Colloidal gold immunochromatography test strip for rapidly detecting giant salamander iridovirus and preparation method of colloidal gold immunochromatography test strip

Abstract

The invention discloses a colloidal gold immunochromatography test strip for detecting giant salamander iridovirus and a preparation method of the colloidal gold immunochromatography test strip. The preparation method comprises the following steps: preparing a specific polyclonal antibody of the giant salamander iridescent, preparing a colloidal gold solution, mixing and incubating the polyclonal antibody and the colloidal gold solution to obtain an antibody-labeled colloidal gold solution, arranging colloid for sticking and fixing on the upper surface of the PVC bottom plate, and preparing the antibody-labeled colloidal gold solution on the upper surface of the PVC bottom plate. A detection line is arranged close to the sample pad on the upper surface of the reaction film, a quality control line is arranged close to the absorbent paper on 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 particle labeled giant salamander iridovirus polyclonal antibody, and the quality control line is coated with a camel anti-rabbit polyclonal antibody. The test strip disclosed by the invention can rapidly detect the giant salamander iridovirus, and has the advantages of ultrafast detection speed, high detection limit sensitivity, strong specificity, environmental friendliness, low cost and simple preparation process.

Description

Technical Field

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

[0002] The giant salamander iridovirus pathogen is taxonomically classified as the Ranavirus genus of the Iridoviridae family. It has the characteristics of a short incubation period (3-5 days), a high mortality rate (mortality rate of more than 90% within 72 hours after infection) and the risk of cross-species transmission. There is currently no commercial vaccine or specific treatment drug.

[0003] At present, the standard detection methods for giant salamander iridovirus include electron microscopy, tissue culture, molecular biology detection and immunoassay. However, electron microscopy (TEM) requires ultra-thin section preparation and professional image interpretation, and the average detection cycle is >48 hours; the cell culture method relies on giant salamander embryonic cell lines (such as GS cells), and virus isolation and culture takes ≥7 days and there are biosafety risks; although molecular biology detection (qPCR / nested PCR) is highly sensitive, it requires precision equipment such as thermal cyclers and is difficult to adapt to the needs of on-site detection in farms; in contrast, colloidal gold immunochromatography technology is a simple and rapid immunological detection method. By fixing specific antibodies on specific areas of nitrocellulose membranes, the sample moves along the membrane and specifically binds to the fixed antibodies by capillary action, and then the color change is displayed by immunocolloidal gold, thereby achieving accurate immunodiagnosis.

[0004] Therefore, developing a rapid, sensitive, accurate and specific real-time detection method for giant salamander iridovirus is of great significance for the safety monitoring of giant salamanders. Summary of the invention

[0005] In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide a colloidal gold test strip for detecting the iridovirus of giant salamander, which can efficiently detect the iridovirus in giant salamander at the aquaculture site.

[0006] The present invention uses rabbit polyclonal antibodies of giant salamander iridovirus as colloidal gold detection antibodies to assemble test strips for on-site detection of giant salamander iridovirus. The test strips are portable and fast, not affected by environmental conditions, and only take 5-10 minutes to detect. There is no cross reaction with non-giant salamander iridoviruses. The sensitivity is high, and the minimum detection concentration of Campylobacter jejuni is 9.94×10 6 The invention can realize the on-site rapid detection of giant salamander iridovirus in giant salamander samples, and is suitable for aquaculture enterprises, inspection and quarantine units and the like.

[0007] In order to achieve the above purpose,Figure 1 As shown, the present invention adopts the following technical solutions:

[0008] 1. A colloidal gold immunochromatographic test strip for detecting giant salamander iris virus, characterized in that:

[0009] The colloidal gold immunochromatographic test strip comprises a PVC base plate, a sample pad, a reaction membrane and absorbent paper.

[0010] The upper surface of the PVC bottom plate is provided with a glue for sticking and fixing, the upper surface of the reaction membrane is provided with a detection line near the sample pad, and 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;

[0011] The detection line is coated with polyclonal antibodies of giant salamander iris virus labeled with colloidal gold particles, and the quality control line is coated with camel anti-rabbit polyclonal antibodies.

[0012] The polyclonal antibody of giant salamander iridovirus is obtained by immunizing New Zealand rabbits with giant salamander iridovirus immunogen and then purifying and expressing the immunogen.

[0013] The specific process is:

[0014] 1) diluting the rabbit serum collected after centrifugation with PBS buffer at a volume ratio of 1:2 to obtain a mixed solution;

[0015] 2) Add Protein A Agarose solution to the above-mentioned mixture to make the final concentration of 10% by volume, shake at room temperature for 1 hour, add the mixture to the empty gravity chromatography column, and repeat the addition 3 times;

[0016] 3) After the liquid is loaded, add TBS buffer 10 times the volume of the gravity chromatography empty column to wash and balance the purification column to remove proteins that are not bound to Protein A Agarose and non-specifically bound to Protein A Agarose;

[0017] 4) After washing, add 100 μL of neutralizing solution per mL of elution solution, pre-add neutralizing solution (1M trimethylamine hydrochloride (Tris-HCl), pH 8.8) to the centrifuge tube, and then use 10 mL (50 mM glycine, pH 2.7) as the elution solution to elute the bound antibody;

[0018] 5) The collected antibodies were added to a dialysis bag and dialyzed in phosphate buffer for 24 hours before use.

[0019] The sample pad is prepared by the following method: soaking the glass cellulose membrane in a sample pad treatment solution, leaving it at room temperature for 1 hour, and drying it for later use;

[0020] The sample pad treatment solution is prepared by the following method: firstly dissolve 0.5-1g polyvinyl pyrrolidone, 0.05-0.1g sodium cholate, 0.05-0.1g sodium caseinate, and 0.5-1mL Tween in a small amount of PBS buffer, and then dilute to 1L with PBS buffer.

[0021] The colloidal gold particle-labeled polyclonal antibody of giant salamander iris virus is prepared by the following steps:

[0022] After preparing the colloidal gold solution, the pH is adjusted, and then the giant salamander iris virus polyclonal antibody is added, mixed and allowed to stand, and then skimmed milk powder is added and allowed to stand and then centrifuged, the supernatant is discarded, and the precipitate is dissolved with the colloidal gold complex solution to obtain the giant salamander iris virus rabbit polyclonal antibody labeled with colloidal gold particles.

[0023] The colloidal gold solution is mainly formed by mixing a chloroauric acid solution with a mass fraction of 1% and a trisodium citrate solution with a mass fraction of 1%. When mixing, the volume ratio of the chloroauric acid solution to the trisodium citrate solution is 1:2. The particle size of the colloidal gold in the colloidal gold solution is 40-50nm.

[0024] Preferably, the colloidal gold solution has a pH value of 6.5 to 7.0.

[0025] Preferably, the particle size of the colloidal gold is 40 to 50 nm.

[0026] Preferably, the precipitate is resuspended with a colloidal gold rehydration solution at 1 / 5 of the volume of colloidal gold.

[0027] The pH is adjusted by adding K 2 CO 3 The pH value is adjusted to 6.5-7.0, the concentration of the polyclonal antibody of giant salamander iris virus is 10 μg / mL, the mass concentration of skimmed milk powder is 10%, the volume of skimmed milk powder added is one tenth of the volume of the colloidal gold solution, and the standing time is 20-30 minutes; the centrifugal force of the centrifugation is 10000xg, the centrifugal temperature is 4°C, and the centrifugal time is 20 minutes;

[0028] The colloidal gold complex solution is a mixture of 1×0.01M PBS solution, 2.5% sucrose by mass, 0.5% BSA by mass and 0.1% Tween 20 by mass, and the volume ratio of the colloidal gold complex solution to the colloidal gold solution is 1:5.

[0029] The innovation of the present invention is to compound the rabbit polyclonal antibody of giant salamander iris virus with colloidal gold, and form a colloidal gold-polyclonal antibody complex through electrostatic adsorption of the antibody and colloidal gold, so as to save the step of gold spraying on the test strip. The complex can maintain the effective combination of colloidal gold particles and antibodies without using the traditional metal spraying process, thereby ensuring the stability and sensitivity of the test strip.

[0030] The concentration of the rabbit polyclonal antibody against giant salamander iris virus used in the T line / detection line of the coating reaction membrane is 1-2 mg / mL, and the concentration of the camel anti-rabbit polyclonal antibody used in the C line / quality control line of the coating reaction membrane is 1-2 mg / mL.

[0031] 2. A method for preparing a colloidal gold immunochromatographic test strip, the preparation method comprising the following steps:

[0032] S1, applying colloidal gold-labeled polyclonal antibody of giant salamander iris virus on a nitrocellulose membrane to obtain a detection line;

[0033] S2, spot-coated camel anti-rabbit polyclonal antibody on nitrocellulose membrane to obtain quality control line;

[0034] S3, soaking the glass cellulose membrane in the sample pad treatment solution, and drying it at room temperature to obtain a sample pad;

[0035] S4. Assemble the reaction membrane, sample pad and absorbent paper on a PVC bottom plate to obtain an antibody colloidal gold immunochromatography test strip.

[0036] Preferably, the PVC bottom plate is made of a non-absorbent PVC plate, one side of the plastic plate is coated with a self-adhesive sticker, and the bottom plate serves to fix and support other components of the test paper.

[0037] In the step S1, the concentration of the colloidal gold-labeled rabbit polyclonal antibody to giant salamander iris virus is 1 to 2.5 mg / ml, and the coating amount on the detection line is 0.5 to 1.5 μL / cm;

[0038] In step S2, the concentration of the camel anti-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. More preferably, the concentration of the giant salamander iris virus rabbit polyclonal antibody used is 2 mg / mL, and the coating amount is 1 μL / cm; the concentration of the camel anti-rabbit polyclonal antibody is 0.5-1 mg / mL, and the coating amount is 1 μL / cm.

[0039] In step S4, the assembly method of the test strip includes: fixing the nitrocellulose reaction membrane to the middle area of ​​the polyvinyl chloride (PVC) substrate, wherein the sample pad and the absorbent filter paper are respectively arranged at the input end and the output end of the PVC substrate; the sample pad covers the end where the reaction membrane detection line (T line) is located with an overlapping width of 2.0±0.5 mm, and the absorbent filter paper covers the end where the reaction membrane quality control line (C line) is located with an overlapping width of 2.0±0.5 mm, so as to form a continuous fluid path of the chromatographic channel.

[0040] 4. A method for detecting giant salamander iris virus in aquaculture using colloidal gold immunochromatographic test strips, the method comprising the following steps:

[0041] D1 immune complex formation: the pre-treated sample solution was mixed with colloidal gold-labeled rabbit-derived giant salamander iris virus polyclonal antibody, and incubated at 37°C for 5-8 minutes to form an antigen-antibody marker complex system;

[0042] D2 chromatography reaction: the mixed solution obtained in step 1 is spotted on the sample pad of the test strip, and migrates to the reaction area of ​​the nitrocellulose membrane through capillary chromatography. When the sample contains the target antigen, a double antibody sandwich complex of "nanoantibody-antigen-colloidal gold labeled polyclonal antibody" is formed in the detection line area;

[0043] D3 color determination: At the detection line (T line), the colloidal gold marker produces a red strip due to the specific binding of antigen and antibody, and its color intensity is positively correlated with the concentration of viral antigen; at the same time, a goat anti-rabbit secondary antibody is fixed at the quality control line (C line) to verify the effectiveness of the chromatography system;

[0044] D4 result interpretation: Qualitative / semi-quantitative detection is achieved through the double-line color development rule. When the T / C double line is colored, it is positive, and only the C line is colored, it is negative. If the double line does not color, it indicates that the reagent is invalid.

[0045] The invention provides a colloidal gold test strip for detecting giant salamander iris virus. The invention utilizes the high specificity reaction of the rabbit polyclonal antibody concentration of giant salamander iris virus and the giant salamander iris virus as well as the immunochromatographic technology of sandwich detection. A sample solution is added to the test strip from a sample pad, and the sample solution flows through the entire test strip through the capillary force. A substance to be detected in the sample is firstly combined with the colloidal gold-labeled rabbit polyclonal antibody concentration in a 96-well plate, and then the mixed solution flows through a reaction membrane, and the solution is sandwich-combined with the giant salamander iris virus rabbit polyclonal antibody on a detection line to form a rabbit polyclonal antibody-antigen-rabbit polyclonal antibody colloidal gold label. The content of giant salamander iris virus in the sample solution to be detected is judged according to whether the detection line is colored or the color depth.

[0046] The present invention is very suitable for on-site rapid detection of giant salamander iris virus and is of great significance for disease prevention and control in aquaculture.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) Simple and fast, the detection process only takes 5-10 minutes, suitable for rapid detection of large quantities of samples; it is also environmentally friendly, low-cost, and has a simple preparation process.

[0049] (2) High specificity and sensitivity. The minimum detection concentration of giant salamander iris virus is 9.94×10 6 PFU / mL, the visual detection limit is 2.1875×10 6 PFU / mL.

[0050] (3) It is easy to carry and simple to prepare. It does not require the preparation of a gold label pad, and the amount of sample required is small, making it suitable for on-site testing.

[0051] (4) It has no cross-reaction with other aquatic viruses and bacteria, has strong specificity and high detection accuracy, and can be used as an alternative or complementary method for the molecular detection of giant salamander iridovirus;

[0052] (5) It has high application value and good commercial prospects, and provides technical support for the rapid detection of giant salamander iris virus in aquaculture sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the scheme of the present invention;

[0054] Figure 2 The invention is the pH optimization for preparing the polyclonal antibody of giant salamander iris virus labeled with colloidal gold;

[0055] Figure 3 The content of skimmed milk powder prepared by the polyclonal antibody of giant salamander iris virus labeled with colloidal gold of the present invention is optimized;

[0056] Figure 4 The antibody content of the colloidal gold-labeled polyclonal antibody of giant salamander iris virus of the present invention is optimized;

[0057] Figure 5 The present invention is to optimize the probe volume of the colloidal gold-labeled polyclonal antibody of giant salamander iris virus;

[0058] Figure 6 It is the optimization of the T line concentration of the colloidal gold test strip of the present invention;

[0059] Figure 7 It is the optimization of the C line concentration of the colloidal gold test strip of the present invention;

[0060] Figure 8 This is a sensitivity test result diagram of the colloidal gold test strip for detecting giant salamander iris virus of the present invention;

[0061] Fig. 9 This is a cross-reaction analysis result diagram of the colloidal gold test strip for detecting giant salamander iris virus of the present invention;

[0062] Fig.10 This is a test result diagram of a real sample of the colloidal gold test strip for detecting giant salamander iris virus of the present invention;

[0063] Fig.11 The present invention is a schematic structural diagram of a gold-labeled test strip for detecting 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.

[0065] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0066] Embodiments of the present invention are as follows:

[0067] Example 1 Preparation of rabbit polyclonal antibodies against giant salamander iris virus

[0068] Immunization steps:

[0069] 1. Primary immunization: Mix 1×108TCID50 / mL of inactivated giant salamander iris virus with complete Freund's adjuvant (CFA) at a volume ratio of 1:1 to prepare an immune suspension, which is injected subcutaneously into the back of the rabbit. The dose is usually 1mL / rabbit.

[0070] 2. Booster immunization: Booster immunization is performed every 2 weeks, using incomplete Freund's adjuvant (IFA) mixed with antigen, for a total of 4-5 immunizations, with the antigen dose being the same as the initial immunization dose

[0071] 3. Antibody titer test: After each immunization, the titer of the polyclonal antibody against giant salamander iris virus in the serum was tested by enzyme-linked immunosorbent assay (ELISA) to confirm the immunization effect.

[0072] 4. Detection method: Coat the ELISA plate with giant salamander iris virus, add diluted rabbit serum, and use camel anti-rabbit ELISA secondary antibody for detection.

[0073] 5. Final blood collection: When the antibody titer reaches the ideal level (usually after 5 immunizations), blood is collected from the rabbit's heart and the serum is collected by centrifugation.

[0074] Purification steps:

[0075] 1. Dilute the rabbit serum collected after centrifugation with PBS at a volume ratio of 1:2;

[0076] 2. Add 10% volume fraction of Protein A Agarose to the above mixture, shake at room temperature for 1 hour, and add to the purification column;

[0077] 3. Add 10 times column volume of PBS to wash the purification column and elute the impurities;

[0078] 4. After washing, add 100uL of neutralizing solution per milliliter of eluent, pre-add 1M Tris-HCL (pH8.8) to the collection tube, and then use 50mM glycine (pH2.7) as the eluent to elute the bound antibody.

[0079] Example 2 Preparation and Optimization of Rabbit Polyclonal Antibodies Labeled with Colloidal Gold Particles

[0080] 1. Solution preparation

[0081] Colloidal gold complexing solution: 1M PBS, 2.5% sucrose, 0.5% BSA, 1% Tween-20.

[0082] 2. Preparation and optimization of colloidal gold markers

[0083] The present invention optimizes the preparation conditions of the colloidal gold-labeled rabbit polyclonal antibody to giant salamander iridescent virus, and the optimization result is determined according to the stability of the colloidal gold-labeled rabbit polyclonal antibody to giant salamander iridescent virus finally obtained and the sensitivity during the test strip detection. The specific optimization conditions are as follows:

[0084] (1) pH optimization:

[0085] 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 10 μL of 1 mg / ml rabbit polyclonal antibody 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 colloidal gold-labeled rabbit polyclonal antibody to giant salamander iridovirus in the test strip test. The sample was diluted to 7*10 using TBS buffer (pH 7.4). 7 PFU / mL. The sample pad of the test strip was vertically added to the sample solution of rabbit polyclonal antibody labeled with colloidal gold particles, TBS buffer was used as a negative control, the timing was started when the liquid flowed, the reaction lasted for 10 to 15 minutes, and the gray value of the CT line of the test strip was measured with a colloidal gold analyzer.

[0086] The pH optimization results are as follows Figure 2 As shown, when K 2 CO 3 When the amount of K is controlled at 6-12 μL, the C and T lines on the test paper will increase with the K 2CO 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.

[0087] (2) Optimization of skimmed milk powder content:

[0088] During the test strip test, when the blocking protein content is low, false positives are found, which are manifested by 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 colloidal gold-labeled polyclonal antibody. The appropriate skimmed milk powder content was determined based on the sensitivity and stability of the final colloidal gold-labeled giant salamander iris virus polyclonal antibody in the test strip test. Figure 3 As shown, considering the reagent dosage, color development effect and sensitivity, 150 μL 10% skimmed milk powder was selected as the ideal blocking protein content.

[0089] (3) Antibody content optimization:

[0090] In order to test whether the content of the rabbit polyclonal antibody to 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, 5, 10, 15, and 20 μL of 1 mg / ml rabbit polyclonal antibody solution to giant salamander iris virus were used to obtain different colloidal gold complexes, and the appropriate amount of rabbit polyclonal antibody to giant salamander iris virus added was determined based on the sensitivity and stability of the final colloidal gold-labeled rabbit polyclonal antibody to giant salamander iris virus in the test strip test. The optimization results of the rabbit polyclonal antibody addition amount are shown in FIG. Figure 4 As shown in the figure, when 10 μg is added, both the quality control line and the detection line of the test strip are dark, indicating that the most suitable addition amount of the rabbit polyclonal antibody in the colloidal gold labeled rabbit polyclonal antibody is 10 μg.

[0091] (4) Optimization of probe addition amount:

[0092] In order to test whether the amount of the colloidal gold particle-labeled rabbit polyclonal antibody of giant salamander iris virus prepared by the present invention has an effect on the test results of the test strip, 5 μL, 7.5 μL, 10 μL, and 12.5 μL of the giant salamander iris virus rabbit polyclonal antibody solution were added to the diluted sample solution. The results of the measurement using the marker prepared by the present invention at different volumes are as follows: Figure 5As 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 7.5uL. This shows that the more appropriate probe addition amount for this test strip is 7.5μL.

[0093] Example 3 Preparation of rabbit polyclonal antibody colloidal gold test strip for detecting giant salamander iris virus

[0094] 1. Preparation of reaction membrane

[0095] Coating of the test line: The prepared rabbit polyclonal antibody to giant salamander iris virus was coated on the reaction membrane to form a test line, and the rabbit polyclonal antibody to giant salamander iris virus was diluted to 2 mg / mL with TBS buffer, and coated on the test line (T line) on the nitrocellulose membrane using an XYZ three-dimensional film spray gold apparatus, with a coating amount of 1 μL / cm, and the coated reaction membrane was placed at 37°C to dry for 12 hours, and dried for use.

[0096] Coating of the quality control line: Coat the camel anti-rabbit polyclonal antibody on the reaction membrane to form a quality control line. Dilute the camel anti-rabbit polyclonal antibody 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. Dry the coated reaction membrane at 37°C for 12 hours and set it aside for use.

[0097] 2. Assembly of test strips

[0098] The reaction membrane, binding release pad, sample pad and absorbent paper prepared above are sequentially pasted on the PVC bottom plate; the assembled test strips are as follows: Fig.11 As shown: the beginning of the sample pad is aligned with the beginning of the PVC bottom plate, the end of the sample pad is connected to the beginning of the reaction membrane, the end of the reaction membrane is connected to the beginning of the absorbent paper, and the end of the absorbent paper 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 pad, and the quality control line is located on the side close to the beginning of the absorbent paper. 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.

[0099] In a specific implementation, the colloidal gold test strip is first provided with a bottom plate, a sample pad is installed on the left side of the bottom plate, a nitrocellulose membrane is installed in the middle of the bottom plate, and absorbent paper is installed on the right side of the bottom plate. Among them, a detection line coated with polyclonal antibodies of giant salamander iris virus and a quality control line coated with sheep anti-immune IgG are provided on the nitrocellulose membrane. The detection judgment standard of the test strip is: the detection line and the quality inspection line are colored at the same time for positive, only the quality inspection line is colored for negative, and the quality inspection line and the detection line are not colored or only the detection line is colored, indicating that the test strip is invalid.

[0100] The principle of the test strip is as follows: when the test result is negative, the test line does not change, and the color of the quality control line becomes redder and darker; when the test result is positive, the colors of both the test line and the quality control line become redder and darker. When there is no change in the test line and the quality control line, or only the test line becomes redder and darker while the quality control line does not change, it is an invalid result, indicating that retesting is required.

[0101] Example 4 Limit test of colloidal gold test strip for detecting giant salamander iris virus

[0102] The present invention optimizes the detection conditions of the giant salamander iris virus colloidal gold test strip, and the optimization results are shown according to the final detection limit. The specific optimization conditions are as follows:

[0103] 1. Optimization of test line concentration:

[0104] In order to test the effect of the detection line concentration of the rabbit polyclonal antibody colloidal gold test strip for detecting giant salamander iris virus prepared by the present invention on the test result 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 rabbit polyclonal antibodies were used for testing.

[0105] The sample pad of the test strip is vertically added to the sample solution of rabbit polyclonal 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. Figure 6 As shown in the figure, when the test strip has a detection line concentration of 2 mg / mL for the rabbit polyclonal antibody of giant salamander iris virus, the quality control line and the test line in the test result are darker, indicating that the more appropriate quality detection line concentration of this test strip is 2 mg / mL.

[0106] 2. Optimization of quality control line concentration:

[0107] In order to test the influence of the quality control line concentration of the colloidal gold test strip of rabbit polyclonal antibody for detecting giant salamander iridovirus prepared by the present invention on the test strip detection result, test strips with different concentrations of quality control lines were used for determination. Camel anti-rabbit polyclonal antibody of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL and 1 mg / mL were used for testing.

[0108] The sample pad of the test strip is vertically added to the sample solution of rabbit polyclonal 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 7As shown in the figure, when the test strip is diluted at a 1:1 ratio and the quality control line concentration is 0.5 mg / mL, the quality control line and the test line in the test result are darker in color. This means that the more appropriate quality control line concentration for this test strip is 0.5 mg / mL.

[0109] 3. Detection limit test

[0110] In order to test the detection limit and sensitivity of the double rabbit polyclonal 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 1.09375×10 6 , 2.1875×10 6 , 4.375×10 6 , 8.75×10 6 , 1.75×10 7 , 3.5×10 7 , 7×10 7 , 1.4×10 8 , 2.8×10 8 , and 5.6×10 8 PFU / mL, forming ten concentration gradients. Take the test strip prepared in Example 2 for detection, vertically drip the sample solution onto the sample 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. Figure 8 The results showed that the vLOD of this method for detecting giant salamander iridovirus was 2.1875×10 6 PFU / mL, LOD is 9.94×10 6 PFU / mL, meeting the national standard detection requirements. Through nonlinear fitting, the IC50 of GISV in this detection method is 1.78×10 8 PFU / mL. This method is 8.75×10 6 PFU / mL-1.4×10 8 The PFU / mL range showed good linear relationship.

[0111] 4. Specificity test

[0112] In order to detect the specificity of the 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 TBS buffer was used as a negative control group to verify potential cross-reactions.

[0113] The test strips prepared by the present invention were used to test different virus-positive serums. Fig. 9 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.

[0114] 5. Matrix Effect

[0115] Matrix effects were evaluated following a simple dilution scheme. Briefly, commercially purchased salamanders were processed, tissue samples were ground, centrifuged, and diluted with TBS to 1:10, 1:100, and 1:1000 dilutions. GSIV solutions were then spiked with a certain concentration. The spiked tissue samples were analyzed using the prepared test strips.

[0116] In this study, tissue samples susceptible to GSIV were collected, homogenized, and centrifuged to ensure consistent processing. The extracted tissue samples were diluted in TBS at ratios of 1:10, 1:100, and 1:1000.

[0117]

[0118] As shown in Table 1, the complex tissue matrix of giant salamander did not seem to have much effect on the test results of the test strip, and the average recovery rate of the test strip remained between 82% and 115% when diluted 10 to 1000 times. These results indicate that the test strip has good reliability in practical applications.

[0119] Example 5 Actual sample detection

[0120] 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 (1×109 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.

[0121] Test results such as Fig.10 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.

[0122] The above specific implementation modes are used to explain the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

[0123] The above description is only a preferred embodiment of the present invention, so all equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims

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

2. The colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 1, characterized in that: The polyclonal antibody of giant salamander iridovirus is obtained by immunizing New Zealand rabbits with giant salamander iridovirus immunogen and then purifying and expressing the immunogen.

3. The colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 1, characterized in that: The sample pad is prepared by the following method: soaking the glass cellulose membrane in a sample pad treatment solution, leaving it at room temperature for 1 hour, and drying it for use; the sample pad treatment solution is prepared by the following method: first dissolving 0.5-1g polyvinyl pyrrolidone, 0.05-0.1g sodium cholate, 0.05-0.1g sodium caseinate, and 0.5-1mL Tween in a small amount of PBS buffer, and then diluting it to 1L with PBS buffer.

4. The colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 1, characterized in that: The colloidal gold particle-labeled giant salamander iridescent virus polyclonal antibody is prepared by the following steps: preparing a colloidal gold solution, adjusting the pH, then adding the giant salamander iridescent virus polyclonal antibody, 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 particle-labeled giant salamander iridescent virus rabbit polyclonal antibody.

5. The colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 4, characterized in that: The colloidal gold solution is mainly formed by mixing a chloroauric acid solution with a mass fraction of 1% and a trisodium citrate solution with a mass fraction of 1%. When mixing, the volume ratio of the chloroauric acid solution to the trisodium citrate solution is 1:

2. The particle size of the colloidal gold in the colloidal gold solution is 40-50nm.

6. The colloidal gold immunochromatographic test strip for detecting giant salamander iris virus according to claim 4, characterized in that: The pH is adjusted by adding K2CO3 to adjust the pH to 6.5-7.0, the concentration of the polyclonal antibody of giant salamander iris virus is 10 μg / mL, the mass concentration of skimmed milk powder is 10%, the volume of skimmed milk powder added is one tenth of the volume of the colloidal gold solution, and the standing time is 20-30 minutes; the centrifugal force of the centrifugation is 10000xg, the centrifugal temperature is 4°C, and the centrifugal time is 20 minutes; The colloidal gold complex solution is a mixture of 1×0.01M PBS solution, 2.5% sucrose by mass, 0.5% BSA by mass and 0.1% Tween 20 by mass, and the volume ratio of the colloidal gold complex solution to the colloidal gold solution is 1:

5.

7. The colloidal gold immunochromatographic test strip for detecting antibodies to the giant salamander iris virus according to claim 1, characterized in that: The concentration of the rabbit polyclonal antibody against giant salamander iris virus used in the detection line of the coating reaction membrane is 1-2 mg / mL, and the concentration of the camel anti-rabbit polyclonal antibody used in the quality control line of the coating reaction membrane is 1-2 mg / mL.

8. The method for preparing the colloidal gold immunochromatographic test strip according to claim 1, characterized in that: The preparation method comprises the following steps: S1, applying colloidal gold-labeled polyclonal antibody of giant salamander iris virus on a nitrocellulose membrane to obtain a detection line; S2, spot-coated camel anti-rabbit polyclonal antibody on nitrocellulose membrane to obtain quality control line; S3, soaking the glass cellulose membrane in the sample pad treatment solution, and drying it at room temperature to obtain a sample pad; S4. Assemble the reaction membrane, sample pad and absorbent paper on a PVC bottom plate to obtain an antibody colloidal gold immunochromatography test strip.

9. The preparation method according to claim 8, characterized in that: In the step S1, the concentration of the colloidal gold-labeled rabbit polyclonal antibody against giant salamander iris virus is 1 to 2.5 mg / ml, and the coating amount on the test line is 0.5 to 1.5 μL / cm; in the step S2, the concentration of the camel anti-rabbit polyclonal antibody is 1 to 2.5 mg / mL, and the coating amount on the quality control line is 0.5 to 1.5 μL / cm; In the step S4, the test strip assembly method comprises: fixing the nitrocellulose reaction membrane to the middle area of ​​a polyvinyl chloride (PVC) substrate, wherein the sample pad and the water-absorbing filter paper are respectively arranged at the input end and the output end of the PVC substrate; The sample pad covers the end of the reaction membrane detection line (T line) with an overlapping width of 2.0±0.5 mm, and the absorbent filter paper covers the end of the reaction membrane quality control line (C line) with an overlapping width of 2.0±0.5 mm, forming a continuous fluid path of the chromatographic channel.

10. Use of the colloidal gold immunochromatographic test strip according to any one of claims 1 to 6 or the colloidal gold immunochromatographic test strip prepared by the preparation method according to any one of claims 7 to 9, characterized in that: Application in the detection of giant salamander iridovirus in aquaculture.

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