A specific detection kit for paratuberculosis of sheep and application thereof

Abstract

The application discloses a specific detection kit for sheep paratuberculosis and application, relates to the field of biological detection, and the kit comprises: an enzyme-labeled plate coated with recombinant paratuberculosis mycobacterium membrane protein antigen, the nucleotide sequence of the recombinin paratuberculosis mycobacterium membrane protein antigen is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2; a blocking solution; a serum diluent; an enzyme-labeled secondary antibody; TMB color developing solution and a termination solution; the specific detection kit for sheep paratuberculosis disclosed by the application adopts the recombinant paratuberculosis mycobacterium membrane protein antigen as a core detection component, the antigen has high specificity, can accurately identify positive serum of paratuberculosis, and effectively distinguishes positive and negative samples; meanwhile, the specific membrane protein of paratuberculosis mycobacterium is prokaryotically expressed and purified 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 application relates to the field of biological detection, and particularly relates to a goat paratuberculosis specific detection kit and application. BACKGROUND

[0002] Paratuberculosis, also known as Johne's disease, is a chronic and wasting infectious disease caused by Mycobacterium avium subspecies paratuberculosis (MAP), which mainly infects ruminants such as cattle and sheep, and is characterized by chronic enteritis, progressive emaciation, diarrhea and decline in production performance, causing huge economic losses to the global livestock industry. Mycobacterium avium subspecies paratuberculosis After sheep are infected with MAP, the incubation period is long, and clinical symptoms appear late, but during this period, bacteria can be continuously discharged into the environment, becoming an important source of infection. Therefore, developing a rapid and accurate early diagnosis technique is crucial for the prevention and control of the disease and the purification of livestock.

[0003] At present, the diagnosis methods of paratuberculosis mainly include etiological detection and serological detection. Although etiological detection (such as bacterial isolation and culture, PCR, etc.) has high specificity, it is time-consuming, complex, and costly, and has low detection rate for subclinical infection or intermittent bacteria discharge animals, which is not suitable for large-scale field screening. Serological detection, especially enzyme-linked immunosorbent assay (ELISA), is the preferred tool for population screening and epidemiological investigation due to its simple operation, high throughput and relatively low cost.

[0004] However, there is a lack of mature domestic paratuberculosis antibody detection commercial kit in the market.

[0005] At the same time, the existing foreign commercial kits are not only expensive, but also have unsatisfactory sensitivity and specificity, and have significant technical defects. SUMMARY

[0006] The present study is aimed 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 fatty acid metabolism process. 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:

[0007] A specific detection kit for sheep paratuberculosis, comprising:

[0008] An enzyme-labeled plate coated with a recombinant paratuberculosis membrane protein antigen, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2;

[0009] Blocking solution;

[0010] Serum diluent;

[0011] Enzyme-labeled secondary antibody;

[0012] TMB developing solution and stopping solution.

[0013] Moreover, the blocking solution is a PBST solution containing 1%-2% BSA.

[0014] Moreover, the serum diluent is a PBST solution containing 1% BSA, and the stopping solution is concentrated sulfuric acid.

[0015] Moreover, the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-sheep IgG with a dilution ratio of 1:10,000.

[0016] Moreover, the antigen coating concentration of the enzyme-labeled plate is 500 ng / well.

[0017] Moreover, the detection condition of the kit comprises: serum dilution multiple is 1:200; antigen coating condition is coating for 3 hours at 37 DEG C; blocking time is 60-90 minutes; reaction time of the serum to be detected and recombinant paratuberculosis mycobacterium membrane protein antigen is 30-60 minutes; secondary antibody incubation time is 30-45 minutes; color development time is 5-10 minutes.

[0018] In another aspect, the application provides the use of the specific detection kit for paratuberculosis of sheep, for preparing a diagnostic product for paratuberculosis of sheep.

[0019] Moreover, when the OD value of the serum sample to be detected is greater than or equal to 0.471, it is determined as positive, and when the OD value is less than or equal to 0.418, it is determined as negative, and when the OD value is greater than 0.471 and less than 0.418, it is determined as suspected positive. 450nm 450nm

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] 1. The kit provided by the application has significant economic advantages while ensuring excellent performance, compared with the imported kit which is expensive and rising in price, so that the majority of breeding enterprises and primary detection units can afford and use it well, greatly reducing the economic burden of prevention and control work, and being conducive to promoting the popularization and application of paratuberculosis detection in the whole country.

[0022] 2. The kit provided by the application takes recombinant paratuberculosis mycobacterium membrane protein antigen as the core detection component, can efficiently detect low-concentration antibodies, and can effectively avoid cross-reactions with serum of common diseases such as sheep colibacillosis, salmonellosis and bovine coronavirus infection, greatly ensuring the accuracy and specificity of the diagnostic results; Western blot verification results show that the antigen only specifically binds with positive serum of paratuberculosis, and has no reaction with negative serum, proving that it has high specificity, can effectively distinguish positive and negative samples, and significantly reduces the risk of false positives and false negatives; sensitivity test shows that in weak positive serum detection, the ELISA method of the application is all positive in 6 detections, showing excellent sensitivity, compared with that, the IDvet kit appears 1 negative and 3 suspected, and the IDEXX kit appears 2 negatives and 4 suspected, indicating that the imported kit has insufficient detection ability for low-concentration antibodies, and is easy to miss detection or cannot be determined clearly; clinical verification shows that 466 field sheep serum samples are detected by using the kit, and the positive detection rate is as high as 41.85%, which is significantly better than that of the IDvet kit (6.22%) and the IDEXX kit (3.43%), highlighting that the method provided by the application has higher sensitivity than the existing kit detection, and is helpful to realize early detection and precise prevention and control of infected animals; sensitivity test proves that even if the serum is diluted to 1:1600 times, the OD value of the kit is still greater than 0.471, which is significantly higher than that of the IDvet kit (0.370) and the IDEXX kit (0.370), proving that the kit provided by the application has higher sensitivity than the existing kit detection, and is helpful to realize early detection and precise prevention and control of infected animals. 450nmThe value is still higher than the positive critical value, indicating that the method has very high detection capability for low abundance antibodies, and is of great significance for the diagnosis of early and latent infection; the specificity test results clearly show that the OD 450nm values of the three other common sheep disease positive sera are lower than the negative critical value, while the paratuberculosis positive serum is clearly higher than the positive critical value, again proving the excellent specificity of the method of the application.

[0023] 3. The serum samples of sheep artificially infected with MAP were used to evaluate whether the ELISA method of the application could accurately detect positive samples in the early stage of infection and at different stages of infection. The test results showed that the method could detect positive serum 75 days after inoculation with MAP, and the positive detection rate significantly increased with the extension of the infection time (such as 46.43% positive rate in 84 serum samples), indicating that it has high sensitivity; by comparing the serum detection results of the artificial infection group and the control group, it was confirmed whether the ELISA method of the application could accurately distinguish paratuberculosis positive and negative samples. In the test, the serum of all time periods of the control group sheep was detected as negative, while the infected sheep showed a high positive rate, proving that the method has excellent specificity; the ELISA method of the application was compared with ID Vet and IDEXX imported commercial kits to evaluate its performance in positive detection rate, earliest detection time and positive sample distribution, etc. The results showed that the positive detection rate (46.43%) of the method of the application was significantly better than the other two kits (ID Vet was 10.71%, and IDEXX was 8.33%), and it could detect positive samples earlier and more extensively. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 SDS-PAGE results of recombinant paratuberculosis mycobacterium membrane protein antigen induced expression;

[0025] Figure 2 Imidazole concentration exploration results of recombinant paratuberculosis mycobacterium membrane protein antigen;

[0026] Figure 3 Western blot results of recombinant paratuberculosis mycobacterium membrane protein antigen;

[0027] Figure 4 Results of ileal submucosal focal granuloma with fibrosis;

[0028] Figure 5 MAP culture identification results (bacterial smear Ziehl-Neelsen acid-fast staining, showing uniform short rod-shaped acid-fast bacilli). DETAILED DESCRIPTION

[0029] Example 1 Construction scheme of prokaryotic expression vector

[0030] The recombinant Mycobacterium paratuberculosis membrane protein antigen gene (GenBank AE016958.1, position 3572243-3573052) is selected as the target gene for the 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.

[0031] The recombinant Mycobacterium paratuberculosis membrane protein antigen gene is fused and cloned into the vector pET-30a by primer synthesis and PCR steps. The His tag is expressed by the E. coli expression system to express the target protein, and expression and purification tests are performed.

[0032] The recombinant Mycobacterium paratuberculosis membrane protein antigen gene is fused and cloned into the vector pET-30a by primer synthesis and PCR steps. The His tag is expressed by the E. coli expression system to express the target protein, and expression and purification tests are performed. Bam H I + target protein + Hin d III, respectively, and the restriction enzyme Bam H I, Hin d III cleavage site. S-Tag is a new type of recombinant protein fusion peptide tag.

[0033] Forward primer: SEQ ID NO: 3;

[0034] Reverse primer: SEQ ID NO: 4.

[0035] Example 2 IPTG-induced expression of recombinant bacteria fusion protein

[0036] Take 250mL and 1000mL of conical flask for high pressure sterilization and then place them in the ultraclean bench for ultraviolet irradiation for 30min. Add 50mL of LB medium containing 50μg / mL kanamycin to the 250mL conical flask, and take the positive bacteria at a ratio of 1:100 and add them to the medium. Incubate at 37℃, 220r / min in a shaking incubator overnight. The next day, take the positive bacteria after shaking (mix the remaining bacteria with 50% glycerol at a ratio of 1:1 and store them in a -80℃ freezer for future use) at a ratio of 1:1000 and add them to 300mL of LB broth containing 50μg / mL of kanamycin. Incubate at 37℃, 180r / min until the OD 600nm is 0.6-0.8. Add IPTG with a final concentration of 1.0mmol / L, and induce at 37℃, 180r / min for 8h. Perform solubility analysis by the following operations:

[0037] (1) Centrifuge the bacteria at 7000r / min for 15min after induction, collect the bacteria and weigh them;

[0038] (2) each 1 g of bacteria was added 10 mL of Binding Buffer for resuspension;

[0039] (3) the bacteria were resuspended and broken by ice bath ultrasonic for 25 min (150 w, 3 s working and 5 s stopping);

[0040] (4) the bacteria broken by ultrasonic were centrifuged at 4 ℃ and 12000 r / min for 15 min to collect the supernatant and the precipitate, and the precipitate was added with Binding Buffer containing 8M urea and left at 4 ℃ overnight;

[0041] (5) 8 μL of the supernatant and the precipitate were taken and added with 2 μL of protein loading buffer (5x), and boiled in a water bath at 100 ℃ for 10 min;

[0042] (6) SDS-PAGE: the protein gel was prepared according to the instructions of the SDS-PAGE gel preparation kit. The control group was set up: E. coli induced and uninduced, E. coli containing pET-30a empty vector induced and uninduced, and supernatant and precipitate of positive bacteria uninduced after ultrasonic breaking, 10 μL of each sample was added and subjected to SDS-PAGE gel electrophoresis, the protein gel was dyed with coomassie brilliant blue staining solution for 20 min, then decolorizing solution was added, and the results were observed after decolorization was completed;

[0043] (7) the expression identification was as shown in Figure 1 (Notes: M: protein molecular weight marker; 1: E. coli whole bacteria uninduced; 2: E. coli whole bacteria induced; 3: pET-30a empty vector uninduced; 4: pET-30a empty vector induced; 5: uninduced breaking supernatant; 6: uninduced breaking precipitate; 7: induced breaking supernatant; 8: induced breaking precipitate). The size of the recombinant protein after induction was 34.2 kDa, which was consistent with the expected size, and the recombinant protein coexisted in the form of solubility and inclusion body. E. coli (lanes 1, 2, 3 and 4) also had protein at the position of 34.2 kDa, but the IPTG induced breaking supernatant (lane 7) was significantly thicker than the uninduced breaking supernatant (lane 5), and the induced breaking precipitate (lane 8) had a more obvious band at the position of 34.2 kDa, which preliminarily indicated that the recombinant protein was successfully expressed in E. coli BL21 (DE3). Subsequently, it was proved by Western bolt that the band in the supernatant and the precipitate was the specific expression product of the target protein.

[0044] Example 3 Ni column affinity chromatography purification, concentration and concentration determination

[0045] After the optimization of the recombinant protein induction expression conditions, the preloaded gravity column of Shengong HyPur T Ni-NTA 6FF (His-Tag) was selected for protein purification. The operation steps are as follows:

[0046] (1) Obtain the supernatant solution containing the recombinant protein according to the optimized induction conditions;

[0047] (2) Filter the supernatant solution using a 0.45 μm filter membrane to remove impurities;

[0048] (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 flow through the column, and repeatedly filter 3 times. Add the filtered flow-through solution to the column and bind at 4°C overnight;

[0049] Wash the purification column with Wash Buffer, and analyze the eluate by SDS-PAGE;

[0050] (5) Place the filtrate collected from 250 mM imidazole into an ultrafiltration tube, and adjust the centrifuge parameters to 4°C, 3000 r / min, and centrifuge for 30 min. When the volume of the solution in the ultrafiltration tube reaches 1 mL, stop the centrifugation, add 9 mL of PBS solution, and continue to centrifuge. Repeat the ultrafiltration three times to complete the buffer replacement of the protein, and finally collect 1 mL of protein concentrate;

[0051] (6) Perform SDS-PAGE analysis, and elute the protein using an imidazole solution with a concentration of 50 mmol / L-500 mmol / L. 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;

[0052] (7) According to the instructions of the BCA protein quantification kit (C503021 BCA method protein concentration determination kit

[0053] Sangon Biotech), draw the protein concentration standard curve y = 1079.7x - 12.079 (x is the absorbance of the protein at OD 562nm ; y is the protein concentration unit μg / mL, R² = 0.9953). According to the BCA protein quantification instructions, measure the absorbance of the purified and ultrafiltered recombinant protein at OD 562nm , which is 1.400. Substitute the standard curve formula to calculate the protein concentration after ultrafiltration, which is 1499.5 μg / mL.

[0054] Example 4: Western bolt analysis of recombinant protein

[0055] The purified and concentrated M. paratuberculosis specific membrane proteins were diluted with protein loading buffer at a ratio of 4:1, then incubated at 100℃ for 10 min in a water bath for later use. Western Blot was performed to detect the M. paratuberculosis specific membrane proteins according to the following procedures:

[0056] (1) SDS-PAGE: The prepared protein samples were loaded into each well for SDS-PAGE at 10 µL per well (the protein samples were electrophoresed to the boundary between the concentrated gel and the separation gel at 90 V for 30 min, and then transferred to 120 V for 60 min);

[0057] (2) Membrane transfer: After the electrophoresis was completed, the upper concentrated gel was removed, and the PVDF membrane was cut to the appropriate size according to the size of the separation gel. The "sponge-filter paper-PVDF membrane-gel-filter paper-sponge" was placed in order. Then, it was placed in a vertical electrophoresis tank with an ice box and membrane transfer solution at a constant current of 250 mA for 60 min;

[0058] (3) Blocking: After the membrane transfer was completed, the PVDF membrane was removed, and it was washed with 1×TBST on a shaker for 5 min for 5 times. After the washing was completed, the PVDF was placed in the blocking solution at 4℃ overnight;

[0059] (4) Primary antibody incubation: The next day, the blocked PVDF membrane was removed, and it was washed with 1×TBST on a shaker for 5 min for 5 times. The PVDF membrane was cut according to the lane position, and it was placed in different antibodies and sera diluted with blocking solution, respectively, and incubated on a shaker at room temperature for 2 h;

[0060] (5) Secondary antibody incubation: The PVDF membrane was washed with 1×TBST on a shaker for 5 min for 5 times, and then it was placed in enzyme-labeled secondary antibody diluted with TBST, and incubated on a shaker at room temperature for 2 h;

[0061] (6) Color development: The PVDF membrane was washed with 1×TBST on a shaker for 5 min for 5 times, and then ECL color development was added in the dark room. The results were observed using an imaging instrument.

[0062] The results of the Western Blot identification of the recombinant protein are shown in Figure 3 (Notes: M: protein molecular weight standard; 1: His mouse tag antibody; 2: M. paratuberculosis positive sheep serum; 3: sheep negative serum), the recombinant protein specifically binds to the mouse His tag antibody and the M. paratuberculosis positive serum at 34.2 kDa, and does not bind to the M. paratuberculosis negative serum.

[0063] Example 5 ELISA test

[0064] (1) Antigen coating: The purified M. paratuberculosis specific membrane proteins were diluted with ELISA coating solution (pH = 9.6 carbonate buffer) to 10 µg / mL, 100 µL / well was added to a 96-well enzyme-labeled plate, and coated at 4℃ for 16 h;

[0065] (2) Blocking: Discard the liquid in the hole, wash with 1 x PBST for 3 times, and the last one is dried. Add blocking solution (PBST solution containing 2% BSA) 200 μL / well, 37°C blocking 2h;

[0066] (3) Incubation of primary antibody: Discard the liquid in the hole, wash with 1 x PBST for 3 times, and the last one is dried. Add diluent (PBST solution containing 1% BSA) to dilute the serum to be tested, 100 μL / well, 37°C incubation for 1h;

[0067] (4) Incubation of secondary antibody: Discard the liquid in the hole, wash with 1 x PBST for 3 times, and the last one is dried. Add diluent (PBST solution containing 1% BSA) to dilute 1:10000 HRP-rabbit anti-goat IgG, 37°C incubation for 30min;

[0068] (5) Color development and determination of OD 450nm : Discard the liquid in the hole, wash with 1 x PBST for 5 times, and the last one is dried. Add substrate TMB solution 100 μL / well, room temperature incubation for 10min, stop color development after termination of liquid (concentrated sulfuric acid) and determine OD 450nm absorbance.

[0069] Under the above conditions, single factor experiment is carried out, as follows:

[0070] 5.1 Optimal antigen coating concentration and serum dilution

[0071] The recombinant protein is coated with coating solution according to 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, 46.875ng / well, two repeated holes for each concentration. The primary antibody (goat paratuberculosis negative and goat paratuberculosis positive serum) is diluted with antibody diluent according to 1:100, 1:200, 1:400, 1:800, two repeated holes for each concentration. Read OD 450nm value to calculate P / N value, the antigen concentration and serum dilution when P / N value is maximum are the best. The results are shown in Table 1, the antigen coating concentration is 500ng / well, the serum dilution is 1:200, and the P / N value is maximum, which is 3.856.

[0072] Table 1 Determination of antigen and serum dilution

[0073]

[0074] 5.2 Determination of optimal coating temperature and time

[0075] Set 4 different coating temperature and time, read the microplate OD 450nm value and calculate P / N value table analysis. The results are shown in Table 2, in 37℃ 1h conditions when coated antigen, P / N value is 3.911, the maximum determined coating 37℃, coating 3h is the best condition.

[0076] Table 2 the best antigen coating temperature and time results

[0077]

[0078] 5.3 determination of the best blocking solution

[0079] Select four kinds of blocking solution read the microplate OD 450nm value table analysis. The results are shown in Table 3, using 2% BSA blocking solution, P / N value is 4.099, the maximum determined 2% BSA blocking solution is the best.

[0080] Table 3 the best blocking solution results

[0081]

[0082] 5.4 optimization of the best blocking time

[0083] According to the above-mentioned indirect ELISA operation steps, select four different blocking time for blocking, read the microplate OD 450nm value table data analysis. The results are shown in Table, the blocking time is 90min, P / N value is 4.311, the maximum determined the best blocking time is 90min.

[0084] Table 4 the best blocking time results

[0085]

[0086] 5.5 the best primary antibody incubation time

[0087] According to the above-mentioned optimization conditions, set 4 serum incubation time, read the microplate OD 450nm value calculation P / N value table analysis. The results are shown in Table 5, in 60min conditions when blocking, P / N value is 4.206, the maximum determined the best time for primary antibody incubation is 60min.

[0088] Table 5 the best serum incubation time results

[0089]

[0090] 5.6 the best dilution of secondary antibody

[0091] According to the above-mentioned optimized conditions, 7 dilution multiples of the secondary antibody were set, and the OD of the microplate reader was read 450nm The P / N value was calculated and analyzed by table. As shown in Table 6, when the dilution multiple of the secondary antibody was 1:10000, the maximum P / N value was 3.965, and the dilution multiple of 1:10000 was determined as the optimal condition.

[0092] Table 6 Optimal dilution multiple of secondary antibody

[0093]

[0094] 5.7 Optimal secondary antibody incubation time

[0095] According to the above-mentioned optimized conditions, 4 secondary antibody incubation times were set for incubation. The OD of the microplate reader was read 450nm The P / N value was calculated and analyzed by table. As shown in Table 7, when the secondary antibody was incubated for 45 min, the maximum P / N value was 5.348, and the incubation time of 45 min was determined as the optimal condition.

[0096] Table 7 Results of optimal secondary antibody incubation time

[0097]

[0098] 5.8 Optimal color development time results

[0099] 4 color development times were set for optimization, and the OD of the microplate reader was read 450nm The data was analyzed. As shown in Table 8, when the color development time was 10 min, the maximum P / N value was 5.341, and the optimal color development time was determined as 10 min.

[0100] Table 8 Results of optimal color development time

[0101]

[0102] 5.9 Determination of critical value

[0103] According to the above-mentioned optimized results, 30 negative serum samples of paratuberculosis sheep were tested, and the OD 450nm value was measured by the microplate reader to establish a table for data analysis. As shown in Table 9, the average OD 450nm value (X) of 30 negative serum samples of sheep was 0.3119, and the standard deviation (SD) was 0.0531. When the OD 450nm ≤0.418 was determined as negative, when the OD 450nm ≥0.471 was determined as positive, and when the OD 450nm was in the interval of 0.471>OD >0.418 was determined as suspicious.

[0104] According to the above-mentioned optimized results, 30 copies of paratuberculosis sheep negative serum were selected for testing, and the OD 450nm value was established to analyze the data. The results are shown in Table 9. The average value (X) was 0.3119, and the standard deviation (SD) was 0.0531. According to the critical value determination principle of "average value ± multiple standard deviation" in statistics, the negative determination threshold was calculated as X + 2SD (0.3119 + 2×0.0531 ≈ 0.418), and the positive determination threshold was calculated as X + 3SD (0.3119 + 3×0.0531 ≈ 0.471). Therefore, the determination criteria are as follows: OD 450nm ≤ 0.418 is negative, OD 450nm ≥ 0.471 is positive, and 0.418 < OD 450nm < 0.471 is suspicious.

[0105] Table 9 Determination of critical values

[0106]

[0107] 5.10 Repetitive test

[0108] (1) Intra-batch repeatability test:

[0109] Three enzyme-coated plates made in the same batch were used to detect the serum at the same time, and the OD 450nm value was analyzed using an enzyme-labeled instrument. According to the formula, the coefficient of variation (CV) was calculated. The results are shown in Table 10. The highest value of intra-batch coefficient of variation was 13.10%, and the lowest value was 4.52%, both of which were lower than 15%.

[0110] Table 10 Results of intra-batch repeatability test

[0111]

[0112] (2) Inter-batch repeatability test:

[0113] Three enzyme-coated plates made in different batches were used to detect the serum at the same time, and the OD 450nm value was analyzed using an enzyme-labeled instrument. According to the formula, the coefficient of variation (CV) was calculated. The results are shown in Table 11. The highest value of inter-batch coefficient of variation was 14.97%, and the lowest value was 6.93%, both of which were lower than 15%.

[0114] Table 11 Results of inter-batch repeatability test

[0115]

[0116] 5.11 Sensitivity test

[0117] According to the optimized conditions, the laboratory preserved paratuberculosis goat positive serum was diluted by 7 times for detection, and the OD value was determined by using an enzyme label instrument. The results are shown in Table 12, when the serum dilution multiple is 1:1600, the OD value is higher than the positive critical value. 450nm 450nm

[0118] Table 12 Sensitivity test results

[0119]

[0120] The ELISA established by the application was compared with the imported commercial kits of ID Vet and IDEXX, and 6 sera of strong positive, medium positive, weak positive and 3 negative sera were detected for 6 times. The results are shown in Table 13, the 6 times detection of the ELISA established by the application showed positive for strong positive, medium positive and weak positive positive serum, and the detection of ID Vet and IDEXX for medium positive and weak positive serum showed different results. It is shown that the ELISA method established by the application has stronger sensitivity.

[0121] Table 13 Positive serum test results

[0122]

[0123] 5.12 Specificity test

[0124] The serum of 3 other common goat diseases (paratuberculosis detection is negative) was detected, and the OD value was determined by using an enzyme label instrument for data analysis. The results are shown in Table 14, the OD value of the 3 other disease positive serum is lower than the negative critical value, and the paratuberculosis positive serum is higher than the positive critical value. 450nm 450nm

[0125] Table 14 Specificity test results

[0126]

[0127] Example 6 Clinical detection

[0128] (1) Field samples

[0129] ​​​​466 sheep serum samples were collected from 466 wild sheep and tested simultaneously using the ELISA method established in this invention, along with imported commercial kits ID Vet and IDEXX. The results are shown in Table 15: the positive rate of the ELISA method established in this invention was 41.85%, while the results for ID Vet and IDEXX kits were 6.22% and 3.43%, respectively. To avoid false positives, 20% (39 sheep) of the 195 sheep that tested positive using the ELISA method established in this invention were randomly collected 30 days later. Serum samples were then tested using the ID Vet kit, IDEXX kit, and the ELISA method established in this invention, respectively. 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 this invention.

[0130] Table 15 Results of field clinical sample testing

[0131]

[0132] (2) Samples from sheep artificially infected with MAP test

[0133] Serum samples from 112 sheep (2 control sheep + 6 infected sheep, 14 samplings each) artificially infected with MAP were collected and simultaneously detected using the ELISA method established in this invention, along with imported commercial kits ID Vet and IDEXX. The results are shown in Table 16: Serum samples from the negative control group (6 sheep, pathological observation...) were all negative at all time points using the three methods; Figure 4 Focal granulomas with fibrosis in the submucosa of the ileum, HE staining); pathogen isolation and identification ( Figure 5 MAP culture and identification results: Ziehl-Neelsen acid-fast staining of bacterial smears showed uniform short rod-shaped acid-fast bacilli; tissue nucleic acid detection confirmed infection; animal experiments were successfully established. The positive rate of 84 serum samples from 6 infected sheep using the ELISA method established in this invention was 46.43%, while the results of ID Vet and IDEXX kits were 10.71% and 8.33%, respectively. The earliest detection by all three methods was 75 days after MAP inoculation. The ID Vet kit showed 9 serological positivity results in 2 sheep; the IDEXX kit showed 7 serological positivity results in only 1 sheep (one of the sheep that tested positive by ID Vet); the ELISA method established in this invention showed 39 serological positivity results distributed among 5 sheep (including the 2 sheep that tested positive by the other two methods).

[0134] Table 16 Detection results of artificially infected samples

[0135]

[0136] Table 17 Detailed results of artificial infection sample detection

[0137]

Claims

1. The application of a specific detection kit for sheep paratuberculosis, characterized in that, Used for preparing diagnostic products for ovine paratuberculosis; when the OD of the serum sample to be tested... 450 A nm value ≥ 0.471 is considered positive, ≤ 0.418 is considered negative, and when 0.471 < OD... 450 A value of nm < 0.418 is considered a suspected positive result; The kit contains an enzyme-labeled plate coated with recombinant Mycobacterium paratuberculosis membrane protein antigen, the nucleotide sequence of which is shown in SEQ ID NO: 1 and the amino acid sequence in which is shown in SEQ ID NO: 2; blocking buffer; serum diluent; enzyme-labeled secondary antibody; substrate TMB chromogenic solution and stop solution.

2. The application of the sheep paratuberculosis specific detection kit as described in claim 1, characterized in that, The blocking solution is a PBST solution containing 1%-2% BSA.

3. The application of the sheep paratuberculosis specific detection kit as described in claim 1, characterized in that, The serum diluent is a PBST solution containing 1% BSA, and the stop solution is concentrated sulfuric acid.

4. The application of the sheep paratuberculosis specific detection kit as described in claim 1, characterized in that, The enzyme-labeled secondary antibody is HRP-labeled rabbit anti-goat IgG, diluted at a ratio of 1:10000.

5. The application of the sheep paratuberculosis specific detection kit as described in claim 1, characterized in that, The antigen coating concentration of the enzyme-labeled plate is 500 ng / well.

6. The application of the sheep paratuberculosis specific detection kit as described in 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 with 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.

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