CN114324859B - Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof - Google Patents
Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof Download PDFInfo
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- CN114324859B CN114324859B CN202111339885.4A CN202111339885A CN114324859B CN 114324859 B CN114324859 B CN 114324859B CN 202111339885 A CN202111339885 A CN 202111339885A CN 114324859 B CN114324859 B CN 114324859B
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Abstract
The invention relates to the technical field of biology, in particular to a coated antigen for detecting a mycoplasma synoviae antibody, a kit and a detection method thereof. Detecting the envelope antigen of the mycoplasma synoviae antibody as recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises the amino acid sequence shown in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein comprises a nucleotide sequence shown in SEQ ID NO: 2. The recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, can be used for detecting the serum antibody of the mycoplasma synoviae in clinic, and can be used for efficiently detecting the mycoplasma synoviae antibody of a farm; the recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, so that the detection method has good reactivities, specificities, sensibility and repeatability, can be used for detecting the serum antibody of the mycoplasma synoviae in clinic, provides a certain guide for the establishment of an immunization program of a mycoplasma synoviae vaccine, and achieves the aim of preventing and controlling the infection of the mycoplasma synoviae.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a coated antigen for detecting a mycoplasma synoviae antibody, a kit and a detection method thereof.
Background
Mycoplasma synoviae (Mycoplasma synoviae, MS) is one of the major pathogenic mycoplasma in birds, whose main infectious group is chickens or turkeys, and which after infection of poultry is MS, is mainly manifested as synovial membrane and tenosynovitis, which are exudative of joints, and can also cause subclinical respiratory symptoms and air sac inflammation. MS infects chickens and turkeys of various ages, but chicks are most infectious and are born for life once infected. MS infection is mostly chronic or recessive infection, has fewer acute cases, and is clinically often shown as mixed infection with NDV, IBV, IBDV pathogenic agents such as escherichia coli and the like. MS is transmitted horizontally mainly by contact between healthy chickens and sick chickens, but also by vertical transmission, through eggs, and is extremely harmful.
At present, prevention and control of MS are mainly through drug prophylaxis and vaccine immunization. In the aspect of vaccine use, foreign parent breeding hens only immunize once for 4-6 weeks of age with a live vaccine, and domestic immunization programs of 'one live one dead' or 'one live two dead' are mainly adopted. The immune effect of the vaccine needs to be effectively detected by antibodies to achieve the aim of preventing and controlling the infection of the mycoplasma synoviae, but the existing detection method has poor antigen specificity and sensitivity, and cannot effectively perform the antibody evaluation of the vaccine.
Disclosure of Invention
In view of the above, it is necessary to provide a coated antigen for detecting a mycoplasma synoviae antibody, a kit and a detection method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a coated antigen for detecting a mycoplasma synoviae antibody, said coated antigen being a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises the amino acid sequence shown in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein comprises a nucleotide sequence shown in SEQ ID NO: 2.
Further, the amino acid sequence of the recombinant EFG protein comprises a sequence which is identical to SEQ ID NO:1 having at least 90% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO:2 having at least 90% homology.
Further, the amino acid sequence of the recombinant EFG protein comprises a sequence which is identical to SEQ ID NO:1 having at least 95% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO:2 having at least 95% homology.
Preferably, the amino acid sequence of the recombinant EFG protein is SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is SEQ ID NO: 2.
Further, the amplification primer sequence of the recombinant EFG protein is shown as SEQ ID NO:3 and SEQ ID NO: 4:
SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；
SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’。
In a second aspect, the invention provides a kit for detecting a mycoplasma synoviae antibody, the kit comprising the recombinant EFG protein as a coating antigen of an ELISA plate.
Further, the kit also comprises an ELISA plate, a coating liquid, a sealing liquid, an ELISA secondary antibody, TMB color development liquid, a stop solution, a washing liquid and a serum dilution liquid.
Further, the coating liquid is carbonate buffer.
Further, the washing solution is PBST.
Furthermore, the enzyme-labeled secondary antibody is goat anti-rabbit enzyme-labeled secondary antibody.
Further, the sealing liquid is 5% skim milk powder.
Further, the serum dilution is 5% nonfat dry milk.
In a third aspect, the invention provides a detection method for detecting a mycoplasma synoviae antibody, comprising the steps of:
S1, performing S1; diluting the coating antigen with coating liquid, then placing the diluted coating antigen into an ELISA plate, and coating overnight;
S2: removing the coating liquid in the step S1, cleaning the coating liquid, then beating the coating liquid to dry, and adding a sealing liquid for sealing;
s3: removing the sealing liquid in the step S2, cleaning the cleaning liquid, then beating the cleaning liquid to dry, adding a serum sample to be tested diluted by a serum diluent, and incubating;
S4: removing the serum sample in the step S3, cleaning the serum sample by using a cleaning solution, then beating the serum sample, adding the diluted enzyme-labeled secondary antibody, and incubating;
s5: removing the enzyme-labeled secondary antibody in the step S4, cleaning the enzyme-labeled secondary antibody by using a cleaning solution, then beating the enzyme-labeled secondary antibody to dry, adding TMB color development solution for light-shielding reaction, and adding a stop solution for stopping the reaction after the reaction is finished;
S6: testing the OD 450nm value of the liquid in the plate hole of the enzyme-labeled plate on the enzyme-labeled instrument, and judging the detection result as positive when the OD 450nm value is more than or equal to 0.3998; and when the OD 450nm value of the sample to be detected is less than 0.3998, the detection result is judged to be negative.
Further, the concentration of the coating antigen in S1 after dilution is 0.8 mug/mL, 100 mug/well of the enzyme-labeled plate is added, and the coating is carried out at 4 ℃ overnight.
Further, washing the washing liquid in the step S2 for 4 times, each time for 2 minutes; 100. Mu.L of blocking solution was added to each well and the wells were blocked at 4℃for 16h.
Further, washing the washing liquid in the step S3 for 4 times, each time for 2 minutes; 100 μl of serum dilution was added per well at 1: the serum sample to be tested after 800-3200 times dilution is incubated for 90min at 37 ℃.
Further, the washing liquid in the step S4 is washed for 4 times, each time for 2 minutes; 100 μl of PBS was added per well according to 1: the enzyme-labeled secondary antibody after 2000 times dilution is incubated for 120min at 37 ℃.
Further, washing the washing liquid in the step S4 for 5 times, each time for 2 minutes; 100 mu L of TMB color development solution is added to each hole, the reaction is carried out in a dark place for 10min, and stop solution is added according to 100 mu L/hole to stop the reaction.
The beneficial effects of the invention are as follows:
(1) The recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, can be used for detecting the serum antibody of the mycoplasma synoviae in clinic, and can be used for efficiently detecting the mycoplasma synoviae antibody of a farm.
(2) The recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, so that the detection method has good reactivity, specificity, sensitivity and repeatability, can be used for detecting the serum antibody of the mycoplasma synoviae in clinic, and provides a certain guide for the establishment of an immunization program of a mycoplasma synoviae vaccine, thereby achieving the purpose of preventing and controlling the infection of the mycoplasma synoviae.
Drawings
FIG. 1 is a diagram showing SDS-PAGE analysis result of purified recombinant EFG protein: in the figure 1, M is a protein molecular mass standard, 1 is broken bacterial supernatant lysate collected by EFG-pSmartI induction, 2 is EFG-pSmartI after passing through a nickel column, 3 is washed by 50mMol imidazole, and 4-7 are eluted by 250mMol imidazole;
FIG. 2 is a graph showing the results of an antigen reactivity analysis of the recombinant EFG protein of example 4;
FIG. 3 is a graph showing the results of antigen-specific analysis of recombinant EFG proteins in example 5: wherein antibody positive serum Po. (+) is a positive standard and neg.(-) is a negative standard;
FIG. 4 is a graph showing the data of the recombinant EFG protein coated antigen sensitivity test of example 6;
FIG. 5 is a graph of data at OD 450nm for clinical serum samples tested against different immune backgrounds in example 8.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further clearly and completely described in the following in conjunction with the embodiments of the present invention. It should be noted that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A coated antigen for detecting a mycoplasma synoviae antibody, said coated antigen being a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein is shown as SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is shown as SEQ ID NO:2, the GC content of the sequence is optimized, and the expression is convenient.
The preparation method of the coating antigen comprises the following steps:
1. EFG gene primer design:
The full-length Sequence of the gene of the mycoplasma synoviae EFG protein (NCBI gene Sequence ID: WP_ 011283211.1) is referred to, and the gene Sequence of the EFG target is obtained after codon optimization. Designing a pair of primers to amplify the EFG target gene sequence, wherein the primer sequences are shown in SEQ ID NO:3 and SEQ ID NO: 4:
SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；
SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’；
The size of the EFG protein target gene sequence is 2088bp;
2. Construction of recombinant plasmids:
The EFG target gene sequence is connected to pSmart-I skeleton carrier, and the recombinant plasmid pSmartI-EFG with correct sequencing is obtained through transformation and purification, and the sequence of the recombinant plasmid pSmartI-EFG is shown as SEQ ID NO:5 is shown in the figure;
3. Expression and purification of recombinant EFG proteins:
3.1 expression of recombinant EFG protein: recombinant plasmid pSmartI-EFG transformed expression bacterium Rosetta with correct sequencing result is aseptically coated on LB/Kan plate culture medium and cultured for 18h at 37 ℃. The monoclonal with positive PCR identification result is inoculated into 10mL LB/Kan liquid culture medium according to the ratio of 1:100, and when the culture is carried out on a shaking table at 37 ℃ and 200r/min until the OD600nm value is about 0.6, IPTG with the final concentration of 0.4mmol/L is added, and the induced expression is carried out at 37 ℃ and 150r/min for 6h. Centrifuging 1mL of bacterial liquid at 8000r/min for 5min, discarding the supernatant, re-suspending with 50 mu L of PBS, adding 50 mu L of 2 Xprotein loading buffer solution, boiling at 100 ℃ for 5min, centrifuging, collecting the supernatant, performing SDS-PAGE analysis, and selecting strains positive in protein expression;
selecting a strain positive in protein expression, culturing overnight in a shaking table, inoculating the strain into a liquid culture medium containing 300mL of LB/Kan according to the ratio of 1:100, and carrying out induced expression under the optimized condition. The bacterial liquid is centrifuged for 5min at 8000r/min, the bacterial cells are blown with sterilized PBS and centrifuged again, and the process is repeated for several times until the supernatant becomes clear. The final bacterial cells were blown off with 30mL sterilized PBS, broken with ice bath ultrasound, procedure: ultrasound was 3 times at intervals of 8s, ten minutes each. Centrifuging the thallus subjected to ultrasonic treatment at 4 ℃ and 12000r/min for 10min, and collecting bacterial supernatant lysate;
3.2 purification of recombinant EFG proteins:
Adding nickel into a purification column, balancing the nickel column by using Tris-HCl buffer solution until the nickel column is completely discolored, adding crushed bacterial supernatant lysate after the nickel column is well balanced, and slowly passing through the column for adsorption. After adsorption, 50mmol imidazole is used for washing impurities, then 250mmol imidazole is used for eluting, so that the purified recombinant EFG protein is obtained, and the result is shown in figure 1, which shows that the purification of the recombinant EFG protein is successful.
Example 2
A kit for detecting a mycoplasma synoviae antibody, the kit comprising a coating antigen, an ELISA plate, a coating liquid, a sealing liquid, an ELISA secondary antibody, a TMB color development liquid, a stop liquid, a washing liquid and a serum dilution liquid, wherein the recombinant EFG protein purified in the embodiment 1 is used as the ELISA plate; the coating liquid is carbonate buffer solution; the washing liquid is PBST; the enzyme-labeled secondary antibody is an enzyme-labeled goat anti-rabbit antibody; the sealing liquid is 5% skimmed milk powder; the serum dilution was 5% nonfat dry milk.
Example 3
The method for detecting mycoplasma synoviae antibody using the kit of example 2, comprising the steps of:
S1, performing S1; diluting the coated antigen with carbonate buffer solution to a concentration of 0.8 mu g/mL, adding 100 mu L/hole into an ELISA plate, and coating at 4 ℃ overnight;
S2: removing the coating liquid in the step S1, cleaning the PBST for 4 times, each time for 2min, beating to dry, adding 100 mu L of 5% skimmed milk powder sealing liquid into each hole, and sealing at 4 ℃ for 16h.
S3: removing the blocking solution in S2, cleaning with PBST for 4 times, each time for 2min, beating to dry, adding 100 μl of 5% skimmed milk powder into each well, and mixing 1: the serum sample to be tested after 800 times dilution is incubated for 90min at 37 ℃;
S4: serum samples from S3 were removed, PBST washed 4 times for 2min each, patted dry, and washed with PBS according to 1: the goat anti-rabbit enzyme-labeled secondary antibody after 2000 times dilution is incubated for 120min at 37 ℃;
S5: removing the enzyme-labeled secondary antibody in the S4, cleaning the PBST for 5 times, each time for 2min, beating to dry, reacting 100 mu L of TMB color development liquid in a dark place for 10min, adding a stop solution according to 100 mu L/hole, and stopping the reaction;
S6: testing the OD 450nm value of the liquid in the plate hole of the enzyme-labeled plate on the enzyme-labeled instrument, and judging the detection result as positive when the OD 450nm value is more than or equal to 0.3998; and when the OD 450nm value of the sample to be detected is less than 0.3998, the detection result is judged to be negative.
Determination of negative-positive threshold: 29 negative sera were tested, 3 wells were repeated for each sample, the mean (X) and Standard Deviation (SD) of the negative OD 450nm values were calculated, the formula was threshold = x+3sd, and the resulting ELISA OD 450nm threshold was 0.3998. When the OD 450nm value of the sample to be detected is larger than 0.3998, the result is judged to be positive; when the OD 450nm value of the sample to be detected is less than 0.3998, the result is judged as negative.
Example 4
Detecting positive standard substances and negative standard substances respectively according to the detection method of the embodiment 3; the positive standard is serum after immunization or maternal antibody serum;
The antibody serum after immunization is prepared by intramuscular injection of attenuated vaccine at the age of 3 weeks, intramuscular injection of inactivated vaccine at the age of 7 weeks and intramuscular injection of inactivated vaccine at the age of 17 weeks, and serum is collected two weeks after immunization;
The maternal antibody serum is 1-day-old chick serum obtained by hatching hen eggs after immunization;
The negative standard is a serum sample which is not subjected to vaccine immunity injection and has the same culture condition.
As shown in FIG. 2, the recombinant EFG protein antigen reacted well with serum antibodies or maternal antibodies after immunization, and did not react with negative standard, indicating good reactivity of the detection method.
Example 5
According to the detection method of example 3, different serum samples were detected, and the detected serum samples were Newcastle Disease Virus (NDV) antibody positive serum, avian Influenza (AIV) antibody positive serum, mycoplasma Gallisepticum (MG) antibody positive serum, escherichia coli (e.coli) antibody positive serum, mycoplasma duck (MA) antibody positive serum, mycoplasma Bovis (MB) antibody positive serum, respectively, and the negative and positive standards in example 4 were used as controls. The detection results are shown in FIG. 3. As can be seen from FIG. 3, the recombinant EFG protein coated antigen of the present invention does not react with other antibody serum, and has good antigen specificity.
Example 6
3 Parts of the positive standard in example 4 were taken and the test method of example 3 was followed, except that the dilution ratio in S3 was 1: as shown in FIG. 4, after the positive standard serum is diluted to 3200 times, the detection invention can still detect the positive antibody, which indicates that the sensitivity of the recombinant EFG protein coated antigen is high.
Example 7
The method of example 3 was followed to perform a lot-to-lot reproducibility test, and the same lot of protein was used to coat the elisa plate, 3 positive sera with different antibody titers were detected, 4 wells were repeated for each sample, OD 450nm values were measured for each well, standard deviations were calculated for each sample, and further the intra-lot variation coefficient (C.V), C.V = (SD/MN) ×100% for each sample was calculated. In the same experimental environment, the ELISA plates are coated at three different time periods, 3 positive serum with different antibody titers are detected respectively, 4 holes are repeated in parallel for each serum, OD 450nm value of each hole is detected, and the variation coefficient between batches of each sample is calculated, and the result is shown in Table 1. As can be seen from the results in Table 1, the coefficient of variation in the intra-batch and inter-batch reproducibility tests was less than 10%, indicating that the method was good in reproducibility and stable in results.
TABLE 1 results of within-and between-lot reproducibility tests of serum samples of different positives
Example 8
Clinical serum samples of different immunization backgrounds were tested according to the test method of example 3, with immunization procedure of 3 week-old intramuscular injection of attenuated vaccine, 7 week-old intramuscular injection of inactivated vaccine, and 17 week-old intramuscular injection of inactivated vaccine. The results of the detection are shown in Table 2 and FIG. 5.
TABLE 2
| Group | Immunization background | Age of day | Positive rate |
| 2018 | Three-phase last 10 weeks | For 27 weeks | 93.75％ |
| 2019 | Three-phase last 10 weeks | For 27 weeks | 100％ |
| 2020 | Three-phase last 10 weeks | For 27 weeks | 100％ |
| 2021 | Three-phase last 10 weeks | For 27 weeks | 100％ |
| 2026 | 12 Weeks after the second time | 19 Weeks of | 100％ |
| 2101 | 12 Weeks after the second time | 19 Weeks of | 93.75％ |
| 2103 | Post-exempt 4 weeks | 11 Weeks of | 100％ |
| 2105 | Post-exempt 1 week | For 30 days | 56.25％ |
| 2106 | Non-immunized | For 16 days | 25％ |
| 2107 | Non-immunized | For 16 days | 0％ |
From fig. 5 and table 2, it can be seen that the detection method of the present invention can specifically detect the level of mycoplasma synoviae antibody, wherein the 10-week positive rate after three-immunity of the spot check is 100%, the 12-week positive rate after two-immunity is 93.75%, the 4-week positive rate after one-immunity is 100%, the 1-week positive rate after one-immunity is only 56.25%, and the 25% positive rate of one of the two non-immunity chicken groups may indicate mycoplasma synoviae infection.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Wenshi food group Co., ltd
University of agricultural in south China
<120> A coated antigen for detecting a mycoplasma synoviae antibody, kit and detection method thereof
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ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggtcacc atcaccatca ccatatgtcg 5100
gactcagaag tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact 5160
cacatcaatt taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc 5220
actcctttaa gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc 5280
ttaagattct tgtacgacgg tattagaatt caagctgatc agacccctga agatttggac 5340
atggaggata acgatattat tgaggctcac agagaacaga ttggtggcca aggatccatg 5400
gcacgtgatt atgatctgaa agattatcgt aacatcggta ttatggcgca tattgatgcg 5460
ggtaaaacca ccaccaccga acgcattctg tatcataccg gtaaaattca taaaatcggt 5520
gaaacacatg atggtgtttc tcagatggat tggatggaac aggaaaaaga acgtggcatt 5580
acaatcacca gcgcagcaac cacagcatat tggaaaaata aacgtattaa catcatcgat 5640
accccgggtc atgttgattt taccgttgaa gttgaacgta gcctgcgtgt tctggatggc 5700
gccgttgcag ttctggatgc acagagcggt gtggaaccgc agaccgaaac cgtgtggcgc 5760
caggcaacca attataaagt tccgcgtatt gtgtatgtta ataaaatgga taaagcaggt 5820
gcggattttg aagcggcagt agcaagcgtt aaaagccgtc tgggtggcaa tgcggttgca 5880
attcagtggc cgatcggtag cgaaagcaat tttaatggca tcattgatct ggttacaatg 5940
accgcgacaa cctataatgg tgaaagtgca gaagaagaat ttccgatgga aattccgacc 6000
gatctgctgg atgtggcaaa agcaaaacgt caggaactgc tggaagcagc ggccaacttt 6060
gatgaagaag ttatgatgat ggtgctggaa ggtgcagatg ttgatattga tacctttaaa 6120
aacaccatcc gtaaagcaac actgaccagc gaattttttc cggtagtttg tggcacgagc 6180
tttaaaaata aaggtgtcaa aaagatgatc gatgcagtgg tggattatct gccgagcccg 6240
ctggatattc cgccgattaa agcatatctg aatgatcagg aaaccgatgt tgtcgcaaca 6300
gatgatggtg aatttgcagc actggcattt aaagttatga ccgatccttt tgttggtagc 6360
ctgacctttt ttcgtgtcta tcgcggtgtt ctggaaaaag gcagctatgt ttataatagc 6420
acgaaagaac agaaagaacg tattggtcgt attctgcaga tgcatgcaaa taatcgtgtt 6480
gaaattgatg aatgtcgtgc aggtgatatt gcagcagcag tgggtctgaa atttaccacc 6540
accggtgata cactggttgg cgaaaaatca cctaaagttg ttctggaaaa aatggttttt 6600
ccggaaccgg ttattagcca ggcactggaa ccggaaagca aagcagcaaa tgaaaaactg 6660
agcctgggtc tgcagaaact gagcgcagaa gatccgacct ttcgtaccta taccgatgaa 6720
gaaaccggtc agaccattat tagcggtatg ggtgaactgc atctggatat tattgttgat 6780
cgtctgaaac gtgaatttgg tgttaaagtt aaagttggtg caccgcaggt tagctatcgt 6840
gaaaccatta ccaaaagcgc agaagttgaa ggtaaacata ttaaacagag cggtggtaaa 6900
ggtcagtatg gtcatgtttg gctgaaattt gaaccgaatc atgatcaggg ttttgaattt 6960
attgataaaa ttgttggtgg taaaattccg aaagaatata ttaaaccgat tcagaaaggt 7020
ctggaagaaa aaatggcagt tggtattctg gcaggttatc cgatgattga tgttaaagca 7080
accctgtttg atggtagcta tcatgatgtt gatagcagcg aactggcata taaaattgca 7140
gcaagcaaag cactgaccaa agcaaaagat ctgattggta ccgttctgct ggaaccgatt 7200
atggatgtta gcgttgttgt tccgagcgat cacatgggtg atgttattgg tgatctgagc 7260
cgtcgtcgtg gtctgattag cgatcaggaa cagcgtaatg atggtgcagt tattgttcgt 7320
gcaaaagttc cgctgagcga aatgtttggt tatagcaccg aactgcgtag catgaccagc 7380
ggtcgtggta cctatcagat gcagtttgat cattatgaaa aatgtccgaa aaatattagc 7440
gatgaaatta ttaaaaaacg taatattcag aataaagatg aagaataact cgagcaccac 7500
caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 7560
gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 7620
tttttgctga aaggaggaac tatatccgga t 7651
Claims (6)
1. A kit for detecting a mycoplasma synoviae antibody, characterized in that the kit comprises a coating antigen for detecting a mycoplasma synoviae antibody, wherein the coating antigen is recombinant EFG protein; the amino acid sequence of the recombinant EFG protein is shown as SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is shown as SEQ ID NO: 2.
2. The kit for detecting a mycoplasma synoviae antibody of claim 1, wherein the amplification primer sequence of the recombinant EFG protein is as set forth in SEQ ID NO:3 and SEQ ID NO: 4:
SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；
SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’。
3. The kit for detecting a mycoplasma synoviae antibody according to claim 1, wherein the kit further comprises an enzyme-labeled plate, a coating liquid, a blocking liquid, an enzyme-labeled secondary antibody, a TMB developing liquid, a stop liquid, a washing liquid, and a serum dilution.
4. A kit for detecting a mycoplasma synoviae antibody according to claim 3, wherein the coating solution is a carbonate buffer; the washing liquid is PBST; the enzyme-labeled secondary antibody is goat anti-rabbit enzyme-labeled secondary antibody; the sealing liquid is 5% skimmed milk powder; the serum dilution was 5% nonfat dry milk.
5. The detection method for detecting the mycoplasma synoviae antibody by using the kit according to any one of claims 1 to 4, characterized by comprising the following steps:
S1, performing S1; diluting the coating antigen with coating liquid, then placing the diluted coating antigen into an ELISA plate, and coating overnight;
S2: removing the coating liquid in the step S1, cleaning the coating liquid, then beating the coating liquid to dry, and adding a sealing liquid for sealing;
s3: removing the sealing liquid in the step S2, cleaning the cleaning liquid, then beating the cleaning liquid to dry, adding a serum sample to be tested diluted by a serum diluent, and incubating;
S4: removing the serum sample in the step S3, cleaning the serum sample by using a cleaning solution, then beating the serum sample, adding the diluted enzyme-labeled secondary antibody, and incubating;
s5: removing the enzyme-labeled secondary antibody in the step S4, cleaning the enzyme-labeled secondary antibody by using a cleaning solution, then beating the enzyme-labeled secondary antibody to dry, adding TMB color development solution for light-shielding reaction, and adding a stop solution for stopping the reaction after the reaction is finished;
S6: testing the OD 450nm value of the liquid in the plate hole of the enzyme-labeled plate on the enzyme-labeled instrument, and judging the detection result as positive when the OD 450nm value is more than or equal to 0.3998; and when the OD 450nm value of the sample to be detected is less than 0.3998, the detection result is judged to be negative.
6. The method according to claim 5, wherein the concentration of the coating solution of the coating antigen in S1 after dilution is 0.8. Mu.g/mL, 100. Mu.L of the coating solution is added to each of the wells of the enzyme-labeled plate, and the coating is carried out at 4 ℃ overnight.
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| CN202111339885.4A CN114324859B (en) | 2021-11-12 | 2021-11-12 | Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5196514A (en) * | 1990-03-06 | 1993-03-23 | The University Of Georgia Research Foundation, Inc. | Method of detecting mycoplasma infection in poultry and compositions therefor |
| CN110221065A (en) * | 2019-05-28 | 2019-09-10 | 宁夏大学 | A kind of fowl Mycoplasma synoviae indirect ELISA testing kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019084833A1 (en) * | 2017-11-01 | 2019-05-09 | 财团法人农业科技研究院 | Composition for preventing or treating mycoplasma synoviae infection |
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2021
- 2021-11-12 CN CN202111339885.4A patent/CN114324859B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5196514A (en) * | 1990-03-06 | 1993-03-23 | The University Of Georgia Research Foundation, Inc. | Method of detecting mycoplasma infection in poultry and compositions therefor |
| CN110221065A (en) * | 2019-05-28 | 2019-09-10 | 宁夏大学 | A kind of fowl Mycoplasma synoviae indirect ELISA testing kit |
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| Title |
|---|
| Identification of major immunogenic proteins of Mycoplasma synoviae isolates;Rebeka Lucijana Bercic等;《Veterinary Microbiology》;第127卷;第147、151页 * |
| Rebeka Lucijana Bercic等.Identification of major immunogenic proteins of Mycoplasma synoviae isolates.《Veterinary Microbiology》.2008,第127卷第147、151页. * |
| 滑液支原体WVU1853株免疫相关膜蛋白的初步分析;包世俊 等;《畜牧兽医学报》;第48卷(第2期);第316-323页 * |
| 鸡滑液囊支原体疫苗研究进展;田亚琴等;《中国家禽》;第42卷(第9期);第96-102页 * |
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