CN116042879A - Kit and detection method for detecting brucella wild strain and vaccine strain - Google Patents
Kit and detection method for detecting brucella wild strain and vaccine strain Download PDFInfo
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Abstract
The invention relates to a kit and a detection method for detecting brucella wild strains and vaccine strains. The kit comprises crRNA (WT) and crRNA (Vac), wherein the crRNA is a combination of the crRNA (WT) and the crRNA (Vac), the sequence of the crRNA (WT) is shown as SEQ ID NO.1, and the sequence of the crRNA (Vac) is shown as SEQ ID NO. 2. Or crRNA is obtained by transcription of crDNA (WT) and crDNA (Vac), and the crDNA sequence is shown as SEQ ID NO.3 and SEQ ID NO. 4. The invention performs genome bioinformatics analysis and differential analysis comparison on the brucella wild strain or the brucella S2 vaccine strain, and discovers that the brucella wild strain or the brucella S2 vaccine strain has obvious difference. And then, crRNA is designed according to the difference, so that the sample to be detected is the wild strain of Brucella or the S2 vaccine strain of Brucella by only using one test strip, and the problem of identification and distinction of the S2 vaccine strain and the wild strain in the prior art is effectively solved.
Description
Technical Field
The invention relates to a kit and a detection method for detecting a brucella wild strain and a vaccine strain, belonging to the technical field of biological detection.
Background
Brucellosis (Brucellosis) is a disease caused by Brucella (Brucella) infection, and is a systemic infectious disease of human and livestock, which is called as "Brucellosis" for short, and also called as Zhonghai achalasia heat, malta heat, wave heat and the like. Brucella hosts are wide, the infectivity is strong, different Brucella species have the capability of cross infection among the hosts, and the Brucella species pose a serious threat to animal husbandry and human health.
Brucella (Brucella) is a gram-negative motionless bacterium, has no capsule (smooth micro capsule), is positive for thixotropic enzymes and oxidase, has absolute aerophilic bacteria, can reduce nitrate, is parasitic in cells, and can survive in a wide variety of livestock bodies. Brucellosis is widely distributed around the world, and reports of human brucellosis are also on an ascending trend in 170 countries and regions around the world, and human brucellosis epidemic situation shows obvious occupational, regional and seasonal properties, and human brucellosis can be related to animals infected by contact. Therefore, brucellosis in animals must be fundamentally prevented and controlled.
On one hand, the traditional pathogen separation culture, serological detection technology and molecular biological detection method have various limitations, such as complicated conventional biochemical identification process, long period and low efficiency; the immunity detection specificity is not strong, and the omission is easy to cause; RT-PCR requires time consuming, cumbersome, expensive equipment, inconvenient to carry and requires manipulation by a professional. On the other hand, the existing kit for Brucella can detect Brucella, but can not distinguish and identify the Brucella wild strain from the vaccine strain. Therefore, there is a need to develop a kit capable of detecting wild strains and vaccine strains of brucella.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a kit and a detection method for detecting wild strains and vaccine strains of brucella.
The technical scheme of the invention is as follows:
a crRNA for detecting a brucella wild strain and/or a vaccine strain, the crRNA being a combination of crRNA (WT) and crRNA (Vac);
the sequence of the crRNA (WT) is shown as SEQ ID NO. 1; the sequence of the crRNA (Vac) is shown as SEQ ID NO. 2.
A crDNA for detecting a wild strain and/or a vaccine strain of brucella, the crDNA being a combination of crDNA (WT) and crDNA (Vac);
the sequence of the crDNA (WT) is shown as SEQ ID NO. 3; the sequence of the crDNA (Vac) is shown in SEQ ID NO. 4.
Detecting DNA of a wild strain and/or a vaccine strain of brucella, said DNA being a combination of DNA (WT) and DNA (Vac);
the sequence of the DNA (WT) is shown as SEQ ID NO. 5; the sequence of the DNA (Vac) is shown in SEQ ID NO. 6.
The crRNA (WT) and the crRNA (Vac) can be obtained through artificial synthesis or can be obtained through transcription by taking crDNA (WT) and crDNA (Vac) as templates, wherein the DNA (WT) is a targeting sequence of the crRNA (WT), and the DNA (Vac) is a targeting sequence of the crRNA (Vac) and is used for detecting and distinguishing a Brucella wild strain and a Brucella S2 vaccine strain.
A kit for detecting a wild strain and/or a vaccine strain of brucella, comprising crRNA or crDNA as described above.
According to the invention, the kit is preferably a colloidal gold test strip kit.
Further preferably, the colloidal gold test strip kit comprises Cas13a protein and a colloidal gold test strip; the colloidal gold test strip comprises a bottom plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and attached to the bottom plate; the nitrocellulose membrane is provided with a first detection line and a second detection line; TAMRA ligand is fixed on the first detection line, and Biotin ligand is fixed on the second detection line.
A method for detecting and identifying Brucella wild strains and/or vaccine strains by using the kit for non-diagnostic purposes comprises the following steps:
(1) Extracting genome of a sample to be detected;
(2) Taking the extracted genome of the sample to be detected as a template, adding the template into a reaction system containing reaction liquid and polymerase, and carrying out RPA amplification;
(3) Respectively establishing a first detection system and a second detection system by taking RPA amplification products, and incubating for 10-60 min at 37 ℃;
(4) Uniformly mixing the first detection system and the second detection system according to the equal volume, adding enzyme-free water to obtain a reaction solution, then dripping the reaction solution to a test strip, and after 5-10 minutes, indicating that a sample to be detected is a wild strain when the first detection line has no strip and the second detection line has a strip; when the first detection line has a strip and the second detection line has no strip, the sample to be tested is an S2 vaccine strain; and when the first detection line and the second detection line are provided with strips, the sample to be tested is not infected by Brucella.
According to the invention, in the step (1), the sample to be tested is whole blood, urine or saliva of sheep, cattle and pigs which are subjected to pre-inactivation treatment at 65-80 ℃ for 10 minutes.
According to a preferred embodiment of the present invention, in step (2), the primer sequence for RPA amplification is:
forward primer:
5'-GAAATTAATACGACTCACTATAGGGCTGCGTCAGCCCGCCATCCACACTCGCCA G-3'；
reverse primer: 5'-TTGCGGGTGGTTATTCCTTTCACCCTTTAC-3'.
According to a preferred embodiment of the present invention, in step (2), the RPA amplification system is: RPA base reaction ball one tube, rehydration buffer 29.5. Mu.L, magnesium acetate buffer 2.5. Mu.L, forward primer 2.5. Mu.L, reverse primer 2.5. Mu.L, the remainder was made up with 8. Mu.L of enzyme free water, and then 5 aliquots were made, one portion per 9. Mu.L, 1. Mu.L of sample to be tested was added per portion, and the total volume was 10. Mu.L.
The amplification conditions were: the reaction temperature is 39-42 ℃ and the incubation time is 5-20 min.
Wherein, RPA base reaction ball and rehydration bufferThe wash and magnesium acetate buffer are both from TwitAmp R Basic kit, RPA Basic reaction ball is spherical solid, contains components such as recombinase, polymerase, etc.
According to a preferred embodiment of the present invention, in step (3), the first detection system is: tris-HCl (400 mM), mgCl 2 (120 mM), T7 Polymerase 0.5. Mu. L, rNTP 0.8. Mu. L, reporter1 (1. Mu.M), crRNA (WT) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 1. Mu.L, RPA amplification product 1. Mu.L, total volume 20. Mu.L; the reporter1 is 5'-DIG-UUUUUUUUUUUUUU-TAMRA-3';
the second detection system is as follows: tris-HCl (400 mM), mgCl 2 (120 mM), reporter2 (1. Mu.M), crRNA (Vac) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 2. Mu. L, T7, polymerase 0.5. Mu. L, rNTP 0.8, 0.8. Mu.L, RPA amplification product 1. Mu.L, total volume 20. Mu.L; the reporter2 is 5'-FAM-UUUUUUUUUUUUUU-Biotin-3'.
According to a preferred embodiment of the invention, in step (4), the volume ratio of the enzyme-free water to the first detection system is 1:3.
The invention is not described in detail in the prior art.
The invention has the technical characteristics that:
the Cas13a protein in the first detection system is combined with crRNA (WT) to form a Cas13a-crRNA (WT) complex, the complex can specifically identify a transcription product of DNA (WT) in an RPA amplification product, if DNA (WT) fragments exist in the RPA amplification product, the Cas13a protein is activated and cuts a reporter1 reporter group, and the cut reporter1 reporter group cannot form a display band with colloidal gold of a first detection line, namely, when the first detection line has no band and a second detection line has a band, the tested sample is a wild strain. If no DNA (WT) fragment exists in the RPA amplification product, the Cas13a protein is not activated, and the reporter1 reporter group is combined with colloidal gold and ligand to form a display band, which indicates that the sample to be detected is negative and is not a wild strain. The Cas13a protein in the second detection system binds to the crRNA (Vac) to form a Cas13a-crRNA (Vac) complex that specifically recognizes the transcript of DNA (Vac) in the RPA amplified product, activates the Cas13a protein if a DNA (Vac) fragment is present in the RPA amplified product, and cleaves the reporter2 reporter group. The cut reporter2 reporter group cannot form a display band with the colloidal gold of the second detection line, namely, when the first detection line has a band and the second detection line has no band, the sample to be tested is an S2 vaccine strain. If the RPA amplification product contains no DNA (WT) and DNA (Vac) fragments, the Cas13a is not activated, and the reporter group is combined with the colloidal gold and the ligand of the detection line, namely, the first detection line and the second detection line are provided with strips, which indicates that the sample to be detected is not infected by Brucella.
The beneficial effects are that:
1. the kit for detecting the wild strain and/or the vaccine strain of the brucella can distinguish whether the sample to be detected is the wild strain or the S2 vaccine strain of the brucella by using only one test strip, and effectively solves the problem of respectively identifying and distinguishing the S2 vaccine strain and the wild strain in the prior art. Compared with the traditional detection method, the detection method provided by the invention has the advantages of strong relative bit variability, low detection limit, high sensitivity, good color development effect, short detection period and capability of achieving visual detection.
2. The detection method provided by the invention combines the RPA amplification technology with the CRISPR system, reduces mutual interference through reasonable proportion, ensures that respective functions can still be accurately and specifically realized among reaction reagents, combines amplification and detection, combines high sensitivity of the RPA amplification technology with high specificity of the CRISPR detection technology, has shorter time consumption in the whole detection process, can complete detection within one hour, has simple operation steps, and provides a brand-new thought and method for purifying brucellosis.
Drawings
FIG. 1 is a graph showing the results of differential gene analysis.
FIG. 2 is a graph showing the results of detecting wild strains and vaccine strains with gradient concentrations by using a colloidal gold test strip kit.
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 described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Cas13a protein, beijing family Biotech Co., ltd.
TwistAmp R Basic kit, available from Twitteddx-merry biotechnology Co.
The WT represents the wild strain and Vac represents the vaccine strain.
Example 1 design and acquisition of crRNA and DNA for detection of Brucella
The gene sequence was downloaded from GenBank by logging in NCBI including: the Brucella vaccine strain S2, brucella wild strain representative strain (M28, ATCC23457, NI, bmWS93, CIT31, B9, QH61, BY38, 20236, RM57, rev.1, A13334, 2308, 9-941, 104M, BD, BAB8416, clpP, MC, BJ1, 1330, QH05, CVI-71) and the like were subjected to genomic bioinformatic analysis and differential analysis comparison, and the analysis results are shown in FIG. 1.
As can be seen from FIG. 1, there is a significant difference between the gene sequences of the Brucella vaccine strain S2 and the Brucella wild strain, and the inventors of the present application have further found that the gene loci for detecting and identifying the Brucella vaccine strain S2 and the Brucella wild strain are DNA (WT) and DNA (Vac) as shown in SEQ ID NO.5 and SEQ ID NO. 6. As can be seen from the sequence comparison, the Brucella vaccine strain S2 has 25 base deletions after 100bp compared with the Brucella wild strain representative strain. The specific base deleted was related to the Brucella wild strain species (FIG. 1).
Then, according to the difference of the gene sequences of the DNA (WT) and the DNA (Vac), the crRNA (WT) and the crRNA (Vac) are designed, the sequences of which are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the crRNA (WT) and the crRNA (Vac) designed by the invention can be specifically matched with the differential fragments of the gene regions of the wild strain and the S2 vaccine strain, so that the corresponding Cas protein nuclease activity is activated, and the reporter group in the system is cut.
The specific procedure for obtaining crRNA is as follows: taking crDNA as a template, respectively carrying out annealing reaction to form double-stranded DNA, then carrying out agarose gel electrophoresis, recovering and purifying DNA fragments by gel, transcribing to generate RNA under the action of T7 RNA polymerase, recovering and purifying to obtain mature crRNA, and sub-packaging and freezing the purified crRNA to-80 ℃; or directly synthesized artificially according to the sequence.
Wherein the sequence of crDNA (WT) is shown as SEQ ID NO. 3; the crDNA (Vac) is shown as SEQ ID NO. 4; the crDNA may be synthesized directly by artificial synthesis according to the sequence.
Annealing system: the DNA oligo to be annealed (synthetic primer) was formulated to 50. Mu.M with sterilized enzyme-free water or re-distilled water.
Dissolve Annealing Buffer for DNA Oligos (5×), mix well for use.
Adding various reagents in the above sequence, and mixing.
The sequence of the T7 primer is as follows: 5'-GAAATTAATACGACTCACTATAGGG-3'.
The annealing reaction was performed by setting a PCR instrument as follows:
the transcription system is as follows: template DNA 1. Mu.g, T7 RNA polymerase mixture 2. Mu.L, NTP Buffer Mix 10. Mu.L, no enzyme water make up, total volume 30. Mu.L.
The transcription conditions were: 37℃for 16h.
Example 3 colloidal gold test strip kit for detecting wild strain and/or vaccine strain of Brucella
The kit of the embodiment comprises a colloidal gold test strip, a colloidal gold test strip card shell, a micropore cup and a TwitAmp R Basic kit for detecting and identifying brucella wildCrRNA (WT) and crRNA (Vac) of the raw strain and vaccine strain (SEQ ID NO. 1-2), cas13a protein, RPA amplification primer, tris-HCl (400 mM), mgCl 2 (120mM)、RNase Inhibitor、reporter1(1μM)、reporter2(1μM)、T7 Polymerase、rNTP。
The colloidal gold test strip comprises a bottom plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and attached to the bottom plate; the nitrocellulose membrane is provided with a first detection line and a second detection line. TAMRA ligand is immobilized on the first detection line, and Biotin ligand is immobilized on the second detection line.
The reporter1 is 5'-DIG-UUUUUUUUUUUUUU-TAMRA-3';
the reporter2 is 5'-FAM-UUUUUUUUUUUUUU-Biotin-3'.
Example 4 method of detecting Brucella colloidal gold test strip Using the kit of example 3
Blood samples containing Brucella suis, brucella caprae and S2 vaccine strain were taken at 20. Mu.L, respectively, and Brucella colloidal gold test strip detection was performed.
The specific method comprises the following steps:
(1) Pre-inactivating the sample to be detected at 80 ℃ for 10 minutes respectively, then adding 20 mu L of NP-40 lysate, vibrating for 15s until the sample is uniformly mixed, and heating the sample in a thermostat at 95-99 ℃ for 10 minutes to obtain genomes of three samples to be detected;
(2) Taking the extracted genome of the sample to be detected as a template, adding the template into a reaction system containing reaction liquid and RPA, and carrying out nucleic acid amplification of a target gene;
RPA amplification reaction system: one tube of RPA base reaction ball, 29.5 mu L of rehydration buffer, 2.5 mu L of magnesium acetate buffer, 2.5 mu L of forward primer, 2.5 mu L of reverse primer, the balance of enzyme-free water and 8 mu L of total volume are complemented, and then 5 equal parts are distributed, each 9 mu L of sample is added, 1 mu L of sample to be tested is added, and RPA reaction of five samples can be amplified simultaneously, and each reaction volume is 10 mu L.
The amplification conditions were: the reaction temperature was 40℃and the incubation time was 15min.
Wherein the RPA groupThe base reaction balls, rehydration buffer and magnesium acetate buffer were all from twist amp R Basic kit.
The RPA amplification primer is selected from the following primer pairs:
forward primer:
5'-GAAATTAATACGACTCACTATAGGGCTGCGTCAGCCCGCCATCCACACTCGCCA G-3'；
reverse primer: 5' -TTGCGGGTGGTTATTCCTTTCACCCTTTAC-3;
RPA amplification conditions: the reaction temperature is 40 ℃ and the incubation time is 10min;
wherein the RPA basic reaction ball, the PBS buffer solution and the magnesium acetate buffer solution are all from TwitAmp R Basic kit; RPA amplification is prior art;
then, the extracted genome is subjected to concentration gradient dilution, wherein the dilution gradient is as follows: 10 6 copies/μL、10 5 copies/μL、10 4 copies/μL、10 3 copies/μL、10 2 copies/μL、10 1 cobies/. Mu.L, followed by enzyme-free water as control (10 0 copy/. Mu.L) to obtain RPA amplification product with gradient concentration;
(3) Taking RPA amplification products with gradient concentration, respectively establishing a first detection system and a second detection system, and incubating for 40min at 37 ℃;
the first detection system is as follows: tris-HCl (400 mM), mgCl 2 (120 mM), T7 Polymerase 0.5. Mu. L, rNTP 0.8.8. Mu. L, reporter1 (0.1. Mu.M), crRNA (WT) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 1. Mu.L, RPA amplification product 1. Mu.L, and total volume 20. Mu.L.
The second detection system is as follows: tris-HCl (400 mM), mgCl 2 (120 mM), reporter2 (2. Mu.M), crRNA (Vac) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 2. Mu. L, T7, polymerase 0.5. Mu. L, rNTP 0.8, 0.8. Mu.L, RPA amplification product 1. Mu.L, total volume 20. Mu.L.
Wherein, reporter1 is 5'-DIG-UUUUUUUUUUUUUU-TAMRA-3';
the reporter2 is 5'-FAM-UUUUUUUUUUUUUU-Biotin-3'.
(4) And uniformly mixing 20 mu L of the first detection system and 20 mu L of the second detection system, adding 60 mu L of enzyme-free water to obtain a reaction solution, dripping the reaction solution into a microporous cup, allowing the reaction solution to be absorbed by a sample pad, sequentially flowing to a first detection line and a second detection line, and obtaining a detection result after 10 minutes.
The detection results of this example are shown in fig. 2. As can be seen from fig. 2, the detection results of the blood sample without S2 vaccine strain and without wild strain genome are that the first detection line and the second detection line both show bands; the detection result of the blood sample with the S2 vaccine strain and without the genome of the wild strain is that the first detection line shows the strip, and the second detection line does not show the strip; the detection result of the blood sample without the S2 vaccine strain and with the genome of the wild strain is that the first detection line does not show the band, and the second detection line shows the band. Meanwhile, FIG. 2 shows that the detection line of the colloidal gold test strip is 10 1 -10 2 The probes/mu L have the advantages of high sensitivity, low price, simple and quick operation and convenient carrying of equipment and instruments.
Claims (10)
1. A crRNA for detecting a wild strain and/or a vaccine strain of brucella, wherein the crRNA is a combination of crRNA (WT) and crRNA (Vac);
the sequence of the crRNA (WT) is shown as SEQ ID NO. 1; the sequence of the crRNA (Vac) is shown as SEQ ID NO. 2.
2. crDNA for the detection of wild strains and/or vaccine strains of brucella, characterized in that the crDNA is a combination of crDNA (WT) and crDNA (Vac);
the sequence of the crDNA (WT) is shown as SEQ ID NO. 3; the sequence of the crDNA (Vac) is shown in SEQ ID NO. 4.
3. A DNA for detecting a wild strain and/or a vaccine strain of brucella, wherein the DNA is a combination of DNA (WT) and DNA (Vac);
the sequence of the DNA (WT) is shown as SEQ ID NO. 5; the sequence of the DNA (Vac) is shown in SEQ ID NO. 6.
4. A kit for detecting a wild strain and/or a vaccine strain of brucella comprising the crRNA of claim 1 or the crDNA of claim 2.
5. The kit for detecting a wild strain and/or a vaccine strain of brucella as claimed in claim 4, wherein the kit is a colloidal gold test strip kit; the colloidal gold test strip kit comprises Cas13a protein and a colloidal gold test strip.
6. The kit for detecting a wild strain and/or a vaccine strain of brucella as claimed in claim 5, wherein the colloidal gold test strip comprises a bottom plate, and a sample pad, a connection pad, a nitrocellulose membrane and an absorption pad which are sequentially bonded to the bottom plate; the nitrocellulose membrane is provided with a first detection line and a second detection line; TAMRA ligand is fixed on the first detection line, and Biotin ligand is fixed on the second detection line.
7. A method for detecting and identifying wild strains and/or vaccine strains of brucella for non-diagnostic purposes using the kit of claim 7, comprising the steps of:
(1) Extracting genome of a sample to be detected;
(2) Taking the extracted genome of the sample to be detected as a template, adding the template into a reaction system containing reaction liquid and polymerase, and carrying out RPA amplification;
(3) Respectively establishing a first detection system and a second detection system by taking RPA amplification products, and incubating for 10-60 min at 37 ℃;
(4) Uniformly mixing the first detection system and the second detection system according to the equal volume, adding enzyme-free water to obtain a reaction solution, then dripping the reaction solution to a test strip, and after 5-10 minutes, indicating that a sample to be detected is a wild strain when the first detection line has no strip and the second detection line has a strip; when the first detection line has a strip and the second detection line has no strip, the sample to be tested is an S2 vaccine strain; and when the first detection line and the second detection line are provided with strips, the sample to be tested is not infected by Brucella.
8. The method according to claim 7, wherein in the step (1), the sample to be tested is whole blood, urine or saliva of sheep, cattle and pigs subjected to pre-inactivation treatment at 65 to 80 ℃ for 10 minutes.
9. The method of claim 7, wherein in step (2), the primer sequences for RPA amplification are:
forward primer:
5'-GAAATTAATACGACTCACTATAGGGCTGCGTCAGCCCGCCATCCACACTCGCCA G-3'；
reverse primer: 5'-TTGCGGGTGGTTATTCCTTTCACCCTTTAC-3';
the RPA amplification system is as follows: one tube of RPA basic reaction ball, 29.5 mu L of rehydration buffer, 2.5 mu L of magnesium acetate buffer, 2.5 mu L of forward primer, 2.5 mu L of reverse primer, and the balance of 8 mu L of enzyme-free water, and then 5 equal parts of the mixture are distributed, wherein each 9 mu L of mixture is added with 1 mu L of sample to be tested, and the total volume is 10 mu L;
the amplification conditions were: the reaction temperature is 39-42 ℃ and the incubation time is 5-20 min.
10. The method of claim 7, wherein in step (3), the first detection system is: tris-HCl (400 mM), mgCl 2 (120 mM), T7 Polymerase 0.5. Mu. L, rNTP 0.8. Mu. L, reporter1 (0.1. Mu.M), crRNA (WT) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 1. Mu.L, RPA amplification product 1. Mu.L, total volume 20. Mu.L; the reporter1 is 5'-DIG-UUUUUUUUUUUUUU-TAMRA-3';
the second detection system is as follows: tris-HCl (400 mM), mgCl 2 (120 mM), reporter2 (2. Mu.M), crRNA (Vac) 1. Mu.L, RNase Inhibitor 1. Mu. L, cas13a protein 2. Mu. L, T7, polymerase 0.5. Mu. L, rNTP 0.8, 0.8. Mu.L, RPA amplification product 1. Mu.L, total volume 20. Mu.L; the reporter2 is 5'-FAM-UUUUUUUUUUUUUU-Biotin-3';
in the step (4), the volume ratio of the enzyme-free water to the first detection system is 1:3.
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| CN116497138A (en) * | 2023-06-20 | 2023-07-28 | 内蒙古大学 | Detection method and kit for identifying and detecting mycoplasma bovis and mycoplasma caprae |
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