Brucella specific antigen and application thereof

Abstract

The present invention relates to the technical field of Brucella detection. The present invention provides a Brucella-specific antigen and its application. The nucleotide sequence of the Brucella-specific antigen is shown in SEQ ID NO.1. The Brucella-specific antigen of the present invention has strong specificity and good use effect. A test strip prepared using the antigen of the present invention can still detect a significant positive reaction when the positive serum concentration is diluted to 1:40; the test strip of the present invention detects Yersinia intestinalis-positive serum and Escherichia coli-positive serum, and the results are both negative, indicating that the test strip has good specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of Brucella detection, in particular to a Brucella-specific antigen and application thereof. Background Art

[0002] Brucellosis, a chronic zoonotic disease caused by the bacterium Brucella, is characterized by abortion, orchitis, and arthritis in male animals, posing a serious threat to the development of the livestock industry and human health. The disease is widespread throughout China and globally, affecting over 170 of the world's 200 countries.

[0003] Brucella's pathogenicity is due to its ability to survive in host monocytes and macrophages, even in conditions such as hypoxia, nutrient deprivation, acidic environments, and the presence of oxidants. Furthermore, Brucella is an intracellular parasite, making it difficult to treat with antibiotics once infection occurs. Even if an infected animal recovers on its own, there is still a risk of continued bacterial excretion.

[0004] In order to find sensitive, specific, and rapid diagnostic methods, domestic and international scholars have conducted extensive research on the diagnosis of brucellosis. Currently, common diagnostic methods for brucellosis include pathogenic methods, molecular biology methods, and immunological methods. However, these methods have disadvantages such as complicated procedures, long time consumption, and high costs. To meet the needs of large-scale grassroots surveys, it is necessary to develop rapid detection methods for multiple animals to enable simple and convenient detection of Brucella. Summary of the Invention

[0005] The purpose of the present invention is to provide a Brucella-specific antigen and application thereof, which can detect Brucella simply and conveniently.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Brucella-specific antigen, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides application of the Brucella specific antigen in preparing a product for detecting Brucella.

[0009] The present invention also provides a colloidal gold immunochromatographic test strip, which includes a sample pad, a colloidal gold binding pad, a chromatography pad and a water-absorbing material. The chromatography pad contains a detection line T and a quality control line C. The detection line T contains a Brucella-specific antigen, and the quality control line C contains a Streptococcus protein G antibody.

[0010] Preferably, the sample pad, colloidal gold binding pad, detection line T, quality control line C, and water absorbing material are arranged in sequence according to the flow direction of the sample.

[0011] Preferably, the preparation method of the colloidal gold binding pad is:

[0012] (1) Mix 1 part of 0.8-1.2% chloroauric acid and 90-110 parts of water in a volume ratio and boil, add 1-2 parts of trisodium citrate, and continue boiling for 10-20 minutes to obtain a colloidal gold solution;

[0013] (2) The colloidal gold solution and streptococcal protein G are mixed, and after 20 to 40 minutes, the mixture is centrifuged at 11,000 to 13,000 rpm for 20 to 40 minutes. The precipitate is resuspended with a resuspension solution and added to the gold label pad to obtain a colloidal gold binding pad.

[0014] Preferably, the chromatography pad is a nitrocellulose membrane.

[0015] The present invention also provides a method for preparing the Brucella colloidal gold immunochromatographic test strip, comprising the following steps:

[0016] (1) Attach a nitrocellulose membrane to a base plate, draw a line with Brucella-specific antigen as the test line T, and draw a line with 0.8-1.2 mg / mL Streptococcus protein G antibody as the quality control line C;

[0017] (2) Affix the colloidal gold binding pad, sample pad, and absorbent paper in sequence to obtain a Brucella colloidal gold immunochromatographic test strip.

[0018] Preferably, the amounts of the Brucella specific antigen and Streptococcus protein G antibody used in step (1) are independently 0.8 to 1.2 μL / cm.

[0019] The present invention provides a Brucella-specific antigen and its application. The nucleotide sequence of the Brucella-specific antigen is shown in SEQ ID NO.1. The Brucella-specific antigen of the present invention has strong specificity and good use effect. A test strip prepared using the antigen of the present invention can still detect a significant positive reaction when the positive serum concentration is diluted to 1:40; the test strip of the present invention detects negative results in Yersinia enteritidis-positive serum and Escherichia coli-positive serum, indicating that the test strip has good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the assembly of a colloidal gold immunochromatographic test strip;

[0021] Figure 2 This is the result of the colloidal gold immunochromatographic reactogenicity test;

[0022] Figure 3 This is the result of the sensitivity test of the colloidal gold immunochromatographic test strip;

[0023] Figure 4 It is a specific test for colloidal gold immunochromatographic test strips. DETAILED DESCRIPTION

[0024] The present invention provides a Brucella-specific antigen, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0025] In the present invention, the amino acid sequence of the Brucella-specific antigen is preferably as shown in SEQ ID NO.2.

[0026] The present invention also provides application of the Brucella specific antigen in preparing a product for detecting Brucella.

[0027] The present invention also provides a colloidal gold immunochromatographic test strip, which includes a sample pad, a colloidal gold binding pad, a chromatography pad and a water-absorbing material. The chromatography pad contains a detection line T and a quality control line C. The detection line T contains a Brucella-specific antigen, and the quality control line C contains a Streptococcus protein G antibody.

[0028] In the present invention, the sample pad, the colloidal gold binding pad, the detection line T, the quality control line C, and the water absorbing material are arranged in sequence according to the flow direction of the sample.

[0029] In the present invention, the preparation method of the colloidal gold binding pad is:

[0030] (1) Mix 1 part of 0.8-1.2% chloroauric acid and 90-110 parts of water in a volume ratio and boil, add 1-2 parts of trisodium citrate, and continue boiling for 10-20 minutes to obtain a colloidal gold solution;

[0031] (2) The colloidal gold solution and streptococcal protein G are mixed, and after 20 to 40 minutes, the mixture is centrifuged at 11,000 to 13,000 rpm for 20 to 40 minutes. The precipitate is resuspended with a resuspension solution and added to the gold label pad to obtain a colloidal gold binding pad.

[0032] In the present invention, the colloidal gold probe content in the colloidal gold conjugate pad is 80-120 μL / cm 2 , more preferably 100 μL / cm 2 .

[0033] In the present invention, the colloidal gold probe is streptococcal protein G.

[0034] In the present invention, the concentration of the chloroauric acid is preferably 1%.

[0035] In the present invention, the amount of water in step (1) is preferably 100 parts.

[0036] In the present invention, the amount of trisodium citrate in step (1) is preferably 1.5 parts.

[0037] In the present invention, the time for continuing boiling is preferably 15 minutes.

[0038] In the present invention, the colloidal gold solution is preferably cooled to room temperature before mixing in step (2).

[0039] In the present invention, the pH value of the colloidal gold solution in step (2) is preferably 7.3 to 7.7, and more preferably 7.5.

[0040] In the present invention, the pH value in step (2) is preferably adjusted with 0.2% K2CO3.

[0041] In the present invention, after the mixing in step (2), the mixture is preferably inverted and mixed once every 4 to 6 minutes, more preferably every 5 minutes.

[0042] In the present invention, the centrifugation time in step (2) is preferably 30 minutes after mixing.

[0043] In the present invention, the centrifugation method in step (2) is preferably centrifugation at 12000 r / min for 30 min.

[0044] In the present invention, the formula of the resuspension in step (2) is preferably 4-6% sucrose, 0.4-0.6% Tween, 1.5-2.5% BSA, 0.08-0.12 M Tris, and more preferably 5% sucrose, 0.5% Tween, 2% BSA, 0.1 M Tris.

[0045] In the present invention, the chromatography pad is a nitrocellulose membrane.

[0046] The present invention also provides a method for preparing the Brucella colloidal gold immunochromatographic test strip, comprising the following steps:

[0047] (1) Attach a nitrocellulose membrane to a base plate, draw a line with Brucella-specific antigen as the test line T, and draw a line with 0.8-1.2 mg / mL Streptococcus protein G antibody as the quality control line C;

[0048] (2) Affix the colloidal gold binding pad, sample pad, and absorbent paper in sequence to obtain a Brucella colloidal gold immunochromatographic test strip.

[0049] In the present invention, the amounts of the Brucella specific antigen and Streptococcus protein G antibody used in the underlining in step (1) are preferably independently 0.8 to 1.2 μL / cm, and more preferably 1 μL / cm.

[0050] In the present invention, the concentration of the streptococcal protein G is preferably 1 mg / mL.

[0051] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] (1) Place 100 mL of deionized water and 1 mL of 1% chloroauric acid in a 250 mL conical flask, heat to boiling, add 1.5 mL of trisodium citrate, and observe that the color of the solution changes from light yellow to black and finally stabilizes to purple-red. Continue boiling for 15 minutes, cool to room temperature, and then dilute to the original volume to obtain a colloidal gold solution.

[0054] (2) Adjust the pH of the colloidal gold solution to 7.5 with 0.2% K2CO3, then add streptococcal protein G to 1 mg / ml. Mix thoroughly by inverting every 5 minutes. After 30 minutes, centrifuge at 12,000 rpm for 30 minutes. Discard the supernatant and resuspend the precipitate in a resuspension solution (5% sucrose, 0.5% Tween, 2% BSA, 0.1 M Tris). Evenly add the prepared colloidal gold probe onto the gold label pad so that 100 μL of colloidal gold probe is contained in 1.0 cm2 of the gold label pad.

[0055] (3) Attach the nitrocellulose membrane to the bottom plate, draw a line with Brucella-specific antigen as the test line T, and draw a line with 1 mg / mL Streptococcus protein G antibody as the quality control line C;

[0056] (4) The colloidal gold binding pad, sample pad and absorbent paper are sequentially attached to the nitrocellulose membrane to obtain a Brucella colloidal gold immunochromatographic test strip.

[0057] Example 2 Determination of serum test results using test strips

[0058] Pipette the diluted serum to be tested and drop it onto the sample pad in the assembled test strip. Let it stand for 3 minutes. If both the T line and the C line are colored, the result can be judged as positive; if the T line does not color and the C line is colored, the sample can be judged as negative; if the C line does not color, the test result of the test strip is judged to be invalid.

[0059] Example 3 Test Strip Sensitivity and Specificity

[0060] Brucella-positive serum was diluted at 1:10, 1:20, 1:40, and 1:80 and dripped onto the sample pad. After 3 minutes, the highest dilution factor at which a positive reaction occurred was observed. The test strip was then tested with Yersinia enterica-positive serum and Escherichia coli-positive blood to observe cross-reactions and specificity.

[0061] Example 4 Detection of clinical samples

[0062] Use the prepared colloidal gold test strips to test 100 sheep serum samples, compare the results with the bengalen red plate agglutination test, and calculate the positive coincidence rate.

[0063] result:

[0064] (1) Detection of sheep serum using colloidal gold immunochromatographic test strips

[0065] Three positive sera (numbered 1 to 3, purchased from the China Veterinary Drug Administration), three negative sera and a blank control group (numbered 7) were tested for their reactivity by colloidal gold immunochromatographic assay. The results were as follows: Figure 2 shown.

[0066] (2) Sensitivity of colloidal gold immunochromatographic test strips

[0067] The results of testing Brucella positive serum at different dilutions were as follows: Figure 3 As shown in the figure, the positive serum of Brucella can still detect obvious positive reaction after being diluted 1:40.

[0068] Among them, 1 to 6 are blank control, Brucella negative serum control, and Brucella positive serum at different dilutions of 1:10, 1:20, 1:40, and 1:80, respectively.

[0069] (3) Specificity results of colloidal gold immunochromatographic test strips

[0070] The results of the test strips for Yersinia intestinalis positive serum and Escherichia coli positive serum were both negative. Figure 4 As shown, it shows that the test strip has good sensitivity and specificity;

[0071] 1 to 5 are blank control, Brucella negative serum, Brucella positive serum, Yersinia enterica positive serum, and Escherichia coli positive serum, respectively.

[0072] (4) Clinical sample test results

[0073] 100 sheep serum samples to be tested were tested by the red bengal plate agglutination test and the above-mentioned colloidal gold method respectively. The positive rate detected by this method was 20%, and the positive rate detected by the red bengal plate agglutination test was 17%. The coincidence rate of the two was 97%.

[0074] As can be seen from the above embodiments, the present invention provides a Brucella-specific antigen and its application, wherein the nucleotide sequence of the Brucella-specific antigen is shown in SEQ ID NO.1. The Brucella-specific antigen of the present invention has strong specificity and good use effect. The test strip prepared using the antigen of the present invention can still detect a significant positive reaction when the positive serum concentration is diluted to 1:40; the test strip of the present invention detects Yersinia enteritidis positive serum and Escherichia coli positive serum, and the results are both negative, indicating that the test strip has good specificity.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Brucella-specific antigen, characterized in that The nucleotide sequence of the Brucella specific antigen is shown in SEQ ID NO.

1.

2. Application of the Brucella specific antigen according to claim 1 in the preparation of a Brucella detection product.

3. A colloidal gold immunochromatographic test strip containing the Brucella-specific antigen according to claim 1, characterized in that: The test strip comprises a sample pad, a colloidal gold binding pad, a chromatography pad and a water-absorbing material; the chromatography pad contains a detection line T and a quality control line C; the detection line T contains Brucella-specific antigens; and the quality control line C contains streptococcal protein G antibodies.

4. The test strip according to claim 3, characterized in that The sample pad, colloidal gold binding pad, detection line T, quality control line C, and water absorbing material are arranged in sequence according to the flow direction of the sample.

5. The test strip according to claim 4, characterized in that The preparation method of the colloidal gold binding pad is as follows: (1) Mix 1 part of 0.8-1.2% chloroauric acid and 90-110 parts of water in a volume ratio and boil, add 1-2 parts of trisodium citrate, and continue boiling for 10-20 minutes to obtain a colloidal gold solution; (2) The colloidal gold solution and streptococcal protein G are mixed, and after 20 to 40 minutes, the mixture is centrifuged at 11,000 to 13,000 rpm for 20 to 40 minutes. The precipitate is resuspended with a resuspension solution and added to the gold label pad to obtain a colloidal gold binding pad.

6. The test strip according to any one of claims 3 to 5, characterized in that: The chromatography pad is a nitrocellulose membrane.

7. The method for preparing the test strip according to any one of claims 3 to 6, characterized in that: The steps include: (1) Attach a nitrocellulose membrane to a base plate, draw a line with Brucella-specific antigen as the test line T, and draw a line with 0.8-1.2 mg / mL Streptococcus protein G antibody as the quality control line C; (2) Affix the colloidal gold binding pad, sample pad, and absorbent paper in sequence to obtain a Brucella colloidal gold immunochromatographic test strip.

8. The preparation method according to claim 7, characterized in that When underlining in step (1), the amounts of the Brucella specific antigen and Streptococcus protein G antibody used are independently 0.8 to 1.2 μL / cm.

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