Primer for rapid detection of caprivirus 2023 in fish based on double signal amplification technology and application thereof

Abstract

The application discloses a primer for rapidly detecting fish rhabdovirus CAPRV2023 on site based on double signal amplification technology and application thereof, and the primer comprises a DNA forward primer F2, a DNA reverse primer R2 and an RNA primer RNA4; the sequences of the DNA forward primer F2, the DNA reverse primer R2 and the RNA primer RNA4 are respectively shown as SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:10; the 5' end of the RNA primer RNA4 is a FAM-labeled reporter group, and the 3' end is a BHQ1-labeled quencher group. The application further discloses application of the primer or kit in rapid detection of fish rhabdovirus CAPRV2023 on site for non-disease diagnosis purposes. The kit can efficiently and rapidly detect samples, has high specificity, and can early discover samples infected with CAPRV2023.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus molecular detection, and particularly relates to a primer for rapidly detecting fish rhabdovirus Carpione rhabdovirus 2023 (CAPRV2023) on site based on a double signal amplification technology and application thereof. BACKGROUND

[0002] Rhabdoviruses are a class of linear single-stranded negative-strand RNA viruses, which have a wide range of hosts and can infect mammals, birds, reptiles, fish, insects and plants. The virus particles are rod-shaped or bullet-shaped, 100-430 nm long and 45-100 nm in diameter. The genome encodes five major structural proteins, namely nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L). Currently, more than 20 species of rhabdoviruses that can infect fish have been reported, which are members of the Novirhabdovirus, Perhabdovirus, Siniperhavirus, Sprivivirus and Scophrhavirus genera. These viruses often cause mass mortality and cause significant economic losses to the fish farming industry. The clinical symptoms of fish rhabdovirus disease mainly include loss of appetite, dark body color, loss of body balance, abdominal distension, and hemorrhage of multiple organs in the body surface and internal organs.

[0003] Trachinotus ovatus, commonly known as gold pompano, has fast growth rate, delicious meat and high economic value, and is one of the important species for deep-sea aquaculture in southern China. With the expansion of intensive aquaculture, the ecological environment of aquaculture is increasingly imbalanced, and pathogenic bacteria are rapidly proliferating, often causing large-scale outbreaks of disease. In recent years, a new type of acute infectious disease has occurred in gold pompano in deep-sea net cage culture in southern China, which has a fast transmission speed, a wide prevalence range, a high incidence and mortality rate, and a mortality rate of more than 85% within 3-7 days, posing a major threat to gold pompano aquaculture. The inventors' team isolated a new rhabdovirus strain from infected gold pompano and named it Carpione rhabdovirus 2023 (CAPRV2023), which is a new member of the rhabdovirus Carpione rhabdovirus and was first discovered to be able to infect marine fish worldwide.

[0004] Due to the lack of antiviral drugs and corresponding vaccines, effective control measures are currently unavailable for this emerging infectious disease. Our team has developed several diagnostic methods for detecting CAPRV2023, including cell culture, in situ hybridization, electron microscopy, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative PCR (TaqMan qPCR), and SYBR Green I qPCR. However, these methods have limitations, primarily being time-consuming, complex, and requiring specialized laboratories and skilled technicians. Therefore, there is an urgent need to develop a simple, rapid, accurate, highly specific, and highly sensitive molecular detection method to detect CAPRV2023 as early as possible, enabling timely and effective prevention and control measures in aquaculture, which is crucial for the healthy and sustainable development of pomfret farming. Summary of the Invention

[0005] The purpose of this invention is to provide a primer for rapid on-site detection of fish rhabdovirus CAPRV2023 based on dual signal amplification technology.

[0006] The present invention also aims to provide a rapid on-site detection method for fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

[0007] The final objective of this invention is to provide the application of the above-described primers or kits in the rapid on-site detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

[0008] The first objective of the present invention can be achieved by the following technical solution: a primer for rapid on-site detection of fish rhabdovirus CAPRV2023 based on dual signal amplification technology, wherein the primer includes a DNA forward primer F2, a DNA reverse primer R2, and an RNA primer RNA4. The sequence of the DNA forward primer F2 is shown in SEQ ID NO: 2, the sequence of the DNA reverse primer R2 is shown in SEQ ID NO: 14, and the sequence of the RNA primer RNA4 is shown in SEQ ID NO: 10. The 5' end of the RNA primer RNA4 is a FAM-labeled reporter group, and the 3' end is a BHQ1-labeled quencher group.

[0009] The RNA primers of this invention have a FAM-labeled reporter group added to the 5' end and a BHQ1-labeled quencher group added to the 3' end to become probes for detecting G protein genes. The FAM and BHQ1 modified RNA primer fluorescent probes are used to display the fluorescent signal generated during the amplification process of the target gene, and the fluorescent signal is proportional to the total amount of DNA bound to the probe.

[0010] The application further provides application of the primer in preparation of a kit for rapidly detecting fish rhabdovirus CAPRV2023 on site.

[0011] The application provides a kit for rapidly detecting fish rhabdovirus CAPRV2023 on site based on double signal amplification technology, and the kit comprises the primer.

[0012] Preferably, the kit further comprises a nucleic acid amplification enzyme system and a signal amplification enzyme system, the nucleic acid amplification enzyme system comprises a polymerase, and the signal amplification enzyme system comprises an endonuclease, and the nucleic acid amplification enzyme system and the signal amplification enzyme system are in a basic dry powder form.

[0013] The double enzyme system can be used for rapidly detecting fish rhabdovirus CAPRV2023 on site based on double signal amplification technology.

[0014] Preferably, the kit further comprises RT-MIX, and the RT-MIX comprises but is not limited to being synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., or other reverse transcription kits such as HiScript II Q RTSuperMix for qPCR (+gDNA wiper) (Nanjing Novelstar) can be used for reverse transcription.

[0015] Preferably, the kit further comprises activating enzymes NTP and RNase-free Water.

[0016] In use, the DNA forward primer, the DNA reverse primer, the RNA primer, the basic dry powder, the RT-MIX and the freeze-drying protective agent can be prepared into pre-packaged freeze-drying balls, and the activating enzymes NTP and RNase-free Water are added for activation.

[0017] Therefore, the kit in the application can comprise a system composed of the DNA forward primer, the DNA reverse primer, the RNA primer, the RT-MIX, the nucleic acid amplification enzyme system and the signal amplification enzyme system, and a reagent prepared from the activating enzymes NTP (nucleoside triphosphate) and RNase-free Water.

[0018] In addition to the DNA forward primer, the DNA reverse primer and the RNA primer, the remaining other components such as the polymerase of the nucleic acid amplification enzyme system, the endonuclease of the signal amplification enzyme system, the activating enzymes NTP, the RNase-free Water and the freeze-drying protective agent are all conventional raw materials on the market, and can be integrated in an RT-Ultradar blank amplification kit, including but not limited to being provided by Suzhou Jingrui Biotechnology Co., Ltd.

[0019] The kit provided by the application can realize 4 copy level DNA detection, in addition to the rapid qualitative detection of fish rhabdovirus CAPRV2023, the early screening of fish rhabdovirus CAPRV2023 can also be realized, the missed detection rate caused by false negative is extremely low, the operation is convenient, and the steps are simple.

[0020] In addition, the kit of the application can further comprise a high-efficiency lysis solution for extracting CAPRV2023 sample DNA, preferably, the high-efficiency lysis solution comprises Tris, NaCl, EDTA, SDS, PVP-40 and a broad-spectrum protease (such as pepsin, trypsin, etc.) with a pH value of 8, and the high-efficiency lysis solution can include but is not limited to purchased from Suzhou Jingrui Biological Co., Ltd.

[0021] The above-mentioned second object of the application can be realized by the following technical scheme: a non-disease diagnosis purpose fish rhabdovirus CAPRV2023 on-site rapid detection method, comprising the following steps:

[0022] Step (S1): collecting CAPRV2023 samples to be tested, adding the high-efficiency lysis solution for high-temperature lysis, and obtaining a DNA template;

[0023] Step (S2): using the DNA of step (S1) as a template, adding the kit for isothermal amplification reaction;

[0024] Step (S3): using the isothermal amplification reaction fluorescence signal Tt value of step (S2) as the CAPRV2023 detection result, when there is no Tt value in the cycle, it indicates that the sample to be tested is not infected with CAPRV2023, and the detection result is negative, when there is a Tt value in the cycle and it is less than 20 min, it indicates that the sample to be tested is infected with CAPRV2023, and the detection result is positive.

[0025] The above-mentioned non-disease diagnosis purpose fish rhabdovirus CAPRV2023 rapid on-site detection method is to perform high-efficiency lysis on the sample to be tested for 10 minutes, add a pre-dispensed freeze-dried ball prepared from a nucleic acid amplification group enzyme system, a signal amplification group enzyme system, an RNA primer probe, a DNA forward primer, a DNA reverse primer, etc., only need to add sample DNA, and then the reaction can be performed on a palm metal bath and a palm fluorescence detector for 10-30 minutes, so that the minute diagnosis is completed.

[0026] In the above-mentioned non-disease diagnosis purpose fish rhabdovirus CAPRV2023 rapid on-site detection method:

[0027] Preferably, the high-temperature lysis temperature in step (S1) is 95 DEG C, and the lysis time is 10 minutes.

[0028] Preferably, the isothermal amplification reaction in step (S2) is carried out at 42 DEG C for 10-30 minutes.

[0029] The application is directed to the conserved region of G protein sequence in the genome of fish rhabdovirus CAPRV2023, and designs RNA primers, DNA forward primers and DNA reverse primers based on enzyme-mediated double amplification nucleic acid rapid detection technology; through isothermal nucleic acid amplification and isothermal signal amplification reaction of the sample to be tested, the fluorescence signal value is finally detected for judgment.

[0030] The above third object of the application can be realized by the following technical solution: application of the above-mentioned primers or kit in rapid on-site detection of fish rhabdovirus CAPRV2023 for non-diagnostic purposes.

[0031] The application has the following advantages:

[0032] (1) The DNA extraction step provided by the application is simplified, and only needs to add a lysis solution and heat treatment for 10 minutes to complete;

[0033] (2) The enzyme-mediated double amplification nucleic acid amplification technology for detecting fish rhabdovirus CAPRV2023 in the application can amplify the target nucleic acid to 10 9 times under the condition of 42 DEG C isothermal condition within 10-30 minutes through integration of nucleic acid amplification group enzymes, and more than 10,000 fluorescence signals can be generated from one nucleic acid amplification product under the condition of 42 DEG C isothermal condition through integration of signal amplification group enzymes, so that high-efficiency and rapid detection of samples can be finally achieved;

[0034] (3) The application detects CAPRV2023 by molecular detection, has strong specificity, and the sensitivity can be as low as 4 copies / μL, so that samples infected with CAPRV2023 can be detected early;

[0035] (4) The application can be used in cooperation with a portable palm metal bath and a palm fluorescence detector, and can be carried to any place such as rural areas for on-site detection, so as to meet various application scenarios of CAPRV2023 in the front line of aquaculture;

[0036] (5) The operation process of the application is simple and suitable for anyone to use, and has wide application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0037] The application will be further described below with reference to the accompanying drawings and embodiments.

[0038] Figure 1 It is the RNA primer screening result of the embodiment 1 of the application;

[0039] Figure 2 It is the high-sensitivity primer group screening experiment result of the embodiment 4 of the application;

[0040] Figure 3 Results of primer set screening experiment for RT reaction of embodiment 5 of the present application;

[0041] Figure 4 Results of specificity experiment of embodiment 6 of the present application;

[0042] Figure 5 Results of sensitivity experiment of embodiment 7 of the present application;

[0043] Figure 6 Flow chart for detecting CAPRV2023 based on enzyme-mediated double amplification nucleic acid amplification in embodiment 8 of the present application. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present application but not to limit the scope of the present application.

[0045] Unless specifically stated, the materials, reagents and equipment used in the present application can be obtained from commercial channels. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The primers and probes, RT-MIX, etc. are recommended but not limited to being synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0046] Preparation of fish rhabdovirus CAPRV2023 G protein gene standard plasmid:

[0047] The plasmid containing the fish rhabdovirus CAPRV2023 G protein gene sequence (amplified using the primers shown in SEQ ID NO: 22 and SEQ ID NO: 23) was linked into pcE3 vector (Ultra-Universal TOPO Cloning Kit, Nanjing Novizen) and transformed into E. coli DH5α for proliferation, then extracted by alkaline lysis method, purified by DNA purification kit, and positive recombinant plasmid standard was obtained.

[0048] Embodiment 1

[0049] The CAPRV2023 positive sample used in the test was from the spleen, liver, feces of Trachinotus ovatus or water sample contaminated with fish rhabdovirus CAPRV2023 G, and the continuous detection by fluorescence quantitative PCR was positive (reference application number 202410994895.9).

[0050] The RNA of the tissue was extracted using the tissue RNA extraction reagent FastPureComplex Tissue Total RNA Isolation Kit (Nanjing Novogene) or high-efficiency lysis solution, and the DNA was reversely transcribed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Nanjing Novogene) or RT-MIX. After the positive sample was determined by fluorescence quantitative detection, it was stored in a refrigerator at -80°C.

[0051] Reaction system:

[0052] In the eight-tube containing the basic dry powder (nucleic acid amplification group enzyme system including polymerase, signal amplification enzyme system including endonuclease), 1 μL of DNA forward primer (10 μM), 1 μL of DNA reverse primer (10 μM), 1 μL of 5'-end FAM-labeled reporter group and 3'-end BHQ1-labeled quencher group RNA primer (1 μM), 6 μL of DNA sample to be tested were added, and then centrifuged, and left at room temperature for 2 min. A solution of 10 μL of activated enzyme NTP (nucleoside triphosphate) and RNase-free water (RNase-free water and activated enzyme NTP were provided by Suzhou Jingrui Biotechnology Co., Ltd.) was added along the wall of the eight-tube, and then centrifuged. Shake and mix for 10 s, and then centrifuge and detect on the machine.

[0053] The DNA forward primer is:

[0054] 5'-ATCCCACCAAGAACACTGCTGAGGGACG-3' (as shown in SEQ ID NO: 2);

[0055] The DNA reverse primer is:

[0056] 5'-AAGCTAATACGACTCACTATAGGGTCCTGATCCATTGTTCTCCAGCAAACTG-3' (as shown in SEQ ID NO: 14);

[0057] The nucleotide sequence of the RNA primer is:

[0058] 5'6-FAM-rApp-UGCGCCUGGACACCAUCCAUGGGGACUU-3`BHQ1 (as shown in SEQ ID NO: 10);

[0059] The fluorescence quantitative PCR instrument (LightCycler 96 (Roach)) has a reaction temperature of 42°C, signal acquisition is performed once for each cycle, a total of 30 cycles, the reaction time is 30 min, and the reporter group is set to FAM.

[0060] Using DNA validated by previous qPCR as a template (standard plasmid of the fish rhabdovirus CAPRV2023G protein gene), Tris-EDTA (TE) was used as a negative control, and RNase-free water (Takara) was used as a blank control for RNA primer screening. Primers F2 and F3 were cross-combined with primers R2 and R3, resulting in F2R2, F2R3, F3R2, and F3R3, respectively. RNA primers RNA1, RNA2, RNA3, RNA4, RNA5, and RNA6 were added to each primer group. Corresponding primer groups were compared, and the RNA primer with the lowest Tt value and the highest endpoint fluorescence value was selected. Simultaneously, based on the RNA primers, the DNA primer screening results for the corresponding four groups were determined, and the RNA primer with the lowest Tt value and the highest endpoint fluorescence value, along with the primer group showing no amplification signal in the negative control, were selected.

[0061] Table 1 shows the primers used in this invention.

[0062]

[0063]

[0064] The results showed that 24 combinations were obtained by combining four primer groups (F2R2, F2R3, F3R2, F3R3) and six RNA primers (RNA1, RNA2, RNA3, RNA4, RNA5, RNA6). Sixteen combinations showed good amplification results. The RNA4 and RNA5 combination had the lowest Tt value and relatively high fluorescence value. No amplification signal was observed in the negative control. Therefore, RNA4 and RNA5 were selected as candidate RNA primers for subsequent experiments. Figure 1 ).

[0065] Example 2

[0066] Using CAPRV2023 (positive recombinant plasmid standard) 10 4 Using the copy plasmid as a template, the RNA4 and RNA5 primers (RNA primers) and forward primers F2 and F3 were fixed according to the sample loading system of Example 1. Reverse primers R1, R2, R3, R4, R5, and R6 were selected for screening. The RNA primers with the smallest Tt value and the highest endpoint fluorescence value, as well as the primer set with no amplification signal in the negative control, were selected.

[0067] The results show that in the DNA reverse primer screening results, the Tt values of F2-R2-RNA4, F2-R5-RNA4, F2-R2-RNA5, F3-R2-RNA5 groups are lower, and the fluorescence values are relatively high, which indicates that the reverse primers R2 and R5 have better effects, and the negative control has no amplification signal, so the reverse primers R2 and R5 are selected for subsequent experiments (Table 2 below).

[0068] Table 2 is the DNA forward primer screening result of Example 2 of the present application

[0069]

[0070]

[0071] Example 3

[0072] The CAPRV2023 10 3 (positive recombinant plasmid standard) copy plasmid is used as a template, the RNA4, RNA5 primers and reverse primers R2 and R5 are fixed, and the forward primers F1, F2, F3, F4, F5 and F6 are selected for screening according to the sample addition system of Example 1. The DNA primers with the smallest Tt value and the highest end-point fluorescence value, and the primers without amplification signal in the negative control are selected.

[0073] The results show that in the DNA reverse primer screening results, the Tt values of F2-R2-RNA4, F2-R5-RNA4, F2-R2-RNA5, F3-R2-RNA5 groups are lower, and the fluorescence values are relatively high, which indicates that the reverse primers R2 and R5 have better effects, and the negative control has no amplification signal, so the reverse primers R2 and R5 are selected for subsequent experiments (Table 3).

[0074] Table 3 is the DNA reverse primer screening result of Example 3 of the present application

[0075]

[0076]

[0077] Example 4

[0078] The CAPRV2023 10 1(positive recombinant plasmid standard) as a template, according to the sample loading system of Example 1, F2-R2-RNA5, F3-R2-RNA5, F4-R2-RNA5, F2-R2-RNA4, F5-R2-RNA4, and F2-R5-RNA4 were screened, and the DNA primer with the lowest Tt value and the highest end-point fluorescence value, and the high-sensitivity primer group without amplification signal in the negative control were selected.

[0079] The results showed that in the screening results of the DNA high-sensitivity primer group, the Tt values of F3-R2-RNA5, F4-R2-RNA5, F2-R2-RNA4, and F2-R5-RNA4 groups were lower, the fluorescence values were higher, and there was no amplification signal in the negative control, so F3-R2-RNA5, F4-R2-RNA5, F2-R2-RNA4, and F2-R5-RNA4 groups were selected for follow-up experiments. Figure 2

[0080] Example 5

[0081] The standard RNA (10 5 copies / μL) sample subjected to qPCR fluorescence quantification was used as a template, and according to the sample loading system of Example 1, F3-R2-RNA5, F4-R2-RNA5, F2-R2-RNA4, and F2-R5-RNA4 were subjected to RT reaction system screening, and the DNA primer with the lowest Tt value and the highest end-point fluorescence value, and the high-sensitivity primer group without amplification signal in the negative control were selected.

[0082] The results showed that in the screening results of the DNA high-sensitivity primer group, the Tt value of the F2-R2-RNA4 group was lower, the fluorescence value was higher, and there was no amplification signal in the negative control, so the F2-R2-RNA4 group was selected for follow-up experiments. Figure 3

[0083] Example 6

[0084] ​​Specific detection was performed on ten kinds of pathogens cDNA or DNA frequently occurring in aquaculture such as Epinephelus akaara frog virus (SGIV), Siniperca chuatsi rhabdovirus (SCRV), Ictalurus nebulosus iridovirus (ISKNV), Infectious hematopoietic necrosis virus (IHNV), Spring viremia of carp virus (SVCV), Tilapia lake virus (TiLV), nervous necrosis virus (NNV), viral hemorrhagic septicemia virus (VHSV), Vibrio harveyi, Strepstococcus iniae, Lactococcus garvieae, Streptococcus dysgalactiae, and Vibrio alginolyticus, using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (Tkara, 9763), with a negative control group (double distilled water) set up, and the specific amplification curve is shown in Figure 4 .

[0085] Only the cDNA of the spleen tissue of the gold pompano infected by CAPRV2023 showed a typical amplification curve, and CAPRV2023 was detected, indicating that the primer and probe combination screened in the present study has high specificity and the detection method has good specificity and high sensitivity. Figure 4 ).

[0086] Example 7

[0087] Reaction system (kit): pre-packaged freeze-dried balls made of basic dry powder (containing polymerase and endonuclease), DNA forward primer F2, DNA reverse primer R2, and RNA primer RNA4, and reagents made of activated enzyme NTP (nucleoside triphosphate) and RNase-free Water.

[0088] Add 50 μL of template DNA (high-efficiency lysis solution, high-temperature lysis temperature 95℃, lysis time 10 minutes, then reverse transcribed into DNA) to the reaction system, cover the lid, shake and mix for 10 s, centrifuge for 10 s, and then detect on the machine. Use a fluorescence quantitative PCR instrument with a reaction temperature of 42℃, signal collection once per cycle, a total of 30 cycles, a reaction time of 30 minutes, and a reporter group set to FAM.

[0089] As a template, the F2-R2-RNA4 whole reaction system sensitivity test was performed according to the sample addition system of Example 1. The minimum was 4 copies, still with a fluorescence signal, and the negative control had no amplification signal, and the minimum sensitivity of F2-R2-RNA4 was determined to be 4 copies. Figure 5 ​

[0090] The standard RNA (10 5 copies / μL) sample was gradient diluted with RNase-free Water to select the copy number of 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 , 4 for machine detection, and the optimal primer set F2-R2-RNA4 obtained by screening was used for sensitivity detection.

[0091] The results showed that the selected primer probe could detect a copy number of 4 with high sensitivity and good machine effect according to the experimental results. Figure 5

[0092] Example 8

[0093] As shown in Figure 6 , the spleen, liver, feces of the laboratory infected Trachurus ovatus or the water sample contaminated with CAPRV2023 elastovirus was used as the sample to be detected, the high-efficiency lysis solution (purchased from Suzhou Jingrui Biological Co., Ltd.) was used for rapid extraction of sample DNA (lysis at 95℃ for 10 minutes), CAPRV2023 DNA (plasmid standard of CAPRV2023 G protein gene fragment) was used as a positive control, TE Buffer was used as a negative control, RNase-free Water was used as a blank control, and the optimal primer set F2-R2-RNA4 obtained by screening was used to judge the effect of the method in actual samples according to the sample addition system, time and temperature of Example 7.

[0094] Twenty-four samples were randomly selected from the laboratory for detection, the lysis solution was used for rapid extraction of sample DNA, and the extracted DNA sample was subjected to isothermal amplification reaction for 10-30 minutes under constant temperature at 42℃, and the remaining samples were used for qPCR detection. The results showed that the detection results were consistent with the qPCR detection results, and the accuracy was 100% (Table 4).

[0095] Table 4 is the results of the present method and the qPCR detection method

[0096]

[0097]

[0098] Note: + is positive, - is negative, and None is not detected.

[0099] ​The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A primer for rapid on-site detection of fish rhabdovirus CAPRV2023 based on dual signal amplification technology, characterized in that, The primers include a DNA forward primer F2, a DNA reverse primer R2, and an RNA primer RNA4. The sequence of the DNA forward primer F2 is shown in SEQ ID NO: 2, the sequence of the DNA reverse primer R2 is shown in SEQ ID NO: 14, and the sequence of the RNA primer RNA4 is shown in SEQ ID NO:

10. The 5' end of the RNA primer RNA4 is a FAM-labeled reporter group, and the 3' end is a BHQ1-labeled quencher group.

2. The application of the primers described in claim 1 in the preparation of a kit for rapid on-site detection of fish rhabdovirus CAPRV2023.

3. A kit for rapid on-site detection of fish rhabdovirus CAPRV2023 based on dual signal amplification technology, characterized in that, The kit includes the primers as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a nucleic acid amplification enzyme system and a signal amplification enzyme system. The nucleic acid amplification enzyme system includes polymerase; the signal amplification enzyme system includes endonuclease. The nucleic acid amplification enzyme system and the signal amplification enzyme system are present in basic dry powder form.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a lysis buffer.

6. A rapid on-site detection method for fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes, characterized in that, Includes the following steps: Step (S1): Collect the CAPRV2023 sample to be tested, add the lysis buffer in claim 5 and perform high-temperature lysis to obtain the DNA template; Step (S2): Using the DNA from step (S1) as a template, add the kit described in claim 4 to perform an isothermal amplification reaction; Step (S3): The Tt (Threshold time) value of the fluorescence signal from the isothermal amplification reaction in step (S2) is used as the detection result of CAPRV2023. When there is no Tt value within the cycle, it means that the sample to be tested is not infected with CAPRV2023 and the detection result is negative. When there is a Tt value within the cycle and it is less than 20 min, it means that the sample to be tested is infected with CAPRV2023 and the detection result is positive.

7. The method for rapid on-site detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes according to claim 6, characterized in that, In step (S1), the high-temperature pyrolysis temperature is 95°C and the pyrolysis time is 10 minutes.

8. The method for rapid on-site detection of CAPRV2023 fish rhabdovirus for non-disease diagnostic purposes according to claim 6, characterized in that, In step (S2), the isothermal amplification reaction is carried out at a constant temperature of 42℃ for 10 to 30 minutes.

9. The application of the primers of claim 1 or the kits of any one of claims 3-5 in the rapid on-site detection of fish rhabdovirus CAPRV2023 for non-disease diagnostic purposes.

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