Double-nano antibody colloidal gold immunochromatography test strip for detecting giant salamander iridovirus and preparation method thereof

Abstract

The invention discloses a double-nano-antibody colloidal gold immunochromatography test strip for detecting giant salamander iridovirus and a preparation method of the double-nano-antibody colloidal gold immunochromatography test strip. The test strip comprises a PVC bottom plate, a sample absorption pad, a reaction film and absorbent paper, colloid for pasting and fixing is arranged on the upper surface of the PVC bottom plate, a detection line is arranged on the portion, close to the sample absorption pad, of the upper surface of the reaction film, a quality control line is arranged on the portion, close to the absorbent paper, of the upper surface of the reaction film, and the quality control line is parallel to the detection line; the detection line is coated with a colloidal gold labeled giant salamander iridovirus nano antibody, and the quality control line is coated with a rabbit anti-camel rabbit polyclonal antibody VHH. The method has the advantages of high sensitivity, high specificity, low cost, simplicity in operation, short detection time, suitability for rapid detection in the market and the like, is very suitable for rapidly detecting the giant salamander iridovirus on site, and has important significance on disease prevention and control of aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of fish virus detection, and in particular relates to a double-nano antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus and a preparation method thereof. Background Art

[0002] Chinese giant salamander, commonly known as baby fish, has attracted much attention for its excellent medicinal, edible and nutritional value. Since the beginning of the 21st century, the country has actively promoted the artificial breeding and domestication of giant salamanders. However, with the continuous expansion of artificial breeding and the deterioration of water environment quality, the problem of pathogen transmission has become more serious, causing huge economic losses to the giant salamander breeding industry. Among them, Chinese Giant Salamander Iridovirμs (CGSIV), also known as Chinese Giant Salamander Ranavirμs (CGSRV), belongs to the genus Ranavirus of the family Iridoviridae. It poses a serious threat to the global giant salamander breeding industry due to its rapid spread, difficulty in control, high mortality rate and difficulty in prevention. Given the current lack of effective CGSIV control methods, it is particularly urgent to develop a detection technology that can quickly identify the virus.

[0003] At present, the standard detection methods for giant salamander iris virus include electron microscopy, tissue culture, molecular biology detection and immunoassay. However, electron microscopy and tissue culture methods are rarely used because they are time-consuming and have high equipment maintenance costs. Although molecular detection methods are highly sensitive, they are limited by the laboratory environment and are not conducive to rapid on-site detection. In contrast, immunoassay is regarded as one of the most promising solutions in the field of aquaculture virus diagnosis due to its advantages such as high feasibility, low cost, high specificity and low professional requirements.

[0004] As a simple and fast immunological detection method, colloidal gold immunochromatography technology fixes specific antibodies on specific areas of nitrocellulose membranes, uses capillary action to move samples along the membrane and specifically bind to fixed antibodies, and then uses immunocolloidal gold or immunoenzyme staining to show color changes, thereby achieving accurate immunodiagnosis. This technology is not only highly specific and sensitive, but also has low detection costs, short detection time, and low technical requirements for personnel.

[0005] However, the antibodies currently used in colloidal gold immunochromatography technology are mostly monoclonal antibodies and polyclonal antibodies, and their high production costs and batch-to-batch differences limit the stability of the test strips. In contrast, nanoantibodies have attracted much attention due to their high stability, low production costs, and ease of acquisition. However, the research on combining dual nanoantibodies as detection antibodies and capture antibodies with immunochromatography technology for detection is still in its infancy, and has not been explored much, let alone applied to the detection of giant salamander iris virus. Summary of the invention

[0006] The development of more efficient, sensitive and effective new products or detection methods for detecting giant salamander iridovirus can provide corresponding theoretical value and practical application significance for the field of aquaculture disease detection, and can also provide insights for the broader application of nano-antibodies. In order to overcome the defects and shortcomings of the prior art, the present invention innovatively provides a double-nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iridovirus and a preparation method thereof.

[0007] The technical solution adopted by the present invention is:

[0008] 1. A double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus:

[0009] It includes a PVC base plate, a sample absorption pad, a reaction membrane and absorbent paper. The reaction membrane is arranged in the middle of the upper surface of the PVC base plate. The sample absorption pad and the absorbent paper are respectively arranged at two ends of the upper surface of the PVC base plate. Part of the sample absorption pad overlaps one end of the upper surface of the reaction membrane, and part of the absorbent paper overlaps the other end of the upper surface of the reaction membrane.

[0010] The upper surface of the PVC base plate is provided with a glue such as a sticker for sticking and fixing, the upper surface of the reaction membrane is provided with a detection line near the sample absorption pad, the upper surface of the reaction membrane is provided with a quality control line near the absorbent paper, and the quality control line is parallel to the detection line; the detection line is coated with a giant salamander iridescent virus nano-antibody labeled with colloidal gold, and the quality control line is coated with a rabbit anti-camel rabbit polyclonal antibody VHH.

[0011] The colloidal gold-labeled giant salamander iridovirus nanobody is prepared by the following steps: adjusting the pH of the colloidal gold solution, adding the giant salamander iridovirus nanobody, mixing and standing, then adding skim milk powder, standing and centrifuging, discarding the supernatant, and dissolving the precipitate with a colloidal gold complex solution to obtain the colloidal gold-labeled giant salamander iridovirus nanobody.

[0012] The amino acid sequence of the giant salamander iris virus nanobody is shown in SEQ ID NO.1-6, which is recorded in Patent 2024117003053.

[0013] The nanobody of giant salamander iris virus is prepared according to the following steps:

[0014] (1) immunizing alpacas with inactivated giant salamander iris virus, collecting peripheral blood lymphocytes of alpacas for mRNA extraction after immunization, synthesizing the first chain of cDNA using the mRNA as a template, and performing PCR amplification using the obtained cDNA as a template to obtain a DNA fragment encoding a nanobody;

[0015] (2) connecting the DNA fragment encoding the nanobody to the pComb3xss vector, transforming into competent cells, and constructing an initial nanobody library; and expanding and culturing the initial library to obtain a nanobody library;

[0016] (3) using the giant salamander iris virus as a coating antigen, and performing enrichment and panning screening on the nanoantibody library to obtain nanoantibody phagemids against the giant salamander iris virus;

[0017] (4) The phagemid is transformed into a host expression bacterium to induce the expression of the nanoantibody, and the nanoantibody sequence targeting the giant salamander iris virus is purified.

[0018] The mRNA extraction described in step (1) is performed using a TRIzol kit.

[0019] The primer sequences used in the PCR amplification described in step (1) are as follows:

[0020] The nucleotide sequence of primer 1 is shown in SEQ ID NO.7;

[0021] The nucleotide sequence of primer 2 is shown in SEQ ID NO.8;

[0022] The nucleotide sequence of primer 3 is shown in SEQ ID NO.9;

[0023] The nucleotide sequence of primer 4 is shown in SEQ ID NO.10;

[0024] The nucleotide sequence of primer 5 is shown in SEQ ID NO.11.

[0025] The initial library described in step (3) is expanded by adding the recovered bacterial solution to the culture medium, shaking at 37°C for 2 hours, adding helper phage to expand the initial library, shaking for 2 hours after adding the helper phage, adding kanamycin, then shaking overnight, and collecting phages by centrifugation the next day to complete the construction of the immune library.

[0026] The nanoantibody library described in step (3) is enriched and screened by three rounds of panning, wherein a monoclonal colony is picked and inoculated into a super broth medium for culture, and then added to an ELISA plate pre-coated with giant salamander iridescent virus, and after washing with shaking at room temperature, an anti-M13-horseradish peroxidase-labeled antibody is added, and the plate is washed with shaking at room temperature again, and 3,3',5,5'-tetramethylbenzidine is added for color development, and the monoclonal colony corresponding to the positive clone well with an absorbance value greater than 1.5 is picked for sequencing.

[0027] The step (4) of transforming the phagemid into the host expression bacteria is to use an Omega plasmid small-scale extraction kit to extract the plasmid of the positive monoclonal colony, then mix the plasmid with TOP10F' competent cells, place them in an ice bath and then heat shock them in 42°C hot water, then place them in an ice bath again, add super broth culture medium, shake at 37°C for one hour to complete the transformation; inoculate the introduced TOP10F' into the culture medium, shake and culture until the absorbance OD600 is 0.8-1.0, add isopropyl thiogalactoside to 1mM, and induce overnight.

[0028] Purification of the nanobody described in step (4) Add BeyoLytic to the overnight cultured bacterial solution TM The bacteria were lysed by a bacterial active protein extraction reagent, and the supernatant was collected by centrifugation; the Ni-NTA matrix was added to the supernatant collected above, and then added to the purification column after shaking at room temperature; then a phosphate equilibration buffer containing imidazole was added to wash the purification column and elute the impurity protein; then a phosphate elution buffer containing imidazole was added to elute the target protein adsorbed on the nickel column; the nano-antibody was added to a dialysis bag and dialyzed in a phosphate buffer.

[0029] The colloidal gold solution is mainly formed by mixing 1% chloroauric acid and 1% trisodium citrate by mass, wherein the volume ratio of chloroauric acid to trisodium citrate is 1:2, and the particle size of the colloidal gold in the colloidal gold solution is 40-50 nm;

[0030] The colloidal gold complex solution is a mixture of 1×0.01M PBS solution and 2.5% sucrose, 0.5% BSA and 0.1% Tween 20 in mass fraction;

[0031] The pH is adjusted by adding K2CO3 to adjust the pH to 6.5-7.0, the concentration of the giant salamander iris virus nanoantibody is 20 μg / mL, the mass fraction of skimmed milk powder is 10%, the volume of skimmed milk powder added is one tenth of the volume of the colloidal gold solution, the standing time is 20-30 minutes, the centrifugal force of the centrifugation is 10000xg, the centrifugal temperature is 4°C, the centrifugal time is 20 minutes, and the volume ratio of the colloidal gold complex solution to the colloidal gold solution is 1:5.

[0032] 2. A method for preparing a double nano-antibody colloidal gold immunochromatographic test strip:

[0033] The method comprises the following steps:

[0034] S1, pre-cooling the nanoantibody of giant salamander iridovirus labeled with colloidal gold;

[0035] S2, applying pre-cooled colloidal gold-labeled giant salamander iridovirus nanoantibodies on the nitrocellulose membrane to obtain a detection line;

[0036] S3, applying rabbit anti-camel rabbit polyclonal antibody on nitrocellulose membrane to obtain quality control line;

[0037] S4, soaking the nitrocellulose membrane pad provided with the detection line described in step S2 and the quality control line described in step S3 in a buffer solution, and then drying it in an oven overnight to obtain a reaction membrane;

[0038] S5. Assemble the reaction membrane, sample absorption pad and water absorption pad described in step S4 on a PVC bottom plate to obtain a double nano-antibody colloidal gold immunochromatography test strip.

[0039] In the step S1, the pre-cooling treatment is to place the colloidal gold-labeled giant salamander iridovirus nanobody in a refrigerator at a temperature of 4°C for standing; in the step S2, the concentration of the colloidal gold-labeled giant salamander iridovirus nanobody is 1-2.5 mg / ml, and the coating amount on the detection line is 0.5-1.5 μL / cm; in the step S3, the concentration of the rabbit anti-camel rabbit polyclonal antibody VHH is 1-2.5 mg / mL, and the coating amount on the quality control line is 0.5-1.5 μL / cm; more preferably, the concentration of the colloidal gold-labeled giant salamander iridovirus nanobody is 2 mg / mL, and the coating amount is 1 μL / cm; the concentration of the rabbit anti-camel rabbit polyclonal antibody VHH is 2 mg / mL, and the coating amount is 1 μL / cm. In step S4, the buffer solution is mainly composed of 1L 0.01M PBS, 1g polyvinyl pyrrolidone, 0.1g sodium cholate, 0.1g sodium caseinate, and 1mL Tween. The soaking time is 30 minutes and the drying temperature is 37°C.

[0040] The assembly is to place the reaction membrane in the middle of the upper surface of the PVC bottom plate, and the sample absorption pad and the water absorbent pad are respectively arranged at the two ends of the upper surface of the PVC bottom plate, wherein a partial area of ​​the sample absorption pad is overlapped on one end of the upper surface of the reaction membrane, and a partial area of ​​the sample absorption pad is close to the detection line on the reaction membrane, and a partial area of ​​the water absorbent paper is overlapped on the other end of the upper surface of the reaction membrane, and a partial area of ​​the water absorbent pad is close to the quality control line on the reaction membrane.

[0041] 3. Application of the double nano-antibody colloidal gold immunochromatographic test strip or the double nano-antibody colloidal gold immunochromatographic test strip prepared by the above preparation method in the detection of giant salamander iris virus in aquaculture.

[0042] 4. A method for detecting giant salamander iris virus in aquaculture using the above-mentioned double nano-antibody colloidal gold immunochromatographic test strip or the double nano-antibody colloidal gold immunochromatographic test strip prepared by the above-mentioned preparation method:

[0043] The method comprises the following steps:

[0044] D1. Add the diluted sample to be tested to the colloidal gold-labeled giant salamander iris virus nanobody in a 96-well plate, and mix with a pipette to obtain a mixed solution; preferably, during the detection, the volume of the diluted sample to be tested is 50 μL, and the amount of the added colloidal gold particle-labeled giant salamander iris virus nanobody is 12.5 μL;

[0045] D2, adding the mixed solution described in step D1 dropwise onto the sample absorption pad, the sample solution flows through the entire test strip by capillary force, the mixed solution flows through the reaction membrane and combines with the giant salamander iris virus nanoantibody sandwich on the detection line to form a nanoantibody-antigen-nanoantibody colloidal gold marker sandwich;

[0046] D3, the nanobody-antigen-nanobody colloidal gold marker described in step D2 is sandwiched on the test line, and the colloidal gold causes the test line to appear red;

[0047] D4. Determine the presence of giant salamander iris virus in the sample based on the color of the detection line.

[0048] The beneficial effects of the present invention are:

[0049] 1. The test strip prepared by the present invention has high sensitivity, strong specificity, low cost, simple operation, short detection time, is suitable for rapid detection in the market, is easy to store, and has a long shelf life.

[0050] 2. The method for detecting giant salamander iridovirus of the present invention is simple, rapid, intuitive, accurate, has a wide range of applications, is low in cost, and is easy to promote and use, and is suitable for screening and on-site monitoring of a large number of samples on the market.

[0051] 3. The present invention fills the gap in the research of double nano antibody test strips in the detection field. The double nano antibody colloidal gold test strips prepared by this method for detecting giant salamander iris virus have a detection limit of 1.05×10 7 TCID 50 / mL, the reaction time is shortened to 10-15min. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graph showing the pH optimization results for the preparation of colloidal gold-labeled giant salamander iris virus nanoantibodies.

[0053] Figure 2 This is a graph showing the optimization results of the skimmed milk powder content of the colloidal gold-labeled giant salamander iris virus nanoantibody.

[0054] Figure 3 This is the optimization result diagram of sample buffer type.

[0055] Figure 4 This is a graph showing the optimization results of the nanobody content.

[0056] Figure 5 Schematic diagram of the amount of added nanoantibody probe for the giant salamander iridovirus labeled with gold.

[0057] Figure 6 This is a structural diagram of the test strip.

[0058] Figure 7 This is a schematic diagram of the test strip detection principle.

[0059] Figure 8 Optimize the result graph for the T line of the test strip.

[0060] Fig. 9 Optimize the C line result graph for the test strip.

[0061] Fig.10 This is the standard curve of the test strip.

[0062] Fig.11 This is a graph showing the test results of different aquatic viruses tested using test strips.

[0063] Fig.12 This is the test result of the test strip on the real sample of giant salamander iris virus. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0065] Embodiments of the present invention are as follows:

[0066] 1. Preparation and optimization of nanoantibodies labeled with colloidal gold particles

[0067] 1. Solution preparation

[0068] The colloidal gold complex solution is mainly composed of 1M PBS, 2.5% sucrose, 0.5% BSA, and 1% Tween-20.

[0069] A nanobody targeting giant salamander iris virus, the amino acid sequence of the nanobody is shown in SEQ ID NO.1-6, and the preparation method comprises the following steps:

[0070] 1) Immunizing alpacas with inactivated giant salamander iris virus, collecting peripheral blood lymphocytes of alpacas for mRNA extraction after immunization, synthesizing the first chain of cDNA with the mRNA as a template, and performing PCR amplification with the obtained cDNA as a template to obtain a DNA fragment encoding a nanobody;

[0071] 2) Connecting the DNA fragment encoding the nanobody to the pComb3xss vector, transforming into competent cells, constructing an initial nanobody library, and expanding the culture to obtain a nanobody library.

[0072] 3) using the giant salamander iris virus as a coating antigen, and performing enrichment and panning screening on the nanobody library to obtain nanobody phagemids against the giant salamander iris virus;

[0073] 4) The phagemid is transformed into a host expression bacterium to induce the expression of the nano-antibody, and the nano-antibody sequence targeting the giant salamander iris virus is purified.

[0074] The colloidal gold-labeled giant salamander iris virus nanobody is prepared by the following steps: the colloidal gold solution is adjusted to pH, and then the giant salamander iris virus nanobody is added, mixed and allowed to stand, and then skimmed milk powder is added for blocking, allowed to stand and then centrifuged, the supernatant is discarded, and the precipitate is fully dissolved and resuspended with a colloidal gold solution to obtain the colloidal gold-labeled giant salamander iris virus nanobody.

[0075] 2. Preparation and optimization of colloidal gold markers

[0076] The invention optimizes the preparation conditions of the nano antibody of giant salamander iris virus labeled with colloidal gold, and the optimization result is determined according to the stability of the nano antibody of giant salamander iris virus labeled with colloidal gold finally obtained and the sensitivity in the test strip detection.

[0077] In the test strip, the test line is the T line and the quality control line is the C line, collectively referred to as the CT line.

[0078] (1) pH optimization

[0079] The pH of the colloidal gold solution was adjusted using a 0.2M potassium carbonate solution. Colloidal gold solutions of different pH values ​​were obtained by adding 6, 8, 10, and 12 μL of potassium carbonate solution, and then 20 μL of 1 mg / ml nanoantibody was added. Different colloidal gold complexes were then obtained according to the above reaction process. The appropriate pH was determined based on the sensitivity and stability of the final colloidal gold-labeled giant salamander iris virus nanoantibody in the test strip test. The sample was diluted to 10 μL using BB buffer (pH 7.4). 8 TCID 50 / mL. The sample pad of the test strip was vertically added to the sample solution of the nano-antibody labeled with colloidal gold particles, and the BB buffer was used as a negative control. The timing was started when the liquid flowed, and the reaction lasted for 10 to 15 minutes. The grayscale value of the CT line of the test strip was measured with a colloidal gold analyzer.

[0080] The pH optimization results are as follows Figure 1 As shown, when K 2 CO 3When the amount of K is controlled at 6-12 μL, the C and T lines on the test paper will increase with the K 2 CO 3 The addition of K 2 CO 3 When the amount of K added was 6 μL, the gray value of the T line of the test strip was the highest, and the false positive effect was the lowest under the negative control, so 6 μL K was selected. 2 CO 3 as the conditions for subsequent experiments.

[0081] (2) Optimization of skimmed milk powder content

[0082] During the test strip test, when the blocking protein content is low, false positives are found, which are manifested as the appearance of a T test line under the negative sample. This line appears after a significant time delay in the test. For the blocking protein content, 50, 100, 150 and 200 μL of 10% skimmed milk powder were added to 1 mL of AuNP solution. The appropriate skimmed milk powder content was determined based on the sensitivity and stability of the final colloidal gold-labeled giant salamander iris virus nanoantibody in the test strip test. Figure 2 As shown in the figure, when the addition amount is 50 μL, the false positive effect in the negative sample in the test paper is obvious. When the addition amount increases, the CT line effect becomes better, but too high an addition amount will reduce the sensitivity. Considering the reagent dosage, color development effect and sensitivity, 100 μL of 10% skim milk powder is selected as the ideal blocking protein content.

[0083] (3) Optimization of sample buffer system

[0084] In order to test whether different buffer systems would affect the test results of the test strips, different buffers were used to dilute the samples. PBS, TBS, and BB buffer (pH 7.4) were used to dilute the samples to 10 8 TCID 50 / mL. The sample pad of the test strip is vertically added to the sample solution of the nano-antibody labeled with colloidal gold particles. The timing starts when the liquid flows, and the reaction lasts for 8 to 10 minutes. The result is analyzed by the color depth of the CT line of the test strip.

[0085] The test strips prepared by the present invention were used to test the results in different buffer systems. Figure 3 As shown in the figure, the gray values ​​of the quality control line and the detection line of the test strip were low when the sample was diluted with PBS and TBS buffer, but the gray values ​​of the quality control line and the detection line were high when the giant salamander iris virus was detected under the condition of BB buffer. This shows that the more suitable buffer system for this test strip is BB buffer.

[0086] (4) Optimization of Nanobody Content

[0087] In order to test whether the content of the nanoantibody of giant salamander iris virus labeled with colloidal gold particles prepared by the present invention has an effect on the test results of the test strip, 10, 15, 20, and 25 μL of 1 mg / ml nanoantibody solution of giant salamander iris virus were used to obtain different colloidal gold complexes, and the appropriate amount of nanoantibody added was determined based on the sensitivity and stability of the nanoantibody of giant salamander iris virus labeled with colloidal gold in the test strip test. The optimization results of the nanoantibody addition amount are shown in FIG. Figure 4 As shown in the figure, when 20 μg is added, the quality control line and the detection line of the test strip are both dark, indicating that the most suitable addition amount of the nanobody in the colloidal gold-labeled nanobody is 20 μg.

[0088] (5) Optimization of probe addition amount

[0089] In order to test whether the amount of the nanoantibody labeled with colloidal gold particles of the giant salamander iris virus prepared by the present invention has an effect on the test results of the test strip, 7.5 μL, 10 μL, 12.5 μL, and 15 μL of the nanoantibody solution of the giant salamander iris virus were added to the diluted sample solution. The results of the measurement using the marker prepared by the present invention at different volumes are shown in FIG. Figure 5 As shown in the figure, the quality control line and the detection line of the test strip are the darkest when the probe addition amount is 12.5uL. This shows that the more appropriate probe addition amount for this test strip is 12.5μL.

[0090] 2. Preparation of Nano-antibody Colloidal Gold Test Strips for Detection of Giant Salamander Iridescent Virus

[0091] The nano-antibody colloidal gold test strip comprises a PVC base plate, a sample absorption pad, a reaction membrane and absorbent paper. A reaction membrane is arranged in the middle of the upper surface of the PVC base plate, and the sample absorption pad and the absorbent paper are respectively arranged at the two ends of the upper surface of the PVC base plate. Part of the sample absorption pad overlaps one end of the upper surface of the reaction membrane, and part of the absorbent paper overlaps the other end of the upper surface of the reaction membrane.

[0092] 1. Preparation of reaction membrane

[0093] (1) Coating of the detection line: The prepared giant salamander iris virus nanoantibody was coated onto the reaction membrane to form a detection line. The giant salamander iris virus nanoantibody was diluted to 2 mg / mL with BB buffer and coated onto the detection line (T line) on the nitrocellulose membrane using an XYZ three-dimensional film spray gold apparatus. The coating amount was 1 μL / cm. The coated reaction membrane was dried at 37°C for 12 h and then dried for use.

[0094] (2) Coating of the quality control line: Coat the rabbit anti-VHH on the reaction membrane to form a quality control line. Dilute the rabbit anti-camel VHH to 2 mg / mL with PBS buffer and coat it on the quality control line (C line) on the nitrocellulose membrane using an XYZ three-dimensional film spray gold instrument. The coating amount is 1 μL / cm. The coated reaction membrane is placed at 37°C to dry for 12 hours and then dried for use.

[0095] 2. Assembly of test strips

[0096] The reaction membrane, binding release pad, sample absorption pad and water absorption pad prepared above are sequentially pasted on the PVC bottom plate; the assembled test strip is as follows: Figure 6 As shown: the beginning of the sample absorption pad is aligned with the beginning of the PVC bottom plate, the end of the sample absorption pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the water absorbent pad, and the end of the water absorbent pad is aligned with the end of the PVC bottom plate. The reaction membrane is provided with a detection line (T line) and a quality control line (C line), both of which are strips perpendicular to the length of the test strip. The detection line is located on the side close to the end of the sample absorption pad, and the quality control line is located on the side close to the beginning of the water absorbent pad. After the test strip is assembled according to the above steps, each part is assembled and pressed tightly, and then cut into small strips of 4 mm width by a machine and packed in a special sealed bag.

[0097] Test strip principle Figure 7 As shown in the figure, when the test result is negative, the test line does not change, and the color of the quality control line turns red and deepens; when the test result is positive, the colors of both the test line and the quality control line turn red and deepen. When there is no change in both the test line and the quality control line, or only the test line turns red and deepens while the quality control line does not change, it is an invalid result, indicating that retesting is required.

[0098] 3. Limit test of double nanoantibody colloidal gold test strip for detecting giant salamander iris virus

[0099] The present invention optimizes the detection conditions of the giant salamander iris virus nanoantibody-colloidal gold test strip, and the optimization result is shown according to the finally obtained detection limit.

[0100] 1. Optimization of quality control line concentration

[0101] In order to test the influence of the quality control line concentration of the nano antibody colloidal gold test strip for detecting giant salamander iris virus prepared by the present invention on the test strip detection result, test strips with quality control lines of different concentrations were used for determination. Quality control lines of 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL were selected for testing.

[0102] The sample pad of the test strip is vertically added to the sample solution of the nano-antibody labeled with colloidal gold particles. The timing starts when the liquid flows, and the reaction lasts for 8 to 10 minutes. The results are analyzed by the color depth of the quality control line and the test line of the test strip. The test strip prepared by the present invention is tested at different quality control line concentrations. Figure 8 As shown in the figure, when the quality control line concentration of the test strip is 2 mg / mL rabbit anti-camel, the quality control line and the test line in the test result are darker, indicating that the more appropriate quality control line concentration of this test strip is 2 mg / mL.

[0103] 2. Optimization of test line concentration

[0104] In order to test the effect of the detection line concentration of the nano antibody colloidal gold test strip for detecting giant salamander iris virus prepared by the present invention on the test results of the test strip, test strips with different detection line concentrations were used for determination. 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL detection lines were used for testing.

[0105] The sample pad of the test strip is vertically added to the sample solution of the nano-antibody labeled with colloidal gold particles. The timing starts when the liquid flows, and the reaction lasts for 8 to 10 minutes. The results are analyzed by the color depth of the quality control line and the test line of the test strip. The test strip prepared by the present invention is measured at different detection line concentrations. The results are shown in FIG. Fig. 9 As shown in the figure, when the test strip has a detection line concentration of 2 mg / mL of the giant salamander iris virus nanoantibody, the quality control line and the test line in the test result are darker, indicating that the more appropriate detection line concentration of this test strip is 2 mg / mL.

[0106] 3. Detection limit test

[0107] In order to test the detection limit and sensitivity of the double nano-antibody colloidal gold test strip for detecting giant salamander iridovirus prepared by the present invention, the giant salamander iridovirus standard was diluted to a maximum of 8×10 8 , 4×10 8 , 2×10 8 , 10 8 , 5×10 7 , 2.5×10 7 , 1.25×10 7 , 6.25×10 6 ,3.125×10 6 , 1.5625×10 6 TCID 50 / mL, forming ten concentration gradients. Take the prepared test strips for detection, vertically drip the sample solution onto the sample absorption pad, start timing when the liquid flows, react for 10 to 15 minutes, and determine the result. Use the logarithm of the standard concentration as the horizontal coordinate and the grayscale of the T line as the vertical coordinate to perform nonlinear fitting using graphpad prism software. In addition, use the standard concentration as the horizontal coordinate and the grayscale intensity of the T line as the vertical coordinate for linear fitting to obtain the linear equation of the standard curve. In this scheme, LOD = 3SB / k, where k is the slope or sensitivity, i is the intercept, and SB is the standard deviation of the blank. For linear regression analysis, it is expressed as y = kx + i. Fig.10 The results showed that the vLOD of this method for detecting giant salamander iridovirus was 1.5625×10 6 TCID 50 / mL, LOD is 1.05×10 7 TCID 50 / mL, which meets the national standard detection requirements. Through nonlinear fitting, the IC50 of GISV in this detection method is 1.58×10 8 TCID 50 / mL. 7 TCID 50 / mL-2×10 8 TCID 50 It showed a good linear relationship in the range of 1:1.

[0108] 4. Specificity test

[0109] In order to detect the specificity of the double nano-antibody colloidal gold test strip for detecting giant salamander iridovirus prepared by the present invention, giant salamander iridovirus, mandarin fish iridovirus, sea bass iridovirus, and Aeromonas hydrophila were used respectively, and BB buffer was used as a negative control group to verify potential cross-reactions.

[0110] The test strips prepared by the present invention were used to test different virus-positive serums. Fig.11 As shown in the figure, when the test strip was used to detect the giant salamander iridovirus, both the quality control line and the test line showed color, indicating a positive result; while when detecting other viruses and bacteria, only the quality control line darkened in color, indicating a negative result, which was consistent with the result of the negative control group. This indicates that the test strip had no cross-reaction to the detection of mandarin fish iridovirus, sea bass iridovirus, and Aeromonas hydrophila, and could only specifically detect the giant salamander iridovirus, with a strong specificity.

[0111] 4. Actual sample testing

[0112] In order to verify the practicality of this method under specific breeding density conditions, a group analysis was conducted. Four groups of giant salamander samples were established: simulated infection (groups 1 and 2), clinical samples (group 3), and negative control (group 4). Giant salamander samples were purchased from the market, and groups 1 and 2 were intraperitoneally injected with 300 μL (5×10 6 copies / mL) and 500 μL (1×10 7 The tissue samples were collected 7 days after injection. Group 3 included 15 positive clinical samples (1×10 9 The kidneys and spleens of giant salamanders infected with giant salamander iridovirus were taken and the tissues were homogenized and ground, and then tested for practical applicability using test strips.

[0113] Test results such as Fig.12 As shown, when testing negative samples, it can be seen that the T line in the test strip is not colored, which means that the test strip is valid, and positive samples of different concentrations can be detected, indicating that the test results can meet the needs of actual sample testing.

[0114] The genetic sequence involved in the present invention is as follows:

[0115] SEQ ID NO.1:

[0116] Name: Sequence 1

[0117] Amino acid type: AA

[0118] Source: Natural sequence Synthetic Construct

[0119] Source organism: Alpaca

[0120] QAAQVQLVESGGGLVQPGGSLRLSCAASGFSFSRYAMSWVRQAPGKGLERVSEIATGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARSREIRVVGGIVESADFGSWGQGTQVTVSS

[0121] SEQ ID NO.2:

[0122] Name: Sequence 2

[0123] Amino Acid Type: AA

[0124] Source: Natural sequence Synthetic Construct

[0125] Source organism: Alpaca

[0126] QAAQLQLVESGGGLVQPGGSLRLSCAASGFSFSRYAMSWVRQAPGKGLERVSEITNDGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARTREIRVVGGIVESADFGSWGQGTQVTVSS

[0127] SEQ ID NO.3:

[0128] Name: Sequence 3

[0129] Amino Acid Type: AA

[0130] Source: Natural sequence Synthetic Construct

[0131] Source organism: Alpaca

[0132] QAAQLQLVESGGGLVQPGGSLRLSCVASGFSLDDYAVGWFRQAPGKEREGVSCISRFGGMTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAKYSSGYVCPDLFWDGMHYWGKGTLVTVSS

[0133] SEQ ID NO.4:

[0134] Name: Sequence 4

[0135] Amino Acid Type: AA

[0136] Source: Natural sequence Synthetic Construct

[0137] Source organism: Alpaca

[0138] QAAQLQLVESGGGLVEPGGSLQLSCVASGFVFSNYHITWARRVPGKELERVAHLYLDGTTYYSDSVKGRFLTSRDNAENTVYLQMINLKPEDTGVYYCAFIREMPPVADDFFFRDVPYDFWGQGTQVTVSS

[0139] SEQ ID NO.5:

[0140] Name: Sequence 5

[0141] Amino Acid Type: AA

[0142] Source: Natural sequence Synthetic Construct

[0143] Source organism: Alpaca

[0144] QAVQWQLVESGGGWGQPGGSLRLSCAAYGLWYNWYPMGWFRQAPGKEREFVIVQSWSGCRTQYADSVKGIFAMSRDSAKKTVFRQMDSMRPEDTTVYYSACRTSPSSAGTKGDGYAYWGQGTQVTV

[0145] SEQ ID NO.6:

[0146] Name: Sequence 6

[0147] Amino Acid Type: AA

[0148] Source: Natural sequence Synthetic Construct

[0149] Source organism: Alpaca

[0150] QAAQVQLVESGGAAVQPGGSLRLSCVMSGATLAHHAIAWFRQAPGKERERVSCISASGISTKYDGPVKGRFTVSRDNTKNMVYLQMNTLKPEDTANYYCAAGQRYGTTWHEFCTDNNMDYWGKGTLVTVSS

[0151] SEQ ID NO.7:

[0152] Name: Primer 1

[0153] DNA type: unassigned DNA

[0154] Source: Synthetic Construct

[0155] CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTCSEQ ID NO.8:

[0156] Name: Primer 2

[0157] DNA type: unassigned DNA

[0158] Source: Synthetic Construct

[0159] CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGG TGCG

[0160] SEQ ID NO.9:

[0161] Name: Primer 3

[0162] DNA type: unassigned DNA

[0163] Source: Synthetic Construct

[0164] CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCT TGGG

[0165] SEQ ID NO.10:

[0166] Name: Primer 4

[0167] DNA type: unassigned DNA

[0168] Source: Synthetic Construct

[0169] CATGCCATGACTCGCGGCCGGCCTGGCCCTTGCATACTTCATTCGTTC CTG

[0170] SEQ ID NO.11:

[0171] Name: Primer 5

[0172] DNA type: unassigned DNA

[0173] Source: Synthetic ConstructCCACGATTCTGGCCGGCCTGGCCTGAGGAGACRGTGACCTGGGTCC.

Claims

1. A double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus, comprising a PVC base plate, a sample absorption pad, a reaction membrane and absorbent paper, characterized in that: The upper surface of the PVC bottom plate is provided with colloid for sticking and fixing, the upper surface of the reaction membrane is provided with a detection line near the sample absorption pad, the upper surface of the reaction membrane is provided with a quality control line near the absorbent paper, and the quality control line is parallel to the detection line; the detection line is coated with colloidal gold-labeled giant salamander iris virus nanoantibodies, and the quality control line is coated with rabbit anti-camel rabbit polyclonal antibodies.

2. The double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 1, characterized in that: The colloidal gold-labeled giant salamander iridovirus nanobody is prepared by the following steps: adjusting the pH of the colloidal gold solution, adding the giant salamander iridovirus nanobody, mixing and standing, then adding skim milk powder, standing and centrifuging, discarding the supernatant, and dissolving the precipitate with a colloidal gold complex solution to obtain the colloidal gold-labeled giant salamander iridovirus nanobody.

3. The double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 2, characterized in that: The colloidal gold solution is mainly formed by mixing 1% chloroauric acid and 1% trisodium citrate by mass, wherein the volume ratio of chloroauric acid to trisodium citrate is 1:2, and the particle size of the colloidal gold in the colloidal gold solution is 40-50 nm.

4. The double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 2, characterized in that: The colloidal gold complex solution is a mixture of 1×0.01M PBS solution and 2.5% sucrose, 0.5% BSA and 0.1% Tween 20 in mass fraction.

5. The double nano-antibody colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 2, characterized in that: The pH is adjusted by adding K2CO3 to adjust the pH to 6.5-7.0, the concentration of the giant salamander iris virus nanoantibody is 20 μg / mL, the mass fraction of skimmed milk powder is 10%, the volume of skimmed milk powder added is one tenth of the volume of the colloidal gold solution, the standing time is 20-30 minutes, the centrifugal force of the centrifugation is 10000xg, the centrifugal temperature is 4°C, the centrifugal time is 20 minutes, and the volume ratio of the colloidal gold complex solution to the colloidal gold solution is 1:

5.

6. The method for preparing the double nanobody colloidal gold immunochromatographic test strip according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: S1, pre-cooling the nanoantibody of giant salamander iridovirus labeled with colloidal gold; S2, applying pre-cooled colloidal gold-labeled giant salamander iridovirus nanoantibodies on the nitrocellulose membrane to obtain a detection line; S3, applying rabbit anti-camel rabbit polyclonal antibody on nitrocellulose membrane to obtain quality control line; S4, soaking the nitrocellulose membrane pad provided with the detection line described in step S2 and the quality control line described in step S3 in a buffer solution, and then drying it in an oven overnight to obtain a reaction membrane; S5. Assemble the reaction membrane, sample absorption pad and water absorption pad described in step S4 on a PVC bottom plate to obtain a double nano-antibody colloidal gold immunochromatography test strip.

7. The method for preparing the double nanobody colloidal gold immunochromatographic test strip according to claim 6, characterized in that: In the step S1, the pre-cooling treatment is to place the colloidal gold-labeled giant salamander iris virus nanobody in a refrigerator at a temperature of 4°C for standing; in the step S2, the concentration of the colloidal gold-labeled giant salamander iris virus nanobody is 1-2.5 mg / ml, and the coating amount on the detection line is 0.5-1.5 μL / cm; in the step S3, the concentration of the rabbit anti-camel rabbit polyclonal antibody is 1-2.5 mg / mL, and the coating amount on the quality control line is 0.5-1.5 μL / cm; in the step S4, the buffer solution is mainly composed of 1L 0.01M PBS, 1g polyvinyl pyrrolidone, 0.1g sodium cholate, 0.1g sodium caseinate, and 1mL Tween, the soaking time is 30 minutes, and the drying temperature is 37°C.

8. The method for preparing the double nanobody colloidal gold immunochromatographic test strip according to claim 6, characterized in that: The assembly is as follows: the reaction membrane is placed in the middle of the upper surface of the PVC bottom plate, the sample absorption pad and the water absorbent pad are respectively arranged at the two ends of the upper surface of the PVC bottom plate, a partial area of ​​the sample absorption pad is overlapped on one end of the upper surface of the reaction membrane, and a partial area of ​​the sample absorption pad is close to the detection line on the reaction membrane, and a partial area of ​​the water absorbent paper is overlapped on the other end of the upper surface of the reaction membrane, and a partial area of ​​the water absorbent pad is close to the quality control line on the reaction membrane.

9. Use of the double nanobody colloidal gold immunochromatographic test strip according to any one of claims 1 to 5 or the double nanobody colloidal gold immunochromatographic test strip prepared by the preparation method according to any one of claims 6 to 8 in the detection of giant salamander iris virus in aquaculture.

10. A method for detecting giant salamander iris virus in aquaculture using the double nanobody colloidal gold immunochromatographic test strip according to any one of claims 1 to 5 or the double nanobody colloidal gold immunochromatographic test strip prepared by the preparation method according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: D1, adding the diluted sample to be tested into the colloidal gold-labeled giant salamander iris virus nanoantibody to obtain a mixed solution; D2, dropping the mixed solution described in step D1 onto the sample absorption pad, and the mixed solution flows through the reaction membrane to form a nanobody-antigen-nanobody colloidal gold marker sandwich; D3, the nanobody-antigen-nanobody colloidal gold marker described in step D2 is sandwiched on the test line, and the colloidal gold causes the test line to appear red; D4. Determine the presence of giant salamander iris virus in the sample based on the color of the detection line.

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