A sheep fascioliasis specific diagnostic antigen and ELISA diagnostic kit

Abstract

The invention provides a sheep fascioliasis-specific diagnostic antigen and an ELISA diagnostic kit thereof, which relate to the field of immunological detection. The specific candidate diagnostic antigen gene ADP09371-1 of Fasciola hepatica is screened out; after the signal peptide of ADP09371-1 is truncated, codon optimization is performed to prepare the sheep fascioliasis-specific diagnostic antigen; the sheep fascioliasis-specific diagnostic antigen is a recombinant protein ADP09371-2; the ELISA diagnostic kit comprises: an enzyme labeling plate coated with the sheep fascioliasis-specific diagnostic antigen, a PBST buffer, a blocking solution, a secondary antibody, a TMB color developing solution and a stop solution; the ELISA diagnostic kit provided by the invention is simple and fast to operate, has high sensitivity and specificity, can accurately identify and detect the Fasciola hepatica-specific antigen in a sample, and avoids confusion with other similar diseases.

Description

Technical Field

[0001] The invention relates to the field of immunological detection, in particular to a sheep fascioliasis specific diagnostic antigen and an ELISA diagnostic kit thereof. Background Art

[0002] Fasciola hepatica is a zoonotic parasitic disease caused by Fasciola hepatica, which mainly parasitizes in the liver and bile ducts of various ruminants such as cattle and sheep, as well as humans. In animal husbandry, ruminants such as cattle and sheep are important breeding species, and the occurrence of Fasciola hepatica will seriously interfere with the normal physiological functions of these animals. For cattle, once infected with Fasciola hepatica, the normal metabolism and physiological functions of the liver and bile ducts will be destroyed, thus affecting important production indicators such as growth rate, milk production and meat quality of cattle. Similar situations will occur after sheep are infected, such as stunted growth and development, and decreased reproductive capacity. This series of impacts will eventually have a serious negative impact on the overall development of my country's animal husbandry industry. From the perspective of human health, Fasciola hepatica also poses a certain threat. Humans may be infected with Fasciola hepatica when they come into contact with water sources and food contaminated by Fasciola hepatica or when they are in close contact with sick animals. After infection, human patients may experience a variety of symptoms such as abdominal pain, bloating, diarrhea, anemia, etc., which seriously affect the patient's quality of life and physical health. At present, the preventive measures for liver fluke disease are not perfect enough, because no effective liver fluke vaccine has been developed so far.

[0003] In this case, timely diagnosis of liver flukes before they spread widely becomes a key link in preventing and controlling the disease. If liver fluke disease can be diagnosed in time, appropriate treatment measures or isolation measures can be taken to prevent the further spread of the disease. In the development of diagnostic methods for liver fluke disease, screening liver fluke-specific antigen genes is of great significance. Specific antigen genes can provide a basis for the development of more sensitive and specific diagnostic methods for liver fluke disease. This specific antigen gene can help diagnostic methods accurately identify liver fluke disease and avoid confusion with other similar diseases, thereby improving the accuracy and reliability of diagnosis. Summary of the invention

[0004] Based on the above background, the present invention provides a sheep fascioliasis-specific diagnostic antigen and an ELISA diagnostic kit thereof, establishes an indirect ELISA diagnostic method for hepatic fascioliasis, and provides a new and effective means for the diagnosis of hepatic fascioliasis, as follows:

[0005] A sheep fascioliasis-specific diagnostic antigen, characterized in that a diagnostic antigen gene ADP09371-1 is obtained from sheep fascioliasis; the diagnostic antigen gene ADP09371-1 is subjected to signal peptide truncation and then codon optimization to prepare a sheep fascioliasis-specific diagnostic antigen; the sheep fascioliasis-specific diagnostic antigen is a recombinant protein ADP09371-2;

[0006] The nucleotide sequence of the selected diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.1;

[0007] The amino acid sequence of the selected diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.2;

[0008] The nucleotide sequence of the optimized codon is shown in SEQ ID NO.3;

[0009] The amino acid sequence of the recombinant protein ADP09371-2 is shown in SEQ ID NO.4.

[0010] On the other hand, the present invention provides an ELISA diagnostic kit, comprising: an ELISA plate coated with a sheep fascioliasis-specific diagnostic antigen, a PBST buffer, a blocking solution, a secondary antibody, a TMB colorimetric solution, and a stop solution.

[0011] Moreover, the preparation method of the ELISA plate is as follows: the sheep fascioliasis specific diagnostic antigen is diluted and coated with a coating solution, added into the well ELISA plate, and incubated at 37° C. for 2 hours to obtain the ELISA plate.

[0012] Moreover, the concentration of the diagnostic antigen after dilution was 0.75 ng / ul, that is, 1 ul of the diagnostic antigen was added to 1359 ul of the coating solution for dilution.

[0013] Furthermore, the blocking solution was 10% skim milk.

[0014] Furthermore, the secondary antibody was rabbit anti-sheep IgG-HRP.

[0015] Furthermore, the dilution of the secondary antibody was 1:6000.

[0016] Moreover, the kit is used to detect the specific antigen gene of Fasciola hepatica in animal serum, and the dilution of the animal serum is 1:3200. The specific method is as follows:

[0017] (1) Take the ELISA plate;

[0018] (2) Remove the liquid from the ELISA plate, add 100 μL of PBST buffer to each well of the ELISA plate, and wash with shaking for 3 times, 1 min each time;

[0019] (3) Add 100 μL of blocking solution (10% skim milk) to each well, block at 37°C for 2 h, and then wash three times with shaking;

[0020] (4) Add 100 μL of animal serum diluted 1:3200 to each well, incubate at 37°C for 2 h, and then wash three times with shaking;

[0021] (5) Add 100 μL of secondary antibody diluted 1:6000 to each well, incubate at 37°C for 45 min, and wash three times with shaking;

[0022] (6) Add TMB colorimetric solution and incubate for 10 min in a dark environment;

[0023] (7) Add 100 μL of stop solution to each well to terminate the reaction. Measure the OD 450nm value within 15 min. When OD 450nm ≥ 0.3643, it is considered positive; when OD 450nm < 0.3070, it is considered negative; when 0.3070 < OD 450nm < 0.3643, it is considered suspected positive.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention screened out the specific candidate diagnostic antigen gene ADP09371-1 of Fasciola hepatica through proteomics data analysis, and prepared the sheep Fasciola disease specific diagnostic antigen through signal peptide truncation and codon optimization.

[0026] 2. Based on the specific diagnostic antigen of sheep fascioliasis, the present invention provides an ELISA diagnostic kit, which is easy and fast to operate, and has high sensitivity and specificity. It can accurately identify and detect the specific antigen of Fasciola hepatica in the sample, avoiding confusion with other similar diseases.

[0027] 3. The present invention establishes an indirect ELISA diagnostic method for fascioliasis. ELISA (enzyme-linked immunosorbent assay) is a technology widely used in biomedical research and clinical diagnosis, and has the advantages of high sensitivity, strong specificity, and simple operation. The present invention provides a new and effective means for the diagnosis of fascioliasis by establishing an indirect ELISA diagnostic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the agarose gel electrophoresis diagram of the PCR amplification product of gene ADP09371-1;

[0029] Figure 2 Colony PCR identification of the recombinant expression vector pET32a-ADP09371-1;

[0030] Figure 3 For enzyme digestion identification of recombinant expression vectors;

[0031] Figure 4 This is a diagram showing the solubility analysis results of the recombinant protein ADP09371-2;

[0032] Figure 5 This is the SDS-PAGE analysis diagram of the recombinant protein ADP09371-2Ni-NTA affinity purification;

[0033] Figure 6 This is the SDS-PAGE identification diagram of the recombinant protein ADP09371-2 after purification;

[0034] Figure 7 This is a Western blotting analysis of sheep positive serum and recombinant protein ADP09371-2;

[0035] Figure 8 This is a Western blotting analysis of sheep negative serum and recombinant protein ADP09371-2;

[0036] Fig. 9 This is the result diagram of serum dilution sensitivity test;

[0037] Fig.10 This is a diagram of stool test results. DETAILED DESCRIPTION

[0038] Example 1 Codon Optimization

[0039] Through proteomics data analysis, a specific candidate diagnostic antigen gene ADP09371-1 of Fasciola hepatica was screened out; the diagnostic antigen gene ADP09371-1 was subjected to signal peptide truncation and then codon optimization to prepare a specific diagnostic antigen for Fasciola ovis.

[0040] The nucleotide sequence of the selected diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.1;

[0041] The amino acid sequence of the selected diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.2;

[0042] The nucleotide sequence of the optimized codon is shown in SEQ ID NO.3;

[0043] The amino acid sequence of the recombinant protein ADP09371-2 is shown in SEQ ID NO.4.

[0044] Example 2 Target gene cloning (PCR amplification)

[0045] The following 22 primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the target sequence:

[0046] NJ0173766-1-BSND_1: SEQ ID NO.5;

[0047] NJ0173766-1-BSND_2: SEQ ID NO.6;

[0048] NJ0173766-1-BSND_3: SEQ ID NO.7;

[0049] NJ0173766-1-BSND_4::SEQ ID NO.8;

[0050] NJ0173766-1-BSND_5: SEQ ID NO.9;

[0051] NJ0173766-1-BSND_6: SEQ ID NO.10;

[0052] NJ0173766-1-BSND_7: SEQ ID NO.11;

[0053] NJ0173766-1-BSND_8: SEQ ID NO.12;

[0054] NJ0173766-1-BSND_9: SEQ ID NO.13;

[0055] NJ0173766-1-BSND_10: SEQ ID NO.14;

[0056] NJ0173766-1-BSND_11: SEQ ID NO.15;

[0057] NJ0173766-1-BSND_12: SEQ ID NO.16;

[0058] NJ0173766-1-BSND_13: SEQ ID NO.17;

[0059] NJ0173766-1-BSND_14: SEQ ID NO.18;

[0060] NJ0173766-1-BSND_15: SEQ ID NO.19;

[0061] NJ0173766-1-BSND_16: SEQ ID NO.20;

[0062] NJ0173766-1-BSND_17: SEQ ID NO.21;

[0063] NJ0173766-1-BSND_18: SEQ ID NO.22;

[0064] NJ0173766-1-BSND_19: SEQ ID NO.23;

[0065] NJ0173766-1-BSND_20: SEQ ID NO.24;

[0066] NJ0173766-1-BSND_21: SEQ ID NO.25;

[0067] NJ0173766-1-BSND_22: SEQ ID NO. 26.

[0068] 1. Full-length PCR round 1:

[0069] The reaction system is shown in Table 1:

[0070] Table 1 PCR one-round reaction system

[0071]

[0072] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 25 sec, annealing at 62°C for 20 sec, extension at 72°C for 45 sec, 25 cycles; and extension at 72°C for 1 min.

[0073] 2. Full-length PCR round 2:

[0074] The reaction system is shown in Table 2:

[0075] Table 2 PCR second round reaction system

[0076]

[0077] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 25 sec, annealing at 62°C for 20 sec, extension at 72°C for 45 sec, 25 cycles; and extension at 72°C for 1 min.

[0078] The ADP09371-1 gene was amplified by PCR. After the amplification, the amplified products were detected by 1% agarose gel electrophoresis. Figure 1(M: DNA molecular mass standard DL5000; 1. PCR amplified target fragment ADP09371-1). A 939 bp specific band can be clearly seen in the figure, which is consistent with the expected target fragment size.

[0079] Example 3 Ligation of pET-32a vector

[0080] Take a 200μL centrifuge tube, add 3.5μL pET-32a (this vector comes with a His tag), 4μL PCR recovery product and 2.5μL recombinase, centrifuge to mix, connect at 16℃ overnight; place in a 50℃ water bath for 25min, leave for 2-3min to lower the temperature, carry out transformation and bacterial liquid coating experiments, and incubate at 37℃ overnight.

[0081] Example 4 PCR Identification of Recombinant Clones

[0082] A single colony was picked from the overnight plate in Example 3, and colony PCR (colony polymerase chain reaction) was performed using primers PET-32ASEQF / G-PET-SEQF1. Positive bacteria were identified by electrophoresis. Four positive bacteria were randomly selected and cultured overnight in a 4 ml single tube at 37° C. shaker. The plasmid was extracted from the overnight bacterial solution and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the correct plasmid. The positive bacterial solution was sequenced using primers GJY002-A05 / PET-SEQYZF1. The results were as follows: Figure 2 As shown (M: DNA molecular mass standard DL5000; 1-9: PCR amplification products), the figure shows that the pET32a-ADP09371-1 recombinant expression vector was successfully constructed.

[0083] The nucleotide sequences of the PET-32ASEQF and G-PET-SEQF1 primers are shown in SEQ ID NO.27 and SEQ ID NO.28.

[0084] Example 5 Identification of ADP09371-1 gene expression vector

[0085] The recombinant plasmid was extracted using an Axygen plasmid extraction kit, connected to the pET-32a expression vector, and double-digested with Stu I and Xho I restriction endonucleases to obtain a 5845 bp pET-32a fragment and a 939 bp target gene fragment, as shown in Figure 3 As shown (M: DNA molecular mass standard DL5000; 1: pET-32a ADP09371-1 double enzyme digestion product).

[0086] Example 6 Transformation of Recombinant Expression Plasmid into Competent Cells

[0087] The recombinant vector was transformed into Escherichia coli BL21-DE3: 1 μL of the recombinant plasmid was added to 100 μL of competent bacteria, placed on ice for 20 minutes, heat-shocked at 42°C for 90 seconds, then quickly placed on ice for 5 minutes, and then 600 μL of LB culture medium was added; at 37°C, shaken at 220 r / min for 1 hour, centrifuged, smeared on an LB plate containing 50 μg / mL Amp, and cultured upside down at 37°C overnight.

[0088] Example 7 Expression and Identification of IPTG-Induced Recombinant Bacterial Fusion Protein

[0089] Pick a single clone on the LB plate in Example 6, inoculate it into a 4 mL LB culture medium test tube containing 50 μg / mL Amp, and shake it at 37°C and 220 r / min overnight; the next day, inoculate it into 100 mL LB culture medium containing 50 μg / mL Amp at a ratio of 1:100, and shake it at 37°C and 220 r / min until OD600 is 0.5-0.8; take 1 mL of culture, centrifuge it at 10000 r / min for 2 min at room temperature, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS; add IPTG to the remaining culture to 0.2 mM, and shake it at 16°C / 30°C and 220 r / min for 18 h to induce fusion protein expression. Take 1 mL of culture, centrifuge at 10000 r / min for 2 min at room temperature, discard the supernatant, and resuspend the bacterial pellet with 100 μL TBS; centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, resuspend the bacterial pellet with TBS, add PMSF to 1 mM, and take samples of the supernatant and 100 μL TBS-resuspended pellet after ultrasonic disruption; perform 12% SDS-PAGE gel electrophoresis to detect and analyze it, and observe the protein bands by Coomassie Brilliant Blue staining to determine whether the fusion protein is successfully expressed. Figure 4 As shown (M: protein molecular weight standard; 1: 16 degrees induced crushing supernatant; 2: 16 degrees induced crushing precipitate; 3: 30 degrees induced crushing supernatant; 4: 30 degrees induced crushing precipitate), from Figure 4 It can be seen that the supernatant fused with the recombinant protein ADP09371-2 showed a protein band at 52.2 kDa, indicating that the recombinant protein ADP09371-2 was expressed in a soluble form in the supernatant of cell lysis.

[0090] Example 8 Ni column affinity purification of recombinant protein ADP09371-2

[0091] Load the supernatant onto a Ni-NTA affinity chromatography column that has been pre-equilibrated with Ni-NTA Binding-Buffer (the buffer contains 20 mM Tris, 0.5 M NaCl, and 0 mM imidazole, and the pH is adjusted to 8.0); rinse 3-5 CV of Binding-Buffer to remove unbound proteins, elute the target protein with 20 mL Ni-NTA Elution-Buffer (20 mM Tris, 0.5 M NaCL, 25 mM imidazole / 50 mM imidazole / 100 mM imidazole / 500 mM imidazole, pH 8.0) and collect the effluent for SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 5 (M: protein molecular weight standard; 1: sample protein; 2: flow-through; 3: 25mM imidazole eluent; 4: 50mM imidazole eluent; 5: 100mM imidazole eluent; 6-7: 500mM imidazole eluent), from Figure 5 It can be seen that the recombinant protein ADP09371-2 has a relatively single band at 52.2 kDa, and the recombinant protein ADP09371-2 with higher purity is obtained.

[0092] Example 9 Dialysis and Concentration

[0093] The collected effluent (protein solution) was dialyzed into 20 mM Tris, 0.5 M NaCL, pH 8.0 buffer at 1:30 at 4°C overnight. The dialysate was replaced the next day and the dialysis was continued at 4°C for 8 hours. After being concentrated by ultrafiltration, SDS-PAGE analysis was performed. The results are shown in Figure 6 (M: protein molecular weight standard; 1: recombinant protein ADP09371-2). The results showed that the purified protein band was single and had no impurities and high concentration, which was consistent with the expected band size of the experiment.

[0094] Example 10 Western blotting analysis of recombinant protein ADP09371-2

[0095] Western blotting analysis of recombinant protein ADP09371-2 was performed using positive serum from sheep infected with Fasciola hepatica as the primary antibody, and specific bands were obtained, such as Figure 7 As shown, the results showed that the positive serum of sheep infected with Fasciola hepatica had good reactivity with the recombinant protein ADP09371-2.

[0096] Western blotting analysis of recombinant protein ADP09371-2 was performed using negative serum from sheep not infected with Fasciola hepatica as the primary antibody. Figure 8 As shown, no specific bands were found in the figure.

[0097] Example 11 ELISA test

[0098] (1) Dilute the recombinant protein ADP09371-2 with the coating solution and add it to a 96-well ELISA plate, 100 μL per well, at 37°C for 2 h;

[0099] (2) Shake off the liquid in the 96-well ELISA plate and pat dry on absorbent paper. Add 100 μL of PBST buffer to each well and wash 3 times with shaking, 1 min each time;

[0100] (3) Add 100 μL of blocking solution to each well, block at 37°C for 2 h, and then wash three times with shaking;

[0101] (4) Add 100 μL of diluted sheep negative and positive serum to each well and incubate at 37°C for 2 h, then wash three times with shaking;

[0102] (5) Add 100 μL of diluted secondary antibody to each well, incubate for a certain period of time under certain conditions, and then wash three times with shaking;

[0103] (6) Add TMB colorimetric solution and incubate for 10 min in a dark environment.

[0104] (7) Add 100 μL of stop solution to each well to terminate the reaction and measure the OD 450 nm value within 15 min.

[0105] Explore the optimal conditions for the above experiment:

[0106] 1. Determination of the optimal working concentration of antigen and serum

[0107] The recombinant protein ADP09371-2 was diluted to 1.5 ng / μl, 0.75 ng / μl, 0.375 ng / μl and 0.1875 ng / μl with coating solution. Negative and positive sera were diluted in a doubling ratio of 1:100-1:3200, and the OD 450nm values ​​of negative and positive sera were measured by an ELISA instrument to determine the optimal working concentration of antigen and serum (Table 3).

[0108] Table 3 Determination of ADP09371.1 antigen and serum dilution

[0109]

[0110] As can be seen from Table 3, when the antigen dilution was 0.75 ng / μl and the serum dilution was 1:3200, the P / N was the highest (3.957195331).

[0111] Optimization of Antigen Incubation Time

[0112] The recombinant protein ADP09371-2 was coated at 37°C for 1 h, 37°C for 2 h, and 4°C overnight, and the OD 450nm value was measured to determine the optimal incubation conditions for the antigen (Table 4).

[0113] Table 4 Determination of antigen incubation conditions

[0114]

[0115] As can be seen from Table 4, when the antigen incubation conditions were 37°C and 2h, the P / N value was the highest (6.006151872).

[0116] 3. Determination of the Optimal Blocking Solution

[0117] Under the optimal conditions screened above, 1% BSA, 10% skim milk and 5% skim milk were used as blocking solutions, respectively. Blocking was performed at 37°C for 2 h, and the OD 450nm value was measured to determine the optimal blocking solution (Table 5).

[0118] Table 5 Determination of the best blocking solution

[0119]

[0120] It can be seen from Table 5 that when the blocking solution is 10% skim milk, the P / N value is the highest (14.17807999).

[0121] 4. Determination of the optimal incubation time for enzyme-labeled secondary antibodies

[0122] Under the optimal conditions screened above, the incubation time of the enzyme-labeled secondary antibody was set at 37°C for 30 min, 45 min, and 60 min, respectively, and the OD 450 nm value was measured to determine the optimal incubation time of the enzyme-labeled antibody (Table 6).

[0123] Table 6 Determination of secondary antibody incubation time

[0124]

[0125] As can be seen from Table 6, when the secondary antibody incubation time was 45 min, the P / N value was the highest (15.39130435).

[0126] In summary, when the antigen dilution was 0.75 ng / μl, the serum dilution was 1:3200, the antigen incubation conditions were 37°C for 2 h, the blocking solution was 10% skim milk, and the secondary antibody incubation conditions were 37°C for 45 min, the P / N value was the highest, indicating that the specific binding between the antibody in the positive serum and the antigen to be tested was the strongest, and the antibody could efficiently recognize and bind to the antigen.

[0127] 5. Determination of the best color development time

[0128] Under the optimal conditions screened above, the color development time was set to 10 min, 15 min, and 20 min, respectively, and the OD 450 nm value was measured to determine the optimal color development time (Table 7).

[0129] Table 7 Determination of the optimal color development time

[0130]

[0131] It can be seen from Table 7 that when the TMB substrate reaction time is 10 min, the P / N value is the highest (17.60099751), and the optimal time for the color development reaction is 10 min.

[0132] 6. Determination of critical value

[0133] The established indirect ELISA method for Fasciola hepatica was used to detect 17 negative sera, and the average OD450nm value (X) was 0.2494, the standard deviation (SD) was 0.0288, and the positive-negative critical value was X+3SD=0.3358. In order to avoid false positive results, one standard deviation was added or subtracted from the critical value as the suspicious interval. Therefore, when OD 450nm≥0.3647, it can be judged as positive; when OD 450nm<0.3070, it can be judged as negative; when 0.3070<0D450nm<0.3643, it is determined to be suspected positive, and the suspected sample needs to be retested.

[0134] VII. Evaluation of the diagnostic effect of indirect ELISA

[0135] 7.1. Repeatability test:

[0136] The above ELISA method was used to conduct intra-batch and inter-batch repeatability experiments on 6 randomly selected sheep sera. Each serum sample had 3 parallel wells, and the coefficient of variation was calculated according to (standard deviation / mean) × 100%. The results are shown in Table 8. Table 8 shows that the coefficient of variation within and between batches was less than 10%, indicating that this experiment has good repeatability.

[0137] Table 8 Repeatability test results

[0138]

[0139] 7.2 Serum dilution sensitivity test

[0140] The recombinant antigen was used as the diagnostic antigen to coat the ELISA plate, and the positive serum was diluted 1:100-1:204800 for ELISA experiment. The experimental results are as follows Fig. 9As shown in the figure, when the positive serum was diluted to 1:102400, the OD value of the positive serum was lower than the critical value, and when the positive serum was diluted to 1:51200, the detection limit was reached.

[0141] Example 12

[0142] The invention discloses an ELISA diagnostic kit, which comprises: an enzyme label plate coated with a sheep fascioliasis specific diagnostic antigen, a PBST buffer, a blocking solution, a secondary antibody, a TMB color developing solution and a stop solution.

[0143] Furthermore, the preparation method of the ELISA plate is as follows: the sheep fascioliasis specific diagnostic antigen is diluted and coated with a coating solution, added to the well ELISA plate, and incubated at 37° C. for 2 hours to obtain the ELISA plate.

[0144] Furthermore, the concentration of the diagnostic antigen after dilution is 0.75 ng / ul, that is, 1 ul of diagnostic antigen is added to 1359 ul of coating solution for dilution.

[0145] Furthermore, the blocking solution is 10% skim milk.

[0146] Furthermore, the secondary antibody is rabbit anti-sheep IgG-HRP.

[0147] Furthermore, the dilution of the secondary antibody is 1:6000.

[0148] Furthermore, the kit is used to detect the specific antigen gene of Fasciola hepatica in animal serum, and the dilution of the animal serum is 1:3200. The specific method is as follows:

[0149] (1) Take the ELISA plate;

[0150] (2) Remove the liquid from the ELISA plate, add 100 μL of PBST buffer to each well of the ELISA plate, and wash with shaking for 3 times, 1 min each time;

[0151] (3) Add 100 μL of blocking solution (10% skim milk) to each well, block at 37°C for 2 h, and then wash three times with shaking;

[0152] (4) Add 100 μL of animal serum diluted 1:3200 to each well, incubate at 37°C for 2 h, and then wash three times with shaking;

[0153] (5) Add 100 μL of secondary antibody diluted 1:6000 to each well, incubate at 37°C for 45 min, and then wash three times with shaking;

[0154] (6) Add TMB colorimetric solution and incubate for 10 min in a dark environment;

[0155] (7) Add 100 μL of stop solution to each well to terminate the reaction. Measure the OD 450nm value within 15 min. When OD 450nm ≥ 0.3643, it is considered positive; when OD 450nm < 0.3070, it is considered negative; when 0.3070 < OD450nm < 0.3643, it is considered suspected positive.

[0156] Example 13 Clinical Testing

[0157] 15 serum samples were collected from a herdsman's home in Tongshi Sumu, Wushi Banner. In addition, 15 serum samples were collected from two herdsmen's homes in Tuke Town, Wushi Banner (a total of 30 samples) for clinical validation tests to verify the ELISA diagnostic kit provided in Example 12 above, and compared with the Fasciola hepatica Antibody Test Kit purchased from IDEXX (three parallel tests were performed for each sample, and the results were consistent). The results are shown in Tables 9-11:

[0158] Table 9 Test results of Fasciola hepatica antibody test kit

[0159]

[0160] Remark:

[0161] 1. Validity of the results: △ positive control OD value>0.8, △ negative control OD value<0.3;

[0162] 2. S / P value calculation: S / P% = △ sample OD value / △ positive control OD value x 100% (△ OD value is each sample 3. Result determination:

[0163] S / P%<10%: no Fasciola hepatica infection;

[0164] 10%≤S / P%<15%: suspected positive;

[0165] 15%≤S / P%: positive.

[0166] Table 10 ELISA diagnostic kit test results

[0167]

[0168] In Table 9, among the 45 samples, 5 were positive and 2 were suspected positive. After retesting, all were determined to be negative. The results in Table 10 showed that among the 45 samples, 10 were positive and 9 were suspected positive. After retesting, all were determined to be negative. The above results were statistically analyzed in Table 11.

[0169] Table 11 Clinical trial results

[0170]

[0171] It can be seen from Table 9 that for the same batch of samples, the number of positive samples detected by the Fasciola hepatica antibody detection kit imported from INGENASA, Spain, was 5, while the number of positive samples detected by the ELISA diagnostic kit provided by the present invention was 10, the positive rate of the imported Fasciola hepatica antibody detection kit was 11.11% (5 / 45), and the positive rate of the ELISA diagnostic kit provided by the present invention was 22.22% (10 / 45), which was significantly higher than the positive rate of the Fasciola hepatica antibody detection kit, indicating that the ELISA antibody detection kit of the present invention has higher sensitivity and higher diagnostic accuracy in detecting antibodies to Fasciola hepatica disease.

[0172] Ten sheep feces that were positive for ELISA were collected and treated with water-washing and precipitation method. Fasciola hepatica eggs were observed under a microscope. Fig.10 ), which proves the accuracy of the ELISA diagnostic kit provided by the present invention.

Claims

1. An ELISA diagnostic kit, characterized in that: The ELISA diagnostic kit comprises: an ELISA plate coated with a sheep fascioliasis specific diagnostic antigen, a PBST buffer, a blocking solution, a secondary antibody, a TMB color developing solution, and a stop solution; The method for obtaining the sheep fascioliasis specific diagnostic antigen is as follows: Obtaining a diagnostic antigen gene ADP09371-1 from Fasciola sheepis; removing the signal peptide of the diagnostic antigen gene ADP09371-1 and then optimizing the codons to prepare a diagnostic antigen specific for Fasciola sheepis; The sheep fascioliasis specific diagnostic antigen is the recombinant protein ADP09371-2; The nucleotide sequence of the diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.1; The amino acid sequence of the diagnostic antigen gene ADP09371-1 is shown in SEQ ID NO.2; The nucleotide sequence of the optimized codon is shown in SEQ ID NO.3; The amino acid sequence of the recombinant protein ADP09371-2 is shown in SEQ ID NO.4; The preparation method of the ELISA plate is as follows: dilute and coat the sheep fascioliasis-specific diagnostic antigen with a coating solution, add it to the well ELISA plate, and incubate it at 37° C. for 2 hours to obtain the ELISA plate; the concentration of the sheep fascioliasis-specific diagnostic antigen after dilution is 0.75 ng / ul; The kit is used for detecting the specific antigen gene of Fasciola hepatica on animal serum, and the dilution of the animal serum is 1:3200.

2. An ELISA diagnostic kit as claimed in claim 1, characterized in that, The blocking solution is 10% skim milk.

3. An ELISA diagnostic kit as claimed in claim 1, characterized in that, The secondary antibody was rabbit anti-sheep IgG-HRP.

4. An ELISA diagnostic kit as claimed in claim 3, characterized in that, The dilution of the secondary antibody was 1:6000.

5. An ELISA diagnostic kit as claimed in claim 1, characterized in that, The specific method of using ELISA diagnostic kit to detect animal serum is as follows: (1) Take the ELISA plate; (2) Remove the liquid from the ELISA plate, add 100 μL of PBST buffer to each well of the ELISA plate, and wash with shaking for 3 times, 1 min each time; (3) Add 100 μL of 10% skim milk to each well, block at 37°C for 2 h, and then wash three times with shaking; (4) Add 100 μL of animal serum diluted 1:3200 to each well, incubate at 37°C for 2 h, and then wash three times with shaking; (5) Add 100 μL of secondary antibody diluted 1:6000 to each well, incubate at 37°C for 45 min, and then wash with shaking for 3 times; (6) Add TMB colorimetric solution and incubate for 10 min in a dark environment; (7) Add 100 μL of stop solution to each well to terminate the reaction. Measure the OD 450nm value within 15 min. When OD 450nm ≥ 0.3643, it is positive; when OD 450nm < 0.3070, it is negative; when 0.3070 < OD450nm < 0.3643, it is suspected positive.

Images