Composition and kit for detecting lymphocyst virus based on RPA isothermal amplification and immunochromatography technology and application

Abstract

The invention provides a composition and a kit for detecting lymphocyst virus based on RPA isothermal amplification and immunochromatography and application, and relates to the technical field of biological detection, the composition comprises 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, according to the present invention, with reference to the LCDV strain genome sequence, the MCP conserved gene sequence of the LCDV strain is determined, and the RPA primer and the specific probe are designed, such that the lymphocystis virus, the largemouth bass frog iridovirus, the viral hemorrhagic septicemia virus, the infectious hematopoietic necrosis virus, the paralichthys olivaceus rhabdovirus, the flounder nested virus and the lymphocystis virus have specificity; the sensitivity reaches 5pg / reaction; and the amplification product is suitable for the LFD test strip.

Description

Technical Field

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

[0002] Lymphocystis disease (LCD) is the first domestic viral disease of marine fish caused by Lymphocystis disease virus (LCDV), which can infect more than 140 species of fish including the Japanese flounder. The virus belongs to the family Iridoviridae and the genus Lymphocystivirus, and is an enveloped double-stranded DNA virus. It is globally transmitted and can occur throughout the year. The external features are gray-white cauliflower-shaped tumors of varying sizes visible on the skin, fins and tail of the diseased fish, and can invade multiple tissues and organs of the fish, such as the gonads, spleen, head kidney, etc. Although the mortality rate is not high, it can cause the diseased individuals to lose their original commercial value. Since the first outbreak of lymphocystis disease in domestically farmed Japanese flounder in the 1990s, it has caused huge losses to my country's aquaculture industry.

[0003] At present, the methods for detecting lymphocystis virus mainly rely on molecular detection technologies that are highly dependent on instruments, such as PCR, qRT-PCR, ELISA, etc. However, the expensive instruments and professional operations required make it unsuitable for widespread application in the industry. There have been many attempts at rapid detection methods for the virus. Among them, the colloidal gold immunochromatographic test strip based on LCDV envelope protein monoclonal antibodies is a rapid detection method, but it is difficult to apply on a large scale due to the difficulty and high cost of monoclonal antibody preparation. Secondly, there is also the loop-mediated isothermal amplification (LAMP) technology developed based on the LCDV capsid protein gene. This technology is easy to operate and has high amplification efficiency, but the detection results rely on the naked eye observation of precipitation and color changes, which is easily affected by subjective factors and the accuracy needs to be improved. Therefore, it is still necessary to explore more efficient, accurate and cost-effective LCDV rapid detection methods.

[0004] In view of this, the present invention is 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 that there is a lack of a primer composition for detecting lymphocystis virus with rapid reaction, high sensitivity and strong specificity in the prior art.

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

[0010] A sixth object of the present invention is to provide a method for isothermal amplification of the recombinase of 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-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0013] In a first aspect, the present invention provides a composition for detecting lymphocystis virus, comprising an upstream primer MCP-F having a nucleotide sequence as shown in SEQ ID NO:7, a downstream primer MCP-R having a nucleotide sequence as shown in SEQ ID NO:8, and a specific probe MCP-P having 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] Furthermore, 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 a C3 spacer.

[0018] Furthermore, 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 of the following:

[0020] A1. Application in the preparation of products for detecting or assisting in the detection of lymphocystis virus;

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

[0022] A3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples;

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

[0024] A5. Application in the preparation of products 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 also includes RPA reaction reagents, positive controls, negative controls, PBS buffer and colloidal gold test strips required for isothermal amplification of the recombinase;

[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 enzyme-free water;

[0032] Preferably, 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.

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

[0034] B1. Application in the preparation of products for detecting or assisting in the detection of lymphocystis virus;

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

[0036] B3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples;

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

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

[0039] In a sixth aspect, the present invention provides a method for isothermal amplification of lymphocystis virus by recombinase, comprising performing isothermal amplification of a sample by recombinase using the primer-probe combination, the reagent or the 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 to 30 min, preferably 15 min;

[0042] Preferably, the amount of nuclease used in the recombinase isothermal amplification is 0.5 to 6 μL of nuclease per 50 μL of system, preferably 1 μL of nuclease per 50 μL of system.

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

[0044] The present invention provides a composition for detecting lymphocystis virus. The LCDV strain genome sequence is referred to to determine its MCP conservative gene sequence, design RPA primers and specific probes, modify biotin at the 5' end of MCP-R, modify tetrahydrofuran at the 30th base on the MCP-P, and modify the blocking group at the 3' end. Experiments have shown that the composition can detect lymphocystis virus and largemouth bass frog iris virus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, flounder rhabdovirus, and flatfish virus, and has specificity for lymphocystis virus; the sensitivity reaches 5pg / reaction; and its amplified product is suitable for lateral flow test strips (LFD test strips). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0047] Figure 2 A comparison chart of colloidal gold test paper detection established by different primer probes provided in Example 1 of the present invention;

[0048] Figure 3 A comparison chart of colloidal gold signal intensities at different RPA amplification temperatures provided in Example 2 of the present invention;

[0049] Figure 4 A comparison chart of colloidal gold signal intensities at different RPA amplification times provided in Example 2 of the present invention;

[0050] Figure 5 A comparison chart of colloidal gold signal intensities at different nfo enzyme addition amounts provided in Example 3 of the present invention;

[0051] Figure 6 The specific detection results of the LCDV nucleic acid flow immunochromatography detection method provided in Example 6 of the present invention;

[0052] Figure 7 The sensitivity test result of the LCDV nucleic acid flow immunochromatography detection method provided in Example 7 of the present invention;

[0053] Figure 8 The RPA-LFD test results of the clinical sample fins provided in Example 8 of the present invention. DETAILED DESCRIPTION

[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 meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0055] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0056] On the one hand, the present invention provides a composition for detecting lymphocystis virus, comprising an upstream primer MCP-F having a nucleotide sequence as shown in SEQ ID NO: 7, a downstream primer MCP-R having a nucleotide sequence as shown in SEQ ID NO: 8, and a specific probe MCP-P having 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 genome sequence of LCDV strain, the conserved gene sequence of MCP was determined, RPA primers and specific probes were designed, biotin was modified at the 5' end of MCP-R, and tetrahydrofuran was modified at the 30th base on the MCP-P in the direction from the 5' end to the 3' end, and the blocking group at the 3' end was modified. Experiments have shown that the composition can detect lymphocystis virus and largemouth bass frog iris virus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, flounder rhabdovirus, and flounder virus, and has specificity for lymphocystis virus; the sensitivity reaches 5pg / reaction; and its amplified product is suitable for lateral flow test strips (LFD test strips).

[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 can also be selected, such as VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705.

[0060] In some specific embodiments, the blocking group is a 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, there is also provided the use of the above-mentioned composition in any of the following:

[0063] A1. Application in the preparation of products for detecting or assisting in the detection of lymphocystis virus;

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

[0065] A3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples;

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

[0067] A5. Application 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 provided, comprising the above-mentioned composition.

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

[0070] In some specific embodiments, the reagents required for isothermal amplification of the recombinase, RPA reaction reagents, positive control, negative control, PBS buffer and colloidal gold test strips are also included.

[0071] In some specific embodiments, the RPA reaction reagents include 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 an MCP sequence, wherein the MCP sequence is the nucleotide sequence shown in SEQ ID NO:10.

[0074] In some specific embodiments, the negative control is sterile 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 a fifth aspect, the present invention provides the use of the above reagent or the above kit in any of the following:

[0077] B1. Application 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 sample contains lymphocystis virus;

[0079] B3. Use in the preparation of products for identifying and distinguishing lymphocystis virus and other pathogens in samples;

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

[0081] B5. Application in the preparation of products for screening diseases caused by lymphocystis virus.

[0082] In a sixth aspect, the present invention provides a method for isothermal amplification of a recombinase of a lymphocystis virus, comprising performing isothermal amplification of a sample with a recombinase using the primer-probe combination, the reagent, or the kit. The method does not require precision instruments, and only requires 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 7.5-30min, preferably 15min. In some specific embodiments, the amount of nuclease used in the recombinase isothermal amplification is to add 0.5-6μL nuclease per 50μL system. By using nuclease, the amplified product can be applied to LFD detection. The specific nuclease can be selected from nfo enzyme, and the amount used is preferably to add 1μL nuclease per 50μL system.

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

[0085] Through recombinase isothermal amplification (RPA isothermal amplification), the amplified product is visually detected through specific antibodies or probes on the test strip. These antibodies or probes can specifically bind to the specific markers of the amplified product, thereby forming a color change or precipitation visible to the naked eye on the test strip to visually determine the presence or absence of the target nucleic acid. On-site detection can be achieved, reducing the difficulty and cost of operation, and it is easy to operate, fast in response, highly sensitive, and highly specific; experiments have shown that RPA isothermal amplification can be completed in as fast as 7.5 minutes, and the target nucleic acid can be amplified and detected in a short time, improving the detection efficiency.

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

[0087] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0088] Example 1 Design and screening of primer and probe combinations

[0089] The genome sequences of LCDV strains were searched with reference to the NCBI GenBank database, and three sets of primers and probes were designed for the conserved MCP 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, the same amount of LCDV from the same batch was used as a template and the colloidal gold test paper RPA detection method established with three sets of primers and probes was used for detection.

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

[0095] 32.9 μL of RPA recombinase polymerase reaction buffer (from Wuhan Jun Nord Biotechnology Co., Ltd. Basic-DNA Constant Temperature Rapid Amplification Kit, Catalog No.: 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 were added to a single tube of independently packaged RPA recombinase polymerase lyophilized powder (from Wuhan Jun Nord Biotechnology Co., Ltd. Basic-DNA Constant Temperature Rapid Amplification Kit, Catalog No.: A4601-48), and then 5 μL of LCDV was added. DNA template, add 2.5μL of magnesium acetate before preparing the reaction; the temperature of RPA amplification reaction is 39℃, the reaction time is 15min, the reaction product is added to 150μL PBS buffer (pH 7.0), and then 100μL is added to the sample pad of the colloidal gold loading card, the sample pad has gold-labeled mouse anti-FAM antibody, the detection line (T) is sprayed with anti-biotin antibody, and the quality control line (C) is sprayed with goat anti-mouse IgG antibody. The result is read after 10 minutes of reaction.

[0096] The results are as follows Figure 2 As shown in FIG. 1 , at the detection line (T), the MCP-For3 / MCP-Rev3 / MCP-Pro3 detection composition has the strongest colloidal gold signal intensity and the best amplification effect, so 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 shown in FIG. Figure 1 shown.

[0097] Example 2 Optimization of reaction conditions

[0098] 1. RPA amplification temperature optimization

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

[0100] 2. RPA amplification time optimization

[0101] According to the reaction system of Example 1, MCP-For3 / MCP-Rev3 / MCP-Pro3 primers / probes were used, and 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 at an amplification reaction temperature of 43°C, in order to screen out the optimal amplification reaction time. After the amplification, the reaction product was added to 150 μL of PBS buffer (pH 7.0), and then 100 μL was added to the sample pad of the colloidal gold loading card, the sample pad was coated with gold-labeled mouse anti-FAM antibody, the detection line (T) was sprayed with anti-biotin antibody, and the quality control line (C) was sprayed with sheep anti-mouse IgG antibody. The results were read after 10 minutes of reaction. The reaction results are as follows. Figure 4 As shown, it can be seen that when the amplification reaction time is 15 minutes, the detection line (T) band is the clearest.

[0102] Example 3 Screening of nfo enzyme concentration

[0103] Except for the concentration of nfo enzyme, refer to the reaction system of Example 1, use MCP-For3 / MCP-Rev3 / MCP-Pro3 primers / probes, amplify the prepared reaction system at an amplification reaction temperature of 43°C for 15 minutes, and add 0μL, 0.1μL, 0.25μL, 0.5μL, 0.75μL, 1μL, 2μL, 3μL, 4μL, 5μL, 6μL and 7μL 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, add 150μL of PBS buffer (pH value is 7.0) to the reaction product, and then take 100μL and add it to the sample pad of the colloidal gold loading card, the sample pad has gold-labeled mouse anti-FAM antibody, the detection line (T) is sprayed with anti-biotin antibody, and the quality control line (C) is sprayed with sheep anti-mouse IgG antibody. The result is read after 10 minutes of reaction. The reaction results are as follows. Figure 5 As shown, it can be seen that when 1 μL of nfo enzyme is added, the detection line (T) band is the clearest.

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

[0105] This example proposes a kit for detecting lymphocystis virus LCDV using RPA isothermal amplification combined with immunochromatography:

[0106] 1. Reagent A: RPA recombinase polymerase lyophilized powder (from Basic-DNA Constant Temperature Rapid Amplification Kit of Wuhan Junno Biotechnology Co., Ltd., catalog number: A4601-48), each tube represents an independent reaction.

[0107] 2. Reagent B: The components of the RPA reaction mixture are 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 (Basic-DNA Constant Temperature Rapid Amplification Kit purchased from Wuhan Junno Biotechnology Co., Ltd., Catalog No.: A4601-48), 1 μL of nfo enzyme (purchased from YEASEN, Catalog No.: 10323ES80), and 4 μL of sterile enzyme-free water.

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

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

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

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

[0112] The standard positive plasmid was obtained according to the following method:

[0113] The MCP sequence was synthesized and connected to the pcDNA3.1 cloning vector, transformed into E. coli competent cells DH5a, and spread on LB medium plates containing 100 mg / L ampicillin, and cultured at 37°C for 12 to 14 hours. After the positive single colony was expanded, the plasmid was extracted using a plasmid extraction kit, and the plasmid with a positive sequence was named pcDNA3.1-MCP, which is the standard positive plasmid.

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

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

[0116] 1. Preparation of sample DNA template

[0117] The genomic DNA of fish tissues carrying LCDV virus was extracted using a marine animal tissue genomic DNA extraction kit (TIANGEN, model: DP324) and referring to the kit instructions.

[0118] 2. RPA reaction system and reaction procedure

[0119] Using the reagents provided in Example 4, the operation is specifically performed according to the following steps: 42.5 μL of reagent B is added to each dry powder reaction tube (reagent A), and then 5 μL of LCDV DNA template is added to the reaction tube, and finally 2.5 μL of reagent C is added to the reaction tube and fully mixed (for multiple reactions, it is recommended to add reagent C to the inside of the lid of the reaction tube and turn the reaction tube upside down 8-10 times to mix); after mixing, the reaction solution is thrown (or quickly centrifuged) to the bottom of the tube, and then the reaction tube is immediately placed in a constant temperature device and incubated at 43°C for 15 minutes. After the reaction is completed, 150 μL of PBS buffer (reagent D) is added to the product, and then 100 μL is added to the sample pad of the colloidal gold test strip, and the result is read after 10 minutes of reaction.

[0120] Use colloidal gold test strips to judge the results:

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

[0122] When the control line (C) of the colloidal gold test strip develops color but 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 develops color, the test result is invalid.

[0124] Example 6 Analysis of the specificity of LCDV nucleic acid flow immunochromatography detection method

[0125] The DNA of Lymphocystis disease virus (LCDV) and Largemouth Bass Iridovirus (LMBV) were extracted and purified, and the prepared viral haemorrhagic septicaemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), Hiramerhabdovirus (HRV), Chinook salmon bafinivirus isolate Bces-Po19, CSBV Bces-Po19) cDNA was used as a template, and the reagents provided in Example 4 were used to perform RPA amplification according to the method provided in Example 5. The reaction product was added to 150 μL of PBS buffer, and then 100 μL was added to the sample pad of the colloidal gold loading card. The sample pad was coated with gold-labeled mouse anti-FAM antibody, the detection line (T) was sprayed with anti-biotin antibody, and the quality control line (C) was sprayed with goat anti-mouse IgG antibody. The results were read after 10 minutes of reaction. The results are shown in Figure 6 As shown, when only LCDV DNA was used as a template, a specific band appeared in the detection line (T).

[0126] Example 7: Sensitivity analysis of LCDV nucleic acid flow immunochromatography detection method

[0127] The extracted LCDV DNA was diluted with sterile enzyme-free water in a 10-fold ratio to 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 100 fg / μL concentrations, and then the gradient concentration DNA was used as a template, the reagents provided in Example 4 were used, and the RPA amplification reaction was performed according to the method provided in Example 5. The reaction product was added to 150 μL of PBS buffer (pH 7.0), and then 100 μL was added to the sample pad of the colloidal gold loading card. The sample pad was coated with gold-labeled mouse anti-FAM antibody, the detection line (T) was sprayed with anti-biotin antibody, and the quality control line (C) was sprayed with goat anti-mouse IgG antibody. The results were read after 10 minutes of reaction. The results are as follows Figure 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 template amount in the reaction system is 5 μL).

[0128] Example 8 Clinical Sample Detection

[0129] Twenty fin rays of clinical samples of flounder suspected of LCDV infection were selected, and DNA samples were prepared using a marine animal tissue genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP324). The RPA-LFD detection method (Example 5) established by the present invention was used for detection. The results are as follows: Figure 8 As shown, wherein numbers 1 to 20 correspond one to one with numbers 1 to 20 in Table 2; at the same time, the detection method for LCDVMCP gene in the published "Effects of RNA interference on the replication of LCDV-cn in flounder" was used for fluorescence quantitative PCR detection as a control, and the results of fluorescence quantitative PCR detection are shown in Table 2; Figure 8 Compared with the test results in Table 2, there is good consistency between the two in terms of the positive sample detection rate.

[0130] Table 2 Fluorescence quantitative PCR test 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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; Preferably, the fluorescent reporter group is FAM; Preferably, the blocking group is a C3 spacer.

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

3.

4. Use of the composition according to any one of claims 1 to 3 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.

5. A reagent for detecting lymphocystis virus, characterized in that: A composition comprising any one of claims 1 to 3.

6. A kit for detecting lymphocystis virus, characterized in that: The invention comprises the composition according to any one of claims 1 to 3 or the reagent according to claim 5.

7. The kit according to claim 6, characterized in that It also includes RPA reaction reagents, positive controls, negative controls, PBS buffer and colloidal gold test strips required for isothermal amplification of recombinase; Preferably, the RPA reaction reagents include recombinase polymerase buffer, recombinase polymerase, nuclease and magnesium acetate; Preferably, the nuclease is nfo enzyme; Preferably, the positive control is a positive plasmid containing the MCP sequence; Preferably, the negative control is sterile enzyme-free water; Preferably, 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.

8. Use of the reagent according to claim 5 or the kit according to claim 6 or 7 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.

9. A method for isothermal amplification of a recombinase of lymphocystis virus, characterized in that: The method comprises performing isothermal amplification of a sample by a recombinase using the composition according to any one of claims 1 to 3, the reagent according to claim 5, or the kit according to claim 6 or 7; Preferably, the reaction temperature of the recombinase isothermal amplification is 35-47°C, preferably 43°C; Preferably, the reaction time of the recombinase isothermal amplification is 7.5 to 30 min, preferably 15 min; Preferably, the amount of nuclease used in the recombinase isothermal amplification is 0.5 to 6 μL of nuclease per 50 μL of system, preferably 1 μL of nuclease per 50 μL of system.

10. 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 claim 9, and determining whether lymphocystis virus exists in the sample according to the detection line.

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