A composition, kit and application for detecting lymphocystis disease virus based on RPA isothermal amplification and immunochromatography technology

Abstract

The present invention provides a composition, a kit and an application for detecting lymphocystis disease virus based on RPA isothermal amplification and immunochromatography technology, which relates to the field of biological detection technology. It includes an upstream primer MCP-F with a nucleotide sequence as shown in SEQ ID NO:7, a downstream primer MCP-R with a nucleotide sequence as shown in SEQ ID NO:8, and a specific probe MCP-P with a nucleotide sequence as shown in SEQ ID NO:9. By referring to the genomic sequence of the LCDV strain, its MCP conserved gene sequence is determined, and RPA primers and specific probes are designed, which can distinguish lymphocystis disease virus from Micropterus salmoides ranavirus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, flounder rhabdovirus, and Pleuronectiformes taura syndrome virus, and is specific for lymphocystis disease virus; the sensitivity reaches 5 pg / reaction; its amplification product is applicable to the LFD test strip.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a composition, a kit and an application for detecting lymphocystis virus based on RPA isothermal amplification and immunochromatography technology. Background Art

[0002] Lymphocystis disease (LCD) is the first prevalent viral disease of marine fish in China caused by Lymphocystis disease virus (LCDV), which can infect more than 140 species of fish including Paralichthys olivaceus. This virus belongs to the family Iridoviridae, genus Lymphocystivirus, and is an enveloped double-stranded DNA virus. It is globally spread and can occur throughout the year. The external feature is that grayish-white cauliflower-like tumors of different sizes can be seen on the skin, fins and tails of diseased fish, and it can invade multiple tissues and organs such as the gonads, spleen and head kidney of the fish body. Although the lethality rate is not high, it can make the diseased individuals lose their original commercial value. Since the first outbreak of lymphocystis disease in cultured Paralichthys olivaceus in China in the 1990s, it has caused huge losses to the aquaculture industry in China.

[0003] Currently, the methods for detecting lymphocystis virus mainly rely on molecular detection techniques highly dependent on instruments, such as PCR, qRT-PCR, ELISA, etc. However, the required expensive instruments and professional operations make them not suitable for wide application in the industrial front line. For rapid detection methods of this virus, there have been various attempts. Among them, the colloidal gold immunochromatographic test strip based on monoclonal antibodies against the envelope protein of LCDV is a rapid detection means, but limited by the difficult preparation and high cost of monoclonal antibodies, it is difficult to be applied on a large scale. Secondly, there is also the loop-mediated isothermal amplification (LAMP) technology developed based on the capsid protein gene of LCDV. This technology has simple operation and high amplification efficiency, but the detection result depends on the naked eye to observe precipitation and color change, which is easily affected by subjective factors and the accuracy needs to be improved. Therefore, it is still necessary to explore a more efficient, accurate and cost-appropriate rapid detection method for LCDV.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a composition for detecting lymphocystis virus, so as to solve the technical problem in the prior art of lacking a primer composition for detecting lymphocystis virus with rapid reaction, high sensitivity and strong specificity.

[0006] A second object of the present invention is to provide the use of the above-mentioned composition in preparing a product for detecting or assisting in detecting lymphocystis virus, a product for detecting whether a sample contains lymphocystis virus, a product for differentiating lymphocystis virus from other pathogens in a sample, a product for diagnosing or assisting in diagnosing a disease caused by lymphocystis virus, or a product for screening a disease caused by lymphocystis virus.

[0007] A third object of the present invention is to provide a reagent for detecting lymphocystis virus.

[0008] A fourth object of the present invention is to provide a kit for detecting lymphocystis virus.

[0009] A fifth object of the present invention is to provide the use of the above-mentioned reagent or the above-mentioned kit in preparing a product for detecting or assisting in detecting lymphocystis virus, a product for detecting whether a sample contains lymphocystis virus, a product for differentiating lymphocystis virus from other pathogens in a sample, a product for diagnosing or assisting in diagnosing a disease caused by lymphocystis virus, or a product for screening a disease caused by lymphocystis virus.

[0010] A sixth object of the present invention is to provide a recombinase isothermal amplification method for lymphocystis virus.

[0011] A seventh object of the present invention is to provide a method for detecting or assisting in detecting lymphocystis virus for non-therapeutic and non-diagnostic purposes.

[0012] In order to achieve the above objects of the present invention, the following technical solutions are specifically adopted:

[0013] In a first aspect, the present invention provides a composition for detecting lymphocystis virus, comprising an upstream primer MCP-F with a nucleotide sequence as shown in SEQ ID NO: 7, a downstream primer MCP-R with a nucleotide sequence as shown in SEQ ID NO: 8, and a specific probe MCP-P with a nucleotide sequence as shown in SEQ ID NO: 9;

[0014] The 5' end of the MCP-R is modified with biotin, the 30th base on the MCP-P is modified with tetrahydrofuran, and the 3' end is modified with a blocking group.

[0015] Further, the 5' end of the MCP-P is modified with a fluorescent reporter group;

[0016] The fluorescent reporter group is FAM;

[0017] Preferably, the blocking group is C3 spacer.

[0018] Further, the molar ratio of MCP-F: MCP-R: MCP-P is 1:1:0.3.

[0019] In a second aspect, the present invention provides the use of the above-mentioned composition in any one of the following:

[0020] A1. Use in the preparation of a product for detecting or assisting in the detection of lymphocystis virus;

[0021] A2. Use in the preparation of a product for detecting whether a test sample contains lymphocystis virus;

[0022] A3. Use in the preparation of a product for differentiating lymphocystis virus from other pathogens in a sample;

[0023] A4. Use in the preparation of a product for diagnosing or assisting in the diagnosis of a disease caused by lymphocystis virus;

[0024] A5. Use in the preparation of a product for screening diseases caused by lymphocystis virus.

[0025] In a third aspect, the present invention provides a reagent for detecting lymphocystis virus, comprising the above-mentioned composition.

[0026] In a fourth aspect, the present invention provides a kit for detecting lymphocystis virus, comprising the above-mentioned composition or the above-mentioned reagent.

[0027] Furthermore, it further comprises RPA reaction reagents, positive control, negative control, PBS buffer and colloidal gold test strip required for recombinase isothermal amplification;

[0028] Preferably, the RPA reaction reagents include recombinase polymerase buffer, recombinase polymerase, nuclease and magnesium acetate;

[0029] Preferably, the nuclease is nfo enzyme;

[0030] Preferably, the positive control is a positive plasmid containing the MCP sequence;

[0031] Preferably, the negative control is sterile and enzyme-free water;

[0032] Preferably, the sample pad of the colloidal gold test strip is labeled with gold-labeled mouse anti-FAM antibody, the test line is sprayed with anti-biotin antibody, and the quality control line is sprayed with goat anti-mouse IgG antibody.

[0033] In a fifth aspect, the present invention provides the use of the above-mentioned reagent or the above-mentioned kit in any one of the following:

[0034] B1. Use in the preparation of a product for detecting or assisting in the detection of lymphocystis virus;

[0035] B2. Use in the preparation of a product for detecting whether a test sample contains lymphocystis virus;

[0036] B3. Use in the preparation of products for differentiating lymphocystis disease virus from other pathogens in a sample;

[0037] B4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by lymphocystis disease virus;

[0038] B5. Use in the preparation of products for screening diseases caused by lymphocystis disease virus.

[0039] In a sixth aspect, the present invention provides a recombinase isothermal amplification method for lymphocystis disease virus, comprising performing recombinase isothermal amplification on a sample using the above primer-probe composition, the above reagent or the above kit;

[0040] Preferably, the reaction temperature of the recombinase isothermal amplification is 35 - 47 °C, preferably 43 °C;

[0041] Preferably, the reaction time of the recombinase isothermal amplification is 7.5 - 30 min, preferably 15 min;

[0042] Preferably, the usage amount of nuclease in the recombinase isothermal amplification is 0.5 - 6 μL of nuclease added to each 50 μL reaction system, preferably 1 μL of nuclease added to each 50 μL reaction system.

[0043] In a seventh aspect, the present invention provides a method for detecting or assisting in the detection of lymphocystis disease virus for non-therapeutic and non-diagnostic purposes, comprising detecting the amplification product prepared by the above recombinase isothermal amplification method using a colloidal gold test strip, and determining whether lymphocystis disease virus is present in the sample according to the test line.

[0044] A composition for detecting lymphocystis disease virus provided by the present invention, referring to the genomic sequence of the LCDV strain, determining its conserved MCP gene sequence, designing RPA primers and specific probes, modifying biotin at the 5' end of MCP-R, modifying tetrahydrofuran at the 30th base of MCP-P, and modifying a blocking group at the 3' end. Experiments prove that the composition can differentiate lymphocystis disease virus from largemouth bass ranavirus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, flounder rhabdovirus, and turbot-associated virus, and is specific for lymphocystis disease virus; the sensitivity reaches 5 pg / reaction; its amplification product is applicable to a lateral flow test strip (LFD test strip). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0046] Figure 1 Sequence alignment results of MCP-For3 / MCP-Rev3 / MCP-Pro3 provided in Example 1 of the present invention;

[0047] Figure 2 Comparison diagram of colloidal gold test strip detection established by different primer probes provided in Example 1 of the present invention;

[0048] Figure 3 Comparison diagram of colloidal gold signal intensity at different RPA amplification temperatures provided in Example 2 of the present invention;

[0049] Figure 4 Comparison diagram of colloidal gold signal intensity at different RPA amplification times provided in Example 2 of the present invention;

[0050] Figure 5 Comparison diagram of colloidal gold signal intensity at different nfo enzyme addition amounts provided in Example 3 of the present invention;

[0051] Figure 6 Specific detection results of the LCDV nucleic acid flow-through immunochromatographic detection method provided in Example 6 of the present invention;

[0052] Figure 7 Sensitivity detection results of the LCDV nucleic acid flow-through immunochromatographic detection method provided in Example 7 of the present invention;

[0053] Figure 8 RPA-LFD detection results of clinical sample fin rays provided in Example 8 of the present invention. Specific embodiments

[0054] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0055] Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0056] On the one hand, the present invention provides a composition for detecting lymphocystis disease virus, comprising an upstream primer MCP-F with a nucleotide sequence as shown in SEQ ID NO:7, a downstream primer MCP-R with a nucleotide sequence as shown in SEQ ID NO:8, and a specific probe MCP-P with a nucleotide sequence as shown in SEQ ID NO:9; the 5'-end of the MCP-R is modified with biotin, the 30th base on the MCP-P is modified with tetrahydrofuran, and the 3'-end is modified with a blocking group.

[0057] Referring to the genomic sequence of the LCDV strain, its MCP conserved gene sequence was determined, and RPA primers and specific probes were designed. The 5'-end of the MCP-R was modified with biotin. In the direction from the 5'-end to the 3'-end, the 30th base on the MCP-P was modified with tetrahydrofuran, and the 3'-end was modified with a blocking group. Experiments have shown that the composition can distinguish lymphocystis disease virus from largemouth bass ranavirus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, flounder rhabdovirus, and turbot tembusu virus, and is specific for lymphocystis disease virus; the sensitivity reaches 5 pg / reaction; and its amplification product is applicable to a lateral flow dipstick (LFD dipstick).

[0058] In some specific embodiments, the 5'-end of the MCP-P is modified with a fluorescent reporter group.

[0059] Specifically, the fluorescent reporter group is FAM, and other fluorescent reporter groups such as VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460, or LCRED705 can also be selected.

[0060] In some specific embodiments, the blocking group is C3 spacer.

[0061] In order to further improve the amplification efficiency, in some specific embodiments, the molar ratio of MCP-F:MCP-R:MCP-P is 1:1:0.3.

[0062] According to another aspect of the present invention, the above composition is also provided for use in any of the following:

[0063] A1. Use in the preparation of a product for detecting or assisting in the detection of lymphocystis disease virus;

[0064] A2. Use in the preparation of products for detecting whether a test sample contains lymphocystis virus;

[0065] A3. Use in the preparation of products for differentiating lymphocystis virus from other pathogens in a sample;

[0066] A4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by lymphocystis virus;

[0067] A5. Use in the preparation of products for screening diseases caused by lymphocystis virus.

[0068] According to another aspect of the present invention, a reagent for detecting lymphocystis virus is also provided, comprising the above composition.

[0069] According to another aspect of the present invention, a kit for detecting lymphocystis virus is also provided, comprising the above composition or the above reagent.

[0070] In some specific embodiments, it further comprises RPA reaction reagents required for recombinase isothermal amplification, positive control, negative control, PBS buffer and colloidal gold test strip.

[0071] In some specific embodiments, the RPA reaction reagents comprise recombinase polymerase buffer, recombinase polymerase, nuclease and magnesium acetate.

[0072] In some specific embodiments, the nuclease is nfo enzyme.

[0073] In some specific embodiments, the positive control is a positive plasmid containing the MCP sequence. The MCP sequence is the nucleotide sequence shown in SEQ ID NO: 10.

[0074] In some specific embodiments, the negative control is sterile and enzyme-free water.

[0075] In some specific embodiments, the sample pad of the colloidal gold test strip is labeled with gold-labeled mouse anti-FAM antibody, the detection line is sprayed with anti-biotin antibody, and the quality control line is sprayed with goat anti-mouse IgG antibody.

[0076] In the fifth aspect, the present invention provides the application of the above reagent or the above kit in any of the following:

[0077] B1. Use in the preparation of products for detecting or assisting in the detection of lymphocystis virus;

[0078] B2. Use in the preparation of products for detecting whether a test sample contains lymphocystis virus;

[0079] B3. Use in the preparation of products for differentiating lymphocystis virus from other pathogens in a sample;

[0080] B4. Use in the preparation of a product for diagnosing or assisting in the diagnosis of diseases caused by lymphocystis virus;

[0081] B5. Use in the preparation of a product for screening diseases caused by lymphocystis virus.

[0082] In a sixth aspect, the present invention provides a recombinase isothermal amplification method for lymphocystis virus, which includes performing recombinase isothermal amplification on a sample using the above primer-probe composition, the above reagent or the above kit. This method does not require sophisticated instruments and only needs a temperature control device to achieve amplification.

[0083] In order to further improve the amplification efficiency, in some specific embodiments, the reaction temperature of the recombinase isothermal amplification is 35-47 °C, preferably 43 °C. In some specific embodiments, the reaction time of the recombinase isothermal amplification is 7.5-30 min, preferably 15 min. In some specific embodiments, the usage amount of the nuclease in the recombinase isothermal amplification is 0.5-6 μL of nuclease added to each 50 μL system. By using the nuclease, the amplification product can be applied to LFD detection. Specifically, the nuclease can be nfo enzyme, and the preferred usage amount is 1 μL of nuclease added to each 50 μL system.

[0084] According to another aspect of the present invention, there is also a method for detecting or assisting in the detection of lymphocystis virus for non-therapeutic and non-diagnostic purposes, which includes detecting the amplification product prepared by the above recombinase isothermal amplification method using a colloidal gold test strip, and determining whether there is lymphocystis virus in the sample according to the test line.

[0085] Through recombinase isothermal amplification (RPA isothermal amplification), the amplification product is visually detected by specific antibodies or probes on the test strip, and these antibodies or probes can specifically bind to specific labels of the amplification product, thereby forming a visible color change or precipitate on the test strip to visually judge the presence or absence of the target nucleic acid. It can achieve on-site detection, reduce the operation difficulty and cost, and has the advantages of simple operation, rapid reaction, high sensitivity and strong specificity; experiments have proved that the RPA isothermal amplification can be completed in as fast as 7.5 min, and it can complete the amplification and detection of the target nucleic acid in a short time, improving the detection efficiency.

[0086] Among them, the detection or assistance in the detection of lymphocystis virus (LCDV) for non-therapeutic and non-diagnostic purposes specifically includes LCDV research purposes, LCDV epidemiological investigation purposes, environmental monitoring purposes, import and export commodity safety quarantine purposes, vaccine research and development purposes and food safety purposes.

[0087] The technical solution of the present invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0088] Example 1 Design and Screening of Primer and Probe Combinations

[0089] Referring to the NCBI GenBank database, the genomic sequences of LCDV strains were searched, and three sets of primers and probes were designed respectively for the MCP conserved gene sequences of different LCDV strains (Table 1).

[0090] Table 1 Primer and Probe Sequences

[0091]

[0092] All primers and probes were synthesized and labeled by Sangon Biotech (Shanghai) Co., Ltd. The 5' end of the downstream primer was modified with biotin; the 5' end of the probe was modified with FAM, the 30th base from the 5' end was modified with tetrahydrofuran (THF), and the 3' end had a polymerase extension blocking group C3 spacer.

[0093] Then, using the same batch of equal amounts of LCDV as the template, the colloidal gold test strip RPA detection methods established with 3 groups of primer-probe combinations were used for detection respectively.

[0094] RPA amplification reaction system for primer-probe screening:

[0095] Add 32.9 μL of RPA recombinase polymerase reaction buffer (from the Basic-DNA isothermal rapid amplification kit of Wuhan Junnuode Biotechnology Co., Ltd., product number: A4601-48), 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 0.6 μL of probe (10 μM), 1 μL of nfo enzyme, and 4 μL of sterile enzyme-free water into the freeze-dried powder of RPA recombinase polymerase in a single-tube independent package (from the Basic-DNA isothermal rapid amplification kit of Wuhan Junnuode Biotechnology Co., Ltd., product number: A4601-48), then add 5 μL of LCDV DNA template, and finally add 2.5 μL of magnesium acetate before the reaction starts. The temperature of the RPA amplification reaction is 39 °C, the reaction time is 15 min. Add the reaction product to 150 μL of PBS buffer (pH value is 7.0), then take 100 μL and add it to the sample pad of the colloidal gold loading card. There is a gold-labeled mouse anti-FAM antibody on the sample pad, the test line (T) is sprayed with anti-biotin antibody, and the quality control line (C) is sprayed with goat anti-mouse IgG antibody. Read the result after 10 minutes of reaction.

[0096] The results are as Figure 2 shown. At the test line (T), the MCP-For3 / MCP-Rev3 / MCP-Pro3 detection composition has the strongest colloidal gold signal intensity and the best amplification effect. Therefore, this composition was selected for the development of the RPA-LFD detection method and kit. The sequence alignment results of the MCP-For3 / MCP-Rev3 / MCP-Pro3 detection composition are as Figure 1 shown.

[0097] Optimization of reaction conditions in Example 2

[0098] 1. Optimization of RPA amplification temperature

[0099] According to the reaction system of Example 1, using the MCP-For3 / MCP-Rev3 / MCP-Pro3 primer / probe, the prepared reaction system was subjected to RPA amplification at 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, 49°C, 51°C and 53°C respectively, and a metal bath was used for constant temperature amplification for 15 min to screen out the optimal amplification reaction temperature. After the amplification was completed, the reaction product was added to 150 μL of PBS buffer (pH value 7.0), and then 100 μL was taken and added to the sample pad of the colloidal gold loading card. There was a gold-labeled anti-FAM antibody on the sample pad, the test line (T) was sprayed with anti-biotin antibody, and the control line (C) was sprayed with goat anti-mouse IgG antibody. The results were read after 10 minutes of reaction. The reaction results are as Figure 3 shown. It can be seen that when the amplification reaction temperature is 43°C, the band of the test line (T) is the clearest.

[0100] 2. Optimization of RPA amplification time

[0101] According to the reaction system of Example 1, using the MCP-For3 / MCP-Rev3 / MCP-Pro3 primer / probe, at the amplification reaction temperature of 43°C, the prepared reaction system was amplified for 1 min, 2.5 min, 5 min, 7.5 min, 10 min, 12.5 min, 15 min, 17.5 min, 20 min, 25 min and 30 min respectively, in order to screen out the optimal amplification reaction time. After the amplification was completed, the reaction product was added to 150 μL of PBS buffer (pH value 7.0), and then 100 μL was taken and added to the sample pad of the colloidal gold loading card. There was a gold-labeled anti-FAM antibody on the sample pad, the test line (T) was sprayed with anti-biotin antibody, and the control line (C) was sprayed with goat anti-mouse IgG antibody. The results were read after 10 minutes of reaction. The reaction results are as Figure 4 shown. It can be seen that when the amplification reaction duration is 15 min, the band of the test line (T) is the clearest.

[0102] Screening of nfo Enzyme Concentration in Example 3

[0103] Except for the nfo enzyme concentration, refer to the reaction system in Example 1. Use the MCP-For3 / MCP-Rev3 / MCP-Pro3 primers / probe. Amplify the prepared reaction system at an amplification reaction temperature of 43°C for 15 min. Add 0 μL, 0.1 μL, 0.25 μL, 0.5 μL, 0.75 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, and 7 μL of nfo enzyme to the prepared reaction system respectively, in order to screen out the optimal concentration of nfo enzyme in the reaction system. After the amplification is completed, add the reaction product to 150 μL of PBS buffer (pH value is 7.0), then take 100 μL and add it to the sample pad of the colloidal gold loading card. There is a gold-labeled mouse anti-FAM antibody on the sample pad, the detection line (T) is sprayed with anti-biotin antibody, and the quality control line (C) is sprayed with goat anti-mouse IgG antibody. Read the result after reacting for 10 minutes. The reaction result is as Figure 5 shown. It can be seen that when 1 μL of nfo enzyme is added, the band of the detection line (T) is the clearest.

[0104] Example 4 Establishment of an RPA-LFD Detection Kit for Detecting LCDV

[0105] This example presents a kit for detecting lymphocystis disease virus (LCDV) using RPA isothermal amplification combined with immunochromatography technology:

[0106] 1. Reagent A: Lyophilized powder of RPA recombinase polymerase (from the Basic-DNA Isothermal Rapid Amplification Kit of Wuhan Junnuode Biotechnology Co., Ltd., product number: A4601-48), each tube represents an independent reaction.

[0107] 2. Reagent B: The composition of the RPA reaction mixture is as follows: 2 μL of 10 μM upstream primer MCP-For3, 2 μL of 10 μM downstream primer MCP-Rev3, 0.6 μL of 10 μM probe MCP-Pro3, 32.9 μL of RPA recombinase polymerase reaction buffer (purchased from the Basic-DNA Isothermal Rapid Amplification Kit of Wuhan Junnuode Biotechnology Co., Ltd., product number: A4601-48), 1 μL of nfo enzyme (purchased from YEASEN, product number: 10323ES80), 4 μL of sterile and enzyme-free water.

[0108] 3. Reagent C: Magnesium acetate (280 mM) 2.5 μL.

[0109] 4. Reagent D: PBS buffer, pH 7.0.

[0110] 5. Colloidal gold test strip: The sample pad is coated with gold-labeled mouse anti-FAM antibody, the test line (T) is sprayed with anti-biotin antibody, and the control line (C) is sprayed with goat anti-mouse IgG antibody.

[0111] 6. Positive control: Standard positive plasmid with a concentration of 100 ng / μL.

[0112] The standard positive plasmid is obtained by the following method:

[0113] Synthesize the MCP sequence and ligate it to the pcDNA3.1 cloning vector. Transform the competent Escherichia coli cells DH5α and spread them on the LB medium plate containing 100 mg / L ampicillin. Incubate at 37 °C for 12 - 14 h. After expanding the culture of positive single colonies, extract the plasmid using a plasmid extraction kit. Name the plasmid with a positive sequence determination as pcDNA3.1-MCP, which is the standard positive plasmid.

[0114] 7. Negative control: Sterile and enzyme-free water.

[0115] Example 5 Establishment of an RPA-LFD detection method for detecting LCDV

[0116] 1. Preparation of sample DNA template

[0117] Use the marine animal tissue genomic DNA extraction kit (TIANGEN, model: DP324) and refer to the kit instructions to extract the genomic DNA of fish tissues infected with LCDV virus.

[0118] 2. RPA reaction system and reaction procedure

[0119] Use the reagents provided in Example 4 and perform the following steps specifically: Add 42.5 μL of reagent B to each dry powder reaction tube (reagent A), then add 5 μL of LCDV DNA template to the reaction tube, and finally add 2.5 μL of reagent C and mix well (for multiple reactions, it is recommended to add reagent C to the inner side of the reaction tube lid and invert the reaction tube 8 - 10 times to mix); after mixing, centrifuge the reaction solution to the bottom of the tube, and then immediately place the reaction tube in a constant temperature device and incubate at 43 °C for 15 min. After the reaction, add 150 μL of PBS buffer (reagent D) to the product, then take 100 μL and add it to the sample pad of the colloidal gold test strip, and read the result after reacting for 10 minutes.

[0120] Use the colloidal gold test strip to judge the result:

[0121] When both the test line (T) and the control line (C) of the colloidal gold test strip show color, the test result is positive;

[0122] When the control line (C) of the colloidal gold test strip shows color while the test line (T) does not, the test result is negative;

[0123] When neither the test line (T) nor the control line (C) of the colloidal gold test strip shows color, the test result is invalid.

[0124] Example 6 Specificity Analysis of LCDV Nucleic Acid Flow-Through Immunochromatographic Detection Method

[0125] Using the purified DNA of Lymphocystis disease virus (LCDV) and Largemouth Bass Iridovirus (LMBV) and the prepared cDNA of Viral haemorrhagic septicaemia virus (VHSV), Infectious hematopoietic necrosis virus (IHNV), Hiramerhabdovirus (HRV), and Chinook salmon bafinivirus isolate Bces-Po19 (CSBV Bces-Po19) as templates respectively, using the reagents provided in Example 4, and performing RPA amplification according to the method provided in Example 5. Add the reaction product to 150 μL of PBS buffer, and then take 100 μL and add it to the sample pad of the colloidal gold loading card. There is a gold-labeled mouse anti-FAM antibody on the sample pad, the test line (T) is sprayed with anti-biotin antibody, and the control line (C) is sprayed with goat anti-mouse IgG antibody. Read the result after 10 minutes of reaction. The results are as Figure 6 shown. Only when the LCDV DNA is used as the template, a specific band appears in the test line (T).

[0126] Example 7 Sensitivity Analysis of LCDV Nucleic Acid Flow-Through Immunochromatographic Detection Method

[0127] Dilute the extracted DNA of LCDV with sterile and enzyme-free water successively by 10-fold serial dilution to concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. Then, using the DNA with gradient concentrations as templates, use the reagents provided in Example 4 and perform RPA amplification reaction according to the method provided in Example 5. Add the reaction product to 150 μL of PBS buffer (pH value is 7.0), and then take 100 μL and add it to the sample pad of the colloidal gold loading card. There is a gold-labeled mouse anti-FAM antibody on the sample pad, the test line (T) is sprayed with anti-biotin antibody, and the control line (C) is sprayed with goat anti-mouse IgG antibody. Read the result after 10 minutes of reaction. The results are asFigure 7 As shown, the detection limit of the detection method established by the present invention for LCDV DNA is 5 pg / reaction (template concentration is 1 pg / μL, and the amount of template in the reaction system is 5 μL).

[0128] Example 8 Detection of Clinical Samples

[0129] Select 20 fin samples of clinical samples of flounder suspected of being infected with LCDV, and prepare DNA samples by using a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP324). Use the RPA-LFD detection method (Example 5) established by the present invention for detection. The results are as Figure 8 shown, where the numbers 1-20 correspond one by one to the numbers 1-20 in Table 2; at the same time, the fluorescence quantitative PCR detection method for the LCDV MCP gene in the published "Effect of RNA Interference on the Replication of LCDV-cn in Flounder" is selected as a control, and the results of the fluorescence quantitative PCR detection are shown in Table 2; Figure 8 Compared with the detection results in Table 2, there is good consistency in the positive sample detection rate between the two.

[0130] Table 2 Fluorescence Quantitative PCR Detection Results of Clinical Samples (Fin Rays)

[0131]

[0132]

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for detecting lymphocystis virus, characterized in that: It comprises an upstream primer MCP-F whose nucleotide sequence is shown in SEQ ID NO:7, a downstream primer MCP-R whose nucleotide sequence is shown in SEQ ID NO:8, and a specific probe MCP-P whose nucleotide sequence is shown in SEQ ID NO:9; The 5' end of the MCP-R is modified with biotin, the 30th base on the MCP-P is modified with tetrahydrofuran, and the 3' end is modified with a blocking group.

2. The composition according to claim 1, characterized in that The 5' end of the MCP-P is modified with a fluorescent reporter group.

3. The composition according to claim 2, characterized in that The fluorescent reporter group is FAM.

4. The composition according to claim 1, characterized in that The blocking group is C3 spacer.

5. The composition according to claim 1, characterized in that The molar ratio of MCP-F:MCP-R:MCP-P is 1:1:0.

3.

6. Use of the composition according to any one of claims 1 to 5 in any of the following: A1. Application in the preparation of products for detecting or assisting in the detection of lymphocystis virus; A2. Use in the preparation of products for detecting whether a sample contains lymphocystis virus; A3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples; A4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by lymphocystis virus; A5. Application in the preparation of products for screening diseases caused by lymphocystis virus.

7. A reagent for detecting lymphocystis virus, characterized in that: A composition comprising any one of claims 1 to 5.

8. A kit for detecting lymphocystis virus, characterized in that: The method comprises the composition according to any one of claims 1 to 5 or the reagent according to claim 7.

9. The kit according to claim 8, characterized in that It also includes RPA reaction reagents, positive controls, negative controls, PBS buffer and colloidal gold test strips required for recombinase isothermal amplification.

10. The kit according to claim 9, characterized in that The RPA reaction reagents include recombinase polymerase buffer, recombinase polymerase, nuclease and magnesium acetate.

11. The kit according to claim 10, characterized in that The nuclease is nfo enzyme.

12. The kit according to claim 9, characterized in that The positive control is a positive plasmid containing the MCP sequence.

13. The kit according to claim 9, characterized in that The negative control was sterile enzyme-free water.

14. The kit according to claim 9, characterized in that The sample pad of the colloidal gold test strip is marked with gold-labeled mouse anti-FAM antibody, the detection line is sprayed with anti-biotin antibody, and the quality control line is sprayed with goat anti-mouse IgG antibody.

15. Use of the reagent according to claim 7 or the kit according to any one of claims 8 to 14 in any of the following: B1. Application in the preparation of products for detecting or assisting in the detection of lymphocystis virus; B2. Use in the preparation of products for detecting whether a sample contains lymphocystis virus; B3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples; B4. Use in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by lymphocystis virus; B5. Application in the preparation of products for screening diseases caused by lymphocystis virus.

16. A method for isothermal amplification of a recombinase of lymphocystis virus, characterized in that: The method comprises performing recombinase isothermal amplification on a sample using the composition according to any one of claims 1 to 5, the reagent according to claim 7 or the kit according to any one of claims 8 to 14.

17. The recombinase isothermal amplification method according to claim 16, characterized in that: The reaction temperature of the recombinase isothermal amplification is 35-47°C.

18. The recombinase isothermal amplification method according to claim 17, characterized in that: The reaction temperature of the recombinase isothermal amplification is 43°C.

19. The recombinase isothermal amplification method according to claim 16, characterized in that: The reaction time of the recombinase isothermal amplification is 7.5 to 30 minutes.

20. The recombinase isothermal amplification method according to claim 19, characterized in that: The reaction time of the recombinase isothermal amplification is 15 minutes.

21. The recombinase isothermal amplification method according to claim 16, characterized in that: The amount of nuclease used in the recombinase isothermal amplification is 0.5 to 6 μL of nuclease added per 50 μL of system.

22. The recombinase isothermal amplification method according to claim 21, characterized in that: The amount of nuclease used in the recombinase isothermal amplification is 1 μL of nuclease added to every 50 μL of system.

23. A method for detecting or assisting in detecting lymphocystis virus for non-therapeutic and non-diagnostic purposes, characterized in that: The method comprises using a colloidal gold test strip to detect the amplification product prepared by the recombinase isothermal amplification method according to any one of claims 16 to 22, and determining whether lymphocystis virus exists in the sample according to the detection line.

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