Primer probe for detecting fish rhabdovirus CAPRV2023 and application thereof

Abstract

The invention discloses a primer probe for detecting fish rhabdovirus CAPRV2023 and application of the primer probe, the primer probe comprises a forward primer CAP-N-F and a reverse primer CAP-N-R, the probe is CAP-N-PROBE, and the sequences of the forward primer CAP-N-F, the reverse primer CAP-N-R and the probe CAP-N-PROBE are shown as SEQ ID NO.1-3 respectively. The invention also discloses a kit for detecting the fish rhabdovirus CAPRV 2023, and application of the primer probe or the kit in the detection of the fish rhabdovirus CAPRV 2023 with a non-disease diagnosis purpose. According to the primer probe or the kit, the detection time can be shortened, the detection sensitivity is high, the real-time monitoring function is achieved, the specificity is excellent, the operation process is simple, and the pollution risk can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of virus molecular detection, and specifically relates to a primer probe for detecting fish rhabdovirus CAPRV2023 and an application thereof, in particular to a primer probe for detecting fish rhabdovirus CAPRV2023 based on TaqMan real-time fluorescence quantitative PCR and an application thereof. Background Art

[0002] Rhabdoviruses (Rhabdoviridae) are a class of linear negative-sense single-stranded RNA viruses, named for their unique virion morphology, which is usually rod-shaped or bullet-shaped, with a particle size range of (100-430) nm × (45-100) nm. Virus particles are composed of five major structural proteins (L, G, N, P, M), which are responsible for genome replication, viral assembly and budding, and promoting transcription and replication. The Rhabdoviridae family is currently known to include more than 318 members, whose hosts cover vertebrates, invertebrates and plants, showing a wide range of biological adaptability and ecological influence. Among fish viruses, Rhabdoviruses have become the main pathogenic threat in freshwater and marine fish aquaculture due to their wide host range, high toxicity and diverse strains. Studies have reported that there are about 20 rhabdoviruses that infect fish, among which the most harmful to the aquaculture industry include spring viremia virus of carp (SVCV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), flatfish rhabdovirus (HIRRV) and mandarin fish rhabdovirus (SCRV). These viruses mainly belong to five genera of the Rhabdoviridae family, namely Novirhabdovirus, Perhabdovirus, Siniperhavirus, Sprivivirus and Scophrhavirus. However, with the advancement of virus isolation and genomic technology, some new viruses have not yet been clearly classified.

[0003] Trachinotus ovatus, also known as golden pomfret, is one of the important economic fish species in my country's marine fish farming industry. It is mainly distributed in the South my country Sea, East China Sea, Yellow Sea and Bohai Sea, and is also distributed in the Indian Ocean, Australia and Japan. Trachinotus ovatus has the advantages of strong adaptability, rapid growth, and acceptance of artificial compound feed during the farming process, and is deeply favored by farmers. In 2023, the farming of ovatus in my country accounted for a significant proportion of the total marine fish production, second only to large yellow croaker. However, with the expansion of aquaculture scale, disease problems have gradually become prominent, especially the infection of multiple diseases has become the main factor affecting the survival rate of golden pomfret, posing a great challenge to the aquaculture industry.

[0004] In 2023, a new acute infectious disease broke out in the oval pomfret, which showed the characteristics of rapid spread, widespread prevalence, high morbidity and high mortality, causing huge economic losses to the aquaculture industry. The diseased fish mainly showed behavioral symptoms such as lethargy, anorexia, abnormal swimming (such as spinning swimming), accompanied by appearance characteristics such as bleeding at the base of the fins and upper and lower jaws. The anatomy of the diseased fish showed symptoms such as pale gills, accumulation of red ascites in the body cavity, enlarged liver and spleen, and severe congestion and bleeding in the liver, spleen, kidneys and intestines. Etiological and epidemiological investigations found that the disease was caused by a new type of rhabdovirus. Through whole genome sequencing, a new virus was isolated and identified, named CAPRV2023, and it was determined to be a new member of the Rhabdoviridae family. This is the first report of the virus infecting marine farmed fish in the world, and its discovery marks an important progress in the study of rhabdoviruses. As an emerging infectious disease, the outbreak of CAPRV2023 in fish diseases has attracted widespread attention. There is currently no specific therapeutic drug for the virus, and its prevention and control mainly relies on strengthening breeding management, optimizing the breeding environment, and cutting off the transmission pathways of pathogens. Today, due to the lack of efficient diagnostic tools, clinical diagnosis and prevention face many challenges. Developing a molecular diagnostic method for accurate and rapid detection of CAPRV2023 is the focus of current research.

[0005] At present, a SYBRGreen fluorescence quantitative detection technology established by a primer and probe set developed for the CAPRV2023G protein gene has been reported. Among the other types of rhabdoviruses, there are also many research reports on G protein. However, studies have shown that the G protein of another rhabdovirus, carp spring blood virus (SVCV), has certain differences in gene sequences between different virus strains, and the detection method using G protein may have missed detection. The N protein is the protein with the highest proportion in rhabdoviruses, plays an important role in the transcription and replication of the virus, and is highly conservative. Therefore, selecting the N gene as a target to detect CAPRV2023 is an ideal strategy, which provides a new target gene detection method for the detection of the virus. It enables mutual verification with other detection technologies to obtain more accurate data results. The present invention intends to select a conservative gene sequence encoded by the N protein to establish a Taqman fluorescence quantitative PCR detection method, which will help to detect viral infection early, improve the efficiency of disease prevention and control, reduce economic losses, and lay a good foundation for the development of the aquaculture industry of oval pomfret. Summary of the invention

[0006] The purpose of the present invention is to provide a primer probe for detecting fish rhabdovirus CAPRV2023. The combination aims 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 diagnosis purposes.

[0008] The last object of the present invention is to provide the use of the above primer probe or the above kit in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes.

[0009] The above-mentioned first object of the present invention can be achieved by the following technical scheme: a primer probe for detecting fish rhabdovirus CAPRV2023, the primers include a forward primer CAP-NF and a reverse primer CAP-NR, 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 design of the primers and probes is based on the specific gene region of the N gene sequence of the rhabdovirus CAPRV2023, ensuring the accuracy and specificity of the detection, wherein the N gene sequence of the rhabdovirus CAPRV2023 is shown as SEQ ID NO.7, and the sequence of the specific gene region is shown as SEQ ID NO.4.

[0011] Preferably, the probe of the present invention is specially treated in structure, the 5′ end of the probe is labeled with a fluorescent group, and the 3′ end of the probe is connected to a quenching group. This design allows the fluorescent group to be released and emit a signal once the probe binds to the target sequence and is cut during PCR amplification, thereby achieving real-time and quantitative detection. The selection of fluorescent groups may include but is not limited to FAM, HEX, VIC, ROX, JOE, Cy3, TxR or Cy5, etc., while the quenching group may be selected from BHQ1, BHQ2, BHQ3, TAMRA or MGB, etc.

[0012] More preferably, in order to obtain the best detection effect, the present 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 can generate a strong fluorescent signal and effectively reduce background noise, thereby improving the sensitivity and accuracy of the detection.

[0013] The present invention also provides the use of the primer probe for detecting fish rhabdovirus CAPRV2023 in preparing a kit for detecting fish rhabdovirus CAPRV2023.

[0014] The present invention also provides a kit for detecting fish rhabdovirus CAPRV2023, and the kit comprises the primer probe.

[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 a qPCR MIX reaction solution, a positive control and a negative control.

[0017] Preferably, the qPCR MIX reaction solution should contain the following components: 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 the rhabdovirus CAPRV2023, and its preparation method is: extracting RNA of the rhabdovirus CAPRV2023 and reverse transcribing it into cDNA, 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 perform PCR amplification reaction on the template cDNA to obtain a DNA containing the N gene sequence (SEQ ID NO.7), recovering the amplified fragment, and connecting it to the pcE3 vector using a conventional cloning method, which is the positive control.

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

[0020] 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, comprising the following steps:

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

[0022] (2) using cDNA as a template, establishing a real-time fluorescence quantitative PCR reaction system and reaction conditions, performing real-time fluorescence quantitative PCR detection, and obtaining a Ct value and amplification curve of the sample to be tested;

[0023] (3) Determination of test results: Determine whether the sample to be tested is positive or negative for fish rhabdovirus CAPRV2023 based on the Ct value and amplification curve of the sample to be tested.

[0024] The present invention provides a method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes. The method is performed using the above-mentioned primer probe or kit and can be widely used in aquaculture, fishery resource protection and scientific research fields to monitor and evaluate the infection of CAPRV2023 virus in fish.

[0025] In the above-mentioned method for detecting fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes:

[0026] Preferably, the reaction system of real-time fluorescence quantitative PCR in step (2) is: 10 μL of qPCR MIX reaction solution, 1 μL of each forward and reverse primer 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 supplemented with ddH2O to a total volume of 20 μL.

[0027] Preferably, the real-time fluorescence quantitative PCR reaction conditions in step (2) are: 95°C for 30s, 53°C for 30s, and 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, and if the repeated result has no Ct value, it is negative, otherwise it is positive.

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

[0030] Compared with traditional PCR detection, the present invention has the following significant advantages:

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

[0032] (2) Real-time monitoring function: By continuously monitoring the fluorescence signal, real-time online monitoring of the detection process is achieved, so that the increase of the product can be observed intuitively. At the same time, the test results can be directly read through computer software, eliminating the tedious post-reaction processing steps;

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

[0034] (4) Simple operation process: Operators only need to complete three simple steps: liquid 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 have a lower operation threshold and is easier for operators to accept and promote.

[0035] (5) Reduce the risk of contamination: The closed tube detection method avoids the subsequent opening of the PCR product, thereby effectively preventing the generation of false positive test results and reducing aerosol pollution to the environment;

[0036] (6) High detection sensitivity: The detection limit can reach 20 copies, and at 2×10 1 copies-5×10 9 The quantity is accurate within the range of copies. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0038] Figure 1 The results are as follows: The results are as follows: The results are as follows:

[0039] Figure 2 The results are as follows: The results are as follows: The results are as follows:

[0040] Figure 3 The optimized result of the reaction temperature of the fluorescent quantitative PCR reaction in Example 2;

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

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

[0043] Figure 6 Figure 3 shows the amplification curve and electrophoresis results of the sensitivity analysis in Example 3, where Figure A is the TaqMan qPCR sensitivity test. Note: 1 to 10 in Figure A represent 2×10 1 copies / μL-5×10 9 The concentration of plasmid is 5×10 copies / μL. Figure B shows the sensitivity of normal PCR. Note: 1 to 9 in Figure B represent 5×10 1 copies / μL-5×10 9 copies / μL concentration of plasmid, N is the negative control;

[0044] Figure 7It is an amplification curve diagram of specific detection in Example 4; wherein 1: positive control; 2: SGIV; 3: SCRV; 4: IHNV; 5: NNV; 6: Vibrio vulnificus; 7: Vibrio harveyi; 8: Streptococcus iniae; 9: Lactococcus garvei; 10: Streptococcus dysgalactiae; 11: Vibrio alginolyticus; 12: negative control; DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0046] Unless otherwise specified, the materials, reagents and equipment used in the present invention can be obtained from commercial sources. The experimental methods used in the following examples are conventional methods unless otherwise specified. The primers and probes are recommended but not limited to those synthesized by 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 for the specific gene region (as shown in SEQ ID NO.4) of the N gene sequence (as shown in SEQ ID NO.7) of the rhabdovirus CAPRV2023, thereby ensuring the accuracy and specificity of the 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-CTGAGAAAGATTGCCAGCTTCATCCGA-3'BHQ1 (as shown in SEQ ID NO. 3).

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

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

[0055] The rhabdovirus CAPRV2023 was isolated and obtained by the following method: fish tissue with rhabdovirus CAPRV2023 was used to make a tissue homogenate using a grinding rod, diluted with 10 times the volume of sterile PBS and centrifuged at 6000xg for 1 hour, the supernatant was taken, and passed through a 0.22μm pinhole filter to make a virus incubation solution, and the virus incubation solution was used to incubate FHM cells for 1 hour, discarded, and L-15 culture medium containing 10% fetal bovine serum was added to the FHM cells again and continued to be cultured at 28°C for 48 hours. The FHM cells and the culture bottles were placed at -80°C and repeatedly frozen and thawed 3 times, and centrifuged at 5000xg for 5 minutes, and a solution containing a large amount of CAPRV virus was obtained. The method is reproducible and can be used to culture the rhabdovirus CAPRV2023 in large quantities.

[0056] The primer probe 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 primer probe.

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

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

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

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

[0061] The positive control is a pCE3 vector clone of the N protein gene fragment of the rhabdovirus CAPRV2023. The preparation method thereof is as follows: RNA of the rhabdovirus CAPRV2023 is extracted and reversely transcribed into cDNA, a PCR amplification reaction is performed on the template cDNA using a forward primer CAP-N-COLN1F: 5'-ACAAATGGGGGAGATGCGTT-3' (SEQ ID NO.5) and a reverse primer CAP-N-COLN1R: 5'-AGCCTGACAATCCTCTTGGC-3' (SEQ ID NO.6) to obtain a DNA containing a target gene sequence, the amplified fragment (SEQ ID NO.7) is recovered, and the fragment is connected to the pCE3 vector using a conventional cloning method, which is 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 diagnosis purposes provided in this embodiment comprises the following steps:

[0065] (1) Using an RNA extraction kit to extract RNA from the test sample and reverse transcribe it into cDNA;

[0066] The specific implementation method is as follows: 10 mg of spleen and kidney tissue of diseased golden pomfret fish infected with CAPRV2023 was weighed, and RNA was extracted using tissue RNA extraction reagent FastPure Complex Tissue Total RNA Isolation Kit (Nanjing Novozyme), and reverse transcribed into cDNA using HiScript IIQ RT SuperMix for qPCR (+gDNA wiper) (Nanjing Novozyme), and the template cDNA was subjected to PCR amplification reaction using forward primer CAP-N-COLN1F and reverse primer CAP-N-COLN1R to obtain N protein gene sequence DNA (shown in SEQ ID NO.7);

[0067] (2) Template preparation: The plasmid containing the fish rhabdovirus CAPRV2023 N protein gene sequence was connected to the pcE3 vector (Ultra-Universal TOPO Cloning Kit, Nanjing Novogene) and transformed into Escherichia coli DH5α for proliferation. After that, it was extracted by alkaline lysis method and purified by DNA purification kit to obtain positive recombinant plasmid standard. The standard was diluted to 5×10 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 reaction solution were added to the reaction system, and 5×10 4 The positive recombinant plasmid standard was amplified by qPCR.

[0069] The annealing temperatures were set at 51, 53, 55, 57 and 59°C, the primer concentrations were set at 0.2, 0.4, 0.5, 0.6, 0.8 μmol / L, and the probe concentrations were set at 0.03125, 0.0625, 0.125, 0.25, 0.375 μmol / L, respectively. The lowest Ct value and the higher fluorescence intensity increase value 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, respectively, the molar ratio is 8:8:1, and the optimal annealing temperature is 53°C.

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

[0072] (5) The real-time fluorescence PCR reaction conditions are: 95° C. for 30 s, 53° C. for 30 s, and 45 cycles; the fluorescence quantitative PCR detection is performed using the reaction system, and the fluorescence signal is collected at the end of each cycle extension to obtain the amplification curve of the sample;

[0073] (6) Determination of test results: In conventional sample testing, RNA from the spleen and kidney tissues of the diseased golden pomfret fish to be tested is extracted and reverse transcribed into cDNA. The qPCR reaction system and reaction conditions established above are used for qPCR amplification. The amplification result is determined by observing the amplification Ct of the fluorescence quantitative PCR instrument. If the Ct value is ≤35 and a typical amplification curve appears, the result is positive ( Figure 4 Figure A in the figure), that is, the test sample contains fish rhabdovirus strain CAPRV2023; there is no Ct value or amplification curve, and the result is negative ( Figure 4 When the Ct value is >35, repeat the sample. If the repeated result has no Ct value, it is negative; otherwise, it is positive.

[0074] Example 3

[0075] Establishment of quantitative formula and sensitivity test of the kit for the preparation of fluorescent quantitative RT-PCR primers for fish rhabdovirus CAPRV2023:

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

[0077] (2) Preparation of standard curve: The fluorescence quantitative PCR reaction system and reaction conditions described in Example 2 were used to prepare the standard curve with different concentrations (2×10 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 fluorescence quantitative PCR amplification was performed on the 100 copies of the samples to obtain amplification curves of standards with different concentrations.

[0078] The amplification curve of the amplification method was obtained by taking the common logarithm (lgC) of the initial copy number concentration (C) of the standard as the abscissa and the cycle threshold (Ct value) of fluorescence as the ordinate.

[0079] The results show that Figure 5 ), the template concentration in the reaction system was 2×10 1 -5×10 9 There is a good linear relationship in the range of 1.5-2.0, and the logarithm of the initial copy number (copies / μL) is closely related to C t The linear relationship between the values ​​is Y = -3.3132*X + 39.142, and the correlation coefficient is R 2 =0.9993, which is in line with the expected effect of the experimental design.

[0080] (3) The linearity and sensitivity of the fish rhabdovirus CAPRV2023 standard with different concentrations were analyzed by real-time fluorescence PCR. The detection limit of the real-time fluorescence quantitative PCR detection method for fish rhabdovirus CAPRV2023 established in the present invention was 20 copies / μL ( Figure 6 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 fish rhabdovirus CAPRV2023 fluorescence quantitative RT-PCR detection kit:

[0083] Using the method described in Example 2, specific detection was performed on cDNA or DNA of ten pathogens frequently occurring in aquaculture, including grouper frog iridovirus (SGIV), mandarin fish rhabdovirus (SCRV), infectious hematopoietic necrosis virus (IHNV), neural necrosis virus (NNV), Vibrio vulnificus, Vibrio harveyi, Streptococcus iniae, Lactococcus garris, Streptococcus dysgalactiae, and Vibrio alginolyticus. A negative control group (double distilled water) and a positive control (spleen tissue of golden pomfret infected with CAPRV2023) were set up at the same time. The specific amplification curve is shown in FIG. Figure 7 .

[0084] from Figure 7 It can be seen that only the cDNA of the spleen tissue of golden pomfret infected by CAPRV2023 and the positive control showed a typical amplification curve, indicating that the RT-PCR detection method of fish rhabdovirus CAPRV2023 has good specificity and extremely high sensitivity.

[0085] Example 5

[0086] Repeatability test of fish rhabdovirus CAPRV2023 fluorescence quantitative RT-PCR detection kit:

[0087] Using the method established in Example 2, the concentration of 2×10 1 -5×10 9 The positive recombinant plasmid standard with 10 copies / μL concentration was used as a template, and the fluorescence quantitative PCR amplification was repeated 3 times within and between each concentration group. The CT value results of the amplification were statistically analyzed (Table 1). As shown in the table, the intra-group variation coefficient was 0.14% to 0.60%, and the inter-group variation coefficient was 0.04% to 2.28%, both less than 2.5%. This indicates that the quantification is accurate and highly repeatable, and can be used to detect quantitative samples.

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

[0089]

[0090] Example 6

[0091] Application of CAPRV2023 Rhabdovirus for detection of infected fish samples

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

[0093] The results showed that the kit and method established by the present invention detected 44 samples, and the qPCR positive detection rate was 31.82%, while the ordinary PCR positive detection rate was only 20.45% (Table 2). The positive detection rate of qPCR was 11.37% higher than that of the traditional PCR detection method. All collected samples were subjected to cell separation and culture and electron microscopy observation, and the confirmed positive sample results were consistent with the positive sample results detected by the real-time fluorescence quantitative PCR method of the present invention, with a compliance rate of 100%. It shows that the real-time fluorescence quantitative PCR detection kit and method of the present invention can be combined to detect the rhabdovirus CAPRV2023 in clinical fish samples, with strong specificity and accurate detection results.

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

[0095]

[0096]

[0097]

[0098] Note: In the figure, “+” represents positive and “-” represents negative.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which 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 attached 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, 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 5' end of the probe is labeled with a fluorescent group, and the 3' end of the probe is connected to a quenching group. 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. Use of the primer probe according to claim 1 in preparing a kit for detecting fish rhabdovirus CAPRV2023.

4. A kit for detecting fish rhabdovirus CAPRV2023, characterized in that: The kit comprises the primer probe according to claim 1.

5. The 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 diagnosis purposes, characterized in that: The following steps are involved: (1) extracting RNA from the sample to be tested and reverse transcribing it into cDNA; (2) using cDNA as a template, establishing a real-time fluorescence quantitative PCR reaction system and reaction conditions, performing real-time fluorescence quantitative PCR detection, and obtaining a Ct value and amplification curve of the sample to be tested; (3) Determination of test results: Determine whether the sample is positive or negative for fish rhabdovirus CAPRV2023 based on the Ct value and amplification curve of the sample.

7. The method according to claim 6, characterized in that The reaction system of real-time fluorescence quantitative PCR in step (2) is: 10 μL of qPCR MIX reaction solution, 1 μL of each forward and reverse primer 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 supplemented with ddH2O to a total volume of 20 μL.

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

9. The method according to claim 6, characterized in that 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 repeated result has no Ct value, it is negative, otherwise it is positive.

10. Use of the primer probe according to claim 1 or the kit according to any one of claims 3 to 5 in the detection of fish rhabdovirus CAPRV2023 for non-disease diagnosis purposes.

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