Method and kit for simultaneously detecting avian leukosis virus, mycoplasma synoviae and salmonella pullorum antibodies

Abstract

The invention discloses a method and a kit for simultaneously detecting avian leukosis virus, mycoplasma synoviae and salmonella pullorum antibodies, and belongs to the technical field of animal epidemic disease detection. According to the method, an avian leukosis virus p27 protein, a chicken mycoplasma synoviae p50 protein and a truncated GroEL-delta8-1 protein of a dominant antigen GroEL of salmonella pullorum are taken as coating antigens, synchronous detection of the three antibodies is realized by optimizing ELSA reaction conditions, and a kit capable of simultaneously detecting the three antibodies is developed at the same time. The kit has the characteristics of strong specificity, high sensitivity and simplicity and convenience in operation, is suitable for rapid screening and purification of poultry epidemic diseases, and reduces the detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal epidemic detection, in particular to a method for simultaneously detecting antibodies of avian leukosis virus, chicken mycoplasma synoviae and chicken salmonella pullorum and a kit thereof. BACKGROUND

[0002] Avian leukosis is a general term for various tumor diseases of poultry caused by avian leukosis virus (ALV) and viruses in the group of avian sarcoma virus (ASV). The disease shows a variety of benign and malignant tumor epidemics, such as lymphocytic leukemia, myeloblastic leukemia and erythroblastic leukemia. Since Roloff reported chicken lymphosarcoma in 1868, avian leukosis has been distributed all over the world, and natural cases have also occurred in China. Chickens infected with avian leukosis virus grow slowly, cause damage to the immune organs of the chickens and thus cause immunosuppression, indirectly making the chickens more susceptible to infection with other pathogens and immune failure, and multiple tissue tumors can even cause death. The disease can spread the virus through horizontal and vertical transmission, causing serious harm to the world's poultry industry and causing huge economic losses to the poultry industry.

[0003] Mycoplasma synoviae (MS) is an important pathogenic mycoplasma that infects poultry. The disease has a long course, and the sick chickens show poor spirit, rough and disordered feathers, emaciation, preference for lying, low mortality, obvious symptoms of joint swelling, synovial bursa and tendon sheath inflammation, and serious impact on the production capacity of chickens. Sick and virus-carrying chickens are the main source of infection, which can be horizontally and vertically transmitted, but mainly horizontally transmitted through contact, and the infection rate of respiratory tract transmission can reach 100%. MS infection can cause persistent infection of chicken flocks and cause immunosuppression, thereby causing mixed infection of other important infectious diseases such as avian influenza, Newcastle disease, infectious bronchitis, etc. It has been reported that about 70%-80% of poultry can be infected with mycoplasma synoviae, with an average incidence of about 38.4%, and the disease is distributed in countries all over the world, and has become one of the important diseases of intensive chicken farming, seriously endangering the healthy development of the global poultry industry.

[0004] Pullorum disease is caused by Salmonella pullorum (SP) and can occur in chickens of all ages. The main symptoms of the disease in young chickens are acute septicemia and white watery feces, while in adult chickens, the disease is mainly caused by chronic infection, which leads to decreased egg production and increased mortality. The disease is one of the most important infectious diseases in the poultry industry. The transmission of Salmonella pullorum is diverse, not only through horizontal transmission such as contaminated water, feed, and housing, but also vertically through infected hens' reproductive tract to their offspring. This complex transmission network can cause serious harm to the poultry industry.

[0005] Avian leukosis, Mycoplasma synoviae disease, and Pullorum disease are all infectious diseases that can be transmitted horizontally and vertically, causing serious harm to the poultry industry. The most effective measure to control these three diseases is to implement disease purification, mainly by regularly testing breeding flocks, eliminating positive chickens, and continuously purifying to breed infection-free flocks. Among the many detection methods, ELISA detection is widely used in clinical detection due to its sensitivity, specificity, and ease of operation. This method is suitable for primary veterinary departments and chicken farms for diagnosis and serological detection of the disease. Currently, there are commercial avian leukosis antigen or antibody test kits, commercial Mycoplasma synoviae antibody test kits, and commercial Salmonella pullorum antibody single test kits.

[0006] Table 1. Statistics of avian leukosis, Mycoplasma synoviae, and Pullorum disease test kits

[0007] Detection item Technical method Source Avian leukosis antigen detection p27 antigen ELISA Imported / domestic Avian leukosis J subgroup antibody detection ALVJ subgroup antibody ELISA Imported Avian leukosis AB subgroup antibody detection ALVAB antibody ELISA Imported Salmonella pullorum antibody detection SP antibody Plate agglutination Domestic Salmonella pullorum antibody detection SP antibody ELISA Imported / domestic Mycoplasma synoviae antibody detection MS antibody ELISA Imported / domestic

[0008] Avian leukosis, Mycoplasma synoviae disease, and Pullorum disease are all infectious diseases that need to be purified in chicken farms. Currently, the purification of avian leukosis is mainly through the detection of avian leukosis virus antigen p27 in chicken cloaca swabs and avian leukosis antibodies in chicken serum using domestic and foreign ELISA kits. The purification of Mycoplasma synoviae disease is mainly through the detection of Mycoplasma synoviae antibodies in chicken serum using domestic and foreign ELISA kits. The purification of Pullorum disease is mainly through the detection of Salmonella pullorum antibodies in chicken serum using plate agglutination test or ELISA test kits. These kits or methods are designed for single infectious disease detection, and there is no single kit that can detect all three diseases. The separate detection method consumes a large amount of manpower, material resources, and financial resources for clinical detection and purification of avian leukosis, Mycoplasma synoviae disease, and Pullorum disease.

[0009] However, there are significant technical challenges in developing a kit capable of simultaneously, efficiently and accurately detecting antibodies against avian leukosis virus, Mycoplasma synoviae and Salmonella pullorum. The main difficulties are: (1) The three pathogens belong to virus, mycoplasma and bacteria, respectively, and there are compatibility problems in the detection system. The abundance, affinity, subclass distribution and optimal detection conditions of the antibodies induced by avian leukosis virus, Mycoplasma synoviae and Salmonella pullorum infection in serum are different. (2) The antigen epitopes of virus, mycoplasma and bacteria are complex and diverse, and there is a potential risk of non-specific binding or cross-reaction between the antibodies induced by different pathogens and non-specific antibodies present in serum. (3) The spatial steric hindrance effect of different pathogen antibody-antigen complexes, and the possible mutual quenching or background interference of different labeled signals (such as enzymes, fluorescein) in the mixed system increase the complexity and error rate of signal interpretation. Due to the above technical obstacles caused by the differences in pathogen types and the inherent complexity of serum antibody detection, there is currently no commercial kit on the market that can effectively achieve co-detection of ALV, MS and SP antibodies.

[0010] Therefore, it is of great significance for the purification of avian leukosis, Mycoplasma synoviae disease and chicken pullorum in China to develop an avian leukosis, Mycoplasma synoviae and Salmonella pullorum antibody co-detection kit with Chinese independent intellectual property rights and to industrialize its production. SUMMARY

[0011] Based on the above technical problems, the present application develops an ELISA detection method and kit capable of simultaneously detecting avian leukosis virus, Mycoplasma synoviae and Salmonella pullorum antibodies by using avian leukosis virus capsid protein p27, prokaryotic expression protein of Mycoplasma synoviae surface antigen p50 and prokaryotic expression protein of truncated GroEL-Δ8-1 of chicken pullorum Salmonella dominant antigen GroEL as coating antigen. The p27 gene of avian leukosis virus is a highly conserved gene between different subgroups of avian leukosis virus, and the encoded p27 protein is the main component of the viral core shell, which has a high content and accounts for more than 30% of the total viral protein components, and can induce the body to produce specific antibodies. The p50 gene is a surface antigen of Mycoplasma synoviae, which has good immunogenicity and can stimulate the host to produce specific antibody response. Heat-shock protein 60 (GroEL) is an important protein antigen of chicken pullorum Salmonella, which can induce a large amount of antibodies when the chicken pullorum Salmonella invades the body. When the truncated GroEL-Δ8-1 protein is used as a coating protein, the coincidence rate of indirect ELISA with Western-blot and plate agglutination experiment is high.

[0012] In one aspect, the present application provides an antigen combination comprising p27 protein, p50 protein and GroEL-Δ8-1 protein.

[0013] Specifically, the Genbank accession number of the amino acid sequence encoding the p27 protein is M37980, the Genbank accession number of the amino acid sequence encoding the p50 protein is WP_020003242.1, and the Genbank accession number of the amino acid sequence encoding the GroEL-Δ8-1 protein is CP003047.1.

[0014] Specifically, the total concentration of the antigen coating is 1-8 μg / mL, and the mass ratio of p27 protein, p50 protein and GroEL-Δ8-1 protein is 0.25-5:1:0.25-5, preferably, the total concentration of the antigen coating is 3-7 μg / mL, and the mass ratio of p27 protein, p50 protein and GroEL-Δ8-1 protein is 1-3:1:1-3.

[0015] In one aspect, the present application provides a kit for simultaneously detecting antibodies against avian leukosis virus, Mycoplasma synoviae and Salmonella pullorum, the kit comprising any of the antigen combinations described above.

[0016] Specifically, the kit further comprises enzyme-labeled secondary antibody, coating buffer, washing buffer, blocking solution, secondary antibody diluent, color developing solution and termination solution.

[0017] Specifically, the blocking solution is selected from any one or any combination of 2% casein, 5% skim milk, 5% fetal bovine serum, 0.7% gelatin and 1% bovine serum albumin (BSA), the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-chicken IgG, the secondary antibody diluent is 0.5% BSA, the color developing solution is 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution, the coating buffer is carbonate buffer, and the washing buffer is PBST buffer.

[0018] Specifically, the sample is serum, and the dilution of the sample when detecting the sample to be tested is 1:(100-1000), and the dilution of the enzyme-labeled secondary antibody with a stock solution concentration of 2 mg / ml is 1:(1000-18000), preferably, the dilution of the sample is 1:(500-800), and preferably, the dilution of the enzyme-labeled secondary antibody is 1:(2000-8000).

[0019] Specifically, the serum incubation time is 20-120 min, the secondary antibody incubation time in the enzyme-labeled secondary antibody reaction is 10-90 min, the color development time is 2-18 min, the required temperature for blocking is 4-43℃, and the required time for blocking is 0.5-5 h, preferably, the required temperature for blocking is 10-37℃, and the required time for blocking is 1-3 h.

[0020] Specifically, the preparation of the p27 protein, the p50 protein and the GroEL-Δ8-1 protein in the antigen combination is also included, the nucleotide sequences encoding the p27 protein, the p50 protein and the GroEL-Δ8-1 protein are respectively inserted into a prokaryotic expression vector, and the corresponding proteins are obtained after induction, lysis and purification.

[0021] In another aspect, the application also provides the use of the antigen combination and the kit in the preparation of a reagent for detecting avian leukemia and / or mycoplasma synoviae and / or chicken white diarrhea.

[0022] The application combines the current situation that avian leukemia, mycoplasma synoviae and chicken white diarrhea need to be purified at the same time, uses the basic principle of ELISA, and uses the prokaryotic expression proteins of the avian leukemia virus capsid protein p27, the mycoplasma synoviae surface antigen p50 and the truncated GroEL-Δ8-1 of the dominant antigen GroEL of the chicken white diarrhea salmonella as coating antigens to develop an ELISA kit capable of simultaneously detecting avian leukemia, mycoplasma synoviae and chicken white diarrhea. For the first time, three high-specificity antigens are combined, and the optimal coating conditions of p27, p50 and GroEL-Δ8-1 are determined. When the three antigens are mixed and co-coated at the optimal coating concentration (2:1:2 μg / mL) of each independent system, a significant synergistic effect is produced (in the same serum sample, the highest serum titer of co-coating detection is greater than or equal to the highest serum titer of single antigen detection; in the mixed serum sample, the sensitivity of co-coating detection is greater than that of single antigen detection). This phenomenon completely overturns the traditional cognition that co-coating may cause steric hindrance or signal inhibition, and optimizes the reaction conditions of ELISA to establish an indirect ELISA method for detecting avian leukemia, mycoplasma synoviae and chicken white diarrhea. Compared with the currently commonly used kits for separately detecting avian leukemia, mycoplasma synoviae or chicken white diarrhea, the kit of the application can simultaneously detect avian leukemia, mycoplasma synoviae and chicken white diarrhea. As long as the detected chicken is positive, it is eliminated, achieving the purpose of simultaneous purification, and having the characteristics of strong specificity, high sensitivity and simple operation. The kit is suitable for rapid screening and purification of poultry diseases and reduces the detection cost. DETAILED DESCRIPTION

[0023] The present application will be further described with reference to the following specific examples, which are intended to be purely exemplary and not limiting of the scope of the application as defined in the claims.

[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper and lower limits of the ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits are also included.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference for the disclosure and

[0026] 1, Research method: According to the p27 gene of avian leukosis virus, a p27 recombinant prokaryotic expression vector is constructed, the p50 gene of chicken synovial mycoplasma is constructed p50 recombinant prokaryotic expression vector, and the GroEL-Δ8-1 gene of chicken white diarrhea is constructed GroEL-Δ8-1 recombinant prokaryotic expression vector. The fusion p27 protein, p50 protein and GroEL-Δ8-1 protein are induced by IPTG, and the p27 protein, p50 protein and GroEL-Δ8-1 protein are purified by nickel column. For specific plasmid construction and preparation method, see patents CN108709995A (publication date: 2018.10.26), CN108776221A (publication date: 2018.11.09) and patent CN116716320A (publication date: 2023.09.08). The purified p27 protein, p50 protein and GroEL-Δ8-1 protein are used as coating antigens, and by exploring the optimal coating conditions, blocking conditions, serum dilution ratio and action time, the dilution degree and action time of the secondary antibody and the color development time, a method for detecting avian leukosis virus antibody, chicken synovial mycoplasma antibody and chicken white diarrhea salmonella antibody is established, and the effectiveness of the established method is verified.

[0027] ELISA immune detection method:

[0028] 2, Reagent configuration:

[0029] Coating buffer (pH 9.6 0.05M carbonate buffer):

[0030] Reagent Amount Na2CO3 1.59g NaHCO3 2.93g Distilled water 1000 mL

[0031] Washing buffer (pH 7.4 PBST):

[0032] Reagent Amount KH2PO4 0.2g Na2HPO4·12H2O 2.9g NaCl 8.0g KCl 0.2g Tween-20 0.5 mL Distilled water 1000 mL

[0033] Blocking buffer:

[0034]

[0035]

[0036] Sample and secondary antibody dilution (0.5% BSA):

[0037] Reagent Amount Bovine serum albumin (BSA) 0.5g Washing buffer 100 mL

[0038] Method of preparing stop solution (2M H2SO4)

[0039] Reagent Amount Concentrated sulfuric acid 22 mL Water 178 mL

[0040] 3. Method of routine ELISA experiment

[0041] (1) Antigen coating: dilute coating protein to desired concentration with coating buffer (pH 9.6) to 100 μL / well, and incubate at 4°C overnight.

[0042] (2) Washing: wash plate with PBST, 300 μL / well, pat dry, and wash three times.

[0043] (3) Blocking: add 5% skim milk to enzyme-labeled plate, 200 μL / well, and incubate at 37°C for 2 h.

[0044] (4) Washing: repeat step (2).

[0045] (5) Antigen-antibody reaction: dilute serum with 0.5% BSA to 1:200, add to enzyme-labeled plate, 100 μL / well, and incubate at 37°C for 1 h.

[0046] (6) Washing: repeat step (2).

[0047] (7) Enzyme-labeled antibody reaction: dilute horseradish peroxidase-labeled (HRP-labeled) goat anti-chicken IgG stock solution with 0.5% BSA to 1:8000, add to enzyme-labeled plate, 100 μL / well, and incubate at 37°C for 1 h.

[0048] (8) Washing: repeat step (2).

[0049] (9) Color development: add TMB color developing solution into the enzyme labeled plate, 100 μL / well.

[0050] (10) Termination: color development at room temperature for 10 min, add 50 μL termination solution into each well to terminate the reaction.

[0051] (11) Reading: open the enzyme labeled instrument 1 min in advance to preheat, set the parameters of the enzyme labeled instrument (reference wavelength: 620 nm, measurement wavelength: 450 nm, vibration 3 s, interval 2 s), read OD450nm value.

[0052] Example 1, Construction of Avian Leukosis Virus p27 Prokaryotic Expression Vector and Protein Expression and Purification

[0053] According to the sequence of p27 gene in the registered full sequence of avian leukosis virus gene (M37980) in Genbank, design specific primers with enzyme cutting sites, PCR amplify p27 gene fragment, and insert p27 gene into pET-30a prokaryotic expression vector by enzyme cutting and enzyme ligation. Transform pET-30a-p27 with correct sequence into Transetta (DE3). Culture pET-30a-p27 / Transetta (DE3) at 37°C with shaking to OD600 of 0.6 to 0.8, add IPTG to make the final concentration of 1 mmol / L, induce expression at 37°C, 200 rpm for 6 h, centrifuge to collect bacterial cells. Ultrasonic lysis the bacterial cells (ultrasonic power ratio 35%, ultrasonic 2 s, pause 2 s, ultrasonic time (including ultrasonic and pause time) 1800 s), then centrifuge at 12000 r / min for 20 min, collect the supernatant. Affinity chromatography purify the supernatant with Ni ion-containing affinity chromatography column, obtain purified p27 protein.

[0054] Example 2, Construction of Chicken Synovial Bursa Mycoplasma p50 Prokaryotic Expression Vector and Protein Expression and Purification

[0055] According to the synovial bursa mycoplasma p50 protein sequence (NCBI accession number WP_020003242.1) retrieved from NCBI, specific primers with enzyme cutting sites were designed, and p50 gene fragment was amplified by PCR. The p50 gene was inserted into the pET-28a prokaryotic expression vector by enzyme cutting and ligation. Since the TGA codon encoding tryptophan in the synovial bursa mycoplasma gene is the stop codon of E. coli, six base sites need to be mutated from TGA to TGG (positions 305, 317, 878, 1223, 1229, and 1250, respectively). Three pairs of specific primers were designed at the two ends of the target fragment and the mutation sites. One pair of specific primers was selected for PCR using MSp50 full-length gene as the template, and the PCR product was used as the template for the next round of PCR. In this way, three rounds of synonymous mutations at different base sites were completed. The correctly sequenced pET-28a-p50 was transformed into Transetta (DE3). The pET-28a-p50 / Transetta (DE3) was cultured at 37°C with shaking until the OD600 was 0.6 to 0.8. IPTG was added to a final concentration of 1 mmol / L, and the expression was induced at 16°C and 200 rpm for 16 h. The bacterial cells were collected by centrifugation. The bacterial cells were lysed by ultrasonic (ultrasonic power ratio 35%, ultrasonic 2 s, pause 2 s, ultrasonic time (including ultrasonic and pause time) 1800 s). The supernatant was collected by centrifugation at 12000 r / min for 20 min. The supernatant was purified by affinity chromatography using a Ni ion-containing affinity chromatography column to obtain purified p50 protein.

[0056] Example 3, Construction of Prokaryotic Expression Vector of Salmonella Gallinarum GroEL-Δ8-1 and Protein Expression and Purification

[0057] According to the GroEL gene sequence of Salmonella gallinarum (gene ID: CP003047.1) retrieved from Genbank, specific primers with BamHI and XhoI as the upstream and downstream enzyme cutting sites were designed, and GroEL-Δ8-1 gene fragment was amplified by PCR. The GroEL-Δ8-1 gene was inserted into the pGEX-6p-1 prokaryotic expression vector by enzyme cutting and ligation. The correctly sequenced pGEX-6p-1-GroEL-Δ8-1 was transformed into Transetta (DE3), which was cultured at 37°C with shaking until the OD600 was 0.6 to 0.8. IPTG was added to a final concentration of 1 mmol / L, and the expression was induced at 30°C and 200 rpm for 10 h. The bacterial cells were collected by centrifugation. The bacterial cells were lysed by ultrasonic (ultrasonic power ratio 35%, ultrasonic 2 s, pause 2 s, ultrasonic time (including ultrasonic and pause time) 1800 s). The supernatant was collected by centrifugation at 12000 r / min for 20 min. The supernatant was purified by affinity chromatography using a Glutathion SepharoseTM 4B affinity chromatography column to obtain GroEL-Δ8-1 protein.

[0058] Establishment of indirect ELISA detection method and determination of conditions

[0059] (1) Determination of synergistic coating conditions

[0060] First, the optimal coating concentration of p27 for the indirect ELISA method of avian leukosis virus antibody was determined to be 2 μg / mL, the optimal coating concentration of p50 for the indirect ELISA method of chicken synovial bursa mycoplasma antibody was determined to be 1 μg / mL, and the optimal coating concentration of GroEL-Δ8-1 for the indirect ELISA method of chicken salmonella pullorum antibody was determined to be 2 μg / mL. The three proteins were coated at the optimal coating concentration and combined in the same system, so that the total concentration of the antigens was 5 μg / mL and the ratio of p27:p50:GroEL-Δ8-1 was 2:1:2. The combined coating system was used as the coating condition, which was compared with the combination of the above-mentioned single coating antigens. The three antigens p27, p50 and GroEL-Δ8-1 were coated on the enzyme-labeled plate with the coating solution alone and mixed, 100 μL / well. The titer of avian leukosis virus positive serum, chicken synovial bursa mycoplasma positive serum or chicken salmonella pullorum positive serum was detected in each combination, and the avian leukosis virus positive serum, chicken synovial bursa mycoplasma positive serum and chicken salmonella pullorum positive serum were diluted with 0.5% BSA, with dilution ratios of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and 1:25600, respectively, 100 μL / well. The sample diluent was used as the negative control. The rest of the operation was performed according to the routine operation of ELISA, the serum titer value of each antigen coating combination was calculated, and the results are shown in Table 2. In the indirect ELISA experiment, P represents the average absorbance value of the positive control well; N represents the average absorbance value of the negative control well; P / N is the ratio of the OD value of the positive control to the OD value of the negative control, which is used to evaluate the effectiveness of the experiment and determine the positive and negative critical value of the sample to be tested. Generally, if P / N≥2.1, the sample is determined to be positive. The data in the table represent the corresponding absorbance, and the serum titer usually refers to the highest dilution multiple of the serum that produces a positive reaction. The serum titer value reflects the binding efficiency of the antigen and antibody, and the higher and more stable the titer is, the higher the antigen-antibody binding efficiency is at this concentration, which can more accurately reflect the true level of the antibody in the serum.

[0061] Table 2. Detection of ALV / SP / MS positive serum by single coating p27 / p50 / GroEL-Δ8-1 protein and mixed coating of the three

[0062]

[0063]

[0064] As shown in Table 2, it was found that the three antigen coating combinations had higher valence than the single coating combination, P27 protein had the highest dilution valence of 1:3200 in ALV serum, and the three together coating detection reached the highest valence of 1:6400 dilution; the highest valence of SP protein and three together coating detection was 1:25600; the highest valence of P50 protein was 1:800, and the highest valence of three together coating detection was 1:3200. The above results show that under this ratio, three together coating not only does not affect the high recognition of GroEL-Δ8-1 antibody in serum, but also has a synergistic effect on the recognition of P27 and P50 antibodies. The antigen coating concentration of 5 μg / mL and the ratio of p27:p50:GroEL-Δ8-1 of 2:1:2, i.e. p27:p50:GroEL-Δ8-1 of 2:1:2, are selected as the coating ratio with synergistic effect.

[0065] (2) Determination of the best blocking solution and blocking time

[0066] The enzyme-labeled plate was coated under the above determined antigen coating conditions, and 5% skim milk, 1% BSA, 5% standard fetal bovine serum, 0.7% gelatin and 2% casein were used for blocking at 4°C overnight, and the blocking time was set as 0.5h, 1h, 1.5h, 2h, 2.5h and 3h at 37°C, respectively. The layout was carried out according to the chessboard method, and the rest was carried out according to the routine operation of ELISA. The P / N values of the detection of avian leukosis positive serum, chicken synovial mycoplasma positive serum and chicken white dysentery positive serum were calculated respectively to determine the best blocking solution and blocking time, and the results are shown in Tables 3-5.

[0067] Table 3. Detection of ALV antibody blocking condition exploration

[0068]

[0069]

[0070] Table 4. Detection of SP antibody blocking condition exploration

[0071]

[0072] Table 5. Detection of MS antibody blocking condition exploration

[0073]

[0074] The results of the detection of avian leukosis positive serum, chicken synovial mycoplasma positive serum and chicken white dysentery positive serum P / N value were all higher when 2% casein was used for blocking at 37°C for 2h, so the blocking condition was 2% casein at 37°C for 2h.

[0075] (3) Determination of the best serum dilution and enzyme-labeled secondary antibody dilution

[0076] According to the optimized conditions, the serum dilution is 1:100, 1:200, 1:400 and 1:800, and the dilution of the enzyme-labeled secondary antibody is 1:2000, 1:4000, 1:8000 and 1:16000. The P / N values of the positive serum of avian leukosis, the positive serum of chicken synovial bursa mycoplasma and the positive serum of chicken white diarrhea are calculated respectively to determine the optimal serum dilution and enzyme-labeled secondary antibody dilution.

[0077] Table 6. Detection of ALV antibody serum dilution and secondary antibody dilution

[0078]

[0079] Table 7. Detection of SP antibody serum dilution and secondary antibody dilution

[0080]

[0081] Table 8. Detection of MS antibody serum dilution and secondary antibody dilution

[0082]

[0083]

[0084] The P / N values of the positive serum of avian leukosis, the positive serum of chicken synovial bursa mycoplasma and the positive serum of chicken white diarrhea are all the highest when the serum dilution is 1:800 and the enzyme-labeled secondary antibody dilution is 1:2000, so the serum dilution is 1:800 and the enzyme-labeled secondary antibody dilution is 1:2000.

[0085] (4) Determination of the optimal incubation time of the primary antibody (chicken serum)

[0086] Under the optimized reaction conditions, the antigen-antibody reaction time is set to 30 min, 45 min, 60 min, 75 min and 90 min, and the rest of the operation is carried out according to the routine operation of ELISA. The P / N values of the positive serum of avian leukosis, the positive serum of chicken synovial bursa mycoplasma and the positive serum of chicken white diarrhea are calculated respectively to determine the optimal time of antigen-antibody reaction.

[0087] Table 9. Detection of ALV antibody incubation time

[0088]

[0089] Table 10. Detection of SP antibody incubation time

[0090]

[0091] Table 11. Incubation time of MS antibody primary antibody detection

[0092]

[0093] The results showed that the P / N values for detecting ALV positive serum, Mycoplasma synoviae positive serum and chicken white dysentery positive serum were all higher when incubated at 37°C for 30 min. Therefore, the incubation time of 37°C for 30 min was determined as the primary antibody reaction condition.

[0094] (5) Determination of the optimal incubation time of enzyme-labeled secondary antibody

[0095] HRP-labeled goat anti-chicken IgG was reacted at 37°C for 15 min, 30 min, 45 min, 60 min and 75 min, respectively, and the P / N values for detecting ALV positive serum, Mycoplasma synoviae positive serum and chicken white dysentery positive serum were compared.

[0096] Table 12. Reaction time of ALV antibody secondary antibody detection

[0097]

[0098] Table 13. Reaction time of SP antibody secondary antibody detection

[0099]

[0100] Table 14. Reaction time of MS antibody secondary antibody detection

[0101]

[0102] The results showed that the P / N values for detecting ALV positive serum, Mycoplasma synoviae positive serum and chicken white dysentery positive serum were all higher when incubated at 37°C for 45 min. Therefore, the incubation time of 37°C for 45 min was determined as the secondary antibody reaction condition.

[0103] (6) Determination of color development time

[0104] According to the optimized conditions, the color development time was set to 3 min, 5 min, 8 min, 10 min and 15 min, respectively, and the P / N values for detecting ALV positive serum, Mycoplasma synoviae positive serum and chicken white dysentery positive serum were compared.

[0105] Table 15. Color development time of ALV antibody detection

[0106]

[0107] Table 16. Color development time of SP antibody detection

[0108]

[0109] Table 17. Detection of MS antibody color development time exploration

[0110]

[0111]

[0112] The results show that the P / N values of the detection of avian leukemia positive serum, chicken bursa mycoplasma positive serum and chicken white diarrhea positive serum are higher when the color development time is 5 min, so the color development time is determined as 5 min.

[0113] (7) The optimal conditions of the finally determined indirect ELISA detection method

[0114] (a) Antigen coating: dilute the antigen with coating solution at pH 9.6, the total concentration of coating is 5 μg / mL, and p27:p50:GroEL-Δ8-1 is 2:1:2, add 100 μL / well to the 96-well enzyme-labeled plate, and coat overnight at 4°C.

[0115] (b) Washing: wash the plate with PBST, 300 μL / well, pat dry, and wash three times.

[0116] (c) Blocking: add 2% casein to the enzyme-labeled plate, 200 μL / well, and block at 37°C for 2 h.

[0117] (d) Washing: repeat step (2).

[0118] (e) Antigen antibody reaction: dilute the chicken serum (test sample) with 0.5% BSA to 1:800, add to the enzyme-labeled plate, 100 μL / well, and incubate at 37°C for 30 min.

[0119] (f) Washing: repeat step (2).

[0120] (g) Enzyme-labeled antibody reaction: dilute the HRP-labeled goat anti-chicken IgG stock solution with 0.5% BSA to 1:2000, add to the enzyme-labeled plate, 100 μL / well, and incubate at 37°C for 45 min.

[0121] (h) Washing: repeat step (2).

[0122] (i) Color development: add TMB color developing solution to the enzyme-labeled plate, 100 μL / well.

[0123] (j) Termination: terminate the reaction by adding 50 μL of termination solution per well after color development at room temperature for 5 min.

[0124] (k) Reading: Turn on the enzyme label meter 1 minute in advance, set the enzyme label meter parameters (reference wavelength: 620 nm, measurement wavelength: 450 nm, vibration 3 s, interval 2 s), read OD450nm value.

[0125] Example 5, mixed sample detection of avian leukosis positive serum, chicken pullorum positive serum and chicken bursa mycoplasma positive serum

[0126] Avian leukosis positive serum, chicken pullorum positive serum and chicken bursa mycoplasma positive serum were mixed in different proportions of 1:0:0, 0:1:0, 0:0:1, 1:1:0, 1:0:1, 0:1:1, 1:1:1, 1:1:10, 1:10:1, 10:1:1, 1:1:50, 1:50:1, 50:1:1, respectively, and the titers of different mixed sera were detected by the optimized self-built chicken pullorum salmonella antibody ELISA detection method, the self-built avian leukosis virus antibody ELISA detection method, the self-built chicken pullorum salmonella antibody ELISA detection method and the self-built avian leukosis virus antibody, chicken bursa mycoplasma antibody and chicken pullorum salmonella antibody ELISA co-detection method, respectively.

[0127] Table 18. Titer results of ALV / SP / MS positive serum mixed in different proportions detected by different coating antigens

[0128]

[0129]

[0130] The results are consistent with expectations, as shown in Table 18, the titers of the sera detected by the single detection ELISA method are generally lower than those detected by the co-detection ELISA method, and the titers of the sera detected by the co-detection ELISA method do not change significantly, indicating that the established avian leukosis virus antibody, chicken bursa mycoplasma antibody and chicken pullorum salmonella antibody ELISA co-detection method can well detect the three diseases.

[0131] Example 6, clinical serum sample detection

[0132] The 121 clinical serum samples were detected by the two methods (self-built chicken pullorum Salmonella antibody ELISA detection method and self-built avian leukemia virus antibody ELISA detection method) optimized in patent CN108709995A, the method (a recombinant nucleic acid of chicken synovial mycoplasma surface antigen P50 and antibody detection application) disclosed in patent CN116716320A and the avian leukemia virus antibody, chicken synovial mycoplasma antibody and chicken pullorum Salmonella antibody ELISA co-detection method (coated with p27+p50+GroEL-Δ8-1) of the present application, the results were statistically analyzed, and the coincidence rates of different detection methods were calculated.

[0133] Table 19. Coincidence rate of avian leukemia virus antibody ELISA detection method and three antibody co-detection ELISA detection methods

[0134]

[0135] Table 20. Coincidence rate of chicken pullorum Salmonella antibody ELISA detection method and three antibody ELISA co-detection methods

[0136]

[0137] Table 21. Coincidence rate of a recombinant nucleic acid of chicken synovial mycoplasma surface antigen P50 and antibody detection application and three antibody ELISA co-detection methods

[0138]

[0139] As shown in Tables 19-21, the coincidence rates of the self-built avian leukemia virus antibody ELISA detection method (patent CN108709995A), the self-built chicken pullorum Salmonella antibody ELISA detection method (patent CN108709995A), the recombinant nucleic acid of chicken synovial mycoplasma surface antigen P50 and antibody detection application (patent CN116716320A) and the avian leukemia virus antibody, chicken synovial mycoplasma antibody and chicken pullorum Salmonella antibody ELISA co-detection method of the present application were 91.74%, 95.87% and 94.21% respectively, indicating that the indirect ELISA method established by co-coating with p27, p50 and GroEL-Δ8-1 could achieve similar results to the previously established single detection method, and could simultaneously detect avian leukemia virus, chicken synovial mycoplasma and chicken pullorum Salmonella antibodies, and could detect avian leukemia virus, chicken synovial mycoplasma and chicken pullorum Salmonella infection. The results of mixed serum of different proportions further verified that the co-detection sensitivity of p27, p50 and GroEL-Δ8-12:1:2 ratio coating combination was greatly improved, and was superior to single coating.

[0140] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. An antigen combination, characterized in that, This includes p27 protein, p50 protein, and GroEL-Δ8-1 protein.

2. The antigen combination according to claim 1, characterized in that, The GenBank accession number for the amino acid sequence encoding the p27 protein is M37980, the GenBank accession number for the amino acid sequence encoding the p50 protein is WP_020003242.1, and the GenBank accession number for the amino acid sequence encoding the GroEL-Δ8-1 protein is CP003047.

1.

3. The antigen combination according to claim 2, characterized in that, The total concentration of the antigen coating is 1-8 μg / mL, wherein the mass ratio of p27 protein, p50 protein and GroEL-Δ8-1 protein is 0.25-5:1:0.25-5.

4. The antigen combination according to claim 3, characterized in that, The total concentration of the antigen coating is 3-7 μg / mL, wherein the mass ratio of p27 protein, p50 protein and GroEL-Δ8-1 protein is 1-3:1:1-3.

5. A kit for simultaneously detecting antibodies against avian leukosis virus, Mycoplasma synoviae, and Salmonella pullorum, said kit comprising the antigen combination described in any one of 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes enzyme-labeled secondary antibody, coating buffer, washing buffer, blocking solution, secondary antibody dilution solution, chromogenic solution, and stop solution.

7. The reagent kit according to claim 6, characterized in that, The blocking solution is selected from any one or more of 2% casein, 5% skim milk, 5% fetal bovine serum, 0.7% gelatin, and 1% bovine serum albumin. The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-chicken IgG. The secondary antibody dilution solution is 0.5% BSA. The chromogenic solution is TMB chromogenic solution. The coating buffer is carbonate buffer. The washing buffer is PBST buffer.

8. The reagent kit according to claim 6, characterized in that, The sample to be tested is serum. When the kit detects the sample, the sample dilution is 1:100-1000. When the enzyme-labeled secondary antibody has a stock solution concentration of 2 mg / ml, the dilution is 1:1000-18000. Preferably, the sample dilution is 1:500-800, and the enzyme-labeled secondary antibody dilution is 1:2000-8000.

9. The kit according to any one of claims 7 to 8, characterized in that, The serum incubation time for the test samples is 20-120 min, the secondary antibody incubation time for the enzyme-labeled secondary antibody reaction is 10-90 min, the color development time is 2-18 min, the blocking temperature is 4-43℃, and the blocking time is 0.5-5 h. Preferably, the sealing temperature is 10-37℃ and the sealing time is 1-3h.

10. The use of the antigen combination according to any one of claims 1-4, or the kit according to any one of claims 5-9, in the preparation of reagents for the simultaneous identification or detection of avian leukosis, Mycoplasma synoviae, and pullorum disease.

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