Chicken mycoplasma synoviae plate agglutination detection system as well as detection method and application thereof

Abstract

The invention relates to the technical field of reagent detection, and discloses a chicken mycoplasma synoviae plate agglutination detection system as well as a detection method and application thereof. The detection sensitivity is improved, and the revolutionary breakthrough of the detection sensitivity is realized by constructing a'biotinylated recombinant antigen-streptavidin-color microsphere 'cascade signal amplification system; the detection specificity is high, cross misjudgment is effectively avoided, the high-purity and high-specificity genetic engineering recombinant p80 / vlhA fusion protein is adopted as a core recognition element, and interference of non-target impure protein is completely eradicated from an antigen source; the result interpretation is visual and objective, and the detection result is presented in the form of red particles or flocculent agglutination which can be clearly seen by naked eyes, and is clearly compared with a uniform red opacifying background.

Description

Technical Field

[0001] This invention relates to the field of reagent detection technology, specifically to a plate agglutination detection system for Mycoplasma synoviae in chickens, its detection method, and its application. Background Technology

[0002] Mycoplasma synoviae (MS) is a major pathogen that seriously harms the poultry industry, causing arthritis, tenosynovitis, and respiratory symptoms in chickens, leading to decreased egg production and significant economic losses. Therefore, rapid and accurate serological testing for MS is crucial for the eradication and control of this disease.

[0003] Plate agglutination tests are the preferred method for on-site screening of MS due to their simplicity, speed, and intuitiveness. Currently, commercially available MS plate agglutination antigens are typically prepared using whole bacterial antigens stained with crystal violet. However, this traditional technique has the following inherent drawbacks: Low sensitivity: The immunoreactivity of whole bacterial antigens is limited. In chickens with early infection or low antibody levels, it is difficult to form visible agglutination particles, easily leading to false negative results and prolonging the detection window period; Poor specificity: MS shares common antigenic epitopes with Mycoplasma gallisepticum (MG), easily causing cross-reactions and false positives, resulting in misjudgments and unnecessary culling in farms, leading to economic losses; Subjective result interpretation: The agglutination particles are small, and the color of the background liquid greatly interferes with interpretation, making it highly dependent on the operator's experience, resulting in poor repeatability and consistency; Poor antigen stability: Whole bacterial antigens are prone to self-agglutination or titer decline during storage and transportation. Summary of the Invention

[0004] The purpose of this invention is to provide a plate agglutination detection system for Mycoplasma synoviae in chickens, as well as its detection method and application, which has the effects of high sensitivity, high specificity and easy interpretation.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a plate agglutination detection method for detecting Mycoplasma synoviae antibodies, comprising the following steps: S1: Sample preparation: Dilute the chicken serum or whole blood to be tested with sample diluent; S2: Antigen-antibody reaction: The sample treated in S1 is mixed with biotinylated MS specific antigen solution on the reaction plane and shaken to allow the MS antibody in the sample to bind to the antigen; S3: Signal amplification and color development: Add streptavidin-colored latex microsphere complex to the reaction system of S2 and continue shaking; S4: Result interpretation: Observe the aggregation state of the reaction mixture. The presence of visible aggregated particles or flocculent matter indicates a positive result, while maintaining a uniform emulsion state indicates a negative result. The biotinylated MS-specific antigen solution contains a genetically engineered recombinant MS p80 / vlhA fusion protein labeled with NHS-long-arm biotin; the streptavidin-colored latex microsphere complex contains streptavidin covalently coupled colored carboxylated polystyrene microspheres.

[0006] A further feature of the present invention is that the amino acid sequence of the genetically engineered recombinant MS p80 / vlhA fusion protein includes immunodominant fragments from MS p80 protein and vlhA protein, and is linked by a flexible linker peptide (Gly4Ser)3.

[0007] A further setting of the present invention is that the concentration of recombinant MS p80 / vlhA fusion protein in the biotinylated MS specific antigen solution is 0.5-1.5 mg / mL, and the biotin to protein labeling molar ratio is 10:1 to 20:1.

[0008] A further provision of the present invention is that, in the streptavidin-colored latex microsphere composite, the particle size of the colored carboxylated polystyrene microspheres is 0.3 μm ± 0.05 μm, and the coupling density of streptavidin on the microspheres is not less than 5 μg streptavidin / mg microspheres.

[0009] A further embodiment of the present invention is that the streptavidin-colored latex microsphere complex is suspended in a Tris-HCl buffer (pH 8.2) containing 2% trehalose, 1% BSA and 0.05% Tween-20.

[0010] A further provision of the present invention is that the sample diluent is a PBS buffer (pH 7.2) containing 0.15M NaCl and 0.5% gelatin.

[0011] A plate agglutination detection system for Mycoplasma synoviae in chickens, the system comprising: (a) Biotinylated MS-specific antigen solution: containing genetically engineered recombinant MSp80 / vlhA fusion protein labeled with NHS-long arm biotin, dissolved in PBS buffer (pH 7.4) containing 0.1% NaN3; (b) Streptavidin-colored latex microsphere complex: containing colored carboxylated polystyrene microspheres covalently coupled with streptavidin, suspended in a buffer solution at pH 8.2; (c) Sample dilution: PBS buffer (pH 7.2) containing 0.15M NaCl and 0.5% gelatin; (d) Positive control serum: rabbit serum containing antibody against MS p80 / vlhA fusion protein; (e) Negative control serum: SPF chicken serum without MS antibody.

[0012] A further setting of the present invention is that the concentration of recombinant protein in the biotinylated MS specific antigen solution is 1.0 mg / mL, and the biotin to protein labeling molar ratio is 15:1.

[0013] A method for preparing biotinylated MS-specific antigen solution includes the following steps: S1: Expression and purification of genetically engineered recombinant MS p80 / vlhA fusion protein; S2: The fusion protein obtained in S1 was reacted with NHS-long-arm biotin in PBS buffer at pH 7.4. The molar ratio of biotin to protein was 10:1 to 20:1. After the reaction was completed, glycine was added to quench the reaction. S3: Purify and adjust the concentration of biotinylated protein to 0.5-1.5 mg / mL, and store in PBS buffer (pH 7.4) containing 0.1% NaN3.

[0014] The application of a genetically engineered recombinant MS p80 / vlhA fusion protein in the preparation of diagnostic reagents or kits for detecting Mycoplasma synoviae infection in chickens, wherein the amino acid sequence of the fusion protein contains immunodominant fragments from MS p80 protein and vlhA protein, and is linked by a flexible linker peptide (Gly4Ser)3.

[0015] In summary, the present invention has the following beneficial effects: 1. Improved Detection Sensitivity: By constructing a cascade signal amplification system of "biotinylated recombinant antigen - streptavidin - colored microspheres", a revolutionary breakthrough in detection sensitivity has been achieved. For the same standard positive serum sample, the detection system of this invention can achieve a maximum detection dilution of 1:256, while the maximum detection dilution of traditional commercially available plate agglutination antigen is only 1:16. The sensitivity of this invention is 16 times that of traditional methods. This order-of-magnitude improvement enables this invention to detect extremely low concentrations of antibodies, which is particularly beneficial for early diagnosis of infection, detection of weak antibody reaction samples, and evaluation of immunization effects, effectively reducing the false negative rate. 2. High detection specificity, effectively avoiding cross-diagnosis: By using a high-purity, high-specificity genetically engineered recombinant p80 / vlhA fusion protein as the core recognition element, this invention eliminates interference from non-target proteins at the antigen source. In tests with positive sera from common avian diseases such as Mycoplasma gallisepticum (MG), Newcastle disease, and infectious bronchitis, the detection system of this invention showed no cross-reaction, with all results being negative. Under the same conditions, two commercially available traditional antigens showed varying degrees of false positives (up to 60%). This proves that this invention can accurately distinguish MS infection from other avian diseases with similar symptoms, significantly improving diagnostic accuracy and avoiding incorrect culling or unnecessary medication due to misdiagnosis. 3. Extremely simple and fast operation, perfectly adapted to field applications: The entire testing process only requires four steps: "dilution-sample addition-mixing-observation". From sample addition to result interpretation, the total time is only 3-4 minutes, and all operations are completed on a glass slide; stable results can be obtained under a wide range of room temperature conditions from 20-45℃, without the need for complex temperature control equipment; This greatly reduces the technical requirements and environmental limitations for operators, enabling non-professionals (such as farm technicians) to quickly master and independently complete accurate tests, achieving "last mile" coverage from the laboratory to the farm site; 4. The results are intuitive and objective, with good reproducibility: The test results are presented as clearly visible red particles or flocculent aggregates, which contrast sharply with the uniform red milky background. The interpretation criteria are clear ("present" or "absent" aggregates), effectively eliminating the subjective differences in interpreting "suspicious" or weakly positive results in traditional methods. The stable reagent system based on chemical coupling ensures the uniformity of performance between different batches of products, and the results are stable, reliable, and highly reproducible. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0018] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0019] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0020] Example 1: Specific configuration and preparation of the detection system Preparation of biotinylated MS-specific antigen solution: Expression and purification of recombinant MS p80 / vlhA fusion protein: Immunodominant fragments of the p80 and vlhA genes were selected from the standard strain of Mycoplasma synoviae (ATCC25204). After codon optimization, the fragments were linked by the flexible linker peptide (G4S)3 sequence to synthesize the "p80-(G4S)3-vlhA" fusion gene, which was then cloned into the pET-28a(+) vector. After transformation into BL21(DE3) Escherichia coli, expression was induced by 0.5 mM IPTG at 25°C for 16 hours. After cell disruption, the recombinant protein (approximately 90 kDa) with a purity >95% was obtained by Ni-NTA affinity chromatography and Superdex 200 molecular sieve chromatography and stored in PBS (pH 7.4).

[0021] Biotin labeling: Take 1 mg of purified protein and add freshly prepared NHS-long arm biotin (dissolved in DMSO) solution at a molar ratio of 15:1 (biotin:protein). React at room temperature in the dark for 2 hours. Quench with glycine (final concentration 50 mM) for 10 minutes, then purify using a desalting column to remove free biotin. Adjust the protein concentration to 1.0 mg / mL with PBS containing 0.1% NaN3 (pH 7.4), filter through a 0.22 μm filter membrane for sterilization, aliquot, and store at 4°C in the dark to obtain the biotinylated MS specific antigen solution.

[0022] Preparation of streptavidin-red latex microsphere complex: Microsphere activation: Take 1 mL of red carboxylated polystyrene microsphere suspension with a particle size of 0.3 μm (solid content 2.5%), wash twice with 0.1 M MES buffer (pH 6.0) and resuspend; add freshly prepared EDC (final concentration -3.33 mg / mL) and NHS (final concentration -1.67 mg / mL) solutions, and activate at room temperature in the dark for 30 minutes.

[0023] Protein coupling and blocking: After activation, the microspheres were washed and quickly resuspended in 1.5 mL of MES buffer containing 1.5 mg streptavidin (pH 6.0) and reacted at room temperature in the dark for 2 hours. After the reaction, the supernatant was discarded by centrifugation, and the microspheres were washed once with PBS containing 0.1% BSA and 0.05% Tween-20, and then blocked with Tris-HCl (pH 8.2) containing 1% BSA for 1 hour.

[0024] Final preparation: After blocking, the microspheres were washed and resuspended in 3 mL of blocking / storage buffer (0.02 M Tris-HCl containing 2% trehalose, 1% BSA, and 0.05% Tween-20, pH 8.2); the streptavidin coupling density was determined by BCA method to be 6.2 μg / mg microspheres, which meets the requirement of ≥5 μg / mg; the complex was stored at 4℃ protected from light.

[0025] Preparation of other components: Sample dilution solution: Prepare PBS buffer (pH 7.2) containing 0.15 M NaCl and 0.5% gelatin, aliquot and store at 4°C.

[0026] Positive control serum: New Zealand white rabbits were immunized with purified MS p80 / vlhA fusion protein. After multiple booster immunizations, blood was collected and serum was separated. Complement was inactivated by water bath at 56°C for 30 minutes. The serum was then aliquoted and stored at -20°C.

[0027] Negative control serum: Blood was collected from SPF chickens that were free of MS infection. The serum was separated and complement was inactivated. The serum was then aliquoted and stored at -20°C.

[0028] Example 2: Sensitivity Assessment (Comparison with Traditional Methods) Experimental methods: A sample of MS standard positive serum (verified by ELISA and Western Blot, titer confirmed as 1:128) confirmed by an international reference laboratory was serially diluted using SPF chicken negative serum at dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. The following two methods were used for detection: The detection system of the present invention operates according to the method described in claim 1, that is, after the sample is diluted with the sample diluent, it is reacted sequentially with the biotinylated antigen solution and the streptavidin-latex microsphere complex.

[0029] Traditional plate agglutination method: Using a commercially available mainstream brand (marked as "Brand C") MS plate agglutination antigen, strictly follow its instructions and directly mix and react with serially diluted serum samples on a glass plate.

[0030] All reactions were conducted at room temperature (approximately 25°C), and the agglomeration results were observed and independently interpreted by two experienced technicians under double-blind conditions. The criteria for interpreting agglomeration intensity were: "+++" for strong agglomeration (large flocculent particles), "++" for moderate agglomeration (clear particles), "+" for weak agglomeration (fine particles), and "-" for no agglomeration.

[0031] result: The detection system of this invention shows that even at a dilution of up to 1:256, clear and uniform granular agglomerates still appear in the reaction solution, which are interpreted as "++". At a dilution of 1:512, the agglomerates disappear.

[0032] Commercially available brand C antigen: Clear agglutination (+++) at a dilution of 1:8, significantly reduced agglutination (+) at a dilution of 1:16, and complete disappearance of agglutination at dilutions of 1:32 and higher, interpreted as negative.

[0033] in conclusion: Experimental results show that, for the same standard positive serum sample, the highest dilution that the detection system of this invention can detect (1:256) is 16 times higher than the highest detection dilution of traditional commercially available antigens (1:16). This fully demonstrates that by introducing a B-SA signal amplification system, this invention greatly improves the sensitivity of plate agglutination detection, enabling the detection of lower concentrations of antibodies, which is beneficial for accurate diagnosis in early infection or when antibody levels are low.

[0034] Example 3: Specificity Verification Experimental methods: Thirty avian serum samples that were confirmed negative by other methods and tested negative for MS were collected, including 10 samples of Mycoplasma gallisepticum (MG) positive serum, 10 samples of Newcastle disease virus (NDV) positive serum, and 10 samples of infectious bronchitis virus (IBV) positive serum. All samples were tested using the detection system of this invention (method as in Example 2), and positive and negative control sera were used as quality controls.

[0035] Comparative example: Comparison of specificity with traditional antigens To highlight the specific advantages of this invention, five additional serum samples identified as strongly positive for MG (MS negative) were selected and tested using the detection system of this invention and MS plate agglutination antigens from two other commercially available brands (labeled as "Brand A" and "Brand B").

[0036] result: Results of detection of antibodies against non-MS pathogens: The detection system of this invention was used to detect 30 MG, NDV and IBV positive serum samples. All 30 samples were negative. The reaction solution remained in a uniform red milky state with no agglutination. The positive control showed strong agglutination, and the negative control showed no agglutination. The system was working normally.

[0037] Specificity comparison results: The detection system of this invention: all 5 MG-positive serum samples tested negative.

[0038] Commercially available brand A antigen: 3 out of 5 samples showed obvious agglutination (false positive) ranging from "++" to "+++".

[0039] Commercially available brand B antigen: 2 out of 5 samples showed weak agglutination (false positive).

[0040] in conclusion: The detection system of this invention does not cross-react with antibodies against common avian pathogens (MG, NDV, IBV), demonstrating excellent specificity. Further comparative examples show that traditional commercially available plate agglutination antigens (which may use whole bacteria or crude extracted antigens) are prone to cross-reaction with antibodies against closely related pathogens such as MG due to low antigen purity, leading to false positives. This invention uses a high-purity, high-specificity genetically engineered recombinant p80 / vlhA fusion protein as the antigen, eliminating interference from other proteins at the source, thus exhibiting significantly superior specificity compared to traditional products.

[0041] Example 4: Validation of Clinical Sample Detection Accuracy and Response Condition Optimization Clinical sample concordance trial: Methods: Two hundred clinical chicken serum samples were randomly collected from different chicken farms; a blinded method was used, and the following three methods were employed for testing: (1) Gold standard: Western Blot (WB) method, using purified MS p80 / vlhA fusion protein as the detection antigen.

[0042] (2) The detection system of the present invention.

[0043] (3) Commercially available brand C plate agglutination antigen.

[0044] Using the results of Western blot (WB) as the basis for determining true positives and true negatives, the sensitivity, specificity, and overall concordance rate of the system of this invention and traditional methods are calculated.

[0045] result: Conclusion: The detection system of this invention exhibits high consistency with the gold standard Western blotting (WB) method, with an overall concordance rate of 98.0%, significantly higher than the traditional plate agglutination method (91.5%). This demonstrates that the present invention maintains high sensitivity while possessing extremely high accuracy, with low false positive and false negative rates, making it suitable for clinical diagnosis.

[0046] Experiment on the effect of reaction temperature (condition optimization and verification): Methods: One clinically positive serum sample with intermediate titer (confirmed by Western blotting) and one negative serum sample were selected. Reaction slides were placed in a precisely temperature-controlled metal bath or constant-temperature plate, with the reaction temperature gradient set as follows: 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, 45℃, 50℃, and 55℃. At each temperature point, the detection system of this invention was used to detect both samples, and the time, intensity, and background clarity of agglutination were observed and recorded.

[0047] result: Within a temperature range of 20℃ to 45℃, positive samples can produce clear and stable agglutination within 2-3 minutes, while negative samples have a uniform background, making the results easy to interpret.

[0048] When the temperature is below 20℃ (such as 10℃ or 15℃), the reaction rate slows down significantly, and positive samples take more than 5 minutes to show weak agglutination, making interpretation difficult.

[0049] When the temperature is above 45℃ (such as 50℃, 55℃), the reaction solution evaporates too quickly, causing the reaction area to dry out rapidly. This exacerbates the non-specific aggregation of microspheres, and particles appear in negative samples, leading to false positive interpretations.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0051] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. A plate agglutination detection method for detecting Mycoplasma synoviae antibodies, characterized in that, Includes the following steps: S1: Sample preparation: Dilute the chicken serum or whole blood to be tested with sample diluent; S2: Antigen-antibody reaction: The sample treated in S1 is mixed with biotinylated MS specific antigen solution on the reaction plane and shaken to allow the MS antibody in the sample to bind to the antigen; S3: Signal amplification and color development: Add streptavidin-colored latex microsphere complex to the reaction system of S2 and continue shaking; S4: Result interpretation: Observe the aggregation state of the reaction mixture. The presence of visible aggregated particles or flocculent matter indicates a positive result, while maintaining a uniform emulsion state indicates a negative result. The biotinylated MS-specific antigen solution contains a genetically engineered recombinant MSp80 / vlhA fusion protein labeled with NHS-long-arm biotin; the streptavidin-colored latex microsphere complex contains streptavidin covalently coupled colored carboxylated polystyrene microspheres.

2. The plate agglutination detection method for detecting Mycoplasma synoviae antibodies according to claim 1, characterized in that, The amino acid sequence of the genetically engineered recombinant MS p80 / vlhA fusion protein contains immunodominant fragments from MS p80 and vlhA proteins, linked by a flexible linker peptide (Gly4Ser)3.

3. The plate agglutination detection method for detecting Mycoplasma synoviae antibodies according to claim 1, characterized in that, In the biotinylated MS-specific antigen solution, the concentration of recombinant MS p80 / vlhA fusion protein is 0.5-1.5 mg / mL, and the biotin to protein labeling molar ratio is 10:1 to 20:

1.

4. The plate agglutination detection method for detecting Mycoplasma synoviae antibodies according to claim 1, characterized in that, In the streptavidin-colored latex microsphere composite, the colored carboxylated polystyrene microspheres have a particle size of 0.3 μm ± 0.05 μm, and the streptavidin coupling density on the microspheres is not less than 5 μg streptavidin / mg microspheres.

5. The plate agglutination detection method for detecting Mycoplasma synoviae antibodies according to claim 1, characterized in that, The streptavidin-colored latex microsphere complex was suspended in a Tris-HCl buffer (pH 8.2) containing 2% trehalose, 1% BSA and 0.05% Tween-20.

6. The plate agglutination detection method for detecting Mycoplasma synoviae antibodies according to claim 1, characterized in that, The sample diluent was a PBS buffer (pH 7.2) containing 0.15M NaCl and 0.5% gelatin.

7. A plate agglutination detection system for Mycoplasma synoviae in chickens, characterized in that, The system includes: (a) Biotinylated MS-specific antigen solution: containing genetically engineered recombinant MS p80 / vlhA fusion protein labeled with NHS-long arm biotin, dissolved in PBS buffer (pH 7.4) containing 0.1% NaN3; (b) Streptavidin-colored latex microsphere complex: containing colored carboxylated polystyrene microspheres covalently coupled with streptavidin, suspended in a buffer solution at pH 8.2; (c) Sample dilution: PBS buffer (pH 7.2) containing 0.15M NaCl and 0.5% gelatin; (d) Positive control serum: rabbit serum containing antibody against MS p80 / vlhA fusion protein; (e) Negative control serum: SPF chicken serum without MS antibody.

8. The chicken synoviae mycoplasma plate agglutination detection system according to claim 7, characterized in that, The concentration of recombinant protein in the biotinylated MS-specific antigen solution was 1.0 mg / mL, and the biotin to protein labeling molar ratio was 15:

1.

9. A method for preparing biotinylated MS-specific antigen solution, characterized in that, Includes the following steps: S1: Expression and purification of genetically engineered recombinant MS p80 / vlhA fusion protein; S2: The fusion protein obtained in S1 was reacted with NHS-long-arm biotin in PBS buffer at pH 7.

4. The molar ratio of biotin to protein was 10:1 to 20:

1. After the reaction was completed, glycine was added to quench the reaction. S3: Purify and adjust the concentration of biotinylated protein to 0.5-1.5 mg / mL, and store in PBS buffer (pH 7.4) containing 0.1% NaN3.

10. The application of a genetically engineered recombinant MS p80 / vlhA fusion protein in the preparation of diagnostic reagents or kits for detecting Mycoplasma synoviae infection in chickens, characterized in that, The amino acid sequence of the fusion protein includes immunodominant fragments from MS p80 and vlhA proteins, linked by a flexible linker peptide (Gly4Ser)3.

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