CN112485423A - Universal rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof - Google Patents
Universal rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof Download PDFInfo
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Abstract
The invention provides a universal rapid detection kit for the content of mycoplasma hyopneumoniae antigens and application thereof, belonging to the technical field of biological engineering. The kit comprises an ELISA plate coated by a swine mycoplasma antigen and rabbit anti-EF-Tu serum; the rabbit anti-EF-Tu serum is obtained by immunizing a New Zealand rabbit with recombinant EF-Tu protein, collecting blood and separating serum, wherein the sequence of the recombinant EF-Tu protein is shown in SEQ ID NO. 4. The kit has the advantages of short time consumption and high accuracy, and can detect the content of the mycoplasma hyopneumoniae antigen in the inactivated vaccine.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a universal rapid detection kit for the content of mycoplasma hyopneumoniae antigens and application thereof.
Background
The mycoplasma is the smallest prokaryotic organism capable of independently living in nature, and the mycoplasma separated so far reaches more than 190 species, is distributed all over the world, has various types, has wide influence and great harm. The mycoplasma suis is an important pathogenic microorganism in veterinary medicine, the pathogenesis of the mycoplasma is generally slow, the morbidity and the mortality are low, and huge economic loss can be caused to production. For example, Mycoplasma hyopneumoniae (MPS) caused by Mycoplasma hyopneumoniae (Mhp) widely occurs around the world, clinical symptoms mainly include cough and asthma, generally, the death rate of pigs is not high, but the feed conversion rate is seriously reduced, and the growth and development are poor for a long time, so that huge economic loss is caused. Other health hazards to pigs include mycoplasma hyorhinis (which can cause clinical symptoms such as multiple serositis and arthritis and cause the reduction of pig growth performance), mycoplasma hyofloccosum (which can cause slight lesions of bronchi), and mycoplasma hyosynoviae (which can cause arthritis of piglets over 10 weeks old).
At present, vaccination is a key measure for preventing and controlling mycoplasma infection of swine, and in the process of vaccine development, the antigen content is an important quality control index and is closely related to the actual efficacy of the vaccine. For the determination of the content of the mycoplasma hyopneumoniae antigen, a standard Color Change Unit (CCU) counting method is generally adopted, but the method detects the amount of live mycoplasma during culture and harvest, so the method is only suitable for quality evaluation of live vaccine products and is not suitable for effective antigen content detection of inactivated vaccine production and finished vaccine, and meanwhile, the determination period of the method is long, and the method is also influenced by detection serum, a detection container, parallel number determination, sampling time and the like.
The prior art lacks a method for efficiently and accurately detecting the content of mycoplasma hyopneumoniae antigens, which can be used for inactivated vaccines.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a universal rapid detection kit for the content of the mycoplasma hyopneumoniae antigen, so as to overcome the defects that the traditional quantitative detection method for mycoplasma takes long time, has low accuracy and cannot detect the content of the mycoplasma hyopneumoniae antigen in inactivated vaccines.
The invention also aims to provide a method for detecting the content of the mycoplasma hyopneumoniae antigen by using the kit for non-diagnosis, which has the advantages of short time consumption, high accuracy and capability of detecting the content of the mycoplasma hyopneumoniae antigen in the inactivated vaccine.
The purpose of the invention is realized by adopting the following technical scheme.
A universal rapid detection kit for the content of swine mycoplasma antigen comprises an ELISA plate coated by swine mycoplasma antigen and rabbit anti-EF-Tu serum; the rabbit anti-EF-Tu serum is obtained by immunizing a New Zealand rabbit with recombinant EF-Tu protein, collecting blood and separating serum, wherein the sequence of the recombinant EF-Tu protein is shown in SEQ ID NO. 4.
In the invention, the ELISA plate is coated by using a mycoplasma hyopneumoniae lysate.
In the invention, the recombinant EF-Tu protein is obtained by inducing and expressing a recombinant bacterium carrying an EF-Tu protein coding gene.
In the invention, the ELISA plate is coated by using a mycoplasma hyopneumoniae lysate and then is sealed by using a PBS (phosphate buffer solution) containing gelatin.
In the invention, the kit also comprises a swine mycoplasma antigen bacteria liquid standard, a PBST solution, a goat anti-rabbit HRP-IgG, a developing solution and a stop solution.
In the invention, the PBST solution is PBS buffer solution containing Tween-20 with the volume percentage concentration of 0.05 percent; the goat anti-rabbit HRP-IgG is purchased from Dr. warrior Biometrics, and the product number is BA 1054.
In the present invention, the mycoplasma hyopneumoniae includes mycoplasma hyopneumoniae, mycoplasma hyorhinis, mycoplasma hyofloccosum, and mycoplasma hyosynoviae.
The invention also provides a method for detecting the content of the mycoplasma hyopneumoniae antigen by using the kit for non-diagnosis purposes, which comprises the following steps:
(1) arranging a sample hole to be detected and a blank control sample hole in an ELISA plate coated by a swine mycoplasma antigen, adding the sample to be detected into the sample hole to be detected, adding a PBS (phosphate buffer solution) into the blank control sample hole, then adding rabbit anti-EF-Tu serum into each hole, and washing with a PBST solution after incubation;
(2) adding goat anti-rabbit HRP-IgG into each hole, and incubating;
(2) after PBST solution is washed, TMB substrate color development liquid is added;
(3) adding a stop solution to stop the reaction, and reading a light absorption value at 450 nm;
(4) and calculating the content of the mycoplasma hyopneumoniae antigen according to the standard curve.
The invention also provides application of the kit in detecting the content of the antigen in the swine mycoplasma culture or swine mycoplasma vaccine product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the universal rapid detection method for the content of the mycoplasma hyopneumoniae antigen realizes the determination of different mycoplasma contents, the detection time is only 2-4 hours, and compared with the common CCU counting method, the method greatly shortens the production period and improves the production efficiency.
(2) The invention can detect the content of mycoplasma hyopneumoniae antigen in the culture bacterial liquid or the live vaccine, and can detect the content of antigen in the concentrated antigen and the inactivated vaccine.
(3) The method is simple to operate, good in repeatability, high in accuracy and simple in result judgment and calculation.
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FIG. 1 identification of rabbit anti-EF-Tu serum, 1: SDS-PAGE electrophoresis of Mhp 168 whole bacterial protein; 2: western Blot identification of rabbit anti-EF-Tu serum and Mhp 168 holobacterin protein.
FIG. 2 shows a standard curve of the standard substance, the abscissa is the logarithmic value of the dilution factor (base number is 2), and the ordinate is B/B0.
FIG. 3 correlation analysis of RP values with CCU measurement results, with the abscissa representing the logarithm of CCU (base 10) and the ordinate representing the RP value.
FIG. 4 is a standard curve of different Mycoplasma hyorhinis, wherein A is a standard curve of Mycoplasma hyorhinis, B is a standard curve of Mycoplasma hyorhinis, C is a standard curve of Mycoplasma hyosynoviae, and the abscissa is the logarithmic value of the dilution factor (base number of 2) and the ordinate is B/B0.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Reagents and sources used in the examples: escherichia coli DH5a was purchased from Takara; freund's complete adjuvant and Freund's incomplete adjuvant, IPTG, Tween20 were purchased from Sigma-Aldrich, USA; the 96-well detachable elisa plate was purchased from Solarbio; the BCA protein concentration determination kit and the TMB color development solution (product number is P0209) are purchased from Biyuntian biotechnology Co., Ltd; the goat anti-rabbit HRP-IgG is purchased from doctor Ded Bio of Wuhan (product number BA1054), and other used chemical reagents are all analytically pure.
EXAMPLE 1 Rabbit anti-EF-Tu serum was prepared.
EF-Tu sequences of mycoplasma hyopneumoniae, mycoplasma hyorhinis, mycoplasma hyofloccosum and mycoplasma hyosynoviae are downloaded from an NCBI website respectively, and homology analysis of amino acid sequences is performed by using DNAMAN software, so that the EF-Tu sequences of the four mycoplasma are relatively conserved and have homology of 91.58%. According to genome information (Gen Bank accession number CP002274) of mycoplasma hyopneumoniae (Mhp)168 strain published on Gen Bank, prokaryotic expression primers P1 and P2 of various EF-Tu homologous regions are designed to amplify fragments with higher homology of mycoplasma hyopneumoniae, mycoplasma hyorhinotracheale, mycoplasma hyofloccosum and mycoplasma hyosynoviae in EF-Tu sequences.
The nucleotide sequence of P1 (SEQ ID NO:1) is as follows: 5'-CGCGGATCCTTTGATCGTTCCAAAGAACAT-3' are provided.
The nucleotide sequence of P2 (SEQ ID NO:2) is as follows: 5'-CCGCTCGAGAATAATTTCGGTAACTGTTCC-3' are provided.
Mhp 168 strain genome DNA is used as a template, P1 and P2 are used as primers, a 1170bp target gene is amplified by PCR, and escherichia coli XL10 competent cells are transformed after double digestion and connection. The recombinant plasmid is extracted for sequencing, the sequence of the target gene is shown as SEQ ID NO. 3, and the sequence of the encoded recombinant EF-Tu protein is shown as SEQ ID NO. 4. The target gene is inserted into a pET-32a vector, the recombinant plasmid with correct sequencing is transformed into BL21 competent cells, and prokaryotic expression is carried out after IPTG induction. Purifying the recombinant EF-Tu protein by a nickel column according to a conventional method, performing ultrafiltration liquid exchange on the purified protein, determining the concentration by using a BCA method, adjusting the final concentration to be 2mg/mL, and storing at the temperature of below 70 ℃ after subpackaging for later use.
Selecting 5 healthy New Zealand white rabbits (2-2.5 kg), mixing 2mg/mL recombinant EF-Tu protein with Freund's complete adjuvant at a volume ratio of 1:1, emulsifying, and performing primary immunization on the 5 New Zealand white rabbits (1 mL antigen content) each by subcutaneous multi-point injection on the back. 2mg/mL of recombinant EF-Tu protein and Freund's incomplete adjuvant are mixed according to the volume ratio of 1:1, and are used for secondary immunization, tertiary immunization and quaternary immunization after emulsification, wherein the immunization dose is 1mL (containing 1mg of antigen) per immunization. The interval between two adjacent immunizations is 14 days. One week after the four-immunization, blood is collected from the sterile heart, and serum is separated under the sterile condition to obtain rabbit anti-EF-Tu serum. The antibody specificity of the rabbit anti-EF-Tu serum is detected by Western Blot by taking mycoplasma hyopneumoniae holomycoprotein as a detection antigen. The results are shown in figure 1, the rabbit anti-EF-Tu serum and the mycoplasma hyopneumoniae holomycoprotein have specific reaction, and the specificity of the rabbit anti-EF-Tu serum is proved to be good. Subpackaging rabbit anti-EF-Tu serum in small amount, and storing at low temperature below-70 deg.C for use.
Example 2 establishment of competitive ELISA quantitative detection method
The sample to be detected is mycoplasma hyopneumoniae NJ strain liquid, batch number: 191025, using standard Color Change Unit (CCU) counting method (D.Calus, D.Maes, K.Vrandkx, et al.Validation of ATP luminescence for Rapid and acid characterizationn of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay[J]Journal of Microbiological Methods 2010,83: 335-8CCU/ml, detection period 15 days. The standard Color Change Unit (CCU) count method is abbreviated as CCU count method.
(1) A competitive ELISA quantitative detection method is adopted to detect the mycoplasma hyopneumoniae NJ strain, and the method specifically comprises the following steps:
a. coating: the antigen of the Mycoplasma hyopneumoniae NJ strain was diluted to 10. mu.g/ml with a coating buffer (pH 9.6, 0.05mol/L carbonate buffer) as a coating solution, added to each well of a 96-well microplate in an amount of 100. mu.l/well, and coated overnight at 4 ℃. The preparation method of the mycoplasma hyopneumoniae NJ strain antigen comprises the following steps: the mycoplasma hyopneumoniae NJ strain liquid is inoculated in a KM2 liquid culture medium according to the ratio of 1:10 (volume ratio), the culture is kept still for 48-72h at 37 ℃, the culture is harvested, the centrifugation is carried out for 20min, the precipitate is washed for 2 times by PBS buffer solution, and after the 2 nd washing, the precipitate is resuspended by a small amount of PBS buffer solution. Carrying out ultrasonic cracking to obtain a lysate, namely the antigen of the mycoplasma hyopneumoniae NJ strain, using a BCA kit to carry out protein content determination, and using the lysate as a coating antigen in a competitive ELISA quantitative detection method for preservation at-70 ℃.
b. And (3) sealing: the coating solution was discarded, washed 3 times with PBST solution, and added with 1% (mass percent concentration) gelatin in PBS buffer as a blocking solution, 200. mu.l/well, and allowed to act at 37 ℃ for 2 hours.
c. Sample adding: discarding the confining liquid, washing with PBST solution for 3 times, adding PBS buffer solution into blank control sample wells, adding the sample to be detected into the sample wells to be detected, 50 μ l/well, repeating each sample for 2 times, adding 50 μ l rabbit anti-EF-Tu serum (prepared in example 1) into each well, and incubating for 2h at 37 ℃;
d. adding an enzyme-labeled antibody: discarding the sample adding, washing with PBST solution for 3 times, adding 100 μ l/well of goat anti-rabbit HRP-IgG (purchased from Dr. warrior, Bio Inc., product number BA1054) diluted with PBST solution at a dilution of 1:10000, and incubating at 37 deg.C for 1 h;
e. color development: discarding the enzyme-labeled antibody, washing with PBST solution for 3 times, adding TMB substrate color development solution (purchased from Biyuntian Biotechnology Co., Ltd., product number P0209), 100 μ l/well, and reacting at room temperature in dark for 10 min;
f. and (4) terminating: stopping the reaction with 2M sulfuric acid aqueous solution (stop solution), reading the absorbance at 450nm at 50 μ l/well;
g. and (4) judging a result: calculating y according to the formula of y-B/B0, wherein B0 is the light absorption value of a blank control sample hole, B is the light absorption value of a sample hole to be detected, then calculating x according to the standard curve chart of y-0.0963 x +0.1676, and calculating the relative effectiveness RP value of 1/2xSince the RP value was calculated to be 1.028, the content of mycoplasma hyopneumoniae in the sample was 1.028 × 108CCU/ml。
In the above-mentioned competitive ELISA quantitative determination method, the coating buffer solution contains 1.59g/L Na2CO3And 2.93g/L NaHCO3An aqueous solution of (a); the PBS buffer solution contains 8g/L NaCl, 0.2g/L KCl, and 1.44g/L Na2HPO4And 0.24g/L KH2PO4An aqueous solution of (a); the PBST solution was PBS buffer containing 0.05% (volume percent) Tween-20, where the PBS buffer still contained 8g/L NaCl, 0.2g/L KCl, 1.44g/L Na2HPO4And 0.24g/L KH2PO4An aqueous solution of (a).
(2) Preparation of standard curve chart in competitive ELISA quantitative detection method
Making a standard curve: the concentration to be detected by CCU counting method is 108CCU/ml Mycoplasma hyopneumoniae bacterial solution prepared from PBS buffer solution (containing 8g/L NaCl, 0.2g/L KCl, and 1.44g/L Na)2HPO4And 0.24g/L KH2PO4Aqueous solution of (2) at a dilution factor of 2, 4, 8, 16, 32, 64, 128, 256 to give a series of standards, which were examined according to steps a-f of title (1) of this example to give an absorbance B at 450 nm. A standard curve is plotted with the logarithmic value of the dilution factor (base number 2) as the abscissa and the corresponding B/B0(B0 is the absorbance of the blank sample well and B is the absorbance of each standard well) as the ordinate. The results are shown in fig. 2, where the standard curve is y 0.0963x +0.1676, where x is the logarithm of the dilution factor (base 2), y is B/B0, and the correlation coefficient R is2＝0.9904。
Example 3 repetitive evaluation of detection of Mycoplasma hyopneumoniae NJ Strain by competitive ELISA quantitative detection method
The competitive ELISA quantitative detection method in example 2 was used to detect antigen samples of 5 batches of Mycoplasma hyopneumoniae NJ strain (bacterial solution obtained by culturing Mycoplasma hyopneumoniae NJ strain in KM2 medium) on the same batch of ELISA reaction plates (obtained in step b of the title (1) in example 2), each batch of antigen samples was detected once by using 3 different ELISA reaction plates in the same batch, and the antigen content and the in-plate variation coefficient were calculated.
In addition, 5 batches of antigen samples of the mycoplasma hyopneumoniae NJ strain (bacterial fluid obtained by culturing the mycoplasma hyopneumoniae NJ strain in the KM2 culture medium) were detected by using 3 batches of ELISA reaction plates according to the competitive ELISA quantitative detection method in example 2, and each group of variation coefficients of the 5 batches of antigens were calculated by simultaneously using 3 different batches of ELISA reaction plates for detection of each batch of antigen samples.
The results (table 1) show that the mean values of the coefficient of variation of the repetition between the plates of this method are 3.74% and 4.79%, respectively, and the reproducibility is good.
TABLE 1 repeatability analysis of competitive ELISA quantitative detection method
Example 4 competitive ELISA quantitative assay method for detecting the content of Mycoplasma antigens of live vaccines
By using the competitive ELISA quantitative detection method in example 2, 10 batches of antigen samples of the Mycoplasma hyopneumoniae NJ strain (the Mycoplasma hyopneumoniae NJ strain is cultured in KM2 culture medium) were detected, and the results were compared by using a CCU counting method and correlation analysis. The results are shown in Table 2 and FIG. 3, and the RP value and the CCU counting method detection result are in positive correlation, and the correlation coefficient R20.9902, RP values are interchangeable with CCU countsThe conversion formula is that y is 0.9602x-6.6814(y is RP value, and x is log)10(CCU/ml)), indicating that the method can be used for the rapid assessment of live vaccine titers. Therefore, the competition ELISA quantitative determination method in example 2 can be used in place of the CCU counting method. The competitive ELISA quantitative detection method in the embodiment 2 realizes quantitative determination of the content of the mycoplasma hyopneumoniae antigen, the time consumption is shorter than that of a CCU counting method, the determined antigens in different batches are stable, and the detection efficiency is improved.
TABLE 2 determination of antigen content in multiple batches of antigen samples of Mycoplasma hyopneumoniae NJ strains
| Sample numbering | RP values measured by competitive ELISA | Log from CCU counting method10(CCU/ml) |
| NJ-1 | 1.268 | 8.25 |
| NJ-2 | 1.231 | 8.25 |
| NJ-3 | 1.448 | 8.5 |
| NJ-4 | 1.437 | 8.5 |
| NJ-5 | 1.006 | 8.0 |
| NJ-6 | 1.217 | 8.25 |
| NJ-7 | 1.003 | 8.0 |
| NJ-8 | 1.267 | 8.25 |
| NJ-9 | 1.745 | 8.75 |
| NJ-10 | 1.737 | 8.75 |
Example 5 competitive ELISA quantitative detection method for vaccine product detection
Inoculating the recovered mycoplasma hyopneumoniae NJ strain bacterial liquid to 1500mL of KM2 culture medium according to the volume ratio of 1:9, carrying out amplification culture, and taking part of the bacterial liquid to determine the antigen content by using a CCU counting method when the bacterial liquid grows to a logarithmic phase. Adding thimerosal with final concentration (mass percentage concentration) of 0.005% into the rest bacteria liquid, and inactivating for 24h at 37 ℃. And centrifuging the inactivated bacterial liquid at 12000r/min and 4 ℃ for 30min, and collecting bacterial precipitates. The pellet was washed 2 times with sterile PBS buffer and centrifuged to discard the supernatant. 30mL of PBS buffer solution is added into the precipitate and mixed evenly, and the obtained suspension is Mhp antigen.
Preparing 3 batches of Mhp antigens according to the method, and preparing the mycoplasma hyopneumoniae inactivated vaccine according to the following methods respectively: (1) oil phase: white oil (esol white mineral oil 52, i.e., MARCOL52) and span 80 were mixed in a volume ratio of 94: 6, uniformly mixing, and carrying out high-pressure sterilization to obtain the oil phase. (2) Tween-80: and (5) autoclaving. (3) Water phase: placing 96 volume parts of Mhp antigen at 37 ℃ for about 20min to make the antigen temperature reach about 37 ℃, adding 4 volume parts of Tween 80, fully oscillating (stirring at 10000 r/min) to dissolve the Tween, if not completely dissolved, continuing to place at 37 ℃ for about 20min, and placing at 4 ℃ to remove foams to obtain a water phase. (4) Emulsification: pouring the oil phase into an emulsification container, stirring at 10000r/min, and stirring the water phase with the water phase: oil phase (volume ratio) 1: slowly adding the mixture according to the proportion of 2.5, and finally emulsifying for 1.5-2min at 20000r/min to obtain the mycoplasma hyopneumoniae inactivated vaccine.
The water dropping test is carried out on 3 batches of inactivated vaccines, and the result is not demulsification. The specific method comprises the following steps: and centrifuging 10ml of the mycoplasma hyopneumoniae inactivated vaccine for 10min at 3000r/min, and separating out a water phase of less than 0.5ml, which indicates that the quality of the prepared mycoplasma hyopneumoniae inactivated vaccine is qualified.
3 batches of the mycoplasma hyopneumoniae inactivated vaccine prepared in the example were tested by the competitive ELISA quantitative detection method in example 2. Firstly, mixing the inactivated vaccine and acetone according to the volume ratio of 1:9, standing at room temperature for 10 minutes, then centrifuging at 4 ℃ and 15000g for 15 minutes, removing a liquid phase by suction, washing a precipitate for 2 times by using acetone, drying at room temperature for about 15 minutes, and adding deionized water with a proper volume for redissolution to obtain the demulsified vaccine antigen. The antigen content in the demulsified vaccine antigen was detected by the competitive ELISA quantitative detection method of example 2, the RP values were 1.05, 1.13, and 1.11, respectively, which were 90.23%, 93.2%, and 94.1% of the antigen content before vaccine production, respectively, indicating that the antigen content of 3 batches of vaccine was acceptable. Therefore, the competitive ELISA quantitative detection method in the embodiment 2 realizes the quantitative determination of the antigen content of the inactivated vaccine of mycoplasma pneumoniae of swine, fills the defect that the existing CCU counting method can not quantitatively detect the antigen in the inactivated vaccine, and ensures the quality of the vaccine.
Example 6 comparison of the method of the invention with other ELISA assays
The competitive ELISA quantitative detection method in example 2 was compared with other ELISA detection methods reported in the prior art.
Culturing 2 batches of mycoplasma hyopneumoniae NJ strain liquid samples by adopting a KM2 liquid culture medium, concentrating the total volume to 1/50 of the original volume by adopting a centrifugation method, then centrifuging, and taking supernatant and sediment to respectively determine the antigen content. The antigen content was measured by using the competitive ELISA quantitative detection method (described as the method of the present invention) in example 2 of the present invention, the control method 1 (the method for measuring viable mycoplasma hyopneumoniae bacterial titer disclosed in the invention patent application with application No. 201610620237.9), and the control method 2 (the mycoplasma hyopneumoniae solid-phase competitive ELISA kit disclosed in the invention patent application with application No. 201910370182.4) to test the sample, and simultaneously, the KM2 medium was used as the medium to control the sample for synchronous detection, and whether the medium would have an influence on the detection of the mycoplasma hyopneumoniae content was examined. When the same to-be-detected product is detected by the 3 methods, the relative efficacy RP value is converted by taking the same standard product as a reference. As a result, as shown in table 3, in the control method 1, a large amount of antigen was detected in the supernatant, and when the bacterial solution of mycoplasma hyopneumoniae NJ strain was concentrated by centrifugation, only the bacterial cell was concentrated, and the supernatant was not concentrated, so that the measured value of the precipitate after concentration could not accurately reflect the fold of antigen concentration, and the use was limited; control method 2 was also determined for 1 out of 2 batches of KM2 medium, indicating that the method may have some non-specific effects and that the quality of the medium may be different and may affect the accuracy of the assay; according to the method, only precipitation is detected to be positive, and the supernatant obtained after the concentrated antigen is centrifuged and can not be detected by the KM2 culture medium contrast, so that the method can accurately reflect the actual content of the bacterial antigen and is not influenced by the quality of the culture medium.
TABLE 3 comparison of the results of the methods for detecting the content of Mycoplasma hyopneumoniae antigen
Example 5 evaluation of the Effect of detecting other Mycoplasma by the method of the present invention
Separately culturing HUB-1 strain (NCBI genome accession number is CP002170) and flocculent pig mycoplasma hyorhinis in KM2 culture mediumAnd (3) the mycoplasma hyorhinis (ATCC 27095) is cultured by adopting an ATCC243 culture medium, and the mycoplasma hyorhinis (ATCC 27095) can be harvested after the culture grows well. (1) The corresponding coatingen was prepared from each mycoplasma culture as follows: centrifuging the mycoplasma sample for 20 minutes at 10000g, washing the precipitate for 2 times by using PBS buffer solution, after washing for 2 times, suspending the precipitate again by using a small amount of PBS buffer solution, performing ultrasonic crushing to obtain mycoplasma lysate which is used as coating antigen, determining the content of protein in the mycoplasma lysate by using a BCA kit, subpackaging and storing at-70 ℃. (2) Each mycoplasma culture was prepared as follows: the concentration to be detected by CCU counting method is 108And (3) diluting the CCU/ml mycoplasma hyopneumoniae bacterial liquid by adopting a PBS (phosphate buffer solution) according to the dilution times of 2, 4, 8, 16, 32, 64, 128 and 256 to obtain a series of standard products for preparing corresponding standard curves.
Separately culturing Mycoplasma hyorhinis (HUB-1 strain, NCBI genome accession number is CP002170), Mycoplasma hyorhinis (ATCC27716) and Mycoplasma hyosynoviae (ATCC 27095), and harvesting the sample to be detected after the culture grows well.
The antigen of the mycoplasma hyopneumoniae NJ strain in the method is replaced by the coating antigen of the mycoplasma hyopneumoniae HUB-1 strain, the mycoplasma hyopneumoniae (ATCC27716) or the mycoplasma hyopneumoniae (ATCC 27095) respectively, and the other antigens are not changed, so that the antigen content of a corresponding mycoplasma test sample can be detected, for example, if the coating antigen is the mycoplasma hyopneumoniae HUB-1 strain, the antigen can be used for detecting the mycoplasma hyopneumoniae. First, a calibration curve for each mycoplasma was prepared using a standard for each mycoplasma according to the method of the present invention (fig. 4). Then, each mycoplasma hyopneumoniae sample to be detected is used for detecting the antigen content respectively by the method. The result shows that the detected RP value of the antigen content in the mycoplasma hyorhinis sample is 2.953, the RP value of the antigen content in the mycoplasma hyorhinis sample is 1.842, and the RP value of the antigen content in the mycoplasma hyosynoviae sample is 1.057. The results show that the detection method can be used for detecting the antigen content of the mycoplasma hyopneumoniae and also can be used for detecting the antigen content of the mycoplasma hyorhinis, the mycoplasma hyofloccosum and the mycoplasma hyosynoviae, and has good universality.
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<110> agricultural science and academy of Jiangsu province
<120> general rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof
<130> 20201116
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213> artificial
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<223> P1
<400> 1
cgcggatcct ttgatcgttc caaagaacat 30
<210> 2
<211> 30
<212> DNA
<213> artificial
<220>
<223> P2
<400> 2
ccgctcgaga ataatttcgg taactgttcc 30
<210> 3
<211> 1170
<212> DNA
<213> Mycoplasma hyopneumoniae
<400> 3
tttgatcgtt ccaaagaaca tattaatatt ggaacaattg gtcatgttga ccatggaaaa 60
acaacactaa cagcagcaat ttcaactgtt ttggcaaaaa gaggactcgc ggaggcaaag 120
gattatgcct caattgatgc agctcctgaa gaaaaagcac gaggaattac aattaatacc 180
gcccatattg aatatagcac tgacaaacgg cactatgccc atgttgattg cccaggtcat 240
gccgattata taaaaaatat gatcaccgga gcggcccaaa tggatggagc aattttagtg 300
gttgcagcaa ccgatggccc aatgcctcaa actcgtgaac acattttatt atcaaaacaa 360
gttggtgtcc caaaaatggt tgttttcctt aataaaattg acttacttga aggcgaagaa 420
gaaatggttg atttagttga ggtcgaaatt cgcgaacttc tttcttctta tgattttgat 480
ggcgataata ctccaattat tcggggatct gctcgtgggg cccttgaagg aaaacctgaa 540
tgggaggcaa aagtacttga attgatggac gcagttgatt cttatattga ctcacctgtt 600
cgggaaatgg ataaaccctt tttgatggcc gttgaggatg tttttacaat tacaggacgt 660
ggaaccgtag caacgggaaa agttgaaaga ggtcaagtta aactaaacga agaagtggaa 720
attgtgggtt atcgcgaaga acctaaaaag accgtaatca ccggaattga aatgtttaat 780
aaaaatcttc aaactgcgat ggctggggat aatgccggtg ttctcctacg tggagtagat 840
cgaaaagata tcgaacgtgg tcaggttatt gcaaaaccaa aaacaattat accgcataca 900
aaatttaaag ctgcaattta tgcccttaaa aaagaagaag gtggaaggca tacaccattt 960
ttcaaaaatt ataaaccaca attttatttt cgaactaccg atgtaacagg cggaatcgag 1020
tttgaaccag gtcgcgagat ggtaattccg ggggataatg ttgatcttac cgttgaatta 1080
attgccccaa ttgccgttga gcagggaacc aaattctcga tccgagaagg tggcagaacc 1140
gtgggtgccg gaacagttac cgaaattatt 1170
<210> 4
<211> 390
<212> PRT
<213> Mycoplasma hyopneumoniae
<400> 4
Phe Asp Arg Ser Lys Glu His Ile Asn Ile Gly Thr Ile Gly His Val
1 5 10 15
Asp His Gly Lys Thr Thr Leu Thr Ala Ala Ile Ser Thr Val Leu Ala
20 25 30
Lys Arg Gly Leu Ala Glu Ala Lys Asp Tyr Ala Ser Ile Asp Ala Ala
35 40 45
Pro Glu Glu Lys Ala Arg Gly Ile Thr Ile Asn Thr Ala His Ile Glu
50 55 60
Tyr Ser Thr Asp Lys Arg His Tyr Ala His Val Asp Cys Pro Gly His
65 70 75 80
Ala Asp Tyr Ile Lys Asn Met Ile Thr Gly Ala Ala Gln Met Asp Gly
85 90 95
Ala Ile Leu Val Val Ala Ala Thr Asp Gly Pro Met Pro Gln Thr Arg
100 105 110
Glu His Ile Leu Leu Ser Lys Gln Val Gly Val Pro Lys Met Val Val
115 120 125
Phe Leu Asn Lys Ile Asp Leu Leu Glu Gly Glu Glu Glu Met Val Asp
130 135 140
Leu Val Glu Val Glu Ile Arg Glu Leu Leu Ser Ser Tyr Asp Phe Asp
145 150 155 160
Gly Asp Asn Thr Pro Ile Ile Arg Gly Ser Ala Arg Gly Ala Leu Glu
165 170 175
Gly Lys Pro Glu Trp Glu Ala Lys Val Leu Glu Leu Met Asp Ala Val
180 185 190
Asp Ser Tyr Ile Asp Ser Pro Val Arg Glu Met Asp Lys Pro Phe Leu
195 200 205
Met Ala Val Glu Asp Val Phe Thr Ile Thr Gly Arg Gly Thr Val Ala
210 215 220
Thr Gly Lys Val Glu Arg Gly Gln Val Lys Leu Asn Glu Glu Val Glu
225 230 235 240
Ile Val Gly Tyr Arg Glu Glu Pro Lys Lys Thr Val Ile Thr Gly Ile
245 250 255
Glu Met Phe Asn Lys Asn Leu Gln Thr Ala Met Ala Gly Asp Asn Ala
260 265 270
Gly Val Leu Leu Arg Gly Val Asp Arg Lys Asp Ile Glu Arg Gly Gln
275 280 285
Val Ile Ala Lys Pro Lys Thr Ile Ile Pro His Thr Lys Phe Lys Ala
290 295 300
Ala Ile Tyr Ala Leu Lys Lys Glu Glu Gly Gly Arg His Thr Pro Phe
305 310 315 320
Phe Lys Asn Tyr Lys Pro Gln Phe Tyr Phe Arg Thr Thr Asp Val Thr
325 330 335
Gly Gly Ile Glu Phe Glu Pro Gly Arg Glu Met Val Ile Pro Gly Asp
340 345 350
Asn Val Asp Leu Thr Val Glu Leu Ile Ala Pro Ile Ala Val Glu Gln
355 360 365
Gly Thr Lys Phe Ser Ile Arg Glu Gly Gly Arg Thr Val Gly Ala Gly
370 375 380
Thr Val Thr Glu Ile Ile
385 390
Claims (9)
1. A universal rapid detection kit for the content of mycoplasma hyopneumoniae antigens is characterized by comprising an ELISA plate coated by the mycoplasma hyopneumoniae antigens and rabbit anti-EF-Tu serum; the rabbit anti-EF-Tu serum is obtained by immunizing a New Zealand rabbit with recombinant EF-Tu protein, collecting blood and separating serum, wherein the sequence of the recombinant EF-Tu protein is shown in SEQ ID NO. 4.
2. The universal rapid detection kit for the content of the mycoplasma hyopneumoniae antigen as claimed in claim 1, wherein the elisa plate is coated by using mycoplasma hyopneumoniae lysate.
3. The universal rapid detection kit for the content of the mycoplasma hyopneumoniae antigen according to claim 1 or 2, wherein the recombinant EF-Tu protein is obtained by inducing expression of a recombinant bacterium carrying an EF-Tu protein encoding gene.
4. The universal rapid detection kit for the content of the swine mycoplasma antigen as claimed in claim 3, wherein the elisa plate is coated with a swine mycoplasma lysate and then sealed with a PBS buffer solution containing gelatin.
5. The universal rapid detection kit for the content of the mycoplasma hyopneumoniae antigen according to claim 4, wherein the kit further comprises a mycoplasma hyopneumoniae antigen bacteria liquid standard, a PBST solution, a goat anti-rabbit HRP-IgG, a developing solution and a stopping solution.
6. The universal rapid detection kit for the content of the mycoplasma hyopneumoniae antigen as claimed in claim 5, wherein the PBST solution is PBS buffer solution containing 0.05% Tween-20 by volume percentage; the goat anti-rabbit HRP-IgG is purchased from Dr. warrior Biometrics, and the product number is BA 1054.
7. The universal rapid detection kit for detecting the content of the mycoplasma hyopneumoniae antigen according to claim 6, wherein the mycoplasma hyopneumoniae comprises mycoplasma hyopneumoniae, mycoplasma hyorhinotracheale, mycoplasma hyofloccosum and mycoplasma hyosynoviae.
8. A method for detecting the content of mycoplasma hyopneumoniae antigens for non-diagnostic purposes using the kit of claim 1, comprising the steps of:
(1) arranging a sample hole to be detected and a blank control sample hole in an ELISA plate coated by a swine mycoplasma antigen, adding the sample to be detected into the sample hole to be detected, adding a PBS (phosphate buffer solution) into the blank control sample hole, then adding rabbit anti-EF-Tu serum into each hole, and washing with a PBST solution after incubation;
(2) adding goat anti-rabbit HRP-IgG into each hole, and incubating;
(3) after PBST solution is washed, TMB substrate color development liquid is added;
(4) adding a stop solution to stop the reaction, and reading a light absorption value at 450 nm;
(5) and calculating the content of the mycoplasma hyopneumoniae antigen according to the standard curve.
9. Use of the kit according to claim 1 for detecting the antigen content in a mycoplasma hyopneumoniae culture or a mycoplasma hyopneumoniae vaccine product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011277555.2A CN112485423A (en) | 2020-11-16 | 2020-11-16 | Universal rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011277555.2A CN112485423A (en) | 2020-11-16 | 2020-11-16 | Universal rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN112485423A true CN112485423A (en) | 2021-03-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011277555.2A Pending CN112485423A (en) | 2020-11-16 | 2020-11-16 | Universal rapid detection kit for content of mycoplasma hyopneumoniae antigen and application thereof |
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| Country | Link |
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| CN (1) | CN112485423A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928585A (en) * | 2012-10-31 | 2013-02-13 | 广东现代农业集团研究院有限公司 | Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof |
| CN110045117A (en) * | 2019-05-06 | 2019-07-23 | 江苏南农高科技股份有限公司 | Mycoplasma hyopneumoniae solid phase competitive ELISA kit and its preparation method and application |
| CN110133284A (en) * | 2019-05-07 | 2019-08-16 | 西南大学 | Proteantigen and its encoding gene and they identifying the application in mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody |
| CN110302368A (en) * | 2013-11-21 | 2019-10-08 | 财团法人农业科技研究院 | The composition for preventing and treating mycoplasm hyopneumoniae infection |
-
2020
- 2020-11-16 CN CN202011277555.2A patent/CN112485423A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928585A (en) * | 2012-10-31 | 2013-02-13 | 广东现代农业集团研究院有限公司 | Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof |
| CN110302368A (en) * | 2013-11-21 | 2019-10-08 | 财团法人农业科技研究院 | The composition for preventing and treating mycoplasm hyopneumoniae infection |
| CN110045117A (en) * | 2019-05-06 | 2019-07-23 | 江苏南农高科技股份有限公司 | Mycoplasma hyopneumoniae solid phase competitive ELISA kit and its preparation method and application |
| CN110133284A (en) * | 2019-05-07 | 2019-08-16 | 西南大学 | Proteantigen and its encoding gene and they identifying the application in mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody |
Non-Patent Citations (1)
| Title |
|---|
| YANFEI YU等: "Mycoplasma hyopneumoniae evades complement activation by binding to factor H via elongation factor thermo unstable (EF-Tu)", 《VIRULENCE》, vol. 11, no. 1, pages 1063 * |
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