Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for sheep bacteroides nodules antibody and application thereof

Abstract

The invention relates to an indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for a sheep bacteroides nodules antibody and application thereof, belonging to the technical field of biological detection. In order to solve the technical problems that a bacterium separation identification and PCR (Polymerase Chain Reaction) detection method of bacteroides nodulosus has defects and a serological identification method for bacteroides nodulosus does not exist in the prior art, the invention adopts a sheep bacteroides nodulosus outer membrane protein Omp1D as a coating antigen in an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit; rapid detection and screening of bovine and sheep bacteroides nodules infection can be realized, and the kit has the characteristics of simple operation, rapidness and sensitivity, accurate result, good specificity and repeatability and the like; meanwhile, the indirect ELISA detection kit for the sheep bacteroides nodules antibody has the characteristics of being simple in preparation method, low in cost, suitable for large-scale production and the like, a rapid, simple and sensitive diagnostic kit for detecting bacteroides nodules infection is provided for large-scale breeding of animal husbandry, and a technical support is provided for preventing and controlling bacteroides nodules infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an indirect ELISA detection kit for Bacteroides ovis antibodies and an application thereof. Background Art

[0002] Limb and hoof disease is a general term for limb and hoof lesions caused by various adverse factors, among which hoof disease accounts for more than 90%, including hoof rot, hoof deformation, laminitis, hoof dermatitis, hoof abscess and hoof trauma. Hoof rot is very harmful in hoof diseases of ruminants. Sick animals will have clinical symptoms such as hoof deformation, lameness, difficulty in movement, and loss of appetite. Bacteroides nodosa is one of the important pathogens of sheep hoof rot. Bacteroides nodosa is a Gram-negative, non-spore-forming, obligate anaerobic bacillus that can cause hoof rot in ruminants and is highly contagious in sheep. In the early stage of the disease, sheep hooves will have slight redness and swelling, and redness of the skin can be seen between the toes or the crown of the hoof. As the disease progresses, the redness and swelling will expand, obvious swelling will occur, the hoof will be hot and painful, and the sheep will become lame. Afterwards, the skin of the hoof may ulcerate and necrotize, black or brown rotten areas will appear, there will be an unpleasant odor, and purulent secretions will ooze out. If the infection is not controlled in time, the pathogen may enter the blood circulation and cause systemic infection. At this time, the sheep will have symptoms such as fever, mental depression, loss of appetite, etc. In severe cases, it will cause weight loss, emaciation, and even death. Foot rot not only affects the health of animals, but also has a huge impact on the development of animal husbandry. For example, it causes direct economic losses such as reduced productivity of maternal animals, increased fattening time, reduced milk production, and reduced fur quality. The direct related losses in the UK are as high as 200,000 to 800,000 pounds each year. In countries such as Switzerland, the indirect costs of prevention and control are equal to the direct costs, or even far higher than the direct costs. This seriously hinders the development of pastures and causes huge economic losses.

[0003] Currently, the diagnosis of Bacteroides nodosa infection in clinical practice is mainly based on bacterial isolation and identification and PCR diagnostic technology. Since Bacteroides nodosa is strictly anaerobic and has strict requirements on culture gas conditions, bacterial isolation and identification and PCR diagnostic technology in clinical practice are often at a disadvantage due to competition with other bacterial flora during sample transportation and culture, resulting in long detection time and poor stability of test results. At the same time, PCR technology as a conventional diagnostic method has also shown poor results in epidemiological surveys of early latent infection of Bacteroides nodosa and multi-sample testing, and there is no serological identification method for Bacteroides nodosa. Therefore, it is necessary to expand research on other diagnostic detection methods for Bacteroides nodosa. Summary of the invention

[0004] In order to solve the defects in the bacterial isolation and identification and PCR detection methods of Bacteroides nodosa, and the technical problem that there is no serological identification method for Bacteroides nodosa in the prior art, the present invention provides an indirect ELISA detection kit for sheep Bacteroides nodosa antibodies and its application. By using the recombinant protein of the outer membrane protein Omp1D of Bacteroides nodosa as the coating antigen, a Bacteroides nodosa antibody detection kit with simple and convenient operation, accurate and sensitive results, strong repeatability and specificity, and a method for detecting Bacteroides nodosa antibodies using the kit are established.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide an indirect ELISA detection kit for sheep Bacteroides nodosa antibodies, the kit comprising a solid phase carrier coated with a Bacteroides nodosa recombinant protein Omp1D, a diluent, a washing solution, a standard positive control serum, a standard negative control serum, an enzyme-labeled secondary antibody, a color developing solution and a stop solution; the nucleotide sequence of the Bacteroides nodosa recombinant protein Omp1D is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2, the standard positive control serum is the serum of a rabbit infected with Bacteroides nodosa, and the standard negative control serum is the serum of a rabbit that is not immunized or not infected with Bacteroides nodosa.

[0007] In one embodiment of the present invention, the preparation method of the recombinant protein Omp1D of Bacteroides nodosa is as follows: design and synthesize specific primers for the nucleic acid sequence corresponding to the Omp1D protein of Bacteroides nodosa, use the whole genome DNA of Bacteroides nodosa as a template, use the primers to perform PCR amplification, and recover the target gene fragment; connect the target gene to a cloning vector and transform it into Escherichia coli competent cells to obtain positive clones, extract the plasmid by bacterial culture, perform double enzyme digestion and purification on the plasmid and the expression vector, and obtain the recombinant bacterial culture by connection and transformation; use IPTG to induce expression, and perform ultrasonic fragmentation on the obtained bacterial culture to obtain the Omp1D protein expressed in the form of precipitation, and perform gel cutting and purification after SDS-PAGE to obtain the recombinant protein Omp1D of Bacteroides nodosa.

[0008] In one embodiment of the present invention, the nucleotide sequence of the upstream primer in the primers is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.4.

[0009] In one embodiment of the present invention, the solid phase carrier coated with the recombinant protein Omp1D of Bacteroides nodosa is a 96-well ELISA plate coated with the recombinant protein Omp1D of Bacteroides nodosa, and the specific preparation method is as follows: using 0.01M phosphate buffer as the coating solution, diluting the purified recombinant protein Omp1D to 2μg / mL, adding 100μL / well to the ELISA plate, and coating at 4°C for 12h; the next day, discarding the coating solution and washing with PBST; adding 5% skim milk by mass as the blocking solution at 300μL / well, and standing at 37°C for 2h; discarding the blocking solution, washing with PBST, and drying at room temperature.

[0010] In one embodiment of the present invention, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG, the color developing solution is TMB substrate color developing solution, the stop solution is 2M H2SO4 solution, and the diluent and washing solution contain 0.05% volume ratio Tween-20 in PBS buffer.

[0011] The second object of the present invention is to provide the use of the above-mentioned indirect ELISA detection kit for Bacteroides ovis antibodies in the detection of Bacteroides ovis antibodies for non-diagnostic purposes, characterized in that the use comprises the following steps:

[0012] S1. Incubation of samples: adding serum samples diluted with a diluent to a solid phase carrier coated with the recombinant protein Omp1D of Bacteroides nodosa, incubating, and washing with a washing solution to obtain a primary ELISA plate;

[0013] S2, incubation of secondary antibody: adding the diluted enzyme-labeled secondary antibody to the primary ELISA plate obtained in S1, incubating, washing with a washing solution, and obtaining an intermediate ELISA plate containing the secondary antibody;

[0014] S3, color development: add the color development solution to the intermediate ELISA plate containing the secondary antibody obtained in S2 and color develop in the dark to obtain a color development ELISA plate;

[0015] S4, stop color development: add the stop solution to the color development plate obtained in S3 to stop color development, and measure the OD of the diluted serum sample. 450nm The absorbance value at

[0016] S5. Interpretation of results: If the serum sample OD after dilution 450nm If the value is ≥0.389, it is judged as positive; if the serum sample OD after dilution is 450nm If the value is ≤0.312, it is judged as negative. 450nm If the value is greater than 0.312 and less than 0.389, it is judged as a suspicious sample and needs to be retested.

[0017] In one embodiment of the present invention, the dilution factor of the serum sample in S1 is 1:12800.

[0018] In one embodiment of the present invention, the incubation temperature of the serum sample in S1 is 37° C., and the incubation time is 0.5 h.

[0019] In one embodiment of the present invention, the dilution multiple of the enzyme-labeled secondary antibody in S2 is 1:100000.

[0020] In one embodiment of the present invention, the incubation temperature of the enzyme-labeled secondary antibody in S2 is 37° C., and the incubation time is 1.5 h.

[0021] In one embodiment of the present invention, the color development in S3 is carried out at room temperature, and the color development time is 15 minutes.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses the outer membrane protein Omp1D of Bacteroides nodules as the coating antigen, and establishes an indirect ELISA detection kit for Bacteroides nodules antibodies. Since the outer membrane protein Omp1D of Bacteroides nodules has genetic uniqueness, the kit can realize rapid detection and screening of Bacteroides nodules infection in cattle and sheep, and has the characteristics of simple operation, quick and sensitive, accurate results, good specificity and repeatability. The indirect ELISA detection kit for Bacteroides nodules antibodies established by the present invention is used to detect pathogens such as Staphylococcus aureus and Brucella, and no cross reaction is found, indicating that the kit has strong specificity and good accuracy. At the same time, the indirect ELISA detection kit for Bacteroides nodules antibodies provided by the present invention has the characteristics of simple preparation method, low cost and suitability for large-scale production, and provides a quick, simple and sensitive diagnostic kit for detecting Bacteroides nodules infection for large-scale breeding in animal husbandry, and provides technical support for the prevention and control of Bacteroides nodules infection.

[0024] The polyclonal antibodies obtained by immunizing animals in the present invention can realize the screening of Bacteroides nodosa infection and limb and hoof disease, and have the characteristics of simple operation, good specificity, high sensitivity, strong repeatability and accurate results. Therefore, the polyclonal antibodies or kits containing polyclonal antibodies provided by the present invention can be used to detect Bacteroides nodosa.

[0025] The present invention provides an application of a recombinant protein of Bacteroides nodosa Omp1D in the preparation of a polyclonal antibody for detecting the recombinant protein of Bacteroides nodosa Omp1D, and the recombinant protein of the present invention can be used to prepare a polyclonal antibody for accurately detecting Bacteroides nodosa, and the obtained polyclonal antibody has good repeatability. At the same time, the present invention provides a polyclonal antibody prepared by the recombinant protein of the present invention, which can realize the screening of Bacteroides nodosa infection, and has the characteristics of simple operation, good specificity, high sensitivity, strong repeatability and accurate results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is the SDS-PAGE identification result of the prokaryotic expression analysis of the recombinant protein Omp1D of Bacteroides nodosa; wherein, M is the pre-stained rainbow protein Marker, 1 is the whole bacterial body of the recombinant protein Omp1D without induction by ultrasonic crushing, 2 is the whole bacterial body of the induced empty vector pET-32a by ultrasonic crushing, 3 is the whole bacterial body of the induced recombinant protein Omp1D by ultrasonic crushing, 4 is the supernatant of the induced recombinant protein Omp1D by ultrasonic crushing, and 5 is the precipitate of the induced recombinant protein Omp1D by ultrasonic crushing;

[0027] Figure 2 The SDS-PAGE analysis result of the purified Bacteroides nodosa recombinant protein Omp1D; wherein, M is the pre-stained rainbow protein marker, and 1 is the purified recombinant protein pET-32a-Omp1D;

[0028] Figure 3 The figure shows the result of Western blot identification of the purified Bacteroides nodosa recombinant protein Omp1D using HIS tag antibody as the primary antibody; where M is the pre-stained rainbow protein marker, and 1 is the purified recombinant protein pET-32a-Omp1D;

[0029] Figure 4 The figure is the result of Western blot identification of the purified Bacteroides nodosa recombinant protein Omp1D using the Bacteroides nodosa polyclonal antibody as the primary antibody; wherein M is the pre-stained rainbow protein marker, and 1 is the purified recombinant protein pET-32a-Omp1D;

[0030] Figure 5 This is the result diagram of the indirect ELISA method for determining the titer of polyclonal antibodies;

[0031] Figure 6 This is the screening result diagram of the optimal enzyme-labeled antibody working concentration;

[0032] Figure 7 This is a sensitivity evaluation result diagram of the indirect ELISA detection method provided in Example 3. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0035] In the following examples, the Bacteroides nodosa A198 strain (DSM20708) was purchased from DSMZ, Germany.

[0036] In the present invention, the nucleotide sequence of the gene encoding the recombinant protein Omp1D of Bacteroides nodosa is shown in SEQ ID NO.1:

[0037]

[0038] In the present invention, the amino acid sequence of the recombinant protein Omp1D of Bacteroides nodosa is shown in SEQ ID NO.2:

[0039] .

[0040] Example 1: Preparation of Bacteroides nodosa antigens

[0041] 1. Antigen preparation

[0042] The recombinant protein Omp1D of Bacteroides nodosa (purified recombinant protein Omp1D of Bacteroides nodosa) was obtained by the following method:

[0043] (1) Extraction of whole genome DNA of Bacteroides nodosa

[0044] The bacterial genome extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the whole genome DNA of Bacteroides nodosa.

[0045] (2) Amplification and purification of the Omp1D fragment of the Bacteroides nodosa

[0046] Design upstream and downstream primers (Omp1D-F and Omp1D-R) for the nucleotide sequence (SEQ ID NO.1) encoding the protein Omp1D, use the whole genome DNA of Bacteroides nodosa obtained in step (1) as a template, use Omp1D-F and Omp1D-R to perform PCR amplification on the Omp1D gene fragment to obtain an amplified product, and then purify the amplified product using the Tiangen genome purification kit to obtain a purified product of the target gene.

[0047] Omp1D-F: 5'-CGggatccATGAAACTTTCACACTTATCATTAGC-3' (SEQ ID NO. 3);

[0048] Omp1D-R: 5'-CCGctcgagTTATTTGCCGTGAGAAATCAACCA-3' (SEQ ID NO. 4).

[0049] The reaction system of the above-mentioned PCR amplification is as follows: 2.5 μL of upstream primer, 2.5 μL of downstream primer, 12.5 μL of 2×Taq, 3.5 μL of ddH2O and 4 μL of whole genome DNA of Bacteroides nodosa; the reaction program of the above-mentioned PCR amplification is as follows: 94°C for 2 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 5 cycles; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 25 cycles; 72°C for 5 min.

[0050] (3) Connection

[0051] The purified product of the target gene obtained in step (2) is connected to the cloning vector pET-32a to obtain a recombinant cloning vector.

[0052] The ligation system was 1 μL of pET-32a, 7.0 μL of the purified product of the target gene, 0.5-1 μL of T4 DNA ligase and 1 μL of T4 DNA ligase buffer; the ligation conditions were 16° C. for 16 h.

[0053] (4) Conversion

[0054] The recombinant cloning vector obtained in step (3) was transformed into E. coli BL21 (DE3) competent cells to obtain positive bacteria. After shaking culture at 220 rpm and 37° C., a bacterial solution was obtained, and the plasmid was extracted and sent to a sequencing company (Shanghai Sangon Biotechnology Co., Ltd.) for sequencing to verify whether the target gene was successfully inserted into the vector. The sequencing results showed that the recombinant plasmid contained a gene encoding the recombinant protein Omp1D as shown in SEQ ID NO.1;

[0055] The recombinant plasmid and expression vector pET-32a obtained above were digested with restriction endonucleases BamHI and Xhol (the double digestion system is shown in Table 1, ggatcc is the BamHI digestion site, ctcgag is the Xhol digestion site, the digestion time is 3h, and the temperature is 37°C) and purified, and the sequence fragment of the Omp1D gene obtained after purification was connected with the digestion product of the pET-32a plasmid to obtain the recombinant expression vector pET-32a-Omp1D after connection; the recombinant expression vector pET-32a-Omp1D was transformed into E. coli BL21 (DE3) by conventional transformation to construct a recombinant Escherichia coli expressing the Omp1D protein, and the culture was carried out in LB liquid medium at 220rpm and 37°C with shaking.

[0056] Table 1 Double enzyme digestion system

[0057]

[0058] (5) Induction and purification of recombinant proteins

[0059] ① Induction of recombinant protein:

[0060] The recombinant E. coli solution obtained in step (4) was measured by a spectrophotometer, and the OD 600nm When the pH value was 0.6-0.8, IPTG was added at a final concentration of 1 mmol / mL, and the mixture was induced at 37°C for 16 h to obtain the induced bacterial solution. The precipitate obtained after centrifugation of the bacterial solution was ultrasonically disrupted at a power of 50 Hz (ultrasound for 5 s, with an interval of 10 s as one cycle), and the ultrasonically disrupted mixture was centrifuged again (4°C, 8000 rpm, 20 min) to obtain the Omp1D protein expressed in the form of a precipitate (the induced recombinant protein pET-32a-Omp1D).

[0061] ②Purification of recombinant protein

[0062] The obtained recombinant protein Omp1D expressed in the form of precipitation was subjected to SDS-PAGE gel cutting and purification to obtain the purified recombinant protein Omp1D of Bacteroides nodosa, and the specific method was as follows: after collecting the precipitate, it was resuspended with an appropriate amount of PBS, protein loading buffer was added thereto, and heated in a 100°C water bath for 15 minutes; a 10% SDS-PAGE protein gel was prepared, a hole comb was inserted into the upper gel, 400 μL of the treated protein sample was added to each gel plate, and SDS-PAGE was performed; after the electrophoresis was completed, the protein gel was cut off and placed in a 0.3M KCl solution pre-cooled in a 4°C refrigerator for 10 minutes; at this time, a silver-white target band appeared, the target band was cut off with a scalpel, and then rinsed with PBS for 3 minutes; the target band was transferred to a clean sealed bag and crushed, the crushed target band was scraped into an EP tube, and 500 μL PBS was oscillated to mix, and then frozen and thawed three times in liquid nitrogen and 4°C. After repeated freezing and thawing, the mixture was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was aspirated. The aspirated supernatant was the purified recombinant protein Omp1D. The protein concentration was detected after purification.

[0063] The empty vector pET-32a induced by IPTG, the recombinant bacteria pET-32a-Omp1D without induction, and the recombinant bacteria pET-32a-Omp1D induced by IPTG were ultrasonically disrupted, and the supernatant and precipitate were separated for SDS-PAGE analysis. The analysis results are shown in the following table. Figure 1 As shown, the results showed that the recombinant protein Omp1D was successfully expressed in the form of precipitation at 94 kDa after induction, the empty vector pET-32a was expressed at 18 kDa after induction, and the uninduced recombinant protein pET-32a-Omp1D was not expressed, which was consistent with the expected results.

[0064] The purified recombinant protein Omp1D of Bacteroides nodosa was analyzed by SDS-PAGE. Figure 2 As shown, the recombinant protein Omp1D of Bacteroides nodosa has a single band at 94 kDa, indicating that the recombinant protein Omp1D of Bacteroides nodosa was successfully purified. The protein concentration of the purified recombinant protein Omp1D was determined to be 1290 μg / mL.

[0065] The purified recombinant protein Omp1D was identified by Western blot using HIS tag antibody as the primary antibody. Figure 3 As shown, there was a single reactive band at about 94 kDa, and the results proved that the recombinant protein Omp1D of Bacteroides nodosa was successfully expressed and had reactogenicity.

[0066] The purified Bacteroides nodosa recombinant protein Omp1D was identified by Western blot using the polyclonal antibody of Bacteroides nodosa prepared in the laboratory as the primary antibody. The results are as follows: Figure 4 As shown, the antigen-antibody reaction result was a single band without other mixed bands, proving that the recombinant protein Omp1D of Bacteroides nodosa was successfully purified.

[0067] 2. Antigen emulsification

[0068] The most important thing in antigen-antibody immunoassay is the emulsification of antigen. The quality of antigen emulsification directly affects the final result of the immunoassay. The specific emulsification experiment steps are as follows:

[0069] The concentration of the purified antigen (ie, recombinant protein Omp1D) was adjusted to 1 mg / mL.

[0070] Selection of immune adjuvant: Freund's adjuvant (Sigma brand adjuvant) is usually used as an immune adjuvant in immunology experiments, which is divided into two parts: Freund's complete adjuvant and Freund's incomplete adjuvant. Freund's complete adjuvant is used for the first immunization of animals to stimulate cellular immunity; Freund's incomplete adjuvant stimulates humoral immunity and is therefore usually used for booster immunization.

[0071] Emulsification step: The antigen and Freund's adjuvant are mixed in a 1:1 ratio during the emulsification process. For the first immunization, 0.5 mL of 1 mg / mL antigen is mixed with 0.5 mL of Freund's complete adjuvant. After mixing, the antigen concentration is adjusted to 0.5 mg / mL, and the amount of antigen for the first immunization is 500 μg.

[0072] 3. Immunization of animals

[0073] The experimental animals were 6-8 week old female rabbits weighing about 2 kg. They were purchased from the Animal Experiment Center and were adaptively raised in the animal room for one week before immunization. The recombinant protein Omp1D emulsified with an equal volume of Freund's complete adjuvant was used to immunize the rabbits by multiple subcutaneous injections on the back. The dose required for basic immunization of each rabbit was 1 mL, and the injection dose at each site was 0.2 mL. The injection sites should be dispersed as much as possible. On the 14th and 28th days after the first immunization, the antigen and Freund's incomplete adjuvant (Sigma F5506) were emulsified in equal proportions and used for booster immunization. On the 7th day after the last immunization, blood was collected from the ear vein, and the antibody titer was determined by indirect ELISA. After the determination, whole blood of the animal was collected by heart blood collection. The collected blood should first be placed in a 37°C incubator and left to stand for about 30 minutes to prevent activation of the complement system in the blood. It should then be transferred to a 4°C environment and left to stand overnight to allow the serum to precipitate. The next day, the completely stratified blood sample should be transferred to a centrifuge tube and centrifuged for 10 minutes at 4°C and 3000rpm / min. The supernatant should be stored in a -80°C refrigerator.

[0074] 4. Determination of antibody titer by indirect ELISA

[0075] Indirect ELISA is one of the most commonly used methods for detecting antibodies in immunology experiments. During the detection, the antigen substance is first combined with the solid phase carrier, and then the primary antibody to be detected is added. After the primary antibody to be detected and the antigen are fully combined, the secondary antibody labeled with horseradish peroxidase is added, and finally the color is developed with a color developing solution. The titer of the antibody to be tested is determined according to the different color depths or by using an enzyme marker. The specific steps are as follows:

[0076] (1) Antigen coating: The purified Bacteroides nodosa recombinant protein Omp1D was diluted to 1 μg / mL with a carbonate coating solution at pH = 9.6 and a concentration of 0.05 M, and added to a 96-well ELISA plate at a volume of 100 μL / well. The Bacteroides nodosa recombinant protein Omp1D was coated overnight at 4°C. The wells without sample were used as the blank control group, the wells with pre-immune serum were used as the negative control group, and the wells with post-immune antiserum were used as the experimental group. The coating solution was discarded on the second day, and the plates were washed 3 times with PBST shaking, each time for 3 minutes.

[0077] (3) Blocking: Add 300 μL of 5% skim milk as blocking solution to each well, block at 37°C for 2 h, discard the blocking solution, and wash with PBST for 3 times with shaking, each time for 3 min.

[0078] (4) Primary antibody incubation: The rabbit pre-immune serum and the test antiserum were diluted with PBST solution at a dilution gradient of 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, etc. The diluted serum was added to the coated ELISA plate at a rate of 100 μL per well. The plates were incubated at 37°C for 1 h. A blank control was set up. The plates were washed with PBST for 3 times with shaking for 3 min each time.

[0079] (5) Secondary antibody incubation: Dilute commercial HRP-labeled goat anti-rabbit IgG with PBST solution at a volume ratio of 1:50,000, 100 μL / well, incubate at 37°C for 1 h, discard the secondary antibody solution, and wash with PBST for 3 times with shaking, 3 min each time.

[0080] (6) Color development: Add 100 μL of TMB substrate color development solution to each well and develop the color for 15 min at room temperature in the dark.

[0081] (7) Stop color development: Add 50 μL of 2 mol / L H2SO4 solution to each well to stop color development and read the OD using an ELISA reader. 450nm The numerical value of .

[0082] Titer determination criteria: P / N (positive OD 450nm / Negative serum OD 450nm The maximum serum dilution factor when the value of ) is greater than or equal to 2 is the titer of the polyclonal antibody. Figure 5 As shown, when the serum dilution was 1:102400, the P / N value was still greater than 2, that is, the titer of the Omp1D polyclonal antibody was 1:102400.

[0083] Example 2: An indirect ELISA kit for detecting Bacteroides ovis antibodies

[0084] This embodiment provides an indirect ELISA detection kit for detecting the Omp1D antibody of Bacteroides ovis, and the kit is composed of a solid phase carrier coated with the recombinant protein Omp1D, a diluent, a washing solution, a standard positive control serum, a standard negative control serum, an enzyme-labeled secondary antibody, a colorimetric solution, and a stop solution.

[0085] The preparation method of the solid phase carrier coated with the recombinant protein Omp1D (96-well ELISA plate coated with the recombinant protein Omp1D of Bacteroides ovis) is as follows: 0.01M phosphate buffer (pH 9.6) is used as the coating solution, the purified Bacteroides ovis recombinant protein Omp1D prepared above is diluted, and 100 μL / well is added to the ELISA plate to ensure that the concentration of the recombinant protein Omp1D of Bacteroides ovis in each well is 2 μg / mL, and the coating is carried out at 4°C for 12 hours; the next day, the coating solution is discarded, 300 μL / well PBST is added for washing, a total of 3 times, each time for 3 minutes, and patted dry after washing; 300 μL / well skim milk with a mass fraction of 5% is added as the blocking solution, and the plate is allowed to stand at 37°C for 2 hours; the blocking solution is discarded, 300 μL / well PBST is added for washing, a total of 3 times, each time for 3 minutes, and patted dry after washing, and the plate is dried at room temperature until no water droplets are observed, and then packed into a packaging bag, a desiccant is added, and vacuum storage is carried out.

[0086] Dilution solution and washing solution: both are PBST solutions, specifically, a PBS solution with a Tween-20 volume fraction of 0.05% obtained by mixing 1 mL of Tween-20 with 2000 mL of PBS solution.

[0087] Enzyme-labeled secondary antibody: HRP-labeled goat anti-rabbit IgG.

[0088] Colorimetric solution: Ready-to-use TMB substrate colorimetric solution (commercial Solebol colorimetric solution PR1200).

[0089] Stop solution: 2M H2SO4 solution, which is prepared by dropwise adding 21.7mL of 98% concentrated sulfuric acid into 178.3mL of distilled water.

[0090] The standard positive control serum is the serum of rabbits infected with Bacteroides nodosa, and the standard negative control serum is the serum of rabbits that are not immunized or infected with Bacteroides nodosa.

[0091] Example 3: Method for detecting Bacteroides ovisum using the kit described in Example 2

[0092] 1. This embodiment provides the following detection method based on the kit provided in Example 2:

[0093] Using 0.01M phosphate buffer (pH 9.6) as the coating solution, the purified Bacteroides nodosa recombinant protein Omp1D was diluted to 2μg / mL and then added to the 96-well ELISA plate strip, 100μL per well, to ensure that the concentration of the Bacteroides nodosa recombinant protein Omp1D in each well was 2μg / mL, and coated at 4°C for 12h; the coating solution was discarded, and PBST was routinely washed; the 96-well ELISA plate coated overnight was blocked, and 5% skim milk was used as the blocking solution, 300μL was added to each well, and the plate was blocked at 37°C for 2h, the blocking solution was discarded, and PBST was routinely washed;

[0094] The serum to be tested, negative serum control, positive serum control and blank control (PBST buffer) were diluted at a dilution factor of 1:12800, and the diluted serum to be tested, negative serum control, positive serum control and blank control (PBST buffer) were added to the coated 96-well ELISA plate at a volume of 100 μL / well, incubated at 37°C for 0.5 h, and then washed with PBST routinely;

[0095] HRP-labeled goat anti-rabbit IgG was diluted at a dilution multiple of 1:100,000 and added to a 96-well ELISA plate at a volume of 100 μL / well. After incubation at 37°C for 1.5 h, routine washing with PBST was performed;

[0096] Take out the TMB substrate colorimetric solution from the 4°C refrigerator and warm it to room temperature. Add 100 μL / well of the colorimetric solution and color for 15 min at room temperature in the dark.

[0097] Add 50 μL / well of stop solution to stop color development and set the microplate reader to read OD 450nm The numerical value of .

[0098] The above-mentioned conventional washing with PBST means that 300 μL of PBST is added to each well for washing each time, for a total of 3 times, each time for 3 minutes.

[0099] The calculation method for the test result judgment standard is as follows:

[0100]

[0101]

[0102] Where N is the negative serum OD 450nm , P is positive serum OD 450nm , = is the positive / negative mean, and SD is the positive / negative standard deviation. 450nm , negative critical value and positive critical value to determine the result: if the OD of the diluted sample 450nm≥ positive critical value, it is judged as positive; if the OD of the diluted sample 450nm ≤ negative critical value, it is judged as negative; if the OD of the diluted sample 450nm If the OD value of the diluted sample is between the positive critical value and the negative critical value, it is considered a suspicious sample and needs to be retested. 450nm ≥ positive critical value, it is judged as positive; if the OD of the diluted sample 450nm ≤ negative critical value, it is judged as negative. 30 negative serum samples with known background were tested and the results were SD=0.077, so the positive critical value is 0.389 and the negative critical value is 0.312.

[0103] If the sample OD 450nm ≥0.389, it was considered positive;

[0104] If the sample OD 450nm If the value is ≤0.312, it is judged as negative;

[0105] If the sample OD 450nm If the value is greater than 0.312 and less than 0.389, it is judged as a suspicious sample and needs to be retested.

[0106] 2. The present invention screened the optimal antigen coating concentration, the optimal negative and positive serum working concentrations, the optimal enzyme-labeled antibody working concentration, the optimal antigen coating solution and coating conditions, the optimal blocking solution and blocking conditions, and the optimal color developing solution working conditions in the above-mentioned detection method.

[0107] (1) Screening of optimal antigen coating concentration and optimal negative and positive serum working concentrations

[0108] On the basis of the detection method provided in this embodiment, the concentrations of the recombinant protein Omp1D of Bacteroides nodosa were diluted to 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL using the coating solution, and the negative and positive sera of Bacteroides nodosa were diluted by 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800 using the diluent, and the goat anti-rabbit enzyme-labeled secondary antibody was diluted by 1:50000, and the P / N value was calculated using the square matrix method, where N is the negative serum OD 450nm , P is positive serum OD 450nm .

[0109] Optimal antigen coating concentration and serum dilution criteria: OD of positive serum 450nm The value is above 1.5, and the OD of negative serum 450nm The best value is below 0.2 and the P / N (positive / negative) value is the largest.

[0110] The screening results are shown in Table 2. When the P / N value was the highest, the optimal antigen coating concentration was 2 μg / mL and the optimal serum working concentration was 1:12800.

[0111] Table 2 Screening results of optimal antigen coating concentration and optimal serum dilution

[0112]

[0113] (2) Screening of the optimal enzyme-labeled antibody working concentration

[0114] On the basis of the detection method provided in this embodiment, the purified recombinant protein Omp1D was coated at an optimal antigen coating concentration of 2 μg / mL, and the standard Bacteroides nodosa negative and positive serum was diluted at an optimal serum dilution multiple of 1:12800, and the enzyme-labeled secondary antibody was diluted at a ratio of 1:2500, 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:100000, 1:160000, 1:320000, 1:640000, 1:1280000, 1:2560000 according to the working range of the instruction manual, and the P / N value was calculated using the square matrix method. Criteria for determining the optimal enzyme-labeled antibody working concentration: the maximum P / N value is the best.

[0115] Filter results such as Figure 6 As shown, when the working concentration of the enzyme-labeled antibody is 1:100000, the P / N value is the largest, so the optimal working concentration of the enzyme-labeled antibody is 1:100000.

[0116] (3) Screening of the best antigen coating solution and coating conditions

[0117] On the basis of the detection method provided in this embodiment, the optimal antigen coating concentration, the optimal negative and positive serum working concentrations and the optimal enzyme-labeled antibody working concentration obtained above were selected, different coating solutions, coating temperatures and coating times were set (see Table 3 for details), each treatment was repeated 3 times, and the P / N value was calculated using the square matrix method. Criteria for determining the optimal antigen coating solution and coating conditions: The maximum P / N (positive / negative) value is the best.

[0118] The screening results are shown in Table 3. It can be seen from the records in Table 3 that at 4°C, using 0.01M phosphate buffer as the coating solution to dilute the antigen and coating for 12 hours, the obtained P / N value is the highest. Therefore, the best antigen coating solution is 0.01M phosphate buffer, and the best coating condition is 4°C for 12 hours.

[0119] Table 3 Screening results of antigen coating solution and coating conditions

[0120]

[0121] (4) Screening of the best blocking solution and blocking conditions

[0122] On the basis of the detection method provided in this embodiment, the optimal antigen coating concentration, the optimal negative and positive serum working concentrations, the optimal enzyme-labeled antibody working concentration, and the optimal antigen coating solution and coating conditions obtained above were selected, and different blocking solutions, blocking temperatures, and blocking times were set (see Table 4 for details). Each treatment was repeated 3 times, and the P / N value was calculated using the square matrix method. Criteria for determining the optimal blocking solution and blocking conditions: The maximum P / N (positive / negative) value is the best.

[0123] The screening results are shown in Table 4. It can be seen from the records in Table 4 that at 37°C, using 5% skim milk as the blocking solution and blocking for 2 hours, the obtained P / N value is the highest. Therefore, the best blocking solution is 5% skim milk, and the best blocking condition is blocking at 37°C for 2 hours.

[0124] Table 4 Screening results of blocking solution and blocking conditions

[0125]

[0126]

[0127] (5) Optimization of the optimal color development solution conditions

[0128] On the basis of the detection method provided in this embodiment, the above-obtained optimal antigen coating concentration, optimal negative and positive serum working concentrations, optimal enzyme-labeled antibody working concentration, optimal antigen coating solution and coating conditions, and optimal blocking solution and blocking conditions were selected, and the color development temperature was set to room temperature or 37°C, and the color development time was set to 5min, 10min, 15min, 20min, 25min and 30min (see Table 5 for details). Each treatment was repeated 3 times, and the P / N value was calculated using the square matrix method. The criterion for determining the optimal color development solution conditions: the maximum value of P / N (positive / negative) is the best.

[0129] The screening results are shown in Table 5. Under room temperature conditions, the P / N value obtained after color development for 15 minutes is the highest. Therefore, the optimal color development condition is color development at room temperature for 15 minutes.

[0130] Table 5 Screening results of color development conditions

[0131]

[0132] 3. Evaluate the repeatability, sensitivity, specificity and compliance of the above-mentioned optimal kit using the above-mentioned optimal indirect ELISA detection method

[0133] (1) Repeatability evaluation

[0134] The repeatability of the best indirect ELISA kit for detecting Bacteroides nodosa recombinant protein Omp1D obtained above was evaluated, specifically by intra-batch and inter-batch repeatability tests.

[0135] Intra-batch repeatability test: Take the same batch of 96-well ELISA plates coated with recombinant protein Omp1D and test three known negative sera and three known positive sera.

[0136] Inter-batch repeatability test: Take different batches of detachable 96-well ELISA plates coated with recombinant protein Omp1D, and test three known negative sera and three known positive sera respectively.

[0137] The basic operation steps of the indirect ELISA kit are the same as the above-mentioned optimal detection method. The average P / N value of each sample in the intra-batch repeatability test and the inter-batch repeatability test is calculated. Standard deviation SD and coefficient of variation CV%, Samples 1 to 3 are known positive samples, and samples 4 to 6 are known negative samples. The test results are shown in Tables 6 and 7.

[0138] From the data recorded in Tables 6 and 7, it can be seen that the CV% values ​​obtained by using the best kit for detection are all less than 10%, proving that the intra-batch and inter-batch repeatability is good.

[0139] Table 6 Results of intra-batch repeatability test of the kit

[0140]

[0141] Table 7 Results of the kit batch reproducibility test

[0142]

[0143] (2) Sensitivity evaluation

[0144] Using the optimal kit provided in this example and the optimal detection method established, the positive serum was diluted from 12800 times and diluted to 1:26214400 by the two-fold dilution method to determine the sensitivity of this method. Calculate the corresponding OD of the positive serum at different dilution times 450nm Value, see the result Figure 7 shown.

[0145] Depend on Figure 7 It can be seen that when the positive serum is diluted to 1:6553600 using the best indirect ELISA method established, the P / N value is less than 0.312, and the result is determined to be negative. Therefore, the sensitivity of the indirect ELISA detection method established in this example is 1:3276800.

[0146] (3) Specificity evaluation

[0147] The optimal kit and the optimal detection method provided in this example were used to detect positive serum of Bacteroides ovis-galli, positive serum of foot-and-mouth disease, positive serum of Brucella, and positive serum of Staphylococcus aureus, and the OD was determined. 450nm The results are shown in Table 8.

[0148] It can be seen from the records in Table 8 that the best kit and the best detection method provided by the present invention are specific.

[0149] Table 8 Specificity test results

[0150]

[0151] (4) Compliance test

[0152] The optimal kit provided in this embodiment and the optimal detection method and PCR identification method established were used to detect 20 clinical sheep serum samples with known results (17 positive sera and 3 negative sera). The PCR identification method was based on the method in the literature (Shao Xiqun, Wang Kejian, Chen Lizhi, et al. Establishment of PCR detection method for Bacteroides nodosa [J]. Specialty Product Research, 2005, (04): 4-8.). The test results are shown in Table 9.

[0153] As shown in Table 9, the conformity of the best kit and the best detection method provided by the present invention is 100%, and the conformity of the traditional PCR method is 94.1%.

[0154] Table 9 Compliance test results

[0155]

[0156] Example 4: Using the best kit and best detection method provided in Example 3 to detect clinical samples

[0157] This example uses the best kit and the best detection method provided in Example 3 to detect 19 clinical serum samples from a sheep farm in Heilongjiang Province. The test results are shown in Table 10.

[0158] Table 10 Clinical sample test results

[0159]

[0160] As shown in Table 10, the best kit and the best detection method provided by the present invention can specifically detect Bacteroides nodosa, and the positive rate is 5.2%.

[0161] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An indirect ELISA kit for detecting Bacteroides ovis-nosus antibodies, characterized in that: The method comprises a solid phase carrier coated with a recombinant protein Omp1D of Bacteroides nodosa, a diluent, a washing solution, a standard positive control serum, a standard negative control serum, an enzyme-labeled secondary antibody, a color developing solution and a stop solution; the nucleotide sequence of the recombinant protein Omp1D of Bacteroides nodosa is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the standard positive control serum is the serum of a rabbit infected with Bacteroides nodosa, and the standard negative control serum is the serum of a rabbit that is not immunized or not infected with Bacteroides nodosa.

2. The indirect ELISA detection kit for Bacteroides ovis-nosus antibody according to claim 1, characterized in that: The preparation method of the recombinant protein Omp1D of Bacteroides nodosa is as follows: specific primers are designed and synthesized for the nucleic acid sequence corresponding to the Omp1D protein of Bacteroides nodosa, the whole genome DNA of Bacteroides nodosa is used as a template, PCR amplification is performed using the primers, and the target gene fragment is recovered; the target gene is connected to a cloning vector and transformed into an Escherichia coli competent cell to obtain a positive clone, a plasmid is extracted by bacterial liquid culture, the plasmid and the expression vector are double-enzyme digested and purified respectively, and a recombinant bacterial liquid is obtained by connection and transformation; IPTG is used for inducing expression, and the obtained bacterial liquid is ultrasonically broken to obtain the Omp1D protein expressed in the form of a precipitate, and the recombinant protein Omp1D of Bacteroides nodosa is obtained after SDS-PAGE and gel cutting and purification.

3. The indirect ELISA detection kit for Bacteroides ovis-nosus antibody according to claim 1, characterized in that: The solid phase carrier coated with the recombinant protein Omp1D of Bacteroides nodosa is a 96-well ELISA plate coated with the recombinant protein Omp1D of Bacteroides nodosa, and the specific preparation method is as follows: using 0.01M phosphate buffer as a coating solution, diluting the purified recombinant protein Omp1D to 2μg / mL, adding 100μL / well to the ELISA plate, and coating at 4°C for 12h; the next day, discarding the coating solution and washing with PBST; adding 5% skim milk by mass as a blocking solution at 300μL / well, standing at 37°C for 2h; discarding the blocking solution, washing with PBST, and drying at room temperature.

4. The indirect ELISA detection kit for Bacteroides ovis-nosus antibodies according to claim 1, characterized in that: The enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG, the color developing solution is TMB substrate color developing solution, the stop solution is 2M H2SO4 solution, and the diluent and washing solution are both PBS buffer containing 0.05% Tween-20 by volume.

5. Use of the indirect ELISA detection kit for Bacteroides ovis nodularis antibodies according to any one of claims 1 to 4 in detecting Bacteroides ovis nodularis antibodies for non-diagnostic purposes, characterized in that: The application comprises the following steps: S1. Incubation of samples: adding serum samples diluted with a diluent to a solid phase carrier coated with the recombinant protein Omp1D of Bacteroides nodosa, incubating, and washing with a washing solution to obtain a primary ELISA plate; S2, incubation of secondary antibody: adding the diluted enzyme-labeled secondary antibody to the primary ELISA plate obtained in S1, incubating, washing with a washing solution, and obtaining an intermediate ELISA plate containing the secondary antibody; S3, color development: add the color development solution to the intermediate ELISA plate containing the secondary antibody obtained in S2 and color develop in the dark to obtain a color development ELISA plate; S4, stop color development: add the stop solution to the color development plate obtained in S3 to stop color development, and measure the OD of the diluted serum sample. 450nm The absorbance value at S5. Interpretation of results: If the serum sample OD after dilution 450nm If the value is ≥0.389, it is judged as positive; if the serum sample OD after dilution is 450nm If the value is ≤0.312, it is judged as negative. 450nm If the value is greater than 0.312 and less than 0.389, it is judged as a suspicious sample and needs to be retested.

6. The use according to claim 5, characterized in that: The dilution factor of the serum sample described in S1 is 1:12800.

7. The use according to claim 5, characterized in that: The incubation temperature of the serum sample in S1 is 37° C., and the incubation time is 0.5 h.

8. The use according to claim 5, characterized in that: The dilution multiple of the enzyme-labeled secondary antibody described in S2 is 1:100000.

9. The use according to claim 5, characterized in that: The incubation temperature of the enzyme-labeled secondary antibody in S2 is 37° C., and the incubation time is 1.5 h.

10. The use according to claim 5, characterized in that: The color development in S3 is carried out at room temperature, and the color development time is 15 minutes.

Images