Sheep paratuberculosis specific detection kit and application

Abstract

The invention discloses a sheep paratuberculosis specific detection kit and application, and relates to the field of biological detection.The kit comprises an elisa plate coated with a recombinant Mycobacterium paratuberculosis membrane protein antigen, the nucleotide sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown as SEQ ID NO: 1, and the amino acid sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown as SEQ ID NO: 2; a confining liquid; a serum diluent; carrying out enzyme labeling on a second antibody; a TMB color developing solution and a stop solution; according to the sheep paratuberculosis specific detection kit disclosed by the invention, the recombinant Mycobacterium paratuberculosis membrane protein antigen is adopted as a core detection component, and the antigen has high specificity and can accurately identify paratuberculosis positive serum and effectively distinguish positive and negative samples; meanwhile, prokaryotic expression and purification are carried out on the specific membrane protein of the mycobacterium paratuberculosis for the first time, and a novel sheep paratuberculosis indirect ELISA diagnosis method with high sensitivity and specificity based on the protein as an antigen is established.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a sheep paratuberculosis specific detection kit and application thereof. Background Art

[0002] Paratuberculosis, also known as Johne's disease, is caused by Mycobacterium paratuberculosis ( Mycobacterium avium subspecies paratuberculosis MAP (Measles, MAP) is a chronic, wasting infectious disease primarily affecting ruminants such as cattle and sheep. It is characterized by chronic enteritis, progressive emaciation, diarrhea, and decreased productivity, causing significant economic losses to the global livestock industry. Sheep infected with MAP have a long incubation period, with clinical symptoms appearing later. However, during this period, they can continuously excrete bacteria into the environment, becoming a significant source of infection. Therefore, the development of rapid and accurate early diagnosis technologies is crucial for preventing and controlling the disease and maintaining herd health.

[0003] Currently, diagnostic methods for paratuberculosis primarily include etiological testing and serological testing. While highly specific, etiological testing (such as bacterial isolation and culture, PCR, etc.) is time-consuming, complex, and costly. Furthermore, it has a low detection rate in animals with subclinical infection or intermittent bacterial excretion, making it unsuitable for large-scale field screening. Serological testing, particularly the enzyme-linked immunosorbent assay (ELISA), has become the preferred tool for population screening and epidemiological surveys due to its ease of use, high throughput, and relatively low cost.

[0004] However, the lack of mature, commercially available domestic paratuberculosis antibody detection kits in the Chinese market forces aquaculture companies and testing institutions to use expensive imported products. These imported kits are not only expensive to purchase but also face the risk of price increases, long lead times, cumbersome import procedures, and even supply disruptions. This severely hinders the widespread and regular implementation of paratuberculosis prevention and control in my country.

[0005] More importantly, the sensitivity and specificity of existing foreign commercial test kits are still not ideal and have significant technical defects. Summary of the Invention

[0006] The present study aims at the specific membrane protein of Mycobacterium avium paratuberculosis (MAP) (GenBank accession number: AAS03514.1; gene accession number: AE016958.1, location 3572243-3573052), which belongs to the enoyl-CoA hydratase family, is one of the cell membrane proteins of MAP, and is an enzyme that hydrates the second and third carbons on acyl-CoA. The enzyme has phorbol ester activity, plays a key role in the process of generating acetyl-CoA and energy from fatty acid metabolism, has the activity of catalyzing the hydration reaction of acyl-CoA, and is involved in the process of fatty acid metabolism. The protein coding gene was cloned and expressed by a prokaryotic expression system, a recombinant expression plasmid was constructed and transformed into Escherichia coli BL21 (DE3), and after IPTG induction expression, the recombinant protein was obtained by Ni column affinity chromatography purification. Western blot analysis showed that the recombinant protein can specifically bind to paratuberculosis positive serum. Based on this, an indirect ELISA detection method is established, and by optimizing the key parameters such as antigen coating concentration and serum dilution ratio, the specific diagnosis of sheep paratuberculosis is realized, and the determination standard is OD 450nm ≥0.471 is positive, which provides a new technical means for the early diagnosis of paratuberculosis, as follows: A specific detection kit for sheep paratuberculosis, comprising: an enzyme-labeled plate coated with a recombinant paratuberculosis membrane protein antigen, the nucleotide sequence of which is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2; a blocking solution; a serum diluent; an enzyme-labeled secondary antibody; TMB developing solution and stopping solution.

[0007] Moreover, the blocking solution is a PBST solution containing 1%-2% BSA.

[0008] Moreover, the serum diluent is a PBST solution containing 1% BSA, and the stopping solution is concentrated sulfuric acid.

[0009] Moreover, the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-sheep IgG, and the dilution ratio is 1:10000.

[0010] Moreover, the antigen coating concentration of the enzyme-labeled plate is 500 ng / well.

[0011] Moreover, the detection conditions of the kit include: serum dilution ratio is 1:200; antigen coating condition is 37℃ coating for 3 hours; blocking time is 60-90 minutes; reaction time of the serum to be tested with recombinant Mycobacterium avium paratuberculosis membrane protein antigen is 30-60 minutes; incubation time of the secondary antibody is 30-45 minutes; color developing time is 5-10 minutes.

[0012] On the other hand, the present invention provides the use of a sheep paratuberculosis-specific detection kit for preparing a sheep paratuberculosis diagnosis product.

[0013] Moreover, when the OD value of the serum sample to be tested is ≥0.471, it is judged as positive, and ≤0.418, it is judged as negative. <OD 450nm When the value was <0.418, it was judged as suspected positive.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Compared with the expensive and ever-increasing imported test kits, the test kit provided by the present invention has significant economic advantages while ensuring excellent performance, making it "affordable and effective" for the majority of breeding enterprises and grassroots testing units, greatly reducing the economic burden of prevention and control work, and conducive to promoting the popularization and application of paratuberculosis testing nationwide.

[0015] 2. The kit provided by the present invention uses recombinant Mycobacterium paratuberculosis membrane protein antigen as the core detection component, can efficiently detect low-concentration antibodies, and can effectively avoid cross-reactions with common disease sera such as sheep Escherichia coli disease, Salmonella typhimurium disease, and bovine coronavirus infection, greatly ensuring the accuracy and specificity of the diagnostic results; Western The blot verification results showed that the antigen only specifically binds to paratuberculosis positive serum and has no reaction with negative serum, confirming its high specificity, which can effectively distinguish positive and negative samples and significantly reduce the risk of false positives and false negatives. The sensitivity test showed that in the detection of weak positive serum, the ELISA method of the present invention was positive in all 6 tests, showing excellent sensitivity. In comparison, the IDvet kit showed 1 negative and 3 suspected results, and the IDEXX kit showed 2 negative and 4 suspected results, indicating that the imported kits have insufficient detection capabilities for low-concentration antibodies, and are prone to missed detection or inability to make a clear judgment. Clinical verification showed that the positive detection rate of 466 wild sheep serum samples tested with this kit was as high as 41.85%, which was significantly better than the IDvet kit (6.22%) and IDEXX kit (3.43%). This highlights that the method provided by the present invention has higher sensitivity than existing kit detection methods, which is conducive to the early detection and precise prevention and control of infected animals. The sensitivity test confirmed that even if the serum was diluted to 1:1600 times, its OD 450nm The value was still higher than the positive critical value, indicating that the method has a very high detection ability for low-abundance antibodies and is of great significance for the diagnosis of early and latent infections; the specificity test results clearly showed that the OD values ​​of the positive sera of three other common sheep diseases were 450nmThe values ​​of the paratuberculosis positive serum were all lower than the negative critical value, while the values ​​of the paratuberculosis positive serum were clearly higher than the positive critical value, which once again strongly demonstrated the excellent specificity of the method of the present invention.

[0016] 3. Serum samples from sheep artificially infected with MAP were used to evaluate the ELISA method of the present invention for its ability to accurately detect positive samples in the early stages of infection and at different stages of infection. The test results showed that the method could detect positive sera as early as 75 days after MAP inoculation, and the positive detection rate significantly increased with the extension of infection time (for example, the positive rate reached 46.43% among 84 sera), indicating its high sensitivity. The serum test results of the artificially infected group and the control group were compared to confirm the ELISA method of the present invention's ability to accurately distinguish between positive and negative paratuberculosis samples. In the experiment, sera from sheep in the control group tested negative at all time periods, while sera from sheep in the infected group showed a high positive rate, demonstrating the excellent specificity of the method. The ELISA method of the present invention was compared with imported commercial kits from ID Vet and IDEXX to evaluate its performance in terms of positive detection rate, earliest detection time, and distribution of positive samples. The results showed that the positive detection rate (46.43%) of the present method was significantly superior to the other two kits (10.71% for ID Vet and 8.33% for IDEXX), and it was able to detect positive samples earlier and more widely. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the SDS-PAGE results of the induced expression of recombinant Mycobacterium paratuberculosis membrane protein antigen; Figure 2 This is the result of exploring the imidazole concentration of recombinant Mycobacterium paratuberculosis membrane protein antigen; Figure 3 This is the Western blot result of recombinant Mycobacterium paratuberculosis membrane protein antigen; Figure 4 Focal granulomas with fibrosis in the ileal submucosa; Figure 5 MAP culture identification results (Ziehl-Neelsen acid-fast staining of bacterial smear, showing uniform short rod-shaped acid-fast bacilli). DETAILED DESCRIPTION

[0018] Example 1 Construction of prokaryotic expression vector A recombinant Mycobacterium paratuberculosis membrane protein antigen gene (GenBank AE016958.1, positions 3572243-3573052) was selected as the target gene for construction of a prokaryotic expression vector. The nucleotide sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0019] The recombinant M. paratuberculosis membrane protein antigen gene is fused and cloned into the vector pET-30a by primer synthesis, PCR and other steps. The fusion His tag is expressed by the E. coli expression system to express the target protein, and expression and purification tests are performed.

[0020] The insertion is in the pET-30a prokaryotic expression vector, and the construction scheme is pET-30a+6*His+S-Tag Bam H I + target protein Hin d III, respectively inserted into the restriction enzyme Bam H I, Hin d III cleavage site. S-Tag is a new type of recombinant protein fusion peptide tag.

[0021] Forward primer: SEQ ID NO: 3; Reverse primer: SEQ ID NO: 4.

[0022] Example 2 IPTG-induced expression of recombinant bacteria fusion protein Take 250mL and 1000mL of conical flask for high pressure sterilization and place it in the ultraclean bench for ultraviolet irradiation for 30min. In a 250mL conical flask, add 50mL of LB medium containing 50μg / mL kanamycin, take the positive bacteria 1:100, add it to the conical flask, and incubate it in a shaking incubator at 37℃, 220r / min overnight. Take the positive bacteria (the remaining bacteria are mixed with 50% glycerol at a ratio of 1:1 and stored in a -80℃ refrigerator for standby) after shaking for 1:1000, add it to 300mL of LB broth containing 50μg / mL of kanamycin, and incubate it at 37℃, 220r / min until the OD 600nm is 0.6-0.8, add IPTG with a final concentration of 1.0mmol / L, and induce it at 37℃, 180r / min for 8h. Perform solubility analysis by the following operations: (1) After induction, centrifuge the bacteria at 7000r / min for 15min and weigh the bacteria; (2) Add 10mL of Binding Buffer to each 1g of bacteria for resuspension; (3) Resuspend the bacteria by ultrasonic crushing in an ice bath for 25min (150w, work for 3s and stop for 5s); (4) Centrifuge the ultrasonic crushed bacteria at 4℃, 12000r / min for 15min to collect the supernatant and the precipitate, and add Binding Buffer containing 8M urea to the precipitate at 4℃ overnight; (5) Take 8 μL of supernatant and 8 μL of precipitate, add 2 μL of protein loading buffer (5×), and boil in a water bath at 100°C for 10 min; (6) SDS-PAGE: Prepare protein gel according to the instructions of the SDS-PAGE gel rapid preparation kit. Set up control groups: E. coli induced and uninduced, E. coli containing the pET-30a empty vector induced and uninduced, and positive bacteria uninduced. After ultrasonic disruption, add 10 μL of the supernatant and precipitate to each well and perform SDS-PAGE gel electrophoresis. Stain the protein gel with Coomassie Brilliant Blue for 20 minutes, then add destaining solution. After destaining, observe the results. (7) Expression identification Figure 1 As shown (Note: M: protein molecular weight standard; 1: whole uninduced E. coli; 2: whole induced E. coli; 3: uninduced pET-30a empty vector; 4: induced pET-30a empty vector; 5: uninduced supernatant; 6: uninduced precipitate; 7: induced supernatant; 8: induced precipitate). The recombinant protein after induction was 34.2 kDa, consistent with the expected size, and was present in both soluble and inclusion bodies. Protein at the 34.2 kDa position was also detected in E. coli (lanes 1, 2, 3, and 4), but the IPTG-induced supernatant (lane 7) was significantly thicker than the uninduced supernatant (lane 5). Furthermore, the induced precipitate (lane 8) showed a more distinct band at the 34.2 kDa position, preliminarily indicating successful expression of the recombinant protein in E. coli BL21(DE3). Subsequent Western blot analysis confirmed that both the supernatant and the precipitate were specifically expressed products of the target protein.

[0023] Example 3 Ni column affinity chromatography purification, concentration and concentration determination After optimizing the induction expression conditions for the recombinant protein, the protein was purified using a pre-packed gravity column of Sangon HyPur T Ni-NTA 6FF (His-Tag). The steps are as follows: (1) Obtaining a supernatant solution containing recombinant protein according to the optimized induction conditions; (2) Filter the supernatant solution using a 0.45 μm filter membrane to remove impurities; (3) Wash the purification column with 5 column volumes of ultrapure water, equilibrate the purification column with 5 column volumes of Binding Buffer, then slowly add the filtered supernatant solution, continue to load the flow-through solution onto the column, repeat the filtration 3 times, add the filtered flow-through solution into the column and bind at 4°C overnight; Elute the purification column with Wash Buffer and reserve the eluate for SDS-PAGE analysis; (5) The filtrate collected from 250 mM imidazole and later was loaded into an ultrafiltration tube, and the centrifuge parameters were adjusted to 4°C, 3000 r / min, and centrifuged for 30 min. When the volume of the solution in the ultrafiltration tube reached 1 mL, the centrifugation was stopped, 9 mL of PBS solution was added, and the centrifugation was continued. This ultrafiltration was repeated three times to complete the replacement of the protein buffer, and finally 1 mL of protein concentrate was collected; (6) SDS-PAGE analysis was performed, and the protein was eluted with an imidazole solution with a concentration of 50 mmol / L-500 mmol / L, and the results are shown in Figure 2 (Note: M: protein molecular weight standard; 1: protein without column; 2: protein after column; 3-9: 50, 100, 150, 200, 250, 300, 500 mmol / L imidazole elution): The maximum amount of target protein can be collected at 250 mmol / L imidazole elution; (7) According to the BCA protein quantitative kit (C503021 BCA method protein concentration determination kit Sangon Biotech) instructions, the protein concentration standard curve y = 1079.7x - 12.079 (x is the absorbance of protein at OD 562nm ; y is the protein concentration unit μg / mL, R² = 0.9953) was drawn. The purified and ultrafiltrated recombinant protein was measured according to the BCA protein quantitative instructions, and the absorbance at OD 562nm was 1.400. Substituting the standard curve formula, the result showed that the protein concentration after ultrafiltration was 1499.5 μg / mL.

[0024] Example 4 Western bolt analysis of recombinant protein The purified and concentrated Mycobacterium paratuberculosis specific membrane protein was diluted and mixed with protein loading buffer at a ratio of 4:1, and then heated at 100°C for 10 min for standby. The Mycobacterium paratuberculosis specific membrane protein was detected by Western Blot according to the following operation: (1) SDS-PAGE: The prepared protein sample was loaded with 10 μL per well for SDS-PAGE. (90V electrophoresis for 30 min to move the protein sample to the boundary between the concentrated gel and the separation gel, and then transfer to 120V electrophoresis for 60 min); (2) Membrane transfer: After electrophoresis, the upper concentrated gel was removed, and the PVDF membrane was cut to the appropriate size according to the size of the separation gel. In the order of "sponge-filter paper-PVDF membrane-gel-filter paper-sponge", put it into a vertical electrophoresis tank with ice box and membrane transfer solution, 250mA constant current membrane transfer for 60 min; (3) Blocking: After the transfer is completed, remove the PVDF membrane and wash it on a shaker with 1×TBST for 5 times, each time for 5 minutes. After washing, place the PVDF in blocking solution at 4°C overnight; (4) Primary antibody incubation: The next day, the blocked PVDF membrane was removed and washed five times with 1×TBST on a shaker for 5 minutes each time. The PVDF membrane was cut according to the lane position and placed in different antibodies and serum diluted in blocking solution, and incubated on a shaker at room temperature for 2 hours. (5) Secondary antibody incubation: The PVDF membrane was washed five times with 1× TBST on a shaker for 5 min each time, and then placed in the enzyme-labeled secondary antibody diluted in TBST and incubated on a shaker at room temperature for 2 h; (6) Color development: The PVDF membrane was washed five times with 1×TBST on a shaker, each time for 5 min. ECL was added dropwise in a dark room for color development and the results were observed using an imager.

[0025] The results of WB identification of recombinant protein are as follows Figure 3 As shown in the table (Note: M: protein molecular weight standard; 1: mouse His tag antibody; 2: sheep paratuberculosis positive serum; 3: sheep paratuberculosis negative serum), the recombinant protein specifically binds to the mouse His tag antibody and sheep paratuberculosis positive serum at 34.2 kDa, but does not bind to sheep paratuberculosis negative serum.

[0026] Example 5 ELISA test (1) Antigen coating: Dilute the purified Mycobacterium paratuberculosis specific membrane protein to 10 μg / mL with ELISA coating solution (carbonate buffer, pH = 9.6), add 100 μL / well to a 96-well ELISA plate, and coat at 4°C for 16 h. (2) Blocking: Discard the liquid in the wells, wash three times with 1× PBST, and pat dry for the final time. Add 200 μL / well of blocking solution (PBST solution containing 2% BSA) and block at 37°C for 2 hours; (3) Primary antibody incubation: Discard the liquid in the wells, wash three times with 1× PBST, and pat dry for the final time. Add diluent (PBST solution containing 1% BSA) to dilute the test serum, 100 μL / well, and incubate at 37°C for 1 hour; (4) Secondary antibody incubation: Discard the liquid in the wells, wash three times with 1×PBST, and pat dry for the final time. Add HRP-rabbit anti-goat IgG diluted 1:10000 in diluent (PBST solution containing 1% BSA) and incubate at 37°C for 30 minutes; (5) Color development and OD measurement 450nm : Discard the liquid in the wells, wash with 1×PBST 5 times, and pat dry for the last time. Add 100μL / well of substrate TMB solution, incubate at room temperature in the dark for 10 minutes, stop the color development with stop solution (concentrated sulfuric acid) and measure the OD 450nm Absorbance value.

[0027] A single-factor experiment was conducted under the above conditions, as follows: 5.1 Antigen coating concentration and optimal serum dilution The recombinant protein was coated with coating solution at 2000ng / well, 1500ng / well, 1000ng / well, 750ng / well, 500ng / well, 375ng / well, 250ng / well, 187.5ng / well, 125ng / well, 93.75ng / well, 62.5ng / well, and 46.875ng / well, with two replicate wells for each concentration. The primary antibody (sheep paratuberculosis negative and sheep paratuberculosis positive serum) was diluted with antibody diluent at 1:100, 1:200, 1:400, and 1:800, with two replicate wells for each concentration. The OD value was read on a microplate reader. 450nm The P / N value was calculated from the values ​​obtained above. The antigen concentration and serum dilution ratio at which the P / N value was the maximum were the best. The results are shown in Table 1. When the antigen coating concentration was 500 ng / well and the serum dilution ratio was 1:200, the maximum P / N value was 3.856.

[0028] Table 1 Determination of antigen and serum dilution

[0029] 5.2 Determination of the optimal coating temperature and time Set 4 different coating temperatures and times and read the OD value on the microplate reader 450nm The results are shown in Table 2. When the antigen was coated at 37°C for 1 hour, the maximum P / N value was 3.911, which confirmed that coating at 37°C for 3 hours was the optimal condition.

[0030] Table 2 Optimal antigen coating temperature and time results

[0031] 5.3 Determination of the optimal blocking solution Select four blocking solutions to read the OD value on the microplate reader 450nm The results were tabulated and analyzed. As shown in Table 3, the maximum P / N value was 4.099 when using 2% BSA blocking buffer. This confirms that 2% BSA is the optimal blocking buffer.

[0032] Table 3 Results of the best blocking solution

[0033] 5.4 Optimization of the optimal closing time According to the above optimized indirect ELISA operation steps, four different blocking time were selected for blocking and the OD value of the microplate reader was read. 450nmThe results are shown in the table. When the sealing time is 90 minutes, the maximum P / N value is 4.311, which determines that the optimal sealing time is 90 minutes.

[0034] Table 4 Optimal closure time results

[0035] 5.5 Optimal primary antibody incubation time According to the above optimized conditions, set 4 serum incubation times and read the OD value of the microplate reader. 450nm The results are shown in Table 5. When the blocking condition is 60 minutes, the maximum P / N value is 4.206, and the optimal incubation time for primary antibody is determined to be 60 minutes.

[0036] Table 5 Optimal serum incubation time results

[0037] 5.6 Optimal dilution of secondary antibody According to the above optimized conditions, set the dilution multiples of 7 enzyme-labeled secondary antibodies and read the OD value of the microplate reader. 450nm The results are shown in Table 6. When the enzyme-labeled secondary antibody dilution factor was 1:10,000, the maximum P / N value was 3.965, confirming that the enzyme-labeled secondary antibody dilution factor of 1:10,000 was the optimal condition.

[0038] Table 6 Optimal dilution of secondary antibodies

[0039] 5.7 Optimal secondary antibody incubation time According to the above optimized conditions, set 4 secondary antibody incubation times for incubation. Read the OD value on the microplate reader. 450nm The results are shown in Table 7. When the secondary antibody was incubated for 45 minutes, the maximum P / N value was 5.348, and the incubation time of the enzyme-labeled secondary antibody was determined to be 45 minutes.

[0040] Table 7 Optimal secondary antibody incubation time results

[0041] 5.8 Optimal color development time results Set 4 color development times for optimization and read the OD value on a microplate reader. 450nm The results are shown in Table 8. When the color development time is 10 minutes, the maximum P / N value is 5.341, and the optimal color development time is determined to be 10 minutes.

[0042] Table 8 Optimal color development time results

[0043] 5.9 Determination of critical value According to the above optimized results, 30 negative serum samples of paratuberculosis sheep were selected for testing and the OD was measured using a microplate reader. 450nm The results are shown in Table 9. The average OD of 30 sheep negative serum samples was 450nm The value (X) is 0.3119 and the standard deviation (SD) is 0.0531. Determine OD 450nm When OD 450nm ≥0.471 was considered positive, and when 0.471>OD 450nm The interval >0.418 was judged as suspected.

[0044] According to the above optimized results, 30 negative serum samples of paratuberculosis sheep were selected for testing and the OD was measured using a microplate reader. 450nm The results are shown in Table 9. After statistical analysis, the mean (X) was 0.3119 and the standard deviation (SD) was 0.0531. Based on the statistical principle of determining critical values ​​using the "mean ± multiple standard deviation" formula, the negative judgment threshold was calculated to be X + 2SD (0.3119 + 2 × 0.0531 ≈ 0.418), and the positive judgment threshold was calculated to be X + 3SD (0.3119 + 3 × 0.0531 ≈ 0.471). Therefore, the judgment criteria are as follows: OD 450nm ≤ 0.418 is negative, OD 450nm ≥ 0.471 is positive, 0.418 <OD 450nm <0.471 is suspected.

[0045] Table 9 Determination of critical values

[0046] 5.10 Repeatability test (1) Intra-batch repeatability test: At the same time, three ELISA plates made from the same batch were used to test the serum, and the OD was measured using an ELISA reader. 450nm Data analysis was performed using the same values. The coefficient of variation (CV) was calculated according to the formula. The results are shown in Table 10. The highest intra-assay coefficient of variation was 13.10%, and the lowest was 4.52%, both below 15%.

[0047] Table 10 Intra-batch repeatability test results

[0048] (2) Inter-batch repeatability test: At the same time, serum was tested using ELISA plates made from three different batches, and OD was measured using an ELISA reader. 450nmData analysis was performed using the same values. The coefficient of variation (CV) was calculated according to the formula. The results are shown in Table 11. The highest inter-assay coefficient of variation was 14.97%, and the lowest was 6.93%, both below 15%.

[0049] Table 11 Results of inter-batch repeatability test

[0050] 5.11 Sensitivity test According to the optimized conditions, the positive serum of paratuberculosis sheep stored in the laboratory was diluted by 7 times and tested, and the OD was measured using a microplate reader. 450nm The results are shown in Table 12. When the serum dilution ratio is 1:1600, OD 450nm The value is higher than the positive cut-off value.

[0051] Table 12 Sensitivity test results

[0052] The ELISA developed in the present invention was compared with imported commercial kits from ID Vet and IDEXX to test six sera, each containing strongly, moderately, and weakly positive paratuberculosis sera, six times. The results, as shown in Table 13, show that the ELISA developed in the present invention showed positive results in all six tests for strongly, moderately, and weakly positive sera, while ID Vet and IDEXX showed different results for moderately and weakly positive sera. This demonstrates that the ELISA method developed in the present invention has greater sensitivity.

[0053] Table 13 Positive serum test results

[0054] 5.12 Specificity Test Detect 3 other common sheep disease sera (paratuberculosis test is negative), use the negative and positive sera of sheep paratuberculosis in the laboratory as control, and use a microplate reader to measure OD 450nm The results are shown in Table 14. The OD values ​​of the three positive sera for other diseases were 450nm The values ​​were all lower than the negative critical value, while those for paratuberculosis positive serum were higher than the positive critical value.

[0055] Table 14 Specificity test results

[0056] Example 6 Clinical Testing (1) Field samples 466 sheep serum samples were collected from 466 sheep in the wild and tested simultaneously using the ELISA method established in the present invention and imported commercial kits from ID Vet and IDEXX. The results, as shown in Table 15, show a positive rate of 41.85% for the ELISA method established in the present invention, while the results for the ID Vet and IDEXX kits were 6.22% and 3.43%, respectively. To avoid false positives, 30 days later, 20% (39) of the 195 sheep that tested positive using the ELISA method established in the present invention were randomly collected and tested using the ID Vet kit, the IDEXX kit, and the ELISA method established in the present invention. The results showed that 31 sheep tested positive using the ID Vet kit, 27 using the IDEXX kit, and 39 using the ELISA method established in the present invention were positive.

[0057] Table 15 Field clinical sample test results

[0058] (2) Sheep artificially infected with MAP test samples Serum samples from 112 sheep artificially infected with MAP (2 control sheep + 6 infected sheep, 14 samples each) were tested using the ELISA method developed in this invention and commercially available kits from ID Vet and IDEXX. The results are shown in Table 16: All three methods tested negative for sera collected from the negative control group of the animal experiment at all time periods. For the artificially infected group (6 sheep, pathological observation showed that the sera were negative for MAP). Figure 4 : Focal granuloma with fibrosis in the ileal submucosa, HE staining); pathogen isolation and identification ( Figure 5 MAP culture identification results: Ziehl-Neelsen acid-fast staining of bacterial smears showed uniform short-rod-shaped acid-fast bacilli; tissue nucleic acid testing identified all infected sheep as infected, and animal experiments were successfully established. The ELISA method established by the present invention had a positive rate of 46.43%, while the results of ID Vet and IDEXX kits were 10.71% and 8.33%, respectively. The earliest detection by the three methods was 75 days after MAP inoculation. The ID Vet kit detected 9 seropositive results in 2 sheep; the IDEXX test detected 7 seropositive results in only 1 sheep (one of the sheep that tested positive by ID Vet); and the ELISA method established by the present invention detected 39 seropositive results in 5 sheep (including the 2 sheep that tested positive by the other two methods).

[0059] Table 16 Test results of artificially infected samples

[0060] Table 17 Detailed results of artificially infected sample testing

Claims

1. A specific detection kit for ovine paratuberculosis, characterized in that: Include: An ELISA plate coated with a recombinant Mycobacterium paratuberculosis membrane protein antigen, wherein the nucleotide sequence of the recombinant Mycobacterium paratuberculosis membrane protein antigen is shown in SEQ ID NO: 1 and the amino acid sequence is shown in SEQ ID NO: 2; Blocking solution; serum diluent; enzyme-labeled secondary antibody; Substrate TMB color development solution and stop solution.

2. A sheep paratuberculosis specific detection kit according to claim 1, characterized in that, The blocking solution is a PBST solution containing 1%-2% BSA.

3. A sheep paratuberculosis specific detection kit according to claim 1, characterized in that, The serum diluent was a PBST solution containing 1% BSA, and the stop solution was concentrated sulfuric acid.

4. A sheep paratuberculosis specific detection kit according to claim 1, characterized in that, The enzyme-labeled secondary antibody was HRP-labeled rabbit anti-goat IgG, and the dilution ratio was 1:10,000.

5. A sheep paratuberculosis specific detection kit according to claim 1, characterized in that, The antigen coating concentration of the ELISA plate was 500 ng / well.

6. A sheep paratuberculosis specific detection kit according to claim 1, characterized in that: The detection conditions of the kit include: serum dilution ratio of 1:200; antigen coating conditions of 37°C for 3 hours; blocking time of 60-90 minutes; reaction time of the test serum and recombinant Mycobacterium paratuberculosis membrane protein antigen of 30-60 minutes; secondary antibody incubation time of 30-45 minutes; and color development time of 5-10 minutes.

7. The use of a sheep paratuberculosis specific detection kit as claimed in claim 1, characterized in that: Used to prepare sheep paratuberculosis diagnostic products.

8. The use of a sheep paratuberculosis specific detection kit according to claim 7, characterized in that: When the OD of the serum sample to be tested 450nm The value ≥0.471 was judged as positive, ≤0.418 was judged as negative, and when 0.471 < OD 450nm When the value was <0.418, it was judged as suspected positive.

Images