values between repeated samples was 0.045 < 0.2, and the coefficient of variation was 0.32%, indicating accurate quantification and high repeatability, which can be used for detecting and quantifying samples.

[0081] Example 6 Clinical application of infected fish samples

[0082] From August 2023 to April 2024, 15 diseased fish samples were collected from seawater farms in various places in Guangdong Province. At the same time, the fluorescence quantitative PCR detection kit and real-time fluorescence quantitative PCR method for rhabdovirus CAPRV2023 established in Examples 1-2 of the present invention were used for detection, and the traditional PCR method was also used for detection.

[0083] The results showed that 10 positive samples were detected by the traditional PCR method, with a positive rate of 66.7%. The kit and method established by the present invention detected 13 positive samples, with a positive rate of 86.7% (Table 1), which was 20.0% higher than that of the traditional PCR detection method. Cell isolation culture and electron microscopy observation were carried out on all the collected samples, and the results of the confirmed positive samples were consistent with those detected by the real-time fluorescence quantitative PCR method of the present invention, with a coincidence rate of 100%. It shows that the combination of the real-time fluorescence quantitative PCR detection kit and method of the present invention can detect the rhabdovirus CAPRV2023 in clinical fish samples, with strong specificity and accurate detection results.

[0084] Table 1 Results of fluorescence quantitative PCR and traditional PCR for detecting clinical samples

[0085]

[0086] The above-described embodiments merely represent several preferred embodiments of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A primer-probe set for real-time fluorescence quantitative PCR detection of fish rhabdovirus CAPRV2023, characterized in that, The primer-probe set includes primers and a probe. The primers include a forward primer CAPRVFq and a reverse primer CAPRVRq. The nucleotide sequence of the forward primer CAPRVFq is as shown in SEQ ID NO.1, the nucleotide sequence of the reverse primer CAPRVRq is as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.

3.

2. The primer-probe set according to claim 1, characterized in that, The 5′ end of the probe is labeled with a fluorescent reporter group, and the 3′ end is labeled with a quenching group. The fluorescent reporter group is FAM, HEX, VIC, ROX, JOE, Cy3, TxR or Cy5, and the quenching group is BHQ1, BHQ2, BHQ3, TAMRA or MGB.

3. A kit for real-time fluorescence quantitative PCR detection of fish rhabdovirus CAPRV2023, characterized in that, It includes the primer-probe set described in claim 1 or 2.

4. The kit according to claim 3, wherein The molar ratio of the forward primer, reverse primer and probe in the kit is 4.8:4.8:

1.

5. The kit according to claim 3, characterized in that, The kit further includes a qPCR MIX reaction solution, a positive control and a negative control. The qPCR MIX reaction solution contains the following components: 5U Taq enzyme, 2.5 mM dNTPs, 25 mM MgCl2 and 2×PCR buffer; the positive control is a T-vector clone of the fish rhabdovirus CAPRV2023 G protein gene fragment, and the sequence of the fish rhabdovirus CAPRV2023 G protein gene fragment is as shown in SEQ ID NO.4, and the negative control is deionized water.

6. Use of the primer-probe set described in claim 1 or 2 in the preparation of a detection kit for fish rhabdovirus CAPRV2023.

7. A detection method for fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes, characterized in that, It includes the following steps: (1) Extract RNA from the sample to be detected and reverse transcribe it into cDNA; (2) Using the cDNA as a template, establish a real-time fluorescence quantitative PCR reaction system and reaction conditions including the primer-probe set described in claim 1, and perform fluorescence quantitative PCR detection with the reaction system and reaction conditions to obtain the amplification curve of the sample to be detected; (3) Judgment of the detection result: After the reaction ends, judge whether it is positive or negative for fish rhabdovirus CAPRV2023 according to the Ct value and amplification curve of the sample to be detected.

8. The detection method of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes according to claim 7, characterized in that, The real-time fluorescence quantitative PCR reaction system described in step (2) is: 10 μL of qPCR MIX reaction solution, 1.2 μL of each of the forward and reverse primers with a concentration of 10 μmol / mL, 0.5 μL of the probe with a concentration of 5 μmol / mL, 1.0 μL of cDNA template, and supplemented with ddH2O to a total volume of 20 μL; the real-time fluorescence quantitative PCR reaction conditions are: 95°C for 3 min; 95°C for 10 s, 55°C for 10 s, 40 cycles.

9. The detection method of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes according to claim 7, characterized in that, When judging the detection result in step (3), if the Ct value ≤ 35 and a typical amplification curve appears, the result is positive, that is, the sample to be detected contains the fish rhabdovirus strain CAPRV2023; without a C t value or no amplification curve, the result is negative, that is, the sample to be detected does not contain the fish rhabdovirus strain CAPRV2023; when the Ct value > 35, re-test the sample. If the re-test result has no Ct value, it is negative, otherwise it is positive.

10. Use of the primer-probe set described in any one of claims 1 to 2, the kit described in any one of claims 3 to 5, or the method described in any one of claims 7 to 9 in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes.

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