LAMP kit for detecting flounder and flatfish calicivirus and detection method thereof

Abstract

The present application belongs to the field of aquaculture, and particularly relates to a LAMP kit for detecting flounder and flatfish calicivirus and a detection method thereof. The present application provides a LAMP detection kit for detecting flounder and flatfish calicivirus, which comprises the following primer sequences: forward outer primer CSBV-F3, reverse outer primer CSBV-B3, forward inner primer CSBV-FIP and reverse inner primer CSBV-BIP. The LAMP kit provided by the present application can obtain experimental results by observing color changes with the naked eye through constant temperature amplification within one hour, and complete detection; and can also be verified again through agarose gel electrophoresis. The method of the present application has the advantages of simple operation, fast speed, low requirement for equipment, and the advantages of high amplification specificity and high sensitivity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of aquaculture, and particularly relates to a LAMP kit for detecting flounder and flatfish calicivirus and a detection method thereof. BACKGROUND

[0002] Chinook Salmon Bafinivirus (CSBV) is one of the pathogenic agents causing "hemorrhagic disease" in flounder and flatfish, which presents symptoms of "bottom plate bleeding, rotten fin and tail, liver bleeding", and leads to a large number of deaths. The virus belongs to the order Nidovirales, the family Tobaniviridae, and the genus Oncotshavirus. For susceptible flounder and flatfish, the mortality rate can be as high as more than 70%. The virus was first isolated and identified in Chinook salmon in Canada in 2014, and in 2019 and 2022, it was found in China's Hebei and Liaoning provinces that it could infect seawater fish, Japanese flounder and turbot. Currently, two new strains of CSBV have been isolated from diseased Japanese flounder and turbot, respectively, and named CSBVBces-Po19 (CSBV Japanese flounder isolate) and CSBV Bces-Sm22 (CSBV turbot isolate). So far, the virus has spread to multiple flounder and flatfish farms in Liaoning and Hebei provinces, causing serious impact on the flounder and flatfish aquaculture industry and becoming a major bottleneck restricting the healthy development of the industry.

[0003] At present, the detection means for CSBV mainly includes PCR method, qRT-PCR method, ELISA method and RNA-Seq sequencing method.(1) PCR method: since the virus was discovered in 2014, the conventional PCR method is the simplest method for identifying the virus.(2) RNA-Seq sequencing method: the unknown virus needs to be incubated in a cell line to produce obvious CPE phenomenon, and then RNA-Seq sequencing is carried out, and then all virus sequences in the database are compared, so as to identify the type of unknown virus; the method requires that the laboratory has the ability to culture cells, so as to infect the virus. Therefore, the requirements for cell culture related conditions are relatively high.(3) ELISA method: the operation is relatively simple, but the preparation is relatively complicated, it is easy to be contaminated to cause false positive, and finally the experimental result is only presented in the form of absorbance value, and the objectivity and repeatability are difficult to guarantee.(4) qRT-PCR method: the required fluorescence quantitative experiment equipment is relatively high in price, the operation (such as keeping the sample amount of parallel samples consistent) and sterile operation conditions of the experimental personnel are relatively high; and the optimal reaction temperature needs to be repeatedly groped in the initial stage of the experiment, and the operator needs to have certain professional knowledge.(5) Digital RT-PCR method: the method has not been used for CSBV virus, but has been applied to the crucian carp coronavirus HB93 and the carp coronavirus HL39 which have close relationship with CSBV; the required instrument is too expensive, and has not been widely popularized in colleges and universities and scientific research institutions; and the related reagent consumables are also expensive, and the sample adding accuracy of the experimental operator is extremely strict.

[0004] Therefore, the above methods have high requirements for experimental conditions, experimental equipment and operation difficulty, so it is difficult to be popularized and applied on a large scale.

[0005] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology proposed by Notom et al. in 2000. The characteristics of LAMP are that four primers are designed for six specified regions of the target gene, including forward inner primer (FIP), reverse inner primer (BIP), forward outer primer (F3) and reverse outer primer (B3). The experimental results can be obtained by observing the color change with the naked eye within one hour of constant temperature amplification, and the results can be further verified by agarose gel electrophoresis. The method has the advantages of simple operation, low requirement for equipment, high amplification specificity and high sensitivity. SUMMARY

[0006] The purpose of the present application is to provide a LAMP kit for detecting turbot and flatfish nested viruses.

[0007] Another object of the present application is to provide a LAMP method for detecting flounder nested virus.

[0008] The LAMP detection kit for detecting flounder nested virus according to the embodiment of the present application comprises the following primer sequences:

[0009] SEQ ID NO. 1: forward outer primer CSBV-F3: 5'-CCCCATTTGACCACACATC-3';

[0010] SEQ ID NO. 2: reverse outer primer CSBV-B3: 5'-TTGAGGAGGAGGGAGTTG-3';

[0011] SEQ ID NO. 3: forward inner primer CSBV-FIP:

[0012] 5'-TGGCAGGAATGAAAGTACCATTATAACTACACTCTTTTTTCACACATG-3';

[0013] SEQ ID NO. 4: reverse inner primer CSBV-BIP:

[0014] 5'-TATGCCTGCTAACAACCTTTCGAGAATCAGGTACGTTGACAA-3'.

[0015] The LAMP detection kit for detecting flounder nested virus according to the embodiment of the present application, wherein the molar ratio of the forward outer primer CSBV-F3, the reverse outer primer CSBV-B3, the forward inner primer CSBV-FIP and the reverse inner primer CSBV-BIP is 1:1:8:8.

[0016] The LAMP detection kit for detecting flounder nested virus according to the embodiment of the present application further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and positive control template, and the reaction system used in the kit is shown in the following table:

[0017]

[0018] The LAMP detection kit for detecting flounder nested virus according to the embodiment of the present application, wherein the flounder nested virus comprises CSBV Bces-Po19 and / or CSBV Bces-Sm22, and the kit has excellent specificity and sensitivity in detecting CSBV Bces-Po19 and CSBV Bces-Sm22.

[0019] The LAMP method for detecting flounder quasivirus according to the embodiment of the present application comprises the following steps of amplifying the flounder quasivirus gene using the following primer sequences:

[0020] forward outer primer CSBV-F3: 5'-CCCCATTTGACCACACATC-3';

[0021] reverse outer primer CSBV-B3: 5'-TTGAGGAGGAGGGAGTTG-3';

[0022] forward inner primer CSBV-FIP:

[0023] 5'-TGGCAGGAATGAAAGTACCATTATAACTACACTCTTTTTTCACACATG-3';

[0024] reverse inner primer CSBV-BIP:

[0025] 5'-TATGCCTGCTAACAACCTTTCGAGAATCAGGTACGTTGACAA-3'.

[0026] The molar ratio of the forward outer primer CSBV-F3, the reverse outer primer CSBV-B3, the forward inner primer CSBV-FIP and the reverse inner primer CSBV-BIP in the LAMP method for detecting flounder quasivirus according to the embodiment of the present application is 1:1:8:8.

[0027] The reaction system used in the LAMP method for detecting flounder quasivirus according to the embodiment of the present application comprises 4xLAMP MasterMix, Bst DNA polymerase, 10 μmol / L forward outer primer CSBV-F3, 10 μmol / L reverse outer primer CSBV-B3, 10 μmol / L forward inner primer CSBV-FIP, 10 μmol / L reverse inner primer CSBV-BIP, ddH2O and positive control template.

[0028] The LAMP method for detecting flounder quasivirus according to the embodiment of the present application comprises the following steps:

[0029] (1) extracting the RNA of flounder quasivirus in the sample to be detected;

[0030] (2) obtaining the template cDNA of flounder quasivirus in the sample to be detected;

[0031] (3) adding the template cDNA obtained in step (2) into the reaction system and performing LAMP amplification under the condition of constant temperature water bath at 65℃.

[0032] In step (2), the following steps are further included:

[0033] (2-1) removing genomic DNA of scophthalmidoid virus of the sample to be detected;

[0034] (2-2) performing a reverse transcription reaction to obtain a template cDNA;

[0035] In step (2-1), the RNA extracted in step (1) is added into a genomic DNA removal reaction system (5x gDNA Eraser Buffer, gDNA Eraser, RNase Free dH2O), and the reaction is performed at 42 DEG C for 2 min, and after the reaction is completed, the sample is placed at 4 DEG C, thereby removing the genomic DNA of the sample to be detected.

[0036] In step (2-2), an RNA reverse transcription kit is used, and the reaction conditions are as follows: 37 DEG C for 15 min, and 85 DEG C for 5 s; and after the reaction is completed, the template cDNA is obtained.

[0037] The application of the LAMP detection kit for detecting scophthalmidoid viruses of the application is especially in the field of aquatic product detection, and specifically, the kit can be used for detecting CSBV Bces-Po19 and CSBV Bces-Sm22, and can effectively detect the tissue samples of fish infected with the above viruses and the tissue samples of naturally diseased fish (for example, Paralichthys olivaceus and Scophthalmus maximus).

[0038] The application achieves the following beneficial effects:

[0039] The LAMP kit provided by the application can obtain the experimental result by naked eye observation of color change through constant temperature amplification within one hour, complete the detection, and further verified by agarose gel electrophoresis. The method has the advantages of simple operation, high speed, low requirement for equipment, high amplification specificity and high sensitivity.

[0040] The LAMP detection method has good specificity and sensitivity, wherein when the content of the cDNA in the reaction system is greater than or equal to 1x10 -2 ng / μL, the LAMP reaction product still turns into dark blue, and the sensitivity can reach 1x10 -2 ng / 20μL reaction system; meanwhile, the application has no amplification reaction to viral hemorrhagic septicemia virus, Paralichthys olivaceus rhabdovirus disease, lymph cyst virus and infectious hematopoietic tissue necrosis virus, and has good specificity. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only show some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative effort based on these drawings.

[0042] Figure 1 The figure shows the color development results and agarose gel electrophoresis results of the design and screening of the LAMP complete reagent in the present application embodiment 1; wherein,

[0043] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control, 1-2 are respectively cDNA templates of CSBV Bces-Po19 and CSBV Bces-Sm22.

[0044] Figure 2 The figure shows the color development results and agarose gel electrophoresis results of the reaction temperature optimization of the LAMP system of the CSBV Scophthalmus maximus isolate (CSBV Bces-Sm22) in the present application embodiment 3;

[0045] Figure 3 The figure shows the color development results and agarose gel electrophoresis results of the reaction temperature optimization of the LAMP system of the CSBV Scophthalmus maximus isolate (CSBV Bces-Sm22) in the present application embodiment 3;

[0046] Figure 4 The figure shows the color development results and agarose gel electrophoresis results of the sensitivity detection of the LAMP system of the CSBV Scophthalmus maximus isolate (CSBV Bces-Sm22) in the present application embodiment 4; wherein,

[0047] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0048] 1-8 are respectively 1×10 3 ng / μL-1×10 - 4ng / μL different gradient concentrations of detection results;

[0049] Figure 5 The figure shows the color development results and agarose gel electrophoresis results of the sensitivity detection of the LAMP system of the CSBV Scophthalmus maximus isolate (CSBV Bces-Sm22) in the present application embodiment 4; wherein,

[0050] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0051] 1-8 are respectively 1×103 ng / μL-1×10 -4 ng / μL different gradient concentration detection results;

[0052] Figure 6 The color development results and agarose gel electrophoresis results of LAMP detection of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application are shown in the schematic diagram.

[0053] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0054] 1-6 are respectively cDNA templates of CSBV Bces-Po19, CSBV Bces-Sm22, VHSV, HIRRV, LCDV and IHNV;

[0055] Figure 7 The color development results and agarose gel electrophoresis results of LAMP detection of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application are shown in the schematic diagram.

[0056] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0057] 1-10 are cDNA templates of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application;

[0058] Figure 8 The color development results and agarose gel electrophoresis results of LAMP detection of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application are shown in the schematic diagram.

[0059] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0060] 1-10 are cDNA templates of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application;

[0061] Figure 9 The color development results and agarose gel electrophoresis results of LAMP detection of the CSBV Pagrus major isolate (CSBV Bces-Sm22) in the embodiment 6 of the application are shown in the schematic diagram.

[0062] M is DL2000 DNA molecular weight marker, negative is negative control, positive is positive control,

[0063] 1-10 are cDNA templates of tissue samples of natural infection CSBV Bces-Po19, and 11 is a cDNA template of healthy tissue samples of B. oxyrhynchus;

[0064] Figure 10 The figure is the color development result and agarose gel electrophoresis result of LAMP detection of natural infection CSBV Bces-Sm22 in Scophthalmus maximus in Example 7 of the present application;

[0065] M is DL2000 DNA molecular weight marker, negative is negative control, and positive is positive control,

[0066] 1-10 are cDNA templates of tissue samples of natural infection CSBV Bces-Sm22 in Scophthalmus maximus, and 11 is a cDNA template of healthy tissue samples of Scophthalmus maximus. DETAILED DESCRIPTION

[0067] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0068] Design and screening of primers in Example 1

[0069] The present application takes different fragments of highly conserved nucleic acid sequences encoding S protein of CSBV Bces-Po19 and CSBV Bces-Sm22 virus as templates, and designs two pairs of primers (kit 1, kit 2) respectively, and the primer sequences are as follows:

[0070]

[0071] The results are shown in Figure 1 The amplification product of the template of the negative control is light blue, and the typical ladder band does not appear in the agarose gel electrophoresis, which is in line with the expectation; the amplification product of the standard positive template is dark blue, and the typical ladder band appears in the agarose gel electrophoresis, which is in line with the expectation; the amplification product of CSBV Bces-Po19 as the template (lane 1) is dark blue, and the typical ladder band appears in the agarose gel electrophoresis, which is in line with the expectation; the amplification product of CSBV Bces-Sm22 as the template (lane 2) is dark blue, and the typical ladder band appears in the agarose gel electrophoresis, therefore, the kit 1 can meet the experimental needs.

[0072] In the detection result of the complete reagent 2, the amplification product of the negative control is dark blue, and the agarose gel electrophoresis has a typical ladder band, and a false positive result appears.

[0073] Therefore, compared with the detection results of the complete reagent 2 and the complete reagent 1, the complete reagent 2 cannot meet the experimental needs because the amplification product with the negative control as a template appears false positive, and therefore, the complete reagent 1 is selected in the present application.

[0074] Therefore, based on the selection of the target sequence of the calicivirus CSBV, four primers for the loop-mediated isothermal amplification (LAMP) method are designed, which are CSBV-F3, CSBV-B3, CSBV-FIP and CSBV-BIP,

[0075] The sequence of the forward outer primer CSBV-F3 is 5'-CCCCATTTGACCACACATC-3';

[0076] The sequence of the reverse outer primer CSBV-B3 is 5'-TTGAGGAGGAGGGAGTTG-3';

[0077] The sequence of the forward inner primer CSBV-FIP is:

[0078] 5'-TGGCAGGAATGAAAGTACCATTATAACTACACTCTTTTTTCACACATG-3';

[0079] The sequence of the reverse inner primer CSBV-BIP is:

[0080] 5'-TATGCCTGCTAACAACCTTTCGAGAATCAGGTACGTTGACAA-3'.

[0081] Example 2: Establishment of the LAMP detection kit for detecting the calicivirus CSBV

[0082] The present example provides a complete reagent for detecting the calicivirus CSBV by the loop-mediated isothermal amplification (LAMP) method, which is composed of the primers selected in Example 1, wherein the primers are CSBV-F3, CSBV-B3, CSBV-FIP and CSBV-BIP,

[0083] The sequence of the forward outer primer CSBV-F3 is 5'-CCCCATTTGACCACACATC-3';

[0084] The sequence of the reverse outer primer CSBV-B3 is 5'-TTGAGGAGGAGGGAGTTG-3';

[0085] The sequence of the forward inner primer CSBV-FIP is:

[0086] 5'-TGGCAGGAATGAAAGTACCATTATAACTACACTCTTTTTTCACACATG-3';

[0087] The sequence of the reverse inner primer CSBV-BIP is:

[0088] 5'-TATGCCTGCTAACAACCTTTCGAGAATCAGGTACGTTGACAA-3'.

[0089] In the kit, each primer is independently packaged, and the molar ratio of CSBV-F3, CSBV-B3, CSBV-FIP and CSBV-BIP is 1:1:8:8.

[0090] The kit further comprises MasterMix, Bst DNA polymerase, 10 μmol / L forward outer primer CSBV-F3, 10 μmol / L reverse outer primer CSBV-B3, 10 μmol / L forward inner primer CSBV-FIP, 10 μmol / L reverse inner primer CSBV-BIP, ddH2O, positive control template.

[0091] Table 1 reaction system

[0092]

[0093] Example 3: Establishment of LAMP method for detecting nested virus CSBV

[0094] 1. Extraction of RNA from nested virus CSBV sample:

[0095] Select reagents, consumables and instruments, including: RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), centrifuge, vortex shaker, ice maker. The operation steps are as follows:

[0096] Take 250 μL of virus suspension into a 1.5 mL centrifuge tube, add 750 μL of lysis solution RZ, and stand at 15-30°C for 5 min;

[0097] Add 200 μL of chloroform, and shake vigorously in the vortex shaker for 15 s. After standing at room temperature for 3 min, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube;

[0098] Add 0.5 times the volume of anhydrous ethanol to the supernatant, mix well, and pour into a centrifugal column. Centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0099] Add 500 μL of deproteinization solution RD (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 s, discard the waste liquid;

[0100] Add 500 μL of rinse solution RW (check if anhydrous ethanol is added before use), stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s, discard the waste liquid;

[0101] Add 500 μL of rinse solution RW (check if anhydrous ethanol is added before use), stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s, discard the waste liquid;

[0102] Centrifuge at 12000 rpm for 2 min to remove the remaining liquid;

[0103] Place the adsorption column in a new 1.5 mL centrifuge tube, open the adsorption column cover, and stand at room temperature for 3-5 min until there is no obvious anhydrous ethanol smell;

[0104] Add 30 μL of RNase-Free ddH2O to the center of the centrifugal column adsorption membrane, stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and obtain the RNA of the sample to be tested.

[0105] 2. Preparation of cDNA of the nested virus CSBV sample to be tested:

[0106] Select reagents, consumables and instruments, including: RNA reverse transcription kit (TAKARA), centrifuge, vortex shaker, ice maker. The operation steps are as follows:

[0107] Step 1: Remove genomic DNA reaction, reaction system see Table 2, reaction conditions: 42℃, 2min; after the reaction is completed, stand at 4℃:

[0108] Table 2 Genomic DNA removal reaction system

[0109]

[0110] Step 2: Perform reverse transcription reaction, reaction system see Table 3, reaction conditions: 37℃, 15min; 85℃, 5s; after the reaction is completed, obtain the template cDNA. Stand at 4℃ for use.

[0111] Table 3 Reverse transcription reaction system

[0112]

[0113] 3. Determination method of reaction product

[0114] The template cDNA was added to the LAMP detection kit of Example 2, and LAMP amplification was performed, and the results were determined by observing the color change of the reaction solution with the naked eye. Then 5 μL of the amplification product was subjected to 2% agarose gel electrophoresis, and the results were determined again by comparing whether a clear and bright band appeared.

[0115] 4. Optimization of reaction temperature

[0116] According to the reaction system of Example 2, the prepared reaction system was reacted in a constant temperature water bath at 63°C, 64°C, 65°C, 66°C and 67°C for 60 min. After the reaction was completed, the results were directly observed by observing the color change of the reaction solution. Then 5 μL of the amplification product was subjected to 2% agarose gel electrophoresis, and the results were observed.

[0117] CSBV Bces-Po19 strain virus Figure 2 ) and CSBV Bces-Sm22 strain virus Figure 3 The results showed that when the reaction temperature was 63°C, 64°C, 65°C, 66°C and 67°C, the amplification product showed a deep blue color, but when the reaction temperature was 65°C, the electrophoresis band of the amplification product was the brightest, therefore, 65°C was the optimal reaction temperature.

[0118] Example 4 Sensitivity of the kit for detecting nested virus CSBV

[0119] The sample to be tested was:

[0120] The cDNA of CSBV Bces-Po19 strain was diluted by 10 times gradient, with 1000 ng / μL of the cDNA of CSBV Bces-Po19 strain as the starting concentration, to obtain CSBV Bces-Po19 cDNA solutions with concentrations of 1 x 10 3 ng / μL, 1 x 10 2 ng / μL, 1 x 10 1 ng / μL, 1 x 10 0 ng / μL, 1 x 10 -1 ng / μL, 1 x 10 -2 ng / μL, 1 x 10 -3 ng / μL, 1 x 10 -4 ng / μL, respectively.

[0121] The cDNA of CSBV Bces-Sm22 strain was diluted by 10 times gradient, with 1000 ng / μL of the cDNA of CSBV Bces-Sm22 strain as the starting concentration, to obtain CSBV Bces-Sm22 cDNA solutions with concentrations of 1 x 10 3 ng / μL, 1 x 10 2 ng / μL, 1 x 10 1 ng / μL, 1 x 10 01 x 10 -1 1 x 10 -2 1 x 10 -3 1 x 10 -4 1 x 10 -2

[0122] The reaction system according to Example 2 and the optimal reaction conditions obtained by Example 3 were used to detect the cDNA of CSBV Bces-Pol9 strain virus and the cDNA of CSBV Bces-Sm22 strain virus, with the cDNA of JFB (JFB) as a negative control.

[0123] The obtained reaction system was reacted in a constant temperature water bath at 65°C for 60 min. After the reaction was completed, the color change of the reaction solution was directly observed to determine the result. Then, 5 μL of the amplification product was used for 2% agarose gel electrophoresis to observe the result.

[0124] The results are shown in Figure 4 Figure 5 When the content of the cDNA in the reaction system was greater than or equal to 1 x 10 -2 ng / μL, the LAMP reaction product still turned dark blue, and a typical ladder band could be clearly observed by agarose gel electrophoresis. The result of agarose gel electrophoresis was consistent with the color development result, indicating that the established LAMP detection method had high sensitivity, and the sensitivity could reach 1 x 10 -2 ng / 20 μL of the reaction system.

[0125] Example 5 Specificity of the kit for detecting the nested virus CSBV

[0126] In this embodiment, cDNA of the following viruses were used as templates: Chinook salmon bafinivirus isolate Bces-Po19 (CSBV Bces-Po19), Chinook salmonbafinivirus isolate Bces-Sm22 (CSBV Bces-Sm22), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus virus (HIRRV), Lymphocystis disease virus (LCDV), and Infectious hematopoietic necrosis virus (IHNV). The amplification was performed according to the LAMP reaction system described in Example 2.

[0127] The reaction results are as follows Figure 6 As shown, only CSBV Bces-Po19 and CSBV Bces-Sm22 are dark blue, indicating a positive reaction; the other virus strains are light blue, indicating a negative reaction.

[0128] Agarose gel electrophoresis revealed typical ladder-like bands for both the CSBV Bces-Po19 and CSBV Bces-Sm22 strains in lane 1 and lanes 3-6, respectively. The agarose gel electrophoresis results were consistent with the dye development results, indicating that the established LAMP detection method has good specificity.

[0129] Example 6: Effectiveness of a kit for detecting nested CSBV virus (re-infected zebrafish)

[0130] This embodiment tested zebrafish infected with CSBV Bces-Po19 and CSBV Bces-Sm22 strains, respectively. The zebrafish used in the experiment were bred in our unit and temporarily held for 10 days before the challenge experiment. A total of 20 healthy zebrafish were selected for the artificial infection experiment with the two strains, that is, 10 healthy zebrafish were artificially infected with each strain. The viscera of diseased turbot collected earlier were homogenized, centrifuged, and the supernatant was filtered through a 0.22 μm membrane. 50 μL of virus suspension was injected into the peritoneal cavity of each zebrafish. Feeding was stopped after challenge, and the disease condition of the fish was observed. Three days after artificial infection, samples were collected from the two groups of 10 zebrafish infected with CSBV Bces-Po19 and CSBV Bces-Sm22 strains, respectively. All their visceral tissues were mixed and ground.

[0131] According to the steps in Example 2, the artificial infected zebrafish cDNA was prepared, and the standard positive template and ultrapure water were used as positive and negative controls, respectively. The LAMP detection method was used to detect the CSBV Bces-Po19 strain and the CSBV Bces-Sm22 strain.

[0132] As shown in Figure 7 , the detection results of the artificially re-infected zebrafish of the CSBV Bces-Po19 strain showed that the amplification product of the negative control template was light blue, the amplification product of the standard positive template was dark blue, and the amplification product of the artificially re-infected zebrafish cDNA of the CSBV Bces-Po19 strain was dark blue (lanes 1-10).

[0133] The results of agarose gel electrophoresis detection showed that the amplification product of the negative control template did not appear typical ladder bands. The amplification product of the standard positive template had typical ladder bands. The amplification product of the artificially re-infected zebrafish cDNA of the CSBV Bces-Po19 strain had typical ladder bands. The results of agarose gel electrophoresis were consistent with the results of dye coloration, indicating that the LAMP detection method established in the application had good effectiveness for fish re-infected with the CSBV Bces-Po19 strain.

[0134] As shown in Figure 8 , the detection results of the artificially re-infected zebrafish of the CSBV Bces-Sm22 strain showed that the amplification product of the negative control template was light blue, the amplification product of the standard positive template was dark blue, and the amplification product of the artificially re-infected zebrafish cDNA of the CSBV Bces-Sm22 strain was dark blue (lanes 1-10).

[0135] The results of agarose gel electrophoresis detection showed that the amplification product of the negative control template did not appear typical ladder bands. The amplification product of the standard positive template had typical ladder bands. The amplification product of the artificially re-infected zebrafish cDNA of the CSBV Bces-Sm22 strain had typical ladder bands. The results of agarose gel electrophoresis were consistent with the results of dye coloration, indicating that the LAMP detection method established in the application had good effectiveness for fish re-infected with the CSBV Bces-Sm22 strain.

[0136] Example 7 Actual application of the kit virus CSBV kit reagent (naturally diseased Paralichthys olivaceus and Scophthalmus maximus)

[0137] This embodiment tested naturally infected turbot exhibiting suspected symptoms for CSBV-Po19 and CSBV-Sm22 strains. The turbot and flounder used in the experiment were collected from aquaculture farms and all showed symptoms of "bottom plate hemorrhage, fin and tail rot, and liver hemorrhage." Ten turbot or flounder with obvious symptoms were sampled from each species, and all their internal organs were mixed and ground.

[0138] cDNA from diseased turbot or diseased flounder was prepared according to the steps in Example 2. Standard positive template and ultrapure water were used as positive and negative controls, respectively. The CSBV Bces-Po19 strain and CSBVBces-Sm22 strain were detected by LAMP assay.

[0139] result Figure 9 As shown, the detection results of turbot infected with CSBV Bces-Po19 strain under natural conditions showed that the amplification product with negative control as template was light blue, the amplification product with standard positive template was dark blue, the amplification product of cDNA of turbot infected with CSBV Bces-Po19 strain under natural conditions was dark blue (lanes 1-10), and the amplification product of healthy turbot was light blue (lane 11).

[0140] Agarose gel electrophoresis revealed that the amplification products using the negative control as template did not exhibit typical ladder-like bands. The amplification products using the standard positive template showed typical ladder-like bands. The amplification products of cDNA from turbot infected with the CSBV Bces-Po19 strain under natural conditions in lanes 1-10 showed typical ladder-like bands, while the amplification products of cDNA from healthy turbot in lane 11 did not show typical ladder-like bands. The agarose gel electrophoresis results were consistent with the dye development results, indicating that the LAMP detection method established in this invention has good effectiveness against turbot infected with the CSBV Bces-Po19 strain under natural conditions.

[0141] The results are as follows Figure 10 As shown, the detection results of turbot infected with CSBV Bces-Sm22 strain under natural conditions showed that the amplification product of the negative control template was light blue, the amplification product of the standard positive template was dark blue, the amplification product of cDNA of turbot infected with CSBV Bces-Sm22 strain under natural conditions was dark blue (lanes 1-10), and the amplification product of healthy turbot was light blue (lane 11).

[0142] The results of agarose gel electrophoresis detection show that: the amplification product of the negative control template does not appear typical ladder band. The amplification product of the standard positive template has typical ladder band. The amplification product of the cDNA of the sciaenops ocellatus infected with the CSBV Bces-Sm22 strain under natural conditions in lanes 1-10 has typical ladder band, and the amplification product of the cDNA of the healthy sciaenops ocellatus in lane 11 does not appear typical ladder band. The results of agarose gel electrophoresis are consistent with the results of dye coloration, indicating that the LAMP detection method established in the application has good effectiveness for the sciaenops ocellatus infected with the CSBV Bces-Sm22 strain under natural conditions.

[0143] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A LAMP detection kit for detecting shelling viruses in flatfish, characterized in that, The kit comprises the following primer sequences: Forward outer primer CSBV-F3: 5'-CCCCATTTGACCACACATC-3'; Reverse outer primer CSBV-B3: 5'-TTGAGGAGGAGGGAGTTG-3'; Forward inner primer CSBV-FIP: 5'-TGGCAGGAATGAAAGTACCATTATAACTACACTCTTTTTTCACACATG-3'; Reverse inner primer CSBV-BIP: 5'-TATGCCTGCTAACAACCTTTCGAGAATCAGGTACGTTGACAA-3'; The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template.

2. The LAMP detection kit for detecting flounder and flatfish calicivirus according to claim 1, characterized by, The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises 4xLAMP MasterMix, Bst DNA polymerase, ddH2O and a positive control template. The kit further comprises

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