A Clostridium perfringens epsilon antibody detection test strip, kit and its application

Abstract

The present invention belongs to the technical field of biomedical detection, and specifically relates to a detection test strip for Clostridium perfringens epsilon antibody and its application. An epsilon toxin protein, the amino acid sequence of which is shown in SEQ ID NO: 4. The present invention also discloses a detection test strip for Clostridium perfringens epsilon antibody. The antigen is labeled at the test line of the detection test strip, and the antigen is the epsilon toxin protein; the secondary antibody is labeled at the quality control line, and the secondary antibody is a secondary antibody of rabbit anti-SPA. The gold-labeled pad is prepared by mixing a colloidal gold solution and SPA protein to obtain a gold-labeled SPA solution, and spraying the gold-labeled SPA solution on the gold-labeled pad. The colloidal gold test strip provided by the present invention can detect Clostridium perfringens epsilon antibody within 10 minutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to a Clostridium perfringens epsilon antibody detection test strip, a kit and their applications. Background Art

[0002] Clostridium perfringens (C. perfringens), also known as Clostridium welchii or Bacillus perfringens, is a Gram-positive anaerobic spore-forming bacillus widely present in nature and the gastrointestinal tract of animals. The pathogenic factors of C. perfringens are mainly alpha, beta, epsilon, iota, CPE, and NetB exotoxins. According to the six exotoxins secreted by it, C. perfringens is divided into types A, B, C, D, E, F, and G. Among the four main exotoxins, the epsilon toxin produced by type B and D C. perfringens is a severe pathogenic factor. C. perfringens is a conditional pathogen. When animals ingest drinking water or feed contaminated with C. perfringens, the normal peristalsis and normal flora of the intestine will inhibit its growth and reproduction. However, when the intestinal flora is dysregulated and the gastrointestinal function is disordered, C. perfringens will multiply in large numbers and produce toxins. Among them, epsilon can specifically bind to its target cell membrane receptor, form pores, and damage the integrity of the cell membrane, resulting in cell edema, necrosis, and apoptosis. Epsilon mainly acts on organs such as the nervous system, heart, and kidneys, causing severe consequences such as acute central nervous system damage and renal failure, and causing necrotic enteritis or enterotoxemia in animals such as lambs, sheep, goats, cattle, and gray squirrels.

[0003] In addition, the toxicity of epsilon is second only to botulinum toxin and tetanus toxin, and it was once listed as a category B bioterrorism agent by the US Centers for Disease Control and Prevention, with important biosafety risks. The disease has an acute onset and often causes death within a few minutes or hours, with a high mortality rate and poor treatment effect, posing a great hazard and causing great losses to the livestock economy around the world. Reducing the harm caused by C. perfringens mainly relies on vaccination prevention, but currently, there is no rapid detection method for the immune effect of C. perfringens vaccine in clinical practice. Therefore, there is an urgent need to develop a test strip that can rapidly detect the immune effect of C. perfringens vaccine for evaluating the immune effect of C. perfringens vaccine. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the lack of a rapid detection method for the immune effect of C. perfringens vaccine in the prior art, and to provide a Clostridium perfringens epsilon antibody detection test strip and its applications.

[0005] To solve the above technical problems, the present invention is carried out by adopting the following technical solutions.

[0006] In the first aspect of the present invention, an ε-toxin protein is provided, and the amino acid sequence of the ε-toxin protein is as shown in SEQ ID NO: 4.

[0007] In the second aspect of the present invention, a test strip for detecting Clostridium perfringens ε antibody is provided, which includes a sample pad, a gold-labeled pad, an NC membrane, a blotting paper and a PV backing board; the sample pad, the gold-labeled pad, the NC membrane and the blotting paper are successively attached to the PV backing board and connected in an overlapping manner, and on the nitrocellulose membrane, a detection line and a quality control line parallel to each other are successively arranged in the direction from the gold-labeled pad to the blotting pad;

[0008] The antigen is marked at the detection line, and the antigen is the ε-toxin protein;

[0009] The secondary antibody is marked at the quality control line, and the secondary antibody is a rabbit anti-SPA secondary antibody;

[0010] The gold-labeled pad is prepared by mixing a colloidal gold solution and an SPA protein to obtain a fusion solution and spraying the fusion solution onto the gold-labeled pad.

[0011] In some technical solutions of the present invention, the sample pad and the gold-labeled pad are statically treated in a gold-labeled pad treatment solution and a sample pad treatment solution.

[0012] In some technical solutions of the present invention, the concentration of the ε-toxin is 0.8 mg / mL - 1.25 mg / mL.

[0013] In some technical solutions of the present invention, the concentration of the rabbit anti-SPA secondary antibody is 0.5 mg / mL - 1.5 mg / mL.

[0014] In some technical solutions of the present invention, the final concentration of the gold-labeled SPA solution is 9.5 μL / mL - 10.5 μL / mL.

[0015] In the third aspect of the present invention, the application of the test strip in preparing a kit for detecting Clostridium perfringens is provided.

[0016] In the fourth aspect of the present invention, a kit for detecting Clostridium perfringens is provided, and the kit includes the test strip and a sample buffer solution.

[0017] In some technical solutions of the present invention, the sample buffer solution is a CB buffer solution or a PB buffer solution.

[0018] In some technical solutions of the present invention, the concentration of the sample buffer solution is 35 mmol / L - 50 mmol / L.

[0019] In the fifth aspect of the present invention, a method for detecting Clostridium perfringens ε antibody is provided, including: using the test strip or the kit.

[0020] In some technical solutions of the present invention, the sample is mixed with a sample buffer solution at a volume ratio of 80:100, and then used for the detection of Clostridium perfringens epsilon antibody.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides an epsilon toxin protein and uses the epsilon toxin protein for the detection of Clostridium perfringens epsilon antibody. In the present invention, the epsilon toxin recombinant protein is coated as the test line (T line), the gold-labeled SPA protein, and the rabbit anti-SPA secondary antibody is coated as the quality control line (C line) to construct a test strip for detecting Clostridium perfringens epsilon antibody. By optimizing each step of the conditions, a colloidal gold immunochromatographic test strip for detecting epsilon antibody is established. The colloidal gold test strip established by the above method can detect Clostridium perfringens epsilon antibody within 10 minutes. The detection test strip for Clostridium perfringens epsilon antibody provided in the present invention has a minimum detection limit of 3 ng / mL epsilon antibody.

[0022] Biological deposit information

[0023] Clostridium perfringens 21-D-5 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 21, 2024, with the deposit number CGMCC No. 30092. The address of the deposit unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. Description of the drawings

[0024] Figure 1 It is a diagram of the identification result of the recombinant plasmid; among them, M represents DNA marker D5000; band 1 represents the recombinant plasmid.

[0025] Figure 2 It is a diagram of the double digestion identification result of the recombinant plasmid; among them, M represents DNA marker D5000, and band 1 represents the recombinant plasmid of pET-28a-epsilon digested by BamHⅠ and XhoⅠ.

[0026] Figure 3 It is a diagram of the PCR identification result of the recombinant plasmid expression bacteria; among them, M represents DNA marker D5000; "+" represents the positive control, "-" represents the negative control; bands 1-8 represent the recombinant expression bacteria BL21(DE3) / pET-28a-epsilon.

[0027] Figure 4It is a figure for the analysis of the expression form of recombinant ε protein and the induction results under different expression conditions. Among them, M represents the molecular weight of the standard protein, and bands 1-4 represent the lysed supernatants after inducing BL21(DE3) / pET-28a-ε at 16°C / 140r / 24h, 25°C / 180r / 18h, 30°C / 200r / 10h, and 37°C / 140r / 24h; bands 5-6 represent the lysed supernatant after inducing BL21(DE3) / pET-28a-ε at 16°C / 140r / 24h and the lysed precipitate after induction.

[0028] Figure 5 It is a figure for the induction expression results of recombinant proteins with different IPTG concentrations. Among them, M is the molecular weight of the standard protein; bands 1-5 represent the supernatants after lysis induced by 0.6 mmol / mL, 0.5 mmol / mL, 0.4 mmol / mL, 0.3 mmol / mL, 0.2 mmol / mL, and 0.1 mmol / mL IPTG.

[0029] Figure 6 It is a figure for the protein purification results. Among them, M is the molecular weight of the standard protein; bands 1-6 represent the washing solutions for 1-6 times; bands 7-11 represent the elution solutions for 1-5 times.

[0030] Figure 7 It is a figure for the Western blot results of recombinant ε protein. Among them, M is the molecular weight of the standard protein, and bands 1-2 represent the ε protein.

[0031] Figure 8 It is a protein standard curve graph.

[0032] Figure 9 It is the ELISA quantitative standard curve of Clostridium perfringens type A ε antibody.

[0033] Figure 10 It is a colloidal gold characterization analysis figure. Among them, Figure 10 (a) in it represents the prepared colloidal gold solution; Figure 10 (b) in it represents the absorbance value of colloidal gold; Figure 10 (c) in it represents the figure of colloidal gold particles observed by transmission electron microscopy; Figure 10 (e) in it represents the particle size of colloidal gold; Figure 10 (f) of it represents the particle size of GNPs-SPA.

[0034] Figure 11 It is a figure for the optimization results of the addition amount of gold-labeled SPA K2CO3. Among them, Figure 11 1-10 in it respectively represent the addition of 1-10 μL of K2CO3 solution.

[0035] Figure 12 It is the optimization result of the minimum stable amount of gold-labeled SPA. Among them, Figure 121-5 represents the concentration of SPA from 1 μL / mL to 10 μL / mL, C represents the control line, and T represents the test line.

[0036] Figure 13 It is a schematic structural diagram of the colloidal gold test strip provided by the present invention.

[0037] Figure 14 It is a graph of the optimization result of the sample buffer; among them, Figure 14 C in it represents the control line, and T represents the test line.

[0038] Figure 15 It is a graph of the optimization result of the buffer concentration; among them, Figure 15 C in it represents the control line, and T represents the test line.

[0039] Figure 16 It is a graph of the optimization result of the test line concentration; among them, Figure 16 C in it represents the control line, and T represents the test line.

[0040] Figure 17 It is the optimization result of the concentration of gold-labeled SPA; among them, Figure 17 C in it represents the control line, and T represents the test line.

[0041] Figure 18 It is a graph of the test result of the sensitivity of the test strip; among them, Figure 18 C in it represents the control line, and T represents the test line.

[0042] Figure 19 It is a graph of the test result of the specificity of the test strip.

[0043] Figure 20 It is a graph of the test result of the stability of the test strip.

[0044] Figure 21 It is a graph of the test results of the test strip for 48 Yulin sheep sera; among them, 1-48 correspond to the test results of 48 Yulin sheep sera.

[0045] Figure 22 It is a graph of the test results of the test strip for 36 Huanxian sheep sera; among them, 1-36 correspond to the test results of 36 Huanxian sheep sera.

[0046] Figure 23 It is a standard curve graph for the quantitative detection of ε-toxin antibody by ELISA kit.

[0047] Figure 24 It is a graph of the numerical values of commercial ELISA kits for detecting clinical samples.

[0048] Figure 25 It is a correlation curve graph of the clinical application results of ELISA kits and colloidal gold immunochromatographic test strips. Detailed implementation method

[0049] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0050] Example 1: Expression of Clostridium perfringens ε toxin protein

[0051] 1. Primer design and synthesis

[0052] According to the ε toxin gene sequence included in GenBank, the ε toxin primer sequence was designed using Primer Premier 5.0, the signal peptide sequence was removed, protective bases were added, BamHI and Xho I were used as restriction sites, the primer sequences were shown in Table 1, and the target gene was amplified using Clostridium perfringens 21-D-5 as a template. Its size was 909 bp and the sequence was shown in SEQ ID NO: 1.

[0053] Table 1 Designed primer sequences

[0054]

[0055] Note: The underlined part in the table indicates restriction site.

[0056] 2. Amplification of ε toxin protein gene and construction of prokaryotic expression vector

[0057] (1) ε toxin protein gene amplification

[0058] In this test, Clostridium perfringens 21-D-5 was used as a template, and the DNA of Clostridium perfringens 21-D-5 was extracted using a genomic DNA small-scale extraction kit (Biyuntian Biotechnology, item number: D0063). PCR amplification and agarose gel electrophoresis were performed, and the size of the target band was verified and then gel recovery was performed. The recovered target fragment and pET-28 (a) empty plasmid were double-digested, and then agarose gel electrophoresis was performed for gel recovery, and then ligated with T4-DNA ligase, and transformed into DH5ɑ Escherichia coli competent state after ligation. After ligation, it was transformed into DH5ɑ Escherichia coli competent state, placed in a 37℃ water bath shaker, and shaken at 200r / min for 45 to 60min; after amplification, 50μL was drawn and coated on a plate containing kanamycin (50μg / mL) for positive clone screening, and incubated in a 37℃ incubator for 12 to 20h, and a single colony was picked for amplification. After amplification, PCR identification was performed and double enzyme digestion was performed to identify the recombinant plasmid. The identification results of the recombinant plasmid and the double enzyme digestion results of the recombinant plasmid are as follows Figure 1 and Figure 2 shown.

[0059] (2) Construction of recombinant expression bacteria

[0060] The recombinant positive plasmid pET-28(a)-etx was transformed into competent Escherichia coli BL21(DE3), and then spread onto nutrient agar plates containing kanamycin (50 μg / mL). Positive colonies were picked and cultured overnight. The positive strain BL21(DE3) / pET-28a-ε was transferred to LB liquid medium containing kanamycin (50 μg / mL) at a ratio of 1:100 and cultured at 37 °C until the logarithmic phase (OD was 0.6 - 0.8), and the bacterial solution showed a cloudy state. The PCR identification results of the recombinant expression bacteria BL21(DE3) / pET-28a-ε are as Figure 3 shown.

[0061] (3) Determination of the optimal induction temperature and time: Add IPTG solution with a final concentration of 0.6 mmol / L, and perform ultrasonic lysis and disruption at 16 °C / 140 r / 24 h, 25 °C / 180 r / 18 h, 30 °C / 200 r / 10 h, 37 °C / 220 r / 8 h (the resuspended bacterial solution was ultrasonically disrupted at a power of 1200, ultrasonically disrupted for 3 min, with a 3 s pause every 3 s of ultrasonic disruption, and the ultrasonic disruption was completed in an ice-water bath); after ultrasonic treatment, centrifuge at 10000 r / min at 4 °C for 5 min to collect the precipitate and supernatant. Take the supernatant and precipitate of the ultrasonically lysed bacteria for SDS-PAGE electrophoresis respectively to analyze the protein expression form.

[0062] Set six final concentrations of IPTG to 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.6 mmol / L, select the optimal temperature from the previous step, with an induction time of 8 h, collect the bacteria for ultrasonic lysis, take the supernatant and precipitate of the ultrasonically lysed bacteria for SDS-PAGE respectively, select the positive bacteria with supernatant expression, and perform induction expression and SDS-PAGE electrophoresis according to the optimal temperature, optimal induction expression time and IPTG concentration explored above.

[0063] The results are as Figure 4 and Figure 5 shown, showing that the expression mode is soluble expression, the protein size is 36 KD, and the optimal expression conditions are a final concentration of IPTG of 0.1 mmol / mL, 16 °C / 140 r / 24 h.

[0064] (4) Induce and culture 500 mL of the recombinant bacteria under the above optimal expression conditions, centrifuge to collect the bacterial cells, resuspend them in 15 mL of PBS, ultrasonically disrupt them on ice bath, centrifuge at high speed at 4°C (12,000 r / 20 min) to collect the supernatant, purify the epsilon toxin protein according to the instructions of the His-tag protein purification kit, determine the protein concentration using the BCA method, aliquot and store in 1.5 mL centrifuge tubes with the addition of glycerol at a final concentration of 10%, and store at -80°C. At the same time, use 6×His mouse monoclonal antibody as the primary antibody and goat anti-mouse as the secondary antibody for Western Blotting verification.

[0065] The standard curve of the epsilon toxin protein is as Figure 8 shown, the amino acid sequence is as SEQ ID NO:4, and the determination result of the epsilon toxin protein concentration is as Figure 6 shown, the protein concentration is 4.67 mg / mL. As Figure 7 shown, the Western blot result shows a good immune response.

[0066] Example 2: Preparation of ovine Clostridium perfringens epsilon positive serum

[0067] Immunize 2 healthy female goats weighing 40 kg with the prepared detoxified epsilon toxin protein. At the first immunization injection, take 5 mL of the detoxified epsilon toxin at 1 mg / mL and fully emulsify it with an equal volume of Freund's complete adjuvant, and inject it at multiple points in the neck and dorsal side. After an interval of half a month, take the same dose of the detoxified epsilon toxin and fully emulsify it with an equal volume of incomplete Freund's adjuvant, and perform the second immunization in the same injection method. Immunize once every two weeks. 10 days after the 4th immunization, collect blood from the jugular vein, detect the antiserum concentration, and perform a large amount of blood collection 15 days after immunization. The control goats are injected with an equal volume of normal saline. Before each immunization, 5 mL of blood needs to be taken from the jugular vein, the serum is separated, and stored at -20°C for convenient detection and comparison of the changes in antibody levels later.

[0068] Use the ovine Clostridium perfringens epsilon antibody ELISA quantitative detection kit to draw a quantitative standard curve according to the standard positive serum provided by the kit, as Figure 9 shown, y = 0.012x + 0.014; R 2 = 0.99. Measure the changes in the epsilon antibody concentrations of the serum before the first immunization and the first, second, third, and fourth sera after immunization, as shown in Table 2. The results show that the concentration of the maternal antibodies carried by the goats is relatively low. After the first immunization and the second immunization, the changes in the epsilon antibody levels of the immunized goats are not obvious. After the third immunization, the antibody levels increase significantly, and the antibody level at the fourth time is 165.6 ng / mL.

[0069] Table 2 Detection of ovine epsilon antibody levels

[0070] Number of immunizations <![CDATA[OD 562nm > Concentration of ε antibody (ng / mL) Before the first immunization 0.0759 26.29 First immunization 0.0846 29.97 Second immunization 0.1080 39.86 Third immunization 0.2180 86.35 Fourth immunization 0.4055 165.60

[0071] Example 3: Preparation of Colloidal Gold Solution and Preparation of Colloidal Gold-Labeled SPA Solution

[0072] All colloidal gold containers used should be pickled with acid. The siliconized glass containers required for firing colloidal gold solution include 250 mL conical flasks, 50 mL measuring cylinders, 200 mL brown wide-mouth bottles, 50 mL glass bottles, etc. Prepare 800 mL of aqua regia (concentrated nitric acid: concentrated hydrochloric acid = 1:3). Pour the aqua regia into the above glass containers, basically level with the bottle mouth, do not fully tighten the bottle cap, and place it in the fume hood away from light for 24 h. After pouring out the aqua regia, wash it 15 times with ultrapure water, seal it, and dry it in the oven.

[0073] (1) When firing the colloidal gold solution: First, add 100 mL of ultrapure water, boil for 5 min and then pour it out. Then add 99 mL of ultrapure water and 1 mL of 1% HAuCl4, boil and stir. After boiling, add 3 mL of trisodium citrate, observe the color change (whether it is light yellow, light black, black, purplish red, red). After the color stabilizes, continue to boil for 3 min, cool to room temperature, and make up to 100 mL. Pour it into a 200 mL brown wide-mouth bottle, place it in a 4°C refrigerator, and take 200 μL of the fired colloidal gold solution into a 96-well culture plate. Use a spectrophotometer to measure the OD of the colloidal gold 450 -OD 600 - the maximum absorption peak value, and retain the change curve of the OD value. The OD value corresponding to the peak is the measured OD value of the colloidal gold. The size of the OD value represents the concentration of the colloidal gold, providing a reference basis for the best labeling conditions of SPA. When determining the gold-labeled SPA, the closer the change in the colloidal gold concentration is to the concentration before gold labeling, the more stable the colloidal gold after gold labeling. As shown in (a)-(f) in Figure 10 , the lower the concentration compared to before, the less stable the colloidal gold and the larger the number of dead gold.

[0074] (2) Determination of the labeling conditions for colloidal gold-labeled SPA:

[0075] Determination of the best pH value condition of SPA. Take 13 RNase-free 1.5 mL EP tubes, add 1 mL of colloidal gold solution to each tube, and add 0 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 12 μL of 0.2 mol K2CO3 solution respectively. Add 20 μL of protein and invert to mix evenly, then add 80 μL of 10% NaCl and observe the color change. Observe the color change of the colloidal gold solution. If the color change is not obvious, further centrifuge at 12000 r / min for 10 min and mix quickly by ultrasound, and observe the color change. The results are as shown in Figure 11As shown, when the addition amount of K2CO3 is 1 - 6 μL, the solution color is purple and the colloidal gold shows slight flocculation; as the addition amount of K2CO3 increases, the solution color gradually turns red. When 10 μL of K2CO3 is added, the solution color is wine red, which is the same as the color of the successfully prepared colloidal gold. Therefore, the pH after adding 10 μL of K2CO3 to 1 mL of colloidal gold solution is the optimal labeling pH for SPA colloidal gold labeling.

[0076] (3) Optimization of the optimal labeling amount of SPA protein: Select the pH value at the optimal labeling in the above steps. For 13 RNase - free 1.5 mL EP tubes, add 1 μL, 2 μL, 3 μL, 4 μL, 5 μL of 1 mg / mL SPA protein to each tube, add 80 μL of 10% NaCl solution, and let it stand at room temperature for 1 h to observe the color change. However, after comparison, the color change is not obvious, and the test strip is directly used to detect and compare the changes between the two. The results are as Figure 12 shown. The optimization results of the minimum stable labeling amount of gold - labeled SPA protein show that when the addition amount of SPA protein is 1 μg / mL, the color of the T line is the lightest; when it is 2 - 3 μg / mL, the colors of the C and T lines are stable; when it is 4 - 5 μg / mL, the T line is redder, but the color of the C line is lighter. Therefore, the optimal labeling amount of SPA protein is 2 μg / mL.

[0077] Colloidal gold labeling is carried out under the conditions of the optimal labeling amount and the optimal pH of SPA protein, and the size and gold - labeling situation of the colloidal gold are observed under a transmission electron microscope. As Figure 10 shown in (a) - (f) below, the transmission electron microscope observation results show that the colloidal gold is well fired, uniformly dispersed in size under the electron microscope. After colloidal gold is labeled with SPA, a halo appears around the colloidal gold particles.

[0078] Example 4: Assembly of colloidal gold test strip and establishment of diagnostic method

[0079] 1. Assembly of colloidal gold test strip

[0080] Immerse the gold - labeled pad and the sample pad in the gold - labeled pad and sample pad treatment solutions respectively, and let them stand overnight at 4°C. For the colloidal gold - labeled SPA solution prepared in Example 3, use a three - dimensional colloidal gold scribing instrument to spray and spread the colloidal gold on the treated gold - labeled pad, and dry it at room temperature; and use the reverse suction scribing method to draw the expressed ε - toxin onto the nitrocellulose membrane (NC membrane) as the test line (T line), and draw the rabbit anti - SPA secondary antibody (1 mg / mL) onto the NC membrane as the quality control line (C line). Subsequently, carry out the assembly and cutting of the test strip. As Figure 13 shown, on the PV backing board, paste the sample pad, the gold - labeled pad, the NC membrane, and the absorbent paper in sequence from left to right, with an overlap of 1.2 mm - 2.5 mm, and cut it into a test strip with a width of 0.4 mm, and assemble it into a plastic cartridge.

[0081] Gold label pad treatment solution: Na2B4O7, 0.38135 g; BSA, 0.5 g; Tween-20, 0.15 mL; NaN3, 0.015 g; made up to 50 mL with ultrapure water.

[0082] Sample pad treatment solution: BSA, 0.5 g; Tween-20, 0.15 mL; sucrose, 2.5 g; NaN3, 0.015 g; made up to 50 mL with ultrapure water.

[0083] 2. Optimization of test strip

[0084] (1) Confirmation of the best sample buffer solution: Under the conditions of the optimal pH and the optimal amount of gold-labeled SPA, 6 kinds of buffer solutions were selected. The sample addition amount in 100 μL of the sample buffer solution was 80 μL, that is, the proportion of the buffer solution was 80%. They were CB buffer solution, PB buffer solution, BB buffer solution, Tris-HCL buffer solution, PBS buffer solution, and trisodium citrate as the sample buffer solution. Using positive serum as the sample, keeping other same buffer conditions, observe the color development of the C line and T line of the test strip.

[0085] As Figure 14 shown, the color development of the T line and C line of the CB buffer solution is the best, the color development of the PB buffer solution is better, the color development of the T line and C line of the PBS buffer solution is lighter, and the C line of the rest of the buffer solutions does not show color. Select the CB buffer solution as the best buffer solution.

[0086] Optimal concentration of the sample buffer solution: The sample buffer solution also has an impact on the color development of the test strip at different concentrations. Therefore, this study further optimized the concentration of the CB buffer solution. Five concentration gradients of 50 mmol / L, 40 mmol / L, 30 mmol / L, 20 mmol / L, and 10 mmol / L were set respectively. Using positive serum as the sample, observe the color development of the C line and T line of the test strip. As Figure 15 shown, with the decrease of the concentration of the CB buffer solution, compared with the background of the white NC membrane, the color development of the C line becomes weaker and the color development of the T line becomes stronger. When the concentration of the CB buffer solution is 40 mmol / L, both the C line and T line show good color development. The optimal buffer solution concentration is the CB buffer solution containing 40 mmol / L.

[0087] (2) Determination of the optimal coating concentration of ε-toxin on the T line: Coat different concentrations of ε-toxin of 0.12 mg / mL, 0.38 mg / mL, 0.56 mg / mL, 0.83 mg / mL, and 1.25 mg / mL on the T line, add 300 μL of 60 ng / L positive serum into the sample adding hole, and observe the color development of the test strip within 15 minutes.

[0088] As Figure 16As shown, when the concentration of ε-toxin protein ≥ 0.83 mg / mL, the T line shows good color development. When the concentration is lower than this value, the color development of the T line becomes weaker and weaker. The optimal coating concentration of the T line is 0.83 mg / mL.

[0089] (3) Confirmation of the concentration of gold-labeled SPA: Under the above optimal conditions, using positive serum as the sample, in the same buffer system, add 0 μL, 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of gold-labeled SPA respectively, and observe the color development of the T line with the NC membrane as the background.

[0090] As Figure 17 shown, when the amount of gold-labeled SPA is 1 - 8 μL, with the increase of the amount of gold-labeled SPA, the color development of the T line gradually strengthens. When the amount of gold-labeled SPA ≥ 10 μL, the color development of the T line is saturated and the color development signal intensity remains unchanged. The optimal final addition concentration of gold-labeled SPA is 10 μL / mL.

[0091] 3. Sensitivity test

[0092] After determining the optimal coating concentrations of the T line and the C line, the sheep-derived ε-toxin polyclonal antibody with a known titer is serially diluted, and the color development of the T line is observed within 15 minutes to determine the lowest antibody concentration that can be detected by the T line.

[0093] The results of the sensitivity test are as Figure 18 shown, and the lowest detection limit is 3 ng / mL ε antibody.

[0094] 4. Specificity test

[0095] Take 300 μL of the positive sera of Mycobacterium pseudotuberculosis, Staphylococcus aureus, Mannheimia haemolytica, and Arcanobacterium pyogenes preserved in the Infectious Diseases Laboratory of the College of Veterinary Medicine, Northwest A&F University, and drop them onto the sample pad of the prepared test strip respectively, and observe whether the T line shows color. If it does not show color, it indicates that the test strip has good specificity.

[0096] The results are as Figure 19 shown, and the test strip has good specificity and is negative for the positive sera of Mycobacterium pseudotuberculosis, Staphylococcus aureus, Mannheimia haemolytica, and Arcanobacterium pyogenes.

[0097] 5. Stability test: Seal the prepared colloidal gold test strip in a sealed bag containing a desiccant, and place it at 4 °C, room temperature, and 37 °C respectively for 7 days, 30 days, 60 days, and 90 days. Detect the color development of the T line and the C line within 15 minutes for the standard positive serum and the standard negative serum.

[0098] The results of the stability test are as Figure 20As shown, when the test strip was stored at 4°C for 15 days and compared with that stored at 4°C for 45 days, the C and T lines showed good color development. However, when the test strip was stored at 4°C for 180 days, the color development signals of the C and T lines decreased, indicating that with the increase of storage time, the color development of the C and T lines gradually decreased. When comparing 45 days at 4°C with 45 days at 25°C, and 180 days at 4°C with 180 days at 25°C, the results showed that with the increase of the storage temperature of the test strip, the color development signals of the C and T lines of the test strip decreased. This test strip can be temporarily stored at room temperature, but it is more suitable for dry and low-temperature storage at 4°C.

[0099] Example 5: Parallel comparison test between an antibody detection test strip and an ELISA kit in clinical application

[0100] Randomly collect 2 mL of blood from the jugular veins of 78 sheep in a sheep farm, separate the serum, and store it at -20°C. Detect the sera of these 78 sheep with the colloidal gold test strip prepared according to the optimized conditions of each step (as shown in Figure 21 、 Figure 22 ), and use the detection result of a commercial ELISA kit as a control (as shown in Figures 23 - 24 ).

[0101] As shown in Figure 25 , compare the detection results, analyze the correlation between the two. Exclude the clinical samples outside the detection range of the test strip. By comparing the correlation between the results of ELISA and colloidal gold immunochromatographic test strips of the remaining 64 clinical samples, draw a standard curve. The results showed that the regression equation was y = 0.71926x + 13.43775, and R 2 = 0.93, showing a good correlation between the two. However, when the antibody concentration in the clinical sample was close to vLOD, the test strip was prone to false negatives. When analyzing with Image J software, the gray value of the T line was too low for the antibody concentration, which was prone to cause an error slightly deviating from the actual concentration, resulting in a relatively low R2 for the two results.

[0102] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. An epsilon toxin protein, characterized in that, The amino acid sequence of the epsilon toxin protein is shown in SEQ ID NO:

4.

2. A detection test strip for Clostridium perfringens epsilon antibody, comprising a sample pad, a gold-labeled pad, an NC membrane, a blotting paper and a PV backing board; the sample pad, the gold-labeled pad, the NC membrane and the blotting paper are successively attached to the PV backing board and connected in an overlapping manner, characterized in that, On the NC membrane, a detection line and a quality control line parallel to each other are sequentially provided in the direction from the gold conjugate pad to the absorbent paper; The antigen is labeled at the detection line, and the antigen is the epsilon toxin protein described in claim 1; The secondary antibody is labeled at the quality control line, and the secondary antibody is a secondary antibody of rabbit anti-SPA; The gold conjugate pad is prepared by mixing a colloidal gold solution and SPA protein to obtain a gold-labeled SPA solution, and spraying the gold-labeled SPA solution onto the gold conjugate pad.

3. The test strip according to claim 2, wherein, The concentration of the epsilon toxin is 0.8 mg / mL - 1.25 mg / mL.

4. The test strip according to claim 2, wherein The concentration of the secondary antibody of rabbit anti-SPA is 0.5 mg / mL - 1.5 mg / mL.

5. The test strip according to claim 2, wherein The final concentration of the gold-labeled SPA solution is 9.5 μL / mL - 10.5 μL / mL.

6. Use of the test strip according to any one of claims 2-5 in the preparation of a kit for detecting Clostridium perfringens.

7. A detection kit for Clostridium perfringens epsilon antibody, characterized in that, The kit includes the test strip according to any one of claims 2-5 and a sample buffer solution.

8. The detection kit according to claim 7, characterized in that, The sample buffer solution is CB buffer or PB buffer.

9. The detection kit according to claim 7, wherein The concentration of the sample buffer solution is 35 mmol / L - 50 mmol / L.

Images