Mycoplasma hyopneumoniae ELISA serum antibody detection method

Abstract

The invention discloses a mycoplasma hyopneumoniae ELISA (enzyme-linked immunosorbent assay) serum antibody detection method. The mycoplasma hyopneumoniae ELISA serum antibody detection method comprises the following steps: coating an ELISA plate with an antigen, sealing, incubating serum, incubating a second antibody and developing a substrate, wherein the antigen is purified Mhp336 protein. The invention also discloses a system for diagnosing swine mycoplasmal pneumonia. The mycoplasma hyopneumoniae ELISA serum antibody detection method disclosed by the invention has the characteristics of good repeatability, high specificity and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting Mycoplasma hyopneumoniae ELISA serum antibodies. Background Art

[0002] Mycoplasma pneumonia is a chronic, consumptive respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp) infection, with coughing and panting as the main symptoms. It is highly contagious, has high morbidity and low mortality, and has caused significant economic losses to the pig industry worldwide. Establishing an accurate, sensitive and rapid serum antibody detection method for Mhp is helpful to understand the prevalence of Mhp in pigs, and then take corresponding prevention, treatment and comprehensive control measures.

[0003] The most widely used method for detecting serum antibodies to Mycoplasma hyopneumoniae is enzyme-linked immunosorbent assay (ELISA). The most commonly used one on the market is the indirect ELISA antibody detection kit developed by IDEXX, which uses the whole bacteria of Mycoplasma hyopneumoniae as the coating antigen, which causes the kit to cross-recognize antibodies produced by other mycoplasmas in the environment when detecting serum antibodies to Mycoplasma hyopneumoniae, resulting in low specificity. Feng et al. (Feng ZX, Shao GQ, Liu MJ, Wang HY, Gan Y, Wu XS. Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection [J]. Veterinary Microbiology, 2010, 143 (2-4): 410-416.) established an indirect ELISA antibody detection method based on P97R1 protein for detecting IgA antibodies in the nasal cavity. This detection method has the problems of sampling difficulty and difficulty in standardization of the sampling process. Our laboratory has previously established a method for detecting Mycoplasma hyopneumoniae ELISA serum antibodies in convalescent sera based on Mhp366 protein (DingH, Wen Y, Xu Z, Zhou B, Tlili C, Tian Y, Wang Z, Ning Y, Xin J. Development of an ELISA for distinguishing convalescent sera with Mycoplasma hyopneumoniaeinfection from hyperimmune sera responses to bacterin vaccination in pigs[J]. Vet Med Sci, 2021, 7(5): 1831-1840. / Tian Y, Xu Z, Wen Y, Yang M, Ning Y, Wang Z, DingH. Development of an indirect ELISA for detection of anti-Mycoplasmahyopneumoniae IgG in naturally infected pathogen-induced convalescent sera[J]. BMC Vet Res, 2021, 17(1): 123.) is used to detect antibodies in the serum of pigs naturally infected with Mycoplasma hyopneumoniae. The sensitivity and specificity of this method are better than those of IDEXX's kit, but its sensitivity still needs to be further improved. Summary of the invention

[0004] The problem to be solved by the present invention is to provide a Mycoplasma hyopneumoniae ELISA serum antibody detection method in view of the defect that the sensitivity of Mycoplasma hyopneumoniae ELISA serum antibody in the prior art needs to be further improved. The Mycoplasma hyopneumoniae ELISA serum antibody detection method provided by the present invention has the characteristics of high sensitivity, specificity and repeatability, and can reduce the amount of antigen used, shorten the color development time, and has potential application prospects.

[0005] The present invention mainly solves the above technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides a method for detecting Mycoplasma hyopneumoniae ELISA serum antibodies, which comprises coating an antigen on an ELISA plate, blocking, serum incubation, secondary antibody incubation and substrate color development, wherein the antigen is a purified Mhp336 protein.

[0007] In some embodiments of the present invention, the amino acid sequence of the Mhp336 protein is shown in SEQ ID NO:3.

[0008] In some preferred embodiments of the present invention, the Mhp336 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2.

[0009] In some embodiments of the present invention, the concentration of the coated antigen is 0.05-0.5 μg / mL, preferably 0.05-0.2 μg / mL, and more preferably 0.05 μg / mL.

[0010] In some embodiments of the present invention, the blocking solution is selected from 1% OVA, 1% gelatin, 10% FBS, 2.5% skim milk powder, PBST and 1% BSA; preferably, the blocking solution is selected from 1% OVA, 2.5% skim milk powder and PBST; more preferably, the blocking solution is 2.5% skim milk powder.

[0011] In the present invention, the 1% OVA is ovalbumin diluted with PBS, so that the concentration of ovalbumin is 1%; the 1% gelatin is gelatin diluted with PBS, so that the final concentration of gelatin is 1%; the 10% FBS is FBS diluted with PBS, so that the final concentration of FBS is 10%; the 2.5% skim milk powder is skim milk powder diluted with PBS, so that the final concentration of skim milk powder is 2.5%; the PBST is PBS with a final concentration of 0.05% Tween 20 added; 1% BSA is bovine serum albumin (BSA) diluted with PBS, so that the final concentration of BSA is 1%.

[0012] In some embodiments of the present invention, the blocking time is 0.5-1.5 h, preferably 0.5-1 h, and more preferably 1 h.

[0013] In some embodiments of the present invention, the serum incubated with the serum is the sample to be tested.

[0014] In some embodiments of the present invention, the serum incubation includes a serum dilution step, wherein the serum dilution multiple is 1:50-1:8000, preferably 1:500-1:2000, and more preferably 1:500;

[0015] In some embodiments of the present invention, the serum incubation time is 0.5-1.5 h, preferably 0.5-1 h, and more preferably 0.5 h.

[0016] In some embodiments of the present invention, the secondary antibody used in the secondary antibody incubation is HRP-labeled rabbit anti-pig IgG;

[0017] In some embodiments of the present invention, the secondary antibody incubation includes a step of diluting the secondary antibody, wherein the secondary antibody dilution multiple is 1:5000-1:80000, preferably 1:5000-1:20000, and more preferably 1:10000;

[0018] In some embodiments of the present invention, the secondary antibody incubation time is 0.5-2 h, preferably 0.5-1.5 h, and more preferably 1 h.

[0019] In some embodiments of the present invention, the substrate takes 5-20 minutes to develop color, preferably 5-10 minutes, and more preferably 5 minutes.

[0020] In some embodiments of the present invention, the temperature of the serum incubation and the secondary antibody incubation is 37°C.

[0021] In some embodiments of the present invention, the result judgment condition is when the OD of the sample to be tested is 450nm When the value was ≥0.332, it was judged as positive, OD 450nm A value < 0.332 was considered negative.

[0022] A second aspect of the present invention provides a system for diagnosing porcine mycoplasma pneumonia, the system comprising the following modules:

[0023] An input module, which is used to input the data of the sample to be tested, wherein the data of the sample to be tested includes the detection data obtained by the Mycoplasma hyopneumoniae ELISA serum antibody detection method as described in the first aspect;

[0024] An analysis module, which is used to analyze the data of the sample to be tested to obtain an analysis result; wherein, when the data of the sample to be tested meets the judgment condition, the analysis result is output as "positive"; when the data of the sample to be tested does not meet the judgment condition, the analysis result is output as "negative";

[0025] A judgment module, which judges whether the pig from which the sample comes suffers from swine mycoplasma pneumonia according to the sample data to be tested, and outputs a judgment result; wherein, when the analysis result of the sample data to be tested is "positive", the output judgment result is "Mycoplasma swine pneumonia positive"; when the analysis result of the sample data to be tested is "negative", the output judgment result is "Mycoplasma swine pneumonia negative";

[0026] In some preferred embodiments of the present invention, the judgment condition is when the sample data to be tested is OD 450nm When the value is ≥0.332, the sample to be tested is judged to meet the judgment condition, and the analysis result is output as "positive"; when the sample data to be tested is OD 450nm When the value is <0.332, the sample to be tested is judged not to meet the judgment condition, and the output analysis result is "negative". BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The PCR amplification results of the mhp336 gene; wherein, M: nucleic acid marker; 1: mhp336 gene.

[0028] Figure 2 It is the double enzyme digestion identification of the recombinant plasmid pET-32a(+)-mhp336; wherein, M: nucleic acid marker; 1: pET-32a(+)-mhp336.

[0029] Figure 3 The expression form of Mhp336 recombinant protein was identified by SDS-PAGE; M: protein marker; 1: Escherichia coli BL21 (DE3)-pET32a (+)-mhp336 whole bacteria (before induction); 2: Escherichia coli BL21 (DE3)-pET32a (+)-mhp336 whole bacteria (after induction); 3: Escherichia coli

[0030] BL21(DE3)-pET32a(+)-mhp336 supernatant (after induction); 4: Escherichia coli BL21(DE3)-pET32a(+)-mhp336 precipitate (after induction).

[0031] Figure 4 The purification of Mhp336 recombinant protein was detected by SDS-PAGE; wherein, M: protein marker; 1: supernatant of Escherichia coli BL21(DE3)-pET32a(+)-mhp336; 2: flow-through; 3: Washing buffer; 4-5: Elution bufferⅠ.

[0032] Figure 5 To determine the optimal antigen coating concentration.

[0033] Figure 6 To determine the optimal blocking solution.

[0034] Figure 7 To determine the optimal closing time.

[0035] Figure 8 To determine the optimal serum dilution.

[0036] Fig. 9 To determine the optimal serum incubation time.

[0037] Fig.10 Determination of the optimal dilution for HRP-labeled rabbit anti-pig IgG.

[0038] Fig.11 To determine the optimal secondary antibody incubation time.

[0039] Fig.12 To determine the optimal color development time.

[0040] Fig.13 The results of the specificity test.

[0041] Fig.14 The results of the sensitivity test. DETAILED DESCRIPTION

[0042] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0043] Introduction and Overview

[0044] Various aspects of the present invention will be described below. It will be apparent to those skilled in the art that the present invention may be implemented according to only some or all aspects of the present invention. For purposes of illustration, specific numbering, materials, and configurations are provided herein to enable a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be implemented without specific details. In other examples, well-known features have been omitted or simplified in order not to obscure the present invention.

[0045] Various operations are described herein as multiple discrete steps, in sequence, and are explained in a manner that is most helpful in understanding the invention; however, the sequential description should not be construed as implying that these operations are necessarily order dependent.

[0046] Various embodiments will be described based on the reactants of typical types. It will be apparent to those skilled in the art that the present invention can be implemented using any number of different types of reactants, not just those provided here for illustrative purposes. In addition, it will also be apparent that the present invention is not limited to any specific mixing example.

[0047] In this article, all dilution ratios not otherwise specified are by volume.

[0048] Experimental reagents

[0049] 1. Conventional reagents

[0050]

[0051]

[0052]

[0053] 2. Preparation of main reagents

[0054] ELISA test related reagents

[0055] ①0.05 mol / L carbonate buffer: weigh 0.159 g sodium carbonate and 0.293 g sodium bicarbonate and dissolve them in 90 mL ddH2O, make up to 100 mL, and adjust the pH to 9.6.

[0056] ② Phosphate buffer (PBS): weigh 8 g NaCl, 3.61 g Na2HPO4·12H2O, 0.24 g KH2PO4, and 0.2 g KCl, add 800 mL ddH2O to fully dissolve, then make up to 1000 mL and adjust the pH to 7.0-7.2.

[0057] ③PBST: Take 1L of prepared PBS, add 500μL Tween 20 to make the final concentration of Tween 20 0.05%, mix well and store at 4℃ for later use.

[0058] ④1% BSA: Weigh 1g BSA and add it to 100mL PBS to make the final concentration of BSA 1%. Mix well and store at 4℃ for later use.

[0059] ⑤2.5% skim milk powder: weigh 2.5g skim milk powder and add it to 100mL PBS to make the final concentration of skim milk powder 2.5%. Mix well and store at 4℃ for later use.

[0060] ⑥10% FBS: Add 10 mL of FBS into 90 mL of PBS to make the final concentration of FBS 10%. Mix well and store at 4°C for later use.

[0061] ⑦1% gelatin: Take 1g of gelatin and add it to 100mL of PBS to make the final concentration of gelatin 1%. Mix well and store at 4℃ for later use.

[0062] ⑧1% OVA: Take 1g of OVA and add it to 100mL PBS to make the concentration of OVA 1%. After mixing, store it at 4℃ for later use.

[0063] ⑨ Substrate colorimetric solution A: 2.72 g anhydrous sodium acetate, 0.35 g citric acid monohydrate, 0.06 mL 30% hydrogen peroxide, add ddH2O to dissolve and make up to 100 mL, store at 4°C for later use.

[0064] ⑩ Substrate colorimetric solution B: Take 0.04 g of EDTA·2Na, 0.2078 g of citric acid monohydrate, 10 mL of glycerol, and 0.0391 g of TMB·2HCl, add ddH2O to dissolve and make up to 100 mL, and store at 4°C for later use.

[0065] Stop solution: Take a clean beaker with a capacity of 200mL, add 50mL ddH2O, then add 10.87mL of concentrated sulfuric acid (add it along the wall, drain and stir), and after the temperature drops, make up to 100mL and store at 4℃ for later use.

[0066] 3. Strains

[0067] The recombinant Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 was constructed by our laboratory.

[0068] See Example 2 for strain construction.

[0069] Example 1 Serum collection and identification

[0070] 1. Collection of Blood Samples

[0071] (1) Blood samples from a pig farm with positive Mycoplasma hyopneumoniae: Blood samples were collected from a pig farm in Chongqing. The pigs in the farm only had typical clinical symptoms of Mycoplasma hyopneumoniae such as coughing and panting, and the pigs had not been immunized with Mycoplasma hyopneumoniae vaccine. A total of 20 blood samples were collected.

[0072] (2) Blood samples from a pig farm with negative Mycoplasma pneumoniae: Blood samples were collected from a pig farm in Chongqing. The farm has a good feeding and management model. The pigs have not been immunized with Mycoplasma pneumoniae vaccine and have not shown clinical symptoms of Mycoplasma pneumoniae. In the past two years of serological monitoring, Mycoplasma pneumoniae antibodies were all negative. A total of 60 blood samples were collected.

[0073] 2. Blood Sample Processing

[0074] (1) The collected blood samples were centrifuged at 4°C, 5000 rpm for 10 min, and the supernatant was collected.

[0075] (2) Add glycerol at a final concentration of 50% in a 1:1 ratio, shake and mix, and store at -20°C.

[0076] 3. Serum testing

[0077] (1) The concentration of purified Mhp366-N protein was adjusted to 0.25 μg / mL with 0.5 mol / L carbonate buffer, 100 μL was added to each well, incubated at 37°C for 1 h, then at 4°C overnight, and washed 5 times with PBST, 3 min each time;

[0078] (2) Add 200 μL of 2.5% skim milk powder to each well as blocking solution, block at 37°C for 1 h, and wash with PBST 5 times, 3 min each time;

[0079] (3) Take the pig serum to be tested and dilute it with blocking solution at a ratio of 1:500, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time;

[0080] (4) HRP-labeled rabbit anti-pig IgG was diluted with blocking solution at a ratio of 1:10,000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST 5 times, 3 min each time.

[0081] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 15 min. Then add 50 μL of stop solution and measure OD with a microplate reader within 10 min. 450nm .

[0082] The results showed that 10 samples of pig sera collected from the Mycoplasma hyopneumoniae positive pig farm were Mycoplasma hyopneumoniae antibody positive, and 5 samples were Mycoplasma hyopneumoniae negative; all pig sera collected from the Mycoplasma hyopneumoniae negative pig farm were Mycoplasma pneumoniae antibody negative.

[0083] Example 2 Expression and purification of Mhp336 recombinant protein

[0084] 1. Cultivation of recombinant Escherichia coli BL21(DE3)-pGEX-6P-1-mhp336 (Yaru Ning, Yaoqin Zhou, Zhaodi Wang, Yukang Wen, Zuobo Xu, Yaqin Tian, ​​Mei Yang, Xudong Wang, Yujiao Yang, Honglei Ding*. Elevated Mhp462 antibody induced by natural infection but not invitro culture of Mycoplasma hyopneumoniae[J]. Heliyon, 2020, 6(8): e04832.)

[0085] (1) Take E. coli BL21(DE3)-pGEX-6P-1-mhp336 stored at -80℃ and place it on an ice box. Streak inoculate the Amp + LB plates, cultured overnight at 37°C.

[0086] (2) Pick a single colony from the plate and inoculate 10 mL of Amp + LB liquid medium, culture at 37℃180r / min for 8h.

[0087] 2. Extraction of pGEX-6P-1-mhp336 recombinant plasmid

[0088] The plasmid extraction kit of Guangzhou Meiji Biotechnology Co., Ltd. was used.

[0089] 3. PCR amplification of mhp336 gene

[0090] (1) Primer design and synthesis

[0091] According to the nucleotide sequence of the mhp336 gene, primers were designed using Primer Premier 6.0 software, and BamH I and Xho I restriction sites and corresponding protective bases were introduced into the upstream primer and downstream primer, respectively. Primer sequence: mhp336-F: (SEQ ID NO:4); mhp336-R: (SEQ ID NO: 5). The single underlined bases represent the restriction sites of BamHI and XhoⅠ, and the double underlined bases represent the protective bases.

[0092] (2) PCR amplification of mhp336 gene

[0093] The extracted pGEX-6P-1-mhp336 recombinant plasmid was used as a template and the synthesized primers were used to amplify the target fragment.

[0094] PCR reaction system: DNA template 0.5 μL, mhp336-F 1 μL, mhp336-R 1 μL, Max DNApolymerase 25μL, ddH2O 22.5μL, total reaction system 50μL.

[0095] PCR reaction conditions: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 55 s, 30 cycles; 72°C full extension for 5 min.

[0096] Depend on Figure 1 It can be seen that PCR amplified a 1488 bp mhp336 gene fragment. The full length of the mhp336 gene is 1473 bp, and the amplified fragment removed the stop codon TAA, but added BamHI and XhoI restriction sites and 3 protective bases at both ends, so the full length of the amplified fragment is 1488 bp.

[0097] 4. Gel Recovery of PCR Products

[0098] Gel recovery was performed using the DNA gel recovery kit from Hunan Aikerui Bioengineering Co., Ltd.

[0099] 5. Extraction of pET-32a(+) vector plasmid

[0100] The pET-32a(+) vector plasmid was extracted using the plasmid extraction kit of Guangzhou Meiji Biotechnology Co., Ltd.

[0101] 6. Construction of recombinant plasmid

[0102] (1) Gel recovery of amplified products and vector plasmid were double-digested with BamH I and XhoI, respectively.

[0103] (2) The double enzyme digestion products are recovered by gel recovery.

[0104] (3) Connect the target gene fragment mhp336 to the vector pET-32a(+).

[0105] 7. Transformation of ligation products and screening and identification of positive recombinant plasmids

[0106] (1) The ligation product was transformed into competent E. coli DH5α.

[0107] (2) Extract plasmid.

[0108] (3) The extracted recombinant plasmid was double digested with BamH I and XhoI for identification. The digestion system was: 8 μL of recombinant plasmid, 0.5 μL of BamH I, 0.5 μL of XhoI, 1 μL of 10×Kbuffer, and 10 μL of total reaction system. The reaction system was shaken and mixed, centrifuged to remove the reagent on the inner wall of the tube cap, and digested at 37°C for 1 hour. After the digestion, 5 μL of the digestion product was taken and the digestion result was detected by 1.5% agarose gel electrophoresis.

[0109] like Figure 2 As shown, the recombinant plasmid was cut into two bands, one for the pET-32a(+) vector fragment of about 5900 bp, and the other for the mhp336 gene fragment of 1476 bp (partial bases of the protective bases, BamH I and XhoI restriction sites were removed based on the PCR product). The constructed recombinant plasmid was named pET-32a(+)-mhp336.

[0110] 8. Determination of gene sequence in recombinant plasmid

[0111] The correct positive recombinant plasmid was identified by double enzyme digestion and then sequenced.

[0112] 9. Prokaryotic expression and purification of Mhp336 recombinant protein

[0113] The Mhp336 protein was expressed in prokaryotes and purified, and the purification status of the purified protein was detected by SDS-PAGE electrophoresis.

[0114] Depend on Figure 3 It can be seen that the recombinant bacteria BL21(DE3)-pET32a(+)-mhp336 can express the target protein in two forms, soluble (lane 3) and inclusion body (lane 4) at 16°C.

[0115] Depend on Figure 4 It can be seen that after purification, the purity of the Mhp336 recombinant protein exceeded 90%.

[0116] 10. Dialysis and concentration of Mhp336 recombinant protein

[0117] (1) The purified Mhp336 recombinant protein was dialyzed.

[0118] (2) After the ultrafiltration centrifuge tube is rinsed with ddH2O and PBS, the dialyzed protein is added to the centrifuge tube.

[0119] (3) Centrifuge at 4000 r / min for 30 min, and observe the centrifugation every 10 min until the required volume is reached. Wash the ultrafiltration tube with ddH2O and store it in 20% ethanol.

[0120] 11. Determination of the concentration of Mhp336 recombinant protein

[0121] 10 μL of dialyzed Mhp336 recombinant protein was taken and the Omni-Easy TM The concentration was determined using a ready-to-use BCA protein quantification kit. After the determination, the concentration of the Mhp336 recombinant protein was adjusted to 2 mg / mL, and sterile glycerol was added at a final concentration of 40% in a 1:1 ratio, and the protein was stored at -80°C.

[0122] Example 3 Optimization and determination of ELISA serum antibody detection method for Mycoplasma hyopneumoniae

[0123] 1. Determination of the optimal antigen coating concentration

[0124] (1) The concentration of purified Mhp336 protein was adjusted to 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL using 0.05 mol / L carbonate buffer. 100 μL of diluted Mhp336 protein was added to each well. The cells were incubated at 37°C for 1 h and then at 4°C overnight. The cells were washed with PBST five times, each time for 3 min.

[0125] (2) Add 200 μL of 2.5% skim milk powder to each well, block at 37°C for 1 h, and wash with PBST 5 times, 3 min each time.

[0126] (3) Take 3 portions of Mycoplasma hyopneumoniae-negative serum and 3 portions of Mycoplasma hyopneumoniae-positive serum, dilute them with blocking solution at a ratio of 1:500, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time.

[0127] (4) HRP-labeled rabbit anti-pig IgG was used as the secondary antibody and diluted with 2.5% skim milk powder at a ratio of 1:10,000. 100 μL was added to each well and incubated at 37°C for 1 h. The cells were washed with PBST five times, each time for 3 min.

[0128] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm The OD of negative serum 450nm The average value is set as N, and the OD of each positive serum 450nm The value is S value. The optimal antigen coating concentration is determined based on the S / N value.

[0129] As shown in Table 1 and Figure 5 As shown in the figure, when the antigen coating concentration is 0.05 μg / mL, the S / N value is the largest. Therefore, the optimal antigen coating concentration is determined to be 0.05 μg / mL.

[0130] Table 1. Determination of optimal antigen coating concentration

[0131] Antigen coating concentration (unit: μg / mL) 0.05 0.1 0.2 0.5 1 2 <![CDATA[OD value of positive serum 450nm value]]> 1.502 1.797 2.538 2.716 2.829 2.884 <![CDATA[Negative serum OD 450nm value]]> 0.155 0.203 0.319 0.398 0.855 1.234 S / N value 9.690 8.852 7.956 6.824 3.309 2.337

[0132] 2. Determination of the Optimal Blocking Solution

[0133] (1) Add 100 μL of 0.05 μg / mL Mhp336 protein to each well of the ELISA plate, incubate at 37°C for 1 h, then incubate at 4°C overnight, and wash five times with PBST, each time for 3 min.

[0134] (2) PBST, 1% BSA, 2.5% skim milk powder, 10% FBS, 1% gelatin, PBST, and 1% OVA were used as blocking solutions, 200 μL was added to each well, and the cells were blocked at 37°C for 1 h. The cells were then washed with PBST five times, each time for 3 min.

[0135] (3) Take 3 portions of negative serum and 3 portions of positive serum of Mycoplasma hyopneumoniae, dilute them with the corresponding solution as blocking solution at a ratio of 1:500, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time;

[0136] (4) HRP-labeled rabbit anti-pig IgG was diluted with the corresponding solution as blocking solution at a ratio of 1:10000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST 5 times, 3 min each time.

[0137] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Determine the best blocking solution based on the S / N value.

[0138] As shown in Table 2 and Figure 6 As shown in the figure, when 2.5% skim milk powder is used as the blocking solution, the S / N value is the largest. Therefore, it is determined that the best blocking solution is 2.5% skim milk powder.

[0139] Table 2. Determination of the best blocking solution

[0140] Blocking solution 1% OVA 1% Gelatin 10% FBS 2.5% skim milk powder PBST 1% BSA <![CDATA[Positive serum OD 450nm value]]> 2.270 2.193 2.421 2.290 1.483 2.130 <![CDATA[Negative serum OD 450nm value]]> 0.304 0.306 0.471 0.248 0.182 0.361 S / N value 7.459 7.164 5.137 9.233 8.158 5.898

[0141] 3. Determination of the optimal closure time

[0142] (1) 100 μL of 0.05 μg / mL Mhp336 protein was added to each well of the ELISA plate, incubated at 37°C for 1 h, then incubated at 4°C overnight, and washed five times with PBST, each time for 3 min.

[0143] (2) Add 200 μL of 2.5% skim milk powder to each well, block at 37°C for 0.5 h, 1 h, 1.5 h, and 2 h, and wash with PBST five times, 3 min each time;

[0144] (3) Take 3 portions of Mycoplasma hyopneumoniae-negative serum and 3 portions of positive serum, dilute them with 2.5% skim milk powder at a ratio of 1:500, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time;

[0145] (4) HRP-labeled rabbit anti-pig IgG was diluted with 2.5% skim milk powder at a ratio of 1:10,000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST 5 times, 3 min each time.

[0146] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the best closing time according to the S / N value.

[0147] From Table 3 and Figure 7 It can be seen that when the blocking time is 1 hour, the S / N value is the largest. Therefore, blocking for 1 hour is determined to be the optimal blocking time.

[0148] Table 3. Determination of the optimal sealing time

[0149] Closure time (unit: h) 0.5 1 1.5 2 <![CDATA[OD value of positive serum 450nm value]]> 1.822 2.610 2.818 2.720 <![CDATA[Negative serum OD 450nm value]]> 0.210 0.277 0.454 0.570 S / N value 8.688 9.403 6.210 4.711

[0150] 4. Determination of the optimal serum dilution multiple

[0151] (1) 100 μL of 0.05 μg / mL Mhp336 protein was added to each well of the ELISA plate, incubated at 37°C for 1 h, then at 4°C overnight, and washed five times with PBST, each time for 3 min;

[0152] (2) Block with 2.5% skim milk powder for 1 h and wash with PBST 5 times, 3 min each time;

[0153] (3) Take 3 portions of Mycoplasma hyopneumoniae-negative serum and 3 portions of positive serum, dilute them with 2.5% skim milk powder at a ratio of 1:50, 1:200, 1:500, 1:1000, 1:2000, 1:4000, and 1:8000, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time;

[0154] (4) HRP-labeled rabbit anti-pig IgG was diluted with 2.5% skim milk powder at a ratio of 1:10,000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST 5 times, 3 min each time.

[0155] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the optimal serum dilution multiple based on the S / N value.

[0156] As shown in Table 4 and Figure 8 As shown in the figure, when the serum dilution factor is 1:500, the S / N value is the largest. Therefore, the optimal serum dilution factor is determined to be 1:500.

[0157] Table 4. Determination of the optimal serum dilution multiple

[0158] Serum dilution multiple 1:50 1:200 1:500 1:1000 1:2000 1:4000 1:8000 <![CDATA[OD value of positive serum 450nm value]]> 2.821 2.558 2.095 1.525 1.152 0.703 0.442 <![CDATA[Negative serum OD 450nm value]]> 0.514 0.366 0.209 0.166 0.132 0.122 0.107 S / N value 5.494 6.986 10.046 9.17 8.702 5.754 4.132

[0159] 5. Determination of the Optimal Serum Incubation Time

[0160] (1) Add 100 μL of 0.05 μg / mL Mhp336 protein to each well of the ELISA plate, incubate at 37°C for 1 h, then incubate at 4°C overnight, and wash five times with PBST, each time for 3 min.

[0161] (2) Block with 2.5% skim milk powder for 1 h and wash with PBST 5 times, 3 min each time;

[0162] (3) Serum was diluted with 2.5% skim milk powder at a dilution factor of 1:500, 100 μL was added to each well, and incubated at 37°C for 0.5 h, 1 h, 1.5 h, and 2 h, and washed with PBST five times, 3 min each time;

[0163] (4) HRP-labeled rabbit anti-pig IgG was diluted with 2.5% skim milk powder at a ratio of 1:10,000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST five times, each time for 3 min.

[0164] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the optimal serum incubation time according to the S / N value.

[0165] As shown in Table 5 and Fig. 9 As shown in the figure, when the serum incubation time is 0.5h, the S / N value is the largest. Therefore, the optimal serum incubation time is determined to be 0.5h.

[0166] Table 5. Determination of optimal serum incubation time

[0167] Serum incubation time (unit: h) 0.5 1 1.5 2 <![CDATA[OD value of positive serum 450nm value]]> 1.829 2.085 2.696 2.952 <![CDATA[Negative serum OD 450nm value]]> 0.193 0.275 0.502 0.727 S / N value 9.469 7.573 5.367 4.063

[0168] 6. Determination of the optimal secondary antibody dilution multiple

[0169] (1) Add 100 μL of 0.05 μg / mL Mhp336 protein to each well of the ELISA plate, incubate at 37°C for 1 h, then incubate at 4°C overnight, and wash five times with PBST, each time for 3 min.

[0170] (2) Block with 2.5% skim milk powder for 1 h and wash with PBST 5 times, 3 min each time;

[0171] (3) Serum was diluted with 2.5% skim milk powder at a dilution factor of 1:500, incubated at 37°C for 0.5 h, and washed five times with PBST for 3 min each time;

[0172] (4) HRP-labeled rabbit anti-pig IgG was diluted with blocking solution 2.5% skim milk powder at a ratio of 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000, and 100 μL was added to each well. The cells were incubated at 37°C for 1 h and washed with PBST 5 times, 3 min each time.

[0173] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the best secondary antibody multiple according to the S / N value.

[0174] As shown in Table 6 and Fig.10 As shown in the figure, when HRP-labeled rabbit anti-pig IgG was diluted with 2.5% skim milk powder at 1:10000, the S / N value was the largest. Therefore, the optimal secondary antibody dilution was determined to be 1:10000.

[0175] Table 6. Determination of the optimal secondary antibody dilution factor

[0176] Secondary antibody dilution factor 1:5000 1:10000 1:20000 1:40000 1:80000 <![CDATA[Positive serum OD 450nm value]]> 2.510 1.450 1.269 0.752 0.533 <![CDATA[Negative serum OD 450nm value]]> 0.304 0.159 0.177 0.131 0.106 S / N value 8.224 9.121 7.156 5.765 5.041

[0177] 7. Determination of the Optimal Secondary Antibody Incubation Time

[0178] (1) Add 100 μL of 0.05 μg / mL Mhp336 protein to each well of the ELISA plate, incubate at 37°C for 1 h, then incubate at 4°C overnight, and wash five times with PBST, each time for 3 min.

[0179] (2) Block with 2.5% skim milk powder for 1 h and wash with PBST 5 times, 3 min each time;

[0180] (3) Serum was diluted with 2.5% skim milk powder at a dilution factor of 1:500, incubated at 37°C for 0.5 h, and washed five times with PBST for 3 min each time;

[0181] (4) HRP-labeled rabbit anti-pig IgG was diluted with 2.5% skim milk powder at a dilution factor of 1:10000, 100 μL was added to each well, and incubated at 37°C for 0.5 h, 1 h, 1.5 h, and 2 h, and washed with PBST five times, 3 min each time;

[0182] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the optimal secondary antibody incubation time based on the S / N value.

[0183] As shown in Table 7 and Fig.11 As shown in the figure, when HRP-labeled rabbit anti-pig IgG was incubated for 1 hour, the S / N value was the largest. Therefore, the optimal secondary antibody incubation time was determined to be 1 hour.

[0184] Table 7. Determination of optimal secondary antibody incubation time

[0185] Secondary antibody incubation time (unit: h) 0.5 1 1.5 2 <![CDATA[OD value of positive serum 450nm value]]> 1.462 2.261 1.299 1.081 <![CDATA[Negative serum OD 450nm value]]> 0.162 0.235 0.157 0.145 S / N value 9.023 9.607 8.263 7.445

[0186] 8. Determination of the optimal color development time

[0187] (1) Add 100 μL of 0.05 μg / mL Mhp336 protein to each well of the ELISA plate, incubate at 37°C for 1 h, then incubate at 4°C overnight, and wash five times with PBST, each time for 3 min.

[0188] (2) Block with 2.5% skim milk powder for 1 h and wash with PBST 5 times, 3 min each time;

[0189] (3) Serum was diluted with 2.5% skim milk powder at a dilution factor of 1:500, incubated at 37°C for 0.5 h, and washed five times with PBST for 3 min each time;

[0190] (4) Incubate with HRP-labeled rabbit anti-swine IgG diluted 1:10,000 with 2.5% skim milk powder at 37°C for 1 h, and wash with PBST five times, 3 min each time;

[0191] (5) Add 50 μL of substrate colorimetric solution A and 50 μL of B to each well, mix well, and color at room temperature in the dark for 5 min, 10 min, 15 min, and 20 min. Then add 50 μL of stop solution and measure and record the OD value with a microplate reader within 10 min. 450nm Select the best color development time according to the S / N value.

[0192] As shown in Table 8 and Fig.12 As shown in the figure, when the color development time is 5 minutes, the S / N value is the largest. Therefore, the optimal color development time is determined to be 5 minutes.

[0193] Table 8. Determination of the optimal color development time

[0194] Secondary antibody incubation time (unit: min) 5 10 15 20 <![CDATA[OD value of positive serum 450nm value]]> 1.310 1.819 2.541 2.806 <![CDATA[Negative serum OD 450nm value]]> 0.130 0.217 0.321 0.413 S / N value 10.087 8.390 7.919 6.799

[0195] 9. Optimal reaction conditions for ELISA serum antibody detection of Mycoplasma hyopneumoniae

[0196] (1) The concentration of purified Mhp336 protein was adjusted to 0.05 μg / mL with 0.05 mol / L carbonate buffer, 100 μL was added to each well, incubated at 37°C for 1 h, then at 4°C overnight, and washed 5 times with PBST, 3 min each time;

[0197] (2) Add 200 μL of 2.5% skim milk powder as blocking solution to each well and block at 37°C for 1 h. Wash with PBST 5 times, 3 min each time.

[0198] (3) Take the pig serum to be tested and dilute it with 2.5% skim milk powder at a ratio of 1:500, add 100 μL to each well, incubate at 37°C for 0.5 h, and wash with PBST 5 times, 3 min each time;

[0199] (4) HRP-labeled rabbit anti-pig IgG was used as the secondary antibody, diluted with 2.5% skim milk powder at a ratio of 1:10,000, and 100 μL was added to each well. The cells were incubated at 37°C for 2 h and washed with PBST five times, each time for 3 min.

[0200] (5) Add 50 μL of substrate solution A and B to each well, mix well, and color for 5 min at room temperature in the dark. Then add 50 μL of stop solution and measure and record OD with a microplate reader within 10 min. 450nm value.

[0201] 10. Criteria for determining indirect ELISA reaction conditions

[0202] 60 Mycoplasma hyopneumoniae antibody-negative sera collected from Mycoplasma hyopneumoniae-negative pig farms were selected for ELISA test according to the determined optimal reaction conditions. Three replicates were performed for each serum. The test results are shown in Table 9. The mean value (X) and standard deviation (SD) were calculated, and X+2SD was used as the critical value of positive serum. If the result is greater than or equal to X+2SD, it is judged as a positive serum; if it is less than X+2SD, it is judged as a negative serum. X is 0.216, SD is 0.058, and X+2SD=0.332 is obtained. OD of serum to be tested 450nm When the value was ≥0.332, it was judged as positive, OD 450nm A value of <0.332 was considered negative.

[0203] Table 9. ELISA test results of 60 negative sera

[0204]

[0205] Example 5 Repeatability test, specificity test and sensitivity test of ELISA serum antibody detection method for Mycoplasma hyopneumoniae

[0206] 1. Repeatability of the Mycoplasma hyopneumoniae ELISA serum antibody test

[0207] 1.1 Intra-batch reproducibility

[0208] Three ELISA plates were coated with the same batch of recombinant antigens. Three negative sera and three positive sera were randomly selected. Each serum was repeated five times. ELISA was performed under the same conditions according to the determined optimal reaction conditions. The OD 450nm The coefficient of variation (CV) = [standard deviation (SD) / mean value (X)] × 100%.

[0209] As shown in Table 10, the coefficient of variation of the intra-batch repeatability test was between 2.46% and 5.52%, which was less than 6%, indicating that the coefficient of variation of the same sample in the same batch of ELISA plates coated with antigens was small, and had good intra-batch repeatability.

[0210] Table 10. Intra-batch repeatability test

[0211]

[0212] 1.2 Batch-to-batch reproducibility

[0213] Take 3 ELISA plates coated with different batches, randomly select 3 negative sera and 3 positive sera, and repeat 5 times for each serum. Perform ELISA test according to the determined optimal reaction conditions and measure OD 450nm values, and calculate the coefficient of variation.

[0214] As shown in Table 11, the coefficient of variation of the batch-to-batch repeatability test was between 3.63% and 6.20%, which was less than 7%, indicating that the coefficient of variation of the same sample in different batches of ELISA plates was small, and the sample had good batch-to-batch repeatability.

[0215] Table 11. Inter-batch reproducibility experiment

[0216]

[0217] 2. Specificity of the Mycoplasma hyopneumoniae ELISA serum antibody detection method

[0218] Using negative and positive serum of Mycoplasma hyopneumoniae as control, the determined optimal ELISA reaction conditions were used to detect positive serum of Mycoplasma hyorhinis, Mycoplasma synovium, Mycoplasma floccosum, porcine circovirus type 2, Streptococcus suis type 2, classical swine fever virus, African swine fever virus, porcine reproductive and respiratory syndrome virus, and pseudorabies virus gB protein. Each serum was repeated 3 times and the OD of the corresponding wells was measured.450nm The determined critical value was used to judge the specificity of the established indirect ELISA method.

[0219] As shown in Table 11 and Fig.13 As shown, it was found that the above serum test results were all negative; at the same time, Mycoplasma hyopneumoniae positive serum and negative serum were used as controls, and the test results were positive and negative, respectively, indicating that the established Mycoplasma hyopneumoniae ELISA serum antibody detection method has good specificity.

[0220] Table 12. Specificity test

[0221] serum <![CDATA[OD 450nm Value]]> Result determination Mycoplasma hyopneumoniae positive serum 1 2.463 Positive Mycoplasma hyopneumoniae positive serum 2 2.32 Positive Mycoplasma hyopneumoniae negative serum 1 0.211 Negative Mycoplasma hyopneumoniae negative serum 2 0.178 Negative Mycoplasma hyorhinis positive serum 0.078 Negative Mycoplasma floccosum positive serum 0.032 Negative Mycoplasma synovium positive serum 0.157 Negative Positive serum for Streptococcus suis type 2 0.19 Negative Classical swine fever virus positive serum 0.185 Negative African swine fever virus positive serum 0.092 Negative Porcine reproductive and respiratory syndrome virus positive serum 0.081 Negative Porcine circovirus type 2 positive serum 0.048 Negative Porcine pseudorabies virus gB protein positive serum 0.063 Negative

[0222] 3. Sensitivity of the Mycoplasma hyopneumoniae ELISA serum antibody detection method

[0223] Take 5 positive sera of Mycoplasma hyopneumoniae and dilute them with the determined optimal blocking solution at a ratio of 1:100, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000. The remaining steps are carried out according to the optimal reaction conditions and the OD of the corresponding wells is measured. 450nm The determined critical value was used to judge the sensitivity of the established indirect ELISA method.

[0224] As shown in Table 13 and Fig.14 As shown in Figure 2, with the increase of serum dilution multiple, OD 450nm The value gradually decreased. Five positive sera were still judged as positive at a dilution of 1:4000, and only two sera were judged as positive at a dilution of 1:8000. This shows that the positive serum can be diluted up to 4000 times using this method.

[0225] Table 13. Sensitivity test

[0226]

[0227] Example 6 Detection of clinical samples

[0228] 1. Collection of Clinical Samples

[0229] (1) 63 blood samples were collected from a slaughterhouse, 20 blood samples were collected from a pig farm, and 70 and 73 blood samples were collected from two fattening pig farms, totaling 226 blood samples. None of these pigs had been vaccinated against Mycoplasma pneumonia.

[0230] (2) The blood sample processing is the same as described in 2 of Example 1.

[0231] 2. Clinical sample testing

[0232] (1) The detection of serum samples is the same as described in 3 of Example 1.

[0233] (2) The Mycoplasma hyopneumoniae ELISA serum antibody detection method is used to detect clinical serum samples, and the steps are as described in 9 of Example 4.

[0234] The 226 clinical pig serum samples collected were tested by the established Mycoplasma hyopneumoniae ELISA serum antibody detection method, and the Mycoplasma hyopneumoniae negative and convalescent serum differential diagnosis ELISA method established in the laboratory in the early stage and the Mycoplasma hyopneumoniae antibody detection kit of IDEXX were used for detection. The results are shown in Table 14. The positive coincidence rate of the Mycoplasma hyopneumoniae serum antibody ELISA detection method established by the present invention and the Mycoplasma hyopneumoniae negative and convalescent serum differential diagnosis ELISA method is 68.47%, the negative coincidence rate is 97.39%, and the overall coincidence rate is 82.93%; the positive coincidence rate of the Mycoplasma hyopneumoniae ELISA serum antibody detection method and the Mycoplasma hyopneumoniae antibody detection kit of IDEXX is 85.71%, the negative coincidence rate is 85.35%, and the overall coincidence rate is 85.53%; the positive coincidence rate of the Mycoplasma hyopneumoniae negative and convalescent serum differential diagnosis ELISA method and the Mycoplasma hyopneumoniae antibody detection kit of IDEXX is 83.93%, the negative coincidence rate is 64.97%, and the overall coincidence rate is 74.45%.

[0235] Table 14. Clinical sample test results

[0236]

[0237] Although the content of the present invention is described in conjunction with the present embodiment, it cannot be considered as limiting the scope of the present invention, and the protection scope of the present invention is defined by the attached claims. In addition, various changes or modifications made to the present invention by those skilled in the art within the scope defined by the attached claims also fall within the protection scope of the present invention.

Claims

1. A method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA, characterized in that: The method comprises the steps of coating an antigen on an ELISA plate, blocking, serum incubation, secondary antibody incubation and substrate color development, wherein the antigen is a purified Mhp336 protein.

2. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to claim 1, characterized in that: The amino acid sequence of the Mhp336 protein is shown in SEQ ID NO: 3; Preferably, the Mhp336 protein is encoded by the nucleotide sequence shown in SEQ ID NO:

2.

3. The Mycoplasma hyopneumoniae ELISA serum antibody detection method according to claim 1 or 2, characterized in that: The concentration of the coated antigen is 0.05-0.5 μg / mL, preferably 0.05-0.2 μg / mL, and more preferably 0.05 μg / mL; 4. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 3, characterized in that: The blocking solution is selected from 1% OVA, 1% gelatin, 10% FBS, 2.5% skim milk powder, PBST and 1% BSA; preferably, the blocking solution is selected from 1% OVA, 2.5% skim milk powder and PBST; more preferably, the blocking solution is 2.5% skim milk powder; And / or, the sealing time is 0.5-1.5h, preferably 0.5-1h, more preferably 1h.

5. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 4, characterized in that: The serum incubated with the serum is the sample to be tested; And / or, the serum incubation includes a serum dilution step, wherein the serum dilution factor is 1:50-1:8000, preferably 1:500-1:2000, and more preferably 1:500; And / or, the serum incubation time is 0.5-1.5 h, preferably 0.5-1 h, more preferably 0.5 h.

6. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 5, characterized in that: The secondary antibody used in the secondary antibody incubation is HRP-labeled rabbit anti-pig IgG; And / or, the secondary antibody incubation includes a step of diluting the secondary antibody, wherein the dilution multiple of the secondary antibody is 1:5000-1:80000, preferably 1:5000-1:20000, and more preferably 1:10000; And / or, the secondary antibody incubation time is 0.5-2 h, preferably 0.5-1.5 h, more preferably 1 h.

7. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 6, characterized in that: The substrate color development time is 5-20 min, preferably 5-10 min, more preferably 5 min.

8. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 7, characterized in that: The temperature of the serum incubation and the secondary antibody incubation was 37°C.

9. The method for detecting Mycoplasma hyopneumoniae serum antibodies by ELISA according to any one of claims 1 to 7, characterized in that: The result judgment condition is when the OD of the sample to be tested 450nm When the value was ≥0.332, it was judged as positive, OD 450nm A value of <0.332 was considered negative.

10. A system for diagnosing porcine mycoplasma pneumonia, characterized in that: The system includes the following modules: An input module, which is used to input the sample data to be tested, wherein the sample data to be tested includes the detection data obtained by the Mycoplasma hyopneumoniae ELISA serum antibody detection method according to any one of claims 1 to 8; An analysis module, which is used to analyze the data of the sample to be tested to obtain an analysis result; wherein, when the data of the sample to be tested meets the judgment condition, the analysis result is output as "positive"; when the data of the sample to be tested does not meet the judgment condition, the analysis result is output as "negative"; A judgment module, which judges whether the pig from which the sample comes suffers from swine mycoplasma pneumonia according to the sample data to be tested, and outputs a judgment result; wherein, when the analysis result of the sample data to be tested is "positive", the output judgment result is "Mycoplasma swine pneumonia positive"; when the analysis result of the sample data to be tested is "negative", the output judgment result is "Mycoplasma swine pneumonia negative"; Preferably, the judgment condition is that when the data of the sample to be tested is OD450 nm value ≥ 0.332, the sample to be tested is judged to meet the judgment condition, and the output analysis result is "positive"; when the data of the sample to be tested is OD450 nm value < 0.332, the sample to be tested is judged not to meet the judgment condition, and the output analysis result is "negative".

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