A primer probe for detecting fish rhabdovirus CAPRV2023 and its application

Abstract

This invention discloses a primer probe for detecting fish rhabdovirus CAPRV2023 and its application. The primers include a forward primer CAP-N-F and a reverse primer CAP-N-R, and the probe is CAP-N-PROBE. The sequences of the forward primer CAP-N-F, the reverse primer CAP-N-R, and the probe CAP-N-PROBE are shown in SEQ ID NO. 1 to 3, respectively. This invention also discloses a kit for detecting fish rhabdovirus CAPRV2023, and the application of the above primer probe or kit in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes. The primer probe or kit of this invention can shorten the detection time, has high detection sensitivity, real-time monitoring function, excellent specificity, simple operation procedure, and reduces the risk of contamination.

Description

Technical Field

[0001] This invention belongs to the field of viral molecular detection technology, specifically relating to a primer probe for detecting fish carpionerhabdovirus (CAPRV2023) and its application, particularly a primer probe based on TaqMan real-time quantitative PCR for detecting fish carpionerhabdovirus (CAPRV2023) and its application. Background Technology

[0002] Rhabdoviridae is a family of linear, negative-sense, single-stranded RNA viruses, named for their distinctive viral particle morphology. They typically appear rod-shaped or bullet-shaped, with a particle size ranging from (100–430) nm to (45–100) nm. The viral particle consists of five main structural proteins (L, G, N, P, and M), which are responsible for key functions such as genome replication, viral assembly and budding, and promoting transcription and replication. The Rhabdoviridae family currently contains over 318 known species, with hosts spanning vertebrates, invertebrates, and plants, demonstrating broad biological adaptability and ecological influence. Among fish viruses, rhabdoviridae, due to their wide host range, high virulence, and diverse strains, pose a major pathogenic threat to both freshwater and marine fish farming. Studies have reported approximately 20 types of rhabdoviruses that infect fish, among which the most harmful to aquaculture include carp spring viremia virus (SVCV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), turbot rhabdovirus (HIRRV), and mandarin fish rhabdovirus (SCRV). These viruses mainly belong to five genera within the family Rhabdoviridae: Novirhabdovirus, Perhabdovirus, Siniperhavirus, Sprivivirus, and Scoprhavirus. However, with advancements in virus isolation and genomic technologies, some novel viruses remain unclassified.

[0003] The golden pomfret (Trachinotus ovatus), also known as the oval pomfret, is one of the important economic fish species in my country's marine aquaculture industry. It is mainly distributed in the South my country Sea, East China Sea, Yellow Sea, and Bohai Sea, and also found in the Indian Ocean, Australia, and Japan. The golden pomfret is highly adaptable, grows rapidly, and can tolerate formulated feeds during the aquaculture process, making it popular among fish farmers. In 2023, the farmed golden pomfret accounted for a significant proportion of my country's total marine fish production, second only to the large yellow croaker. However, with the expansion of aquaculture scale, disease problems have gradually become prominent, especially the infection of various diseases, which has become a major factor affecting the survival rate of golden pomfret, posing a significant challenge to the aquaculture industry.

[0004] In 2023, a novel acute infectious disease broke out in oval pomfret, exhibiting rapid spread, widespread prevalence, high morbidity, and high mortality rates, causing significant economic losses to the aquaculture industry. Affected fish primarily exhibit behavioral symptoms such as lethargy, anorexia, and abnormal swimming (e.g., swirling movements), accompanied by external features such as hemorrhage at the base of the fins and in the upper and lower jaws. Anatomical examination revealed pale gills, accumulation of red ascites in the body cavity, enlarged liver and spleen, and severe congestion and hemorrhage in the liver, spleen, kidneys, and intestines. Etiological and epidemiological investigations identified the disease as being caused by a novel rhabdovirus. Through whole-genome sequencing, a novel virus was isolated and identified, named CAPRV2023, confirming its membership in the Rhabdoviridae family. This is the first global report of this virus infecting farmed marine fish, marking a significant advancement in rhabdovirus research. As a newly emerging infectious disease, the outbreak of CAPRV2023 in fish diseases has attracted widespread attention. Currently, there are no specific treatments for this virus; its prevention and control mainly rely on strengthening aquaculture management, optimizing the aquaculture environment, and cutting off the transmission routes of the pathogen. At present, due to the lack of efficient diagnostic tools, clinical diagnosis and prevention face many challenges. Developing an accurate and rapid molecular diagnostic method for detecting CAPRV2023 is a key research focus.

[0005] Currently, a SYBR Green quantitative PCR detection technique based on primers and probes developed targeting the CAPRV2023 G protein gene has been reported. Numerous studies have also been reported on G proteins in other types of rhabdoviruses. However, some studies have shown that the G protein gene sequence of another rhabdovirus—Carp Spring Blood Virus (SVCV)—differs among different viral strains, potentially leading to false negatives when using G protein detection methods. The N protein, being the most abundant protein in rhabdoviruses, plays a crucial role in viral transcription and replication and exhibits high conservation. Therefore, selecting the N gene as a target for CAPRV2023 detection is an ideal strategy, providing a novel target gene detection method for this virus. This allows for cross-validation with other detection techniques, resulting in more accurate data. This invention aims to establish a Taqman quantitative PCR detection method based on the conserved gene sequence encoded by the N protein. This method will help in the early detection of viral infections, improve disease control efficiency, reduce economic losses, and lay a solid foundation for the development of pomfret aquaculture. Summary of the Invention

[0006] The purpose of this invention is to provide a primer and probe combination for detecting fish rhabdovirus CAPRV2023. This combination is designed to achieve efficient and specific detection of CAPRV2023 virus in fish samples through precisely designed primers and probes.

[0007] The present invention also aims to provide a method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

[0008] The final object of the present invention is to provide the application of the above-described primers and probes or the above-described kits in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

[0009] The first objective of the present invention can be achieved by the following technical solution: a primer probe for detecting fish rhabdovirus CAPRV2023, wherein the primers include a forward primer CAP-NF and a reverse primer CAP-NR, and the probe is CAP-N-PROBE. The nucleotide sequence of the forward primer CAP-NF is shown in SEQ ID NO.1, the sequence of the reverse primer CAP-NR is shown in SEQ ID NO.2, and the sequence of the probe CAP-N-PROBE is shown in SEQ ID NO.3.

[0010] The primers and probes are designed based on the specific gene region of the N gene sequence of rhabdovirus CAPRV2023, ensuring the accuracy and specificity of the detection. The N gene sequence of rhabdovirus CAPRV2023 is shown in SEQ ID NO.7, and the sequence of the specific gene region is shown in SEQ ID NO.4.

[0011] Preferably, the probe of the present invention has undergone special structural treatment, with a fluorescent group labeled at the 5′ end and a quenching group attached to the 3′ end. This design allows the fluorescent group to be released and emit a signal once the probe binds to and is cleaved by the target sequence during PCR amplification, thereby achieving real-time and quantitative detection. The fluorescent group can be selected from, but is not limited to, FAM, HEX, VIC, ROX, JOE, Cy3, TxR, or Cy5, while the quenching group can be selected from, such as BHQ1, BHQ2, BHQ3, TAMRA, or MGB.

[0012] More preferably, to achieve optimal detection results, this invention recommends using FAM as a fluorescent reporter group at the 5′ end of the probe and BHQ1 as a quencher group at the 3′ end. This combination generates a strong fluorescence signal and effectively reduces background noise, thereby improving the sensitivity and accuracy of the detection.

[0013] The present invention also provides the application of the above-mentioned primers and probes for detecting fish rhabdovirus CAPRV2023 in the preparation of a kit for detecting fish rhabdovirus CAPRV2023.

[0014] The present invention also provides a kit for detecting fish rhabdovirus CAPRV2023, the kit comprising the aforementioned primers and probes.

[0015] Preferably, the molar ratio of the forward primer, reverse primer, and probe in the kit is 8:8:1.

[0016] Furthermore, the kit also includes qPCR MIX reaction solution, a positive control, and a negative control.

[0017] Preferably, the qPCR MIX reaction solution should contain the following components in the following amounts: 5U Taq enzyme, 2.5mM dNTPs, 25mM MgCl2 and 2×PCR buffer.

[0018] Preferably, the positive control is a T-vector clone of the N protein gene fragment of rhabdovirus CAPRV2023. The preparation method is as follows: RNA of rhabdovirus CAPRV2023 is extracted and reverse transcribed into cDNA. The template cDNA is then amplified by PCR using the forward primer CAP-N-COLN1F: 5'-ACAAATGGGGGAGATGCGTT-3' (SEQ ID NO.5) and the reverse primer CAP-N-COLN1R: 5'-AGCCTGACAATCCTCTTGGC-3' (SEQ ID NO.6) to obtain DNA containing the N gene sequence (SEQ ID NO.7). This amplified fragment is recovered and ligated into the pcE3 vector using conventional cloning methods, thus serving as the positive control.

[0019] Preferably, the negative control is enzyme-free water.

[0020] The second objective of this invention can be achieved through the following technical solution: a method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes, comprising the following steps:

[0021] (1) Extract RNA from the sample to be tested and reverse transcribe it into cDNA;

[0022] (2) Using cDNA as a template, establish a real-time quantitative PCR reaction system and reaction conditions, perform real-time quantitative PCR detection, and obtain the Ct value and amplification curve of the sample to be tested.

[0023] (3) Detection result determination: The Ct value and amplification curve of the sample to be tested are used to determine whether the sample is positive or negative for fish rhabdovirus CAPRV2023.

[0024] The present invention provides a method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes. This method utilizes the above-mentioned primers, probes or kits and can be widely applied in aquaculture, fishery resource protection and scientific research to monitor and assess the infection status of CAPRV2023 virus in fish.

[0025] In the above-mentioned detection methods for fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes:

[0026] Preferably, the reaction system for real-time quantitative PCR in step (2) is as follows: 10 μL of qPCR MIX reaction solution, 1 μL each of forward and reverse primers with a concentration of 10 μmol / mL, 0.125 μL of probe with a concentration of 10 μmol / mL, 1.0 μL of cDNA template, and ddH2O to make up to a total volume of 20 μL.

[0027] Preferably, the real-time fluorescence quantitative PCR reaction conditions in step (2) are: 95℃ for 30s, 53℃ for 30s, for 45 cycles.

[0028] Preferably, in step (3), when the Ct value is ≤35 and a typical amplification curve appears, the result is positive, that is, the sample to be tested contains the fish rhabdovirus strain CAPRV2023; when there is no Ct value or no amplification curve, the result is negative; when the Ct value is >35, the sample is repeated. If the result of the repeated test is negative and there is no Ct value, it is positive otherwise.

[0029] The third objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned primer probe set or the above-mentioned kit in the detection of fish rhabdovirus CAPRV2023 for non-diagnostic purposes.

[0030] Compared to traditional PCR detection, this invention has the following significant advantages:

[0031] (1) Highly efficient detection time: Detection of fish rhabdovirus CAPRV2023 can be completed within 40 minutes. Compared to the traditional PCR technology with a detection time of approximately 1.5 to 2 hours, this innovation significantly shortens the detection time;

[0032] (2) Real-time monitoring function: By continuously monitoring the fluorescence signal, the detection process is monitored online in real time, allowing for a direct observation of the increase in product. Simultaneously, the detection results can be directly read by computer software, eliminating cumbersome post-reaction processing steps.

[0033] (3) Excellent specificity: By introducing fluorescent probes, primers and fluorescent probes can bind specifically to the template at the same time, thereby significantly improving the specificity of detection and ensuring that only CAPRV2023 virus is detected.

[0034] (4) Simple operation process: Operators only need to complete three simple steps: solution preparation, machine operation and result reading. There is no need to perform complicated operations such as gel running and enzyme digestion. This not only simplifies the operation process, but also makes the method more accessible and easier for operators to accept and promote.

[0035] (5) Reduce the risk of contamination: The closed tube detection method avoids the need to open the PCR product in the later stage, thus effectively preventing the generation of false positive results and reducing the pollution of aerosols to the environment.

[0036] (6) High detection sensitivity: The detection limit is up to 20 copies, and it is within 2×10 1 copies-5×10 9 Accurate quantification within the range of copies. Attached Figure Description

[0037] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0038] Figure 1 The results show the optimization of primer concentrations for the real-time PCR reaction in Example 2;

[0039] Figure 2 The results show the optimization of probe concentration for the real-time PCR reaction in Example 2;

[0040] Figure 3 The results show the optimized reaction temperature for the real-time PCR reaction in Example 2;

[0041] Figure 4 The results are the TaqMan qPCR detection control results for positive and negative samples in Example 2;

[0042] Figure 5 This is the TaqMan qPCR standard curve from Example 3;

[0043] Figure 6 Figure A shows the amplification curves and electrophoresis results for sensitivity analysis in Example 3. Figure A shows the TaqMan qPCR sensitivity assay. Note: In Figure A, 1-10 represent 2×10⁻⁶ cells / mL, respectively. 1 copies / μL-5×10 9 Plasmid concentration at copies / μL; Figure B shows the sensitivity of conventional PCR. Note: In Figure B, 1–9 represent 5 × 10⁻⁹ copies / μL, respectively. 1 copies / μL-5×10 9 Plasmid at a concentration of copies / μL, with N as the negative control;

[0044] Figure 7This is an amplification curve for specific detection in Example 4; where 1: positive control; 2: SGIV; 3: SCRV; 4: IHNV; 5: NNV; 6: Vibrio vulnificus; 7: Vibrio harveyi; 8: Streptococcus dolphinus; 9: Lactococcus gasseri; 10: Streptococcus dysgalactiae; 11: Vibrio alginolyticus; 12: negative control; Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0046] Unless otherwise specified, the materials, reagents, and equipment used in this invention are commercially available. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Primers and probes are recommended, but not limited to, synthesis at Sangon Biotech (Shanghai) Co., Ltd.

[0047] Example 1

[0048] The primers and probes for detecting fish rhabdovirus CAPRV2023 provided in this embodiment are as follows:

[0049] Primers and probes were designed targeting the specific gene region (as shown in SEQ ID NO.4) of the N gene sequence of rhabdovirus CAPRV2023 (as shown in SEQ ID NO.7) to ensure the accuracy and specificity of detection.

[0050] Forward primer CAP-NF: 5'-AGAGTTCCCAACCGACAAAG-3' (as shown in SEQ ID NO.1);

[0051] Reverse primer CAP-NR: 5'-CCACCACAAGGAAGGAATACA-3' (as shown in SEQ ID NO.2).

[0052] Specific TaqMan probe: CAP-N-PROBE: 5'-FAM-CTGAGAAAGATTGCCAGCTTCATCCG A-3'BHQ1 (as shown in SEQ ID NO.3).

[0053] The probe is labeled with the fluorescent reporter group FAM at its 5' end and the fluorescent quencher group BHQ1 at its 3' end.

[0054] The probe was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] Rhabdovirus CAPRV2023 was isolated using the following method: Fish tissue containing CAPRV2023 was homogenized using a grinder, diluted 10 times with sterile PBS, centrifuged at 6000xg for 1 hour, and the supernatant was filtered through a 0.22 μm pinhole membrane to prepare a virus incubation solution. FHM cells were incubated with this solution for 1 hour, discarded, and then L-15 culture medium containing 10% fetal bovine serum was added to the FHM cells, which were then incubated at 28°C for 48 hours. The FHM cells, along with the culture flask, were subjected to three freeze-thaw cycles at -80°C and centrifuged at 5000xg for 5 minutes to obtain a solution containing a large amount of CAPRV virus. This method is reproducible and can be used for large-scale culture of CAPRV2023.

[0056] These primers and probes can be used in the preparation of a detection kit for fish rhabdovirus CAPRV2023. For example, this embodiment also provides a kit for detecting fish rhabdovirus CAPRV2023, including the aforementioned primers and probes.

[0057] The molar ratio of the forward primer, reverse primer, and probe in the kit is 8:8:1.

[0058] The kit also includes qPCR MIX reaction solution, a positive control, and a negative control.

[0059] The qPCR MIX reaction solution contains the following components in the following amounts: 5U Taq enzyme, 2.5mM dNTPs, 25mM MgCl2 and 2×PCR buffer.

[0060] Recommended qPCR MIX reaction solution, but not limited to, 2×Taq Pro U+Multiple Probe qPCR Mix (Nanjing Novizan, QN213).

[0061] The positive control is a pCE3 vector clone of the N protein gene fragment of rhabdovirus CAPRV2023. Its preparation method is as follows: RNA of rhabdovirus CAPRV2023 is extracted and reverse transcribed into cDNA. The template cDNA is then amplified by PCR using the forward primer CAP-N-COLN1F: 5'-ACAAATGGGGGAGATGCGTT-3' (SEQ ID NO.5) and the reverse primer CAP-N-COLN1R: 5'-AGCCTGACAATCCTCTTGGC-3' (SEQ ID NO.6) to obtain DNA containing the target gene sequence. This amplified fragment (SEQ ID NO.7) is recovered and ligated into the pCE3 vector using conventional cloning methods, thus serving as the positive control.

[0062] The negative control was enzyme-free water.

[0063] Example 2

[0064] The method for rapid detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes provided in this embodiment includes the following steps:

[0065] (1) RNA was extracted from the test samples using an RNA extraction kit and reverse transcribed into cDNA;

[0066] The specific implementation method is as follows: Weigh 10mg of spleen and kidney tissue from golden pomfret infected with CAPRV2023, extract RNA using the FastPureComplexTissueTotalRNAIsolation Kit (Nanjing Novizan), reverse transcribe it into cDNA using HiScript IIQ RT SuperMix for qPCR(+gDNA wiper) (Nanjing Novizan), and amplify the template cDNA by PCR using forward primer CAP-N-COLN1F and reverse primer CAP-N-COLN1R to obtain the N protein gene sequence DNA (SEQ ID NO.7);

[0067] (2) Template preparation: The plasmid containing the N protein gene sequence of fish rhabdovirus CAPRV2023 was ligated into the pcE3 vector (Ultra-Universal TOPO Cloning Kit, Nanjing Novizan) and transformed into Escherichia coli DH5α for proliferation. After extraction by alkaline lysis, the plasmid was purified using a DNA purification kit to obtain a positive recombinant plasmid standard, which was then diluted to 5×10⁻⁶. 4 copy;

[0068] (3) Using TaqMan probe technology, DNA, forward primer CAP-NF, reverse primer CAP-NR, specific TaqMan probe CAP-N-PROBE, and 2×Taq Pro U+Multiple Probe qPCR Mix were added to the reaction system. A Lightcycle 96 (ROCHE, Switzerland) was used to test 5×10⁻⁶ PCR solutions. 4 The positive recombinant plasmid standard was amplified by qPCR.

[0069] Annealing temperatures of 51, 53, 55, 57, and 59 °C, primer concentrations of 0.2, 0.4, 0.5, 0.6, and 0.8 μmol / L, and probe concentrations of 0.03125, 0.0625, 0.125, 0.25, and 0.375 μmol / L were set respectively. The lowest Ct value and the highest fluorescence intensity increase were used as reference standards to screen the optimal annealing temperature, primer concentration, and probe concentration.

[0070] The results are as follows Figure 1 , Figure 2 , Figure 3 As shown, the concentrations of the forward primer, reverse primer, and probe are 0.5:0.5:0.0625, the molar ratio is 8:8:1, and the optimal annealing temperature is 53℃.

[0071] (4) The optimized real-time quantitative PCR reaction system is as follows: 10 μL of PCR MIX reaction solution, 1 μL each of forward and reverse primers (10 μmol / mL), 0.125 μL of probe (10 μmol / mL), 1.0 μL of cDNA template, and ddH2O to make up to a total volume of 20 μL; set up positive and negative controls; mix the prepared PCR tubes, centrifuge, and place them in a real-time quantitative PCR instrument for reaction.

[0072] (5) The real-time fluorescence PCR reaction conditions are: 95℃ for 30s, 53℃ for 30s, 45 cycles; use the reaction system described above for fluorescence quantitative PCR detection, collect fluorescence signals at the end of each cycle extension, and obtain the amplification curve of the sample;

[0073] (6) Result Interpretation: In routine sample testing, RNA was extracted from the spleen and kidney tissues of the diseased golden pomfret and reverse transcribed into cDNA. Using the established qPCR reaction system and conditions, qPCR amplification was performed. The amplification Ct value was observed using a quantitative real-time PCR instrument to determine the amplification result. If the Ct value was ≤35 and a typical amplification curve appeared, the result was positive. Figure 4 Figure A in the diagram shows that the sample tested contained the fish rhabdovirus strain CAPRV2023; there was no Ct value or no amplification curve, indicating a negative result. Figure 4 (See Figure B in the diagram); if the Ct value is >35, the sample should be redone. If the redo result has no negative Ct value, it is positive otherwise.

[0074] Example 3

[0075] Establishment of quantitative formula and sensitivity test for reagent kit for preparing primers for fish rhabdovirus CAPRV2023 real-time RT-PCR:

[0076] (1) Template preparation: The plasmid containing the fish rhabdovirus CAPRV2023 N protein gene sequence (SEQ ID NO.7) was ligated into the pcE3 vector (Ultra-Universal TOPO Cloning Kit, Nanjing Novizan) and transformed into Escherichia coli DH5α for proliferation. The plasmid was extracted by alkaline lysis and purified by a DNA purification kit to obtain a positive recombinant plasmid standard.

[0077] (2) Preparation of standard curve: Using the fluorescence quantitative PCR reaction system and reaction conditions described in Example 2, different concentrations of standards (2×10⁻⁶) were prepared. 1 5×10 1 5×10 2 5×10 3 5×10 4 5×10 5 5×10 6 5×10 7 5×10 8 5×10 9 Real-time quantitative PCR amplification was performed using copies of the standard to obtain amplification curves for different concentrations of the standard.

[0078] The amplification curve of this amplification method is obtained by plotting the common logarithm (lgC) of the initial copy number concentration (C) of the standard as the x-axis and the cycle threshold (Ct value) at which fluorescence appears as the y-axis.

[0079] The results show ( Figure 5 The template concentration in the reaction system is 2×10⁻⁶. 1 -5×10 9 There is a good linear relationship within the range, and the logarithm of the initial copy number (copies / μL) is related to C. t The linear relationship between the values ​​is Y = -3.3132*X + 39.142, with a correlation coefficient R. 2 =0.9993, which meets the expected results of the experimental design.

[0080] (3) The linearity and sensitivity of fish rhabdovirus CAPRV2023 standards at different concentrations were analyzed by real-time fluorescence PCR. The detection limit of the real-time fluorescence quantitative PCR method for fish rhabdovirus CAPRV2023 established in this invention is 20 copies / μL. Figure 6 As shown in Figure A, the sensitivity is more than 25 times higher than that of the PCR established in our laboratory. Figure 6 (As shown in Figure B).

[0081] Example 4

[0082] Specificity test of the fish rhabdovirus CAPRV2023 real-time RT-PCR detection kit:

[0083] Using the method described in Example 2, specific detection was performed on the cDNA or DNA of ten commonly found pathogens in aquaculture, including grouper frog iridovirus (SGIV), mandarin fish rhabdovirus (SCRV), infectious hematopoietic organ necrosis virus (IHNV), neuronecrosis virus (NNV), Vibrio vulnificus, Vibrio harveyi, Streptococcus dolphinii, Lactococcus gasseri, Streptococcus dysgalactiae, and Vibrio alginolyticus. A negative control group (double-distilled water) and a positive control (CAPRV2023-infected golden pomfret spleen tissue) were also set up. Specific amplification curves are shown in [Figure 2]. Figure 7 .

[0084] from Figure 7 As can be seen, only the cDNA from the spleen tissue of golden pomfret 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.

[0085] Example 5

[0086] Repeatability test of the fish rhabdovirus CAPRV2023 real-time RT-PCR detection kit:

[0087] Using the method established in Example 2, for a concentration of 2×10 1 -5×10 9 Ten concentrations of positive recombinant plasmid standards (copies / μL) were used as templates. Quantitative real-time PCR amplification was performed in triplicate for each concentration group, both within and between groups. The CT values ​​of the amplified plasmids were statistically analyzed (Table 1). As shown in the table, the coefficient of variation within groups ranged from 0.14% to 0.60%, and the coefficient of variation between groups ranged from 0.04% to 2.28%, both less than 2.5%. This indicates accurate quantification and high repeatability, making it suitable for quantitative sample detection.

[0088] Table 1. Results of Repeatability Test in Example 5

[0089]

[0090] Example 6

[0091] Application of rhabdovirus CAPRV2023 in the detection of infected fish samples

[0092] From September to December 2024, 44 samples of oval pomfret were collected from marine aquaculture farms in various parts of Guangdong Province. The samples were simultaneously detected using the fluorescence quantitative PCR detection kit for rhabdovirus CAPRV2023 established in Examples 1 and 2 of this invention, the real-time fluorescence quantitative PCR method, and the conventional PCR method.

[0093] The results showed that the kit and method established in this invention detected 31.82% positive results in qPCR of 44 samples, while the positive result of conventional PCR was only 20.45% (Table 2). The positive result of qPCR was 11.37% higher than that of conventional PCR. All collected samples underwent cell isolation and culture and electron microscopy observation. The results of confirmed positive samples were consistent with the results of positive samples detected by the real-time quantitative PCR method of this invention, with a concordance rate of 100%. This indicates that the real-time quantitative PCR detection kit and method of this invention can detect rhabdovirus CAPRV2023 in clinical fish samples with high specificity and accurate results.

[0094] Table 2. Test results of 44 oval pomfret samples.

[0095]

[0096]

[0097]

[0098] Note: In the diagram, "+" represents positive and "-" represents negative.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A primer probe for detecting fish rhabdovirus CAPRV2023, characterized in that, The primers include a forward primer CAP-NF and a reverse primer CAP-NR, and the probe is CAP-N-PROBE. The nucleotide sequence of the forward primer CAP-NF is shown in SEQ ID NO.1, the sequence of the reverse primer CAP-NR is shown in SEQ ID NO.2, and the sequence of the probe CAP-N-PROBE is shown in SEQ ID NO.

3.

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

3. The application of the primers and probes according to claim 1 in the preparation of a kit for detecting fish rhabdovirus CAPRV2023.

4. A kit for detecting fish rhabdovirus CAPRV2023, characterized in that, The kit includes the primers and probes as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The molar ratio of the forward primer, reverse primer, and probe in the kit is 8:8:

1.

6. A method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes, characterized in that, Includes the following steps: (1) Extract RNA from the sample to be tested and reverse transcribe it into cDNA; (2) Using cDNA as a template, establish a real-time quantitative PCR reaction system and reaction conditions, perform real-time quantitative PCR detection, and obtain the Ct value and amplification curve of the sample to be tested; (3) Result determination: The Ct value and amplification curve of the sample to be tested shall be used to determine whether the sample is positive or negative for fish rhabdovirus CAPRV2023; In step (3), if the Ct value is ≤35 and a typical amplification curve appears, the result is positive, that is, the sample to be tested contains the fish rhabdovirus strain CAPRV2023; if there is no Ct value or no amplification curve, the result is negative; if the Ct value is >35, the sample should be retested. If the retest result has no Ct value and is negative, it is positive otherwise.

7. The method according to claim 6, characterized in that, The reaction system for real-time quantitative PCR in step (2) is as follows: 10 μL of qPCR MIX reaction solution, 1 μL each of forward and reverse primers with a concentration of 10 μmol / mL, 0.125 μL of probe with a concentration of 10 μmol / mL, 1.0 μL of cDNA template, and ddH2O to make up to a total volume of 20 μL.

8. The method according to claim 6, characterized in that, The real-time quantitative PCR reaction conditions in step (2) are: 95℃ for 30s, 53℃ for 30s, for 45 cycles.

9. The use of the primers and probes of claim 1 or the kit of any one of claims 4-5 in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

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