A double rna virus rpa-lfd primer, probe and kit for detecting siniperca duensis

Abstract

The application belongs to the technical field of virus detection, and particularly relates to a RPA-LFD primer, probe and kit for detecting Siniperca duocula double RNA virus. The application provides a RPA-LFD primer probe combination and kit for detecting Siniperca duocula double RNA virus, and establishes a RPA-LFD detection method for Siniperca duocula double RNA virus on the basis. Firstly, RNA of a sample to be detected is extracted, then the extracted RNA is used as an amplification template, RPA amplification reaction is carried out by using the above-mentioned RPA combination to obtain a product, finally, the product obtained by reaction is analyzed by using a lateral chromatographic test strip, and a detection result is read. The RPA-LFD combination, kit and detection method for detecting Siniperca duocula double RNA virus provided by the application can realize RPA detection of Siniperca duocula double RNA virus, can quickly, simply and specifically detect Siniperca duocula double RNA virus, and is suitable for on-site rapid detection in a breeding farm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus detection, and particularly relates to a double RNA virus RPA-LFD primer, probe and kit for detecting Siniperca chuatsi. BACKGROUND

[0002] With the improvement of people's living quality, Siniperca chuatsi gradually becomes a popular and famous fish species due to its advantages such as tender meat, delicious taste and high protein content. The breeding scale of Siniperca chuatsi is continuously expanding, and Siniperca chuatsi has become one of important freshwater aquaculture varieties. Aquabirnavirus (ABV) belongs to the family Birnaviridae, and the virus particle is icosahedral spherical symmetry with a diameter of about 60 nm and no envelope. Among them, infectious pancreatic necrosis virus (IPNV) is a representative species of ABV, which was first discovered in diseased fish of Salmo gairdneri in 1960. IPNV can cause acute infectious diseases in various aquatic animals and cause great economic losses to the aquaculture industry. Since the 19th century, aquatic animal birnavirus disease has frequently broken out, especially infectious pancreatic necrosis virus (IPNV) is the most popular. The mortality rate of juvenile fish suffering from the disease is as high as 95%, which has brought great damage to the aquaculture industry. Therefore, the establishment of the detection method for aquatic animal birnavirus is of great significance for the prevention of the disease.

[0003] A new birnavirus was isolated from diseased Siniperca chuatsi. The main clinical symptoms were lethargy, irregular swimming behavior, and dark body color. The intestine was filled with yellow mucus, the liver was hemorrhagic, and there was ascites. According to routine diagnosis, it belongs to an unreported pathogen. Therefore, it is preliminarily determined as a new pathogen. Subsequently, the pathogen was identified by virus isolation, electron microscopy observation, physicochemical property identification, genome sequence analysis, indirect immunofluorescence, and in vivo challenge experiment. It was found that the pathogen was a new virus homologous to members of the Birnavirus family, and was named Siniperca chuatsi birnavirus (SCBV). The virus has no envelope and has an icosahedral structure. The genome consists of two segments of double-stranded RNA. Segment A encodes VP2, VP3, and VP4 proteins, and segment B encodes VP1 protein (Delmas B, Attoui H, Ghosh S, Malik YS, Mundt E, Vakharia VN, Ictv RC. 2019. ICTV virus taxonomy profile: Birnaviridae. J Gen Virol 100: 5-6. https: / / doi.org / 10.1099 / jgv.0.001185.). The homologous virus of SCBV, Largemouth bass birnavirus (LBBV), was first isolated from the tissues of largemouth bass in 2020, and a nested RT-PCR and TaqMan real-time fluorescent quantitative RT-PCR detection method was established (Luo M J, Li N Q, Lin Q, et al. Establishment and application of a nested RT-PCR method for detecting a new type of largemouth bass birnavirus [J]. Journal of Fishery Sciences of China, 2021, 45(09): 1584-1591.). However, PCR is time-consuming and has low sensitivity, and it is seriously dependent on laboratory equipment for gel electrophoresis. Although qPCR is sensitive, it requires expensive fluorescence instruments, and both methods are not suitable for rapid detection at the grassroots level. Since SCBV is a new virus involved in the outbreak of birnavirus disease, there is no corresponding detection method, so there is an urgent need for a portable, rapid, and sensitive detection method to fill the gap in rapid detection in aquaculture ponds.

[0004] Recombinase polymerase amplification (RPA) is an isothermal DNA amplification technology, which has more advantages compared with other DNA amplification methods, especially its application in non-laboratory environment. The recombinase polymerase reaction has strong specificity, and the amplification of specific oligonucleotide sequences can be realized under the condition of constant temperature of 37-45℃ for 15-30min, and the amplification product can be visualized and distinguished by lateral flow test strip. The technology has very low requirements for hardware equipment, and after optimization, it can realize the amplification reaction without large or expensive hardware equipment, and the reaction time is short, and the sample does not need to be complicatedly treated, and has the advantages of good flexibility and strong practicability. Therefore, if a RPA-LFD detection method for SCBV can be established, it is expected to realize the rapid detection and analysis of mandarin fish double RNA virus nucleic acid. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a recombinase polymerase isothermal amplification combined with immunochromatography test paper (RPA-LFD) primer and probe combination for detecting mandarin fish double RNA virus, which breaks through the limitation of personnel and place of the prior art, shortens the detection time, improves the portability and convenience of the detection equipment, and realizes the rapid diagnosis of diseased fish and the rapid screening of suspected SCBV carrying fish on site.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0007] The first aspect of the present application provides a RPA-LFD primer and probe combination for detecting mandarin fish double RNA virus, wherein the RPA-LFD primer is composed of an upstream primer B5F shown in SEQ ID NO. 1 and a downstream primer B5R shown in SEQ ID NO. 2, and the RPA-LFD probe is a probe P shown in SEQ ID NO. 3. The 5' end of the RPA-LFD probe is labeled with carboxyfluorescein FAM, the 3' end is added with an extension blocking group C3 Spacer, and the 31st base is replaced with tetrahydrofuran.

[0008] In order to realize the rapid and accurate identification and amplification of mandarin fish double RNA virus specific nucleic acid, the present application screens the best primer in the isothermal amplification system, so that the amplification reagent can sensitively, rapidly and accurately detect the mandarin fish double RNA virus, and can maintain stability and reliability during storage and transportation, and realize the rapid detection and analysis of mandarin fish double RNA virus nucleic acid.

[0009] Preferably, the Siniperca chuatsi birnavirus (SCBV) has a NCBI accession number of 171-3443 for the genomic sequence.

[0010] The second aspect of the present application provides a kit for detecting Siniperca chuatsi birnavirus, which comprises the RPA-LFD primer probe combination of the first aspect.

[0011] Preferably, the kit further comprises A Buffer, B Buffer, recombinase binding single-stranded nucleic acid, strand displacement DNA polymerase, single-stranded DNA binding protein (lyophilized enzyme preparation), reaction system buffer or magnesium acetate.

[0012] Further, the A Buffer is 10% PEG, and the B Buffer is a magnesium acetate solution.

[0013] Preferably, the kit further comprises a positive control, which is the genomic RNA of Siniperca chuatsi birnavirus. The kit further comprises a negative control (ddH2O).

[0014] Preferably, the kit further comprises an RT-RPA nucleic acid amplification test strip.

[0015] Preferably, the final concentration of the RPA-LFD primer is 0.4 μM, and the final concentration of the probe is 0.12 μM.

[0016] Preferably, the total reaction system of the kit is 50 μL, which comprises A Buffer 29.4 μL, ddH2O 8.5 μL, B5F 2 μL, B5R 2 μL, probe P 0.6 μL, RNA template 5 μL, and B Buffer 2.5 μL.

[0017] Preferably, the reaction temperature of the kit is 37-45℃, and the reaction time is more than 10 min.

[0018] Further, the reaction temperature of the kit is 39℃, and the reaction time is 20-30 min.

[0019] The present application also provides a use method of the above-mentioned kit for detecting Siniperca chuatsi birnavirus, which comprises the following steps:

[0020] (1) extracting the RNA of the fish sample to be tested;

[0021] (2) using the RNA extracted in step (1) as an amplification template, and performing RPA amplification reaction (37-45℃, more than 10 min) using the above-mentioned total reaction system to obtain a product;

[0022] (3) using lateral flow dipstick to analyze the product obtained in step (2): the test paper strip quality control line and detection line appear strip at the same time, which is positive; the test paper strip quality control line C appears strip, but the detection line T has no strip, which is negative.

[0023] The recombinant enzyme isothermal amplification technology is a novel nucleic acid in-vitro amplification technology under constant temperature conditions, and has the advantages of easy transportation in the form of freeze-dried powder, high sensitivity, strong specificity, fast reaction time, simple operation and the like. The recombinant enzyme isothermal amplification technology fills the gap of traditional culture and temperature change equipment-dependent technology, and can realize instant detection on site in a breeding farm and in a non-laboratory environment. The RPA technology can realize efficient and rapid amplification of trace nucleic acid in vitro in a constant or wide temperature range through a dynamic environment of about 30 min automatic cycle, and then the amplification product is detected by an end-point detection method, so that an ideal detection result can be obtained. The immunochromatography test paper (LFD) has the same principle as the colloidal gold detection test paper, and is composed of a sample pad, a gold label pad, a detection pad, a quality control pad and a water absorption pad. Since the detection result can be observed by naked eyes, the immunochromatography test paper is suitable for clinical and field detection.

[0024] According to the conserved sequence of the A segment of SCBV, a series of primers and probes are designed, and the best primer and probe combination is screened. After optimization of the reaction conditions, a convenient, rapid, specific and highly sensitive SCBV RPA-LFD detection method is established, and the detection limit can be as low as 10 1 copies / uL. The sensitivity can be comparable to the most commonly used qPCR, is much higher than that of ordinary PCR and other RPA-LFD detection methods, and is very suitable for rapid detection and screening of SCBV infection and carrying in fish bodies such as mandarin fish and sea bass in primary units and breeding sites.

[0025] The third aspect of the present application provides the application of the RPA-LFD primer and probe combination of the first aspect or the kit of the second aspect in the preparation of a mandarin fish double RNA virus detection product.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The application provides a primer probe combination and kit for detecting Siniperca duocula double RNA virus based on an RPA-LFD method, and establishes a SCBV RPA-LFD detection method which is convenient, rapid, specific and highly sensitive, and is rapid, sensitive and simple to operate. Meanwhile, the RPA technology is combined with a lateral flow test strip (LFD), and only 5-10 min is needed for reaction at 37-45 DEG C, the amplification product is mixed with enzyme-free water, inserted into the LFD test strip, and the experimental results can be observed by naked eyes in about 5 min, without gel electrophoresis, so that the results can be directly observed, and the operation is very simple, and is suitable for field detection and large-scale screening. In addition, the amplification primer and probe provided by the application are specific and highly sensitive, can accurately detect the Siniperca duocula double RNA virus, and the detection result is stable and has good repeatability; the Siniperca duocula double RNA virus can be detected on site in a single tube, the result can be directly observed, and the result can be rapidly visualized. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a screening result of RPA primers, wherein 1-5 are full negative detection of five pairs of RPA-LFD primers with blank water as a template, and the primer pairs without non-specific amplification are screened out (1-5 are primer pair 1, primer pair 2, primer pair 3, primer pair 4 and primer pair 5 respectively).

[0029] Figure 2 It is an influence of different reaction conditions on RPA-LFD amplification effect; the influence of different reaction temperatures on RPA-LFD amplification effect (a), wherein the reaction temperatures of 1-6 test strips in the figure are blank (negative), 37 DEG C, 39 DEG C, 41 DEG C, 43 DEG C and 45 DEG C respectively; the influence of different reaction times on RPA-LFD amplification effect (b), wherein the reaction times of 1-7 test strips in the figure are blank (negative), 5 min, 10 min, 15 min, 20 min, 25 min and 30 min respectively.

[0030] Figure 3 It is a specificity test result of the RPA-LFD method, wherein 1-6 test strips in the figure are SCBV, LMBV, ISKNV, SVCV, TiLV and blank respectively.

[0031] Figure 4 It is a sensitivity test result of the RPA-LFD method, wherein 1-8 test strips in the figure are plasmid copy numbers 10 7 -10 0 copies / μL, and 9 is blank.

[0032] Figure 5 It is a repeatability detection result of the RPA-LFD method, wherein 1-5 test strips in the figure are plasmid copy numbers 10 5copies / μL of five replicates, 6-10 test strips were 10 3 copies / μL of five replicates. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application are further described below. It should be noted that the description of these embodiments is intended for purposes of illustration only and is not intended to be limiting. Furthermore, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0034] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0035] Example 1: Establishment, specificity, sensitivity and repeatability analysis of SCBV RPA-LFD method

[0036] 1. Experimental materials and instruments

[0037] 1.1. Main instruments and consumables

[0038] Real-time fluorescent quantitative PCR instrument QuantStudio TM 5Purchased from the United States Applied Biosystems Company; electrophoresis instrument and gel phase analysis system purchased from the United States BIO-RAD Company; desktop high-speed refrigerated microcentrifuge, desktop refrigerated high-speed centrifuge purchased from BIOBASE Company; various specifications of pipette purchased from Dalongxingchuang Experimental Instrument (Beijing) Co., Ltd., sterile 0.2mL, 0.5mL, 1mL and 1.5mL centrifuge tubes purchased from Shanghai Kaijin Biotechnology Co., Ltd.

[0039] 1.2. Main reagents

[0040] RT-RPA nucleic acid amplification reagent (test strip type) kit purchased from Amp Future (Changzhou) Biotechnology Co., Ltd., total RNA extraction reagent (OMEGA) box purchased from Guangzhou Feiyang Biological Engineering Co., Ltd.; viral DNA / RNA extraction reagent kit purchased from Hunan Aikuer Biological Engineering Co., Ltd.; other required reagents provided by the laboratory.

[0041] 1.3. Main virus strains

[0042] The DNA or RNA of Siniperca duocula bimavirus (SCBV), largemouth bass frog iridovirus (LMBV), infectious spleen and kidney necrosis virus (ISKNV), spring viremia of carp virus (SVCV) and Luohu virus (TiLV) were prepared and preserved by the laboratory.

[0043] 2. Primer and probe design and screening

[0044] According to the conserved sequence of the A segment of SCBV (NCBI accession number of genome sequence is 171-3443), the recombinase-specific primer and probe were designed according to the RPA instruction, wherein the conserved segment of the upstream primer was designed at 774-801 bp, the conserved segment of the downstream primer was designed at 917-944 bp, and the conserved segment of the probe was designed at 864-909 bp. The RPA nucleic acid amplification technology has certain differences from the conventional PCR primer design. A pair of primers is composed of two oligonucleotides, which specifically recognize the upstream and downstream nucleotide sequences of a nucleic acid target, respectively; the length is between 30-35 nucleotides (nt), and there is no palindromic sequence, continuous single-base repeat sequence and internal secondary structure region in the sequence; the primer T m mismatch is not the main consideration factor in the design; the optimal primer pair needs to be screened out by experiment optimization. The probe design sequence does not overlap with the specific primer recognition site, the length is 46-52 nt, the sequence avoids palindromic sequence, internal secondary structure and continuous repeat base; there are four modification sites, a tetrahydrofuran (THF) is marked at the middle position of ≥ 35 nt from the 5' end, as the recognition site of exonuclease; a fluorescent group is marked upstream of the THF site, and a quencher group is marked downstream, the distance between the two groups is 2-4 nt; the THF is ≥ 15 nt from the 3' end, and a modification group is marked at the 3' end, and a biotin is marked at the 5' end of the downstream primer.

[0045] According to the design principle of RPA primer and probe, RPA primer and RPA-nfo probe were designed and synthesized, and all primers and probes were synthesized by Shanghai Bioengineering Technology Service Co., Ltd. Five pairs of primers and one probe were designed in this embodiment (Table 1).

[0046] First, according to the RT-RPA nucleic acid amplification reagent (test strip type) kit instruction, the amplification efficiency of the five pairs of primers was compared with blank water as the template, so as to screen out the best primer pair B5F / R without non-specific amplification, see Figure 1 . Then, the reaction condition optimization was carried out according to the primer B5F / R.

[0047] Table 1 RPA-LFD primer and probe sequence

[0048]

[0049] 3. Construction of SCBV recombinant plasmid

[0050] PCR amplification was performed with the designed recombinant plasmid primer PCR-F / PCR-R as the template (10 uL PCR amplification system: 2 x PCR Mix 10 uL, Primer F (10 uM) 1 uL, Primer R (10 uM) 1 uL, template 2 uL, and enzyme-free water 6 uL. The amplification conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 1 min, a total of 35 cycles; 72 °C extension for 10 min, 12 °C for 5 min), gel recovery, and the product was connected with pMD19-T vector and then transferred into DH5a competent cells. After 12 h of plating, colony PCR identification was performed. The suspected positive colonies were selected for plasmid extraction and sequencing identification. The positive recombinant plasmid was determined for concentration by a nucleic acid concentration detector and then stored at -20 °C for standby use.

[0051] 4. Optimization of RPA-LFD reaction system and conditions

[0052] RT-RPA reaction was performed according to the kit instructions with the RNA of SCBV as the template. The total reaction system was 50 uL. First, A Buffer 29.4 uL, ddH2O 8.5 uL, upstream primer (F) 2 uL, downstream primer (R) 2 uL, probe (P) 0.6 uL, and RNA template 5 uL were added to the reaction tube. B Buffer 2.5 uL was added to the tube cover, and after thorough mixing, the reaction EP tube was placed in a constant temperature instrument or water bath, and the reaction temperature and time were set. The constant temperature amplification reaction was performed at a specific temperature. After the reaction was completed, 10 uL of the product was diluted 20 times with enzyme-free water, mixed, and then 80 uL was added to the LFD (Anpu future) for immediate observation. The experimental results could be observed by naked eye after about 5 min. Only the test strip quality control zone (C line) appeared a band, while the detection zone (T line) had no band, which was considered negative. The test strip with both the quality control zone (C line) and the detection zone (T line) was considered positive.

[0053] In order to determine the optimal reaction temperature of RPA-LFD, incubation was performed at different temperatures for 30 min. The temperatures were set at 37 °C, 39 °C, 41 °C, 43 °C, and 45 °C, respectively. The results are shown in Figure 2 a. After determining the optimal temperature, the reaction tube was placed in a 39 °C water bath and incubated for 5, 10, 15, 20, 25, and 30 min, respectively. The optimal reaction time was determined by observing the results. The results are shown in Figure 2 b.

[0054] Finally, the reaction conditions of the SCBV RPA-LFD detection method established in this embodiment were as follows: primer final concentration 0.4 uM, probe final concentration 0.12 uM, reaction temperature 39 °C, and reaction time 20-30 min.

[0055] 5. Specificity test

[0056] In order to determine the specificity of the method, the RNA or DNA of LMBV, ISKNV, SVCV and TiLV, a total of four common fish pathogens, were selected as templates for amplification reaction with SCBV, and the amount was 5 μL. RPA-LFD detection was performed to evaluate the specificity of the method, and sterile ddH2O was used as a negative control. The test results are shown in Figure 3 . It can be seen that Figure 3 RPA-LFD can only detect the A segment gene of SCBV, and has no cross reaction with other viruses. It is shown that the established RPA-LFD method has good specificity.

[0057] 6. Sensitivity test

[0058] In order to determine the minimum detection amount of RPA-LFD method, recombinant plasmid was constructed according to the A segment gene of SCBV as standard, which was diluted by ten times to seven concentrations, i.e. 6.2 x 10 7 -6.2 x 10 0 copies / uL. The detection was performed by using the optimal reaction condition, and the results are shown in Figure 4 . It is shown that RPA-LFD method can detect SCBV within 10 min, and 10 0 -10 2 Each dilution was repeated for 8 times, and was used to evaluate the detection limit of SCBV RPA-LFD test. The results show that RPA-LFD method can detect SCBV at 10 1 copies / uL and 10 2 copies / uL, and cannot detect SCBV at 10 1 copies / uL. It can be determined that the detection limit is 10 1 copies / uL.

[0059] 7. Reproducibility test

[0060] In order to verify the reproducibility of RPA-LFD detection results, 10 5 copies / uL and 10 3 copies / uL plasmid standard were selected, and 50 μL system was repeated for 5 times. The test results are shown in Figure 5 . It is shown that the method has good reproducibility.

[0061] Example 2: Establishment of SCBV RPA-LFD kit

[0062] The SCBV RPA-LFD kit is established based on the RPA detection technology. The kit includes specific amplification primers B5F / R and probe RPA-Probe (sequences are shown in Table 1). The final concentration of the primers is 0.4 μM, and the final concentration of the probe is 0.12 μM. In addition, the kit also includes corresponding RT-RPA basic fluorescence universal reaction reagents and reaction buffer, such as 10% PEG, magnesium acetate solution, freeze-dried enzyme preparation of recombinant enzymes, polymerase and single-stranded DNA binding protein, etc. These common reagents are well known to those skilled in the art. In addition, there can be positive and negative controls. The positive control should be able to completely amplify the target gene fragment corresponding to the primer pair during the amplification process; the negative control is ddH2O.

[0063] Preferably, the total reaction system of the kit is 50 μL, including A Buffer (10% PEG) 29.4 μL, ddH2O 13.5 μL, B5F 2 μL, B5R 2 μL, probe (P) 0.6 μL, RNA template 5 μL, B Buffer (magnesium acetate solution) 2.5 μL. The reaction temperature of the kit is 39°C, and the reaction time is 20-30 min.

[0064] The use method of the kit is:

[0065] (1) Extracting the RNA of the fish to be tested;

[0066] (2) Using the RNA extracted in step (1) as the amplification template, and performing RPA amplification reaction (39°C, 20-30 min) using the above total reaction system to obtain the product;

[0067] (3) Using the lateral flow test strip to analyze the product obtained in step (2): if the quality control line and the detection line of the test strip appear bands at the same time, it is positive; if the quality control line C of the test strip appears a band, but the detection line T does not appear a band, it is negative.

[0068] The value of the kit lies in that the detection sample can be the gill mucus, liver, spleen, kidney, gill and other internal organs of the fish. Through the most simple and specific primer and probe detection, the recombinant enzyme amplification technology is used to rapidly and accurately amplify and identify the specific nucleic acid of the double RNA virus of the mandarin fish. It is not only stable, convenient and accurate for detection, but also greatly improves the sensitivity and specificity of virus diagnosis. Therefore, putting this kit into production practice can simplify the operation steps, shorten the detection time, reduce the operation time of the detection personnel, improve the detection efficiency, and is suitable for large-scale screening.

[0069] Example 3: Comparison of SCBV RPA-LFD and qPCR for detection of clinical samples

[0070] The present embodiment collected 48 clinical tissue samples of Siniperca chuatsi in Foshan, Guangdong in 2023 for blind detection. The SCBV RPA-LFD kit constructed in Example 2 and qPCR method (25uL amplification system: 2.78x one step RT-qPCR Mix 9uL, Primer F (10uM) 0.5uL, Primer R (10uM) 0.5uL, Probe (10uM) 0.5uL, template 5uL, enzyme-free water 9.5uL; amplification condition: 95℃ 3min; 95℃ 10s, 60℃ 30s, 40 cycles, collect fluorescence signal in the second step of each cycle.) were used to identify the 48 samples at the same time, and the detection effects of the two methods were compared. As can be seen from Table 2, among the 48 clinical samples, 12 samples were detected as positive for SCBV by RPA-LFD, and the positive rate was 25%, which was consistent with the result of qPCR. At the same time, RPA-LFD and fluorescence quantitative PCR had the same diagnostic sensitivity of 100% and diagnostic specificity of 100%. It can be seen that the detection result of the RPA-LFD method of the present application is reliable, and the time is shorter and the operation is simpler.

[0071] Table 2 Detection results of two methods for clinical Siniperca chuatsi samples

[0072]

[0073] Note: CR% is the coincidence rate of the two detection methods.

[0074] As can be seen from the above, the present application provides an RPA-LFD primer probe combination and kit for detecting Siniperca chuatsi double RNA virus, and on this basis, an RPA-LFD detection method for Siniperca chuatsi double RNA virus is established. First, the RNA of the sample to be tested is extracted, then the extracted RNA is used as an amplification template, and the above-mentioned RPA composition is used for RPA amplification reaction to obtain a product, and finally the product obtained by the reaction is analyzed using a lateral flow test strip, if the test strip quality control line and detection line appear strip at the same time, it is positive, if the test strip quality control line appears strip, but the detection line has no strip, it is negative. The RPA-LFD composition, kit and detection method for detecting Siniperca chuatsi double RNA virus provided by the present application can realize the RPA detection of Siniperca chuatsi double RNA virus, and can quickly, simply and specifically detect Siniperca chuatsi double RNA virus, and is suitable for on-site rapid detection in breeding farms.

[0075] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. An RPA-LFD primer-probe combination for detecting a double RNA virus in mandarin fish, characterized in that, The RPA-LFD primers consist of the upstream primer B5F shown in SEQ ID NO.1 and the downstream primer B5R shown in SEQ ID NO.2, and the RPA-LFD probe is the probe P shown in SEQ ID NO.

3.

2. A kit for detecting a double RNA virus in mandarin fish, characterized in that, The kit includes the RPA-LFD primer-probe combination as described in claim 1.

3. The kit for detecting mandarin fish double RNA virus according to claim 2, characterized in that, The kit also includes A Buffer, B Buffer, recombinase for binding single-stranded nucleic acids, strand displacement DNA polymerase, single-stranded DNA binding protein, and reaction system buffer or magnesium acetate; A Buffer is 10% PEG, and B Buffer is magnesium acetate solution.

4. A kit for detecting double RNA virus in mandarin fish according to claim 2, characterized in that, The kit also includes a positive control, which is the genomic RNA of a mandarin fish double RNA virus.

5. A kit for detecting double RNA virus in mandarin fish according to claim 2, characterized in that, The kit also includes RT-RPA nucleic acid amplification test strips.

6. A kit for detecting double RNA virus in mandarin fish according to claim 2, characterized in that, The final concentration of the RPA-LFD primers was 0.4 μM, and the final concentration of the probe was 0.12 μM.

7. A kit for detecting double RNA virus in mandarin fish according to claim 3, characterized in that, The total reaction volume of the kit is 50 μL, including 29.4 μL of A Buffer, 8.5 μL of ddH2O, 2 μL of B5F, 2 μL of B5R, 0.6 μL of probe P, 5 μL of RNA template, and 2.5 μL of B Buffer; A Buffer is 10% PEG, and B Buffer is magnesium acetate solution.

8. The application of the RPA-LFD primer-probe combination according to claim 1, or the kit according to any one of claims 2-7, in the preparation of a mandarin fish double RNA virus detection product.

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