CN1715422A - Novel Mycobacterium bovis and the method for quick identification of mycobacterium tuberculosis and application - Google Patents
Novel Mycobacterium bovis and the method for quick identification of mycobacterium tuberculosis and application Download PDFInfo
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Abstract
Novel Mycobacterium bovis and the method for quick identification of mycobacterium tuberculosis and application.Though the clinical samples smear for microscopic examination is simple, economical, quick at present, is not that each clinical samples (particularly sputum) can both successfully detect tubercule bacillus, can't differentiate that it is tubercule bacillus or other mycobacterium.Method of the present invention: adding has Mycobacterium bovis Vallee strain gene group DNA to amplify the fragment of 294bp, primer with Mycobacterium bovis can only go out goal gene from the DNA of Mycobacterium bovis, adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify the fragment of 294bp, primer with mycobacterium tuberculosis can only amplify goal gene from the DNA of mycobacterium tuberculosis, identical upstream primer and two discrepant downstream primers, two amplification gene fragments are 294bp, 67 ℃ annealing temperature.Present method is used for the direct detection at pure cultures of bacteria and clinical sample.
Description
Technical field:
The present invention relates to a kind of novel and discrimination method of Mycobacterium bovis and mycobacterium tuberculosis fast.
Background technology:
Tuberculosis is the chronic infectious disease that a kind of people and multiple animal suffer from altogether, this sick pathogenic bacteria is a mycobacterium tuberculosis, occurring in nature exists the tubercule bacillus of people's tuberculosis (or human-like) mycobacterium, Mycobacterium bovis (or ox type) and three types of avain tuberculosis (or fowl type) mycobacterium, and they all have virulence to people and many animals.Bacillus tuberculosis bovis not only can make ox morbidity, can also infringer and multiple animal; The Bacillus tuberculosis is except that causing people's tuberculosis, and is all dangerous to animals such as monkey, ox, pig, cats; The avain tuberculosis bacillus is as the same, and it can make some animal beyond the fowl suffer from tuberculosis, and as far back as nineteen forty-seven, the U.S. has just found the first routine people's fowl type tuberculosis.Up to now, except that the people, have at least 66 kinds of birds such as 50 kinds of Mammalss such as ox, horse, sheep, pig, monkey, deer, dog, cat and chicken, goose, duck, turkey, ostrich, sparrow that tuberculous record is arranged.Milk cow is to propagate tuberculosis to give the most dangerous human animal, because it and people's relation is closer, and the morbidity of bovine tuberculosis is quite high, contacts for a long time with sick ox, or the edible accidentally people who contains tubercule bacillus milk might infect tuberculosis.An external report shows that in 100 tuberculosis patients, it is from animal infected that 26 examples are arranged.Wherein, accounted for 20 examples unexpectedly below 15 years old, they all with drink the milk that contains tubercule bacillus accidentally or contact relevant closely with animal.This shows that prapes is the contagium of other animal tuberculosis maximums.
At present, the discrimination method that is used for diagnosis lungy and pathogenic agent kind fails to obtain satisfied the solution for a long time always.Generally come it is carried out tentative diagnosis clinically according to symptom, direct smear microscopy, tuberculin (OT) tuerculoderma, resistance test and biochemical test analysis.Also used widely in practice though the clinical samples smear for microscopic examination is simple, economical, quick, but be not that each clinical samples (particularly sputum) can both successfully detect tubercule bacillus, microscopy need repeat 6-8 time sometimes, microscopy promptly enables to detect antiacid mycobacterium, can't differentiate that also it is tubercule bacillus or other mycobacterium.Can only depend on to lungy making a definite diagnosis that tubercule bacillus is cultivated and the biochemical identification of bacterial type, and the cultivation of tubercule bacillus not only positive rate is low, and time-consuming, generally need 2-3 month, be subjected to influence simultaneously again such as factors such as sampling, medication, pollutions.Use in recent years that serology detects and the dna molecule hybridize method detects tuberculosis, corresponding raising arranged on susceptibility, but the immunoenzyme specificity not too high, be prone to false positive results such as cross-immune reaction.And the molecular hybridization complex operation, the isotopic labeling probe needs special facility and protection, and susceptibility still can not satisfy the requirement of clinical detection.
Summary of the invention
The technical problem to be solved in the present invention:
Need the shortcoming that the time is long and discriminating is difficult at Mycobacterium bovis and mycobacterium tuberculosis, the invention solves the problem that Mycobacterium bovis and mycobacterium tuberculosis bacterial type are differentiated fast.
The technical solution adopted for the present invention to solve the technical problems is:
The method of a kind of quick discriminating Mycobacterium bovis and mycobacterium tuberculosis, adding has Mycobacterium bovis Vallee strain gene group DNA to amplify the fragment of 294bp, primer with Mycobacterium bovis can only go out goal gene from the DNA of Mycobacterium bovis, adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify the fragment of 294bp, primer with mycobacterium tuberculosis can only amplify goal gene from the DNA of mycobacterium tuberculosis, identical upstream primer and two discrepant downstream primers, two amplification gene fragments are 294bp, 67 ℃ annealing temperature.
The method of described quick discriminating Mycobacterium bovis and mycobacterium tuberculosis: the segmental method that described adding has Mycobacterium bovis Vallee strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and TB-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
The method of described quick discriminating Mycobacterium bovis and mycobacterium tuberculosis: the segmental method that described adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and MT-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
The method of a kind of quick discriminating Mycobacterium bovis and mycobacterium tuberculosis is in the application of the direct context of detection of pure cultures of bacteria and clinical sample.
The invention has the beneficial effects as follows:
1, present, the PCR method of domestic application is merely able to distinguish mycobacterium tuberculosis mixture and anonymous mycobacteria, and can not distinguish Mycobacterium bovis and bacillus tuberculosis typus humanus.The present invention not only can differentiate Mycobacterium bovis and anonymous mycobacteria, and can differentiate Mycobacterium bovis and bacillus tuberculosis typus humanus, has solved the problem that Mycobacterium bovis and bacillus tuberculosis typus humanus differentiate.
Advantages such as that round pcr has is highly sensitive, high specificity, height easy and simple to handle, quick, repeated and automated operation can detect the atomic mycobacterium tuberculosis dna of quantity in 1-2 hour, its susceptibility can reach 10 tuberculosis thalline.Tubercule bacillus PCR detects, and selecting goal gene and design of primers is the key of this detection technique.Because tubercule bacillus has many kinds of variability, so primer must be chosen to be at conservative DNA section and just is unlikely omission, mycobacterium has many kinds simultaneously, its every kind mycobacterium has different floras again, can cause human wherein the part of only being lungy, many mycobacteriums are not pathogenic or cause non-tuberculous disease.Therefore, the primer of design must have specificity preferably to the tubercule bacillus of causing a disease, and just is unlikely false positive to occur.The 65KD antigen gene is that Mycobacterium is common, therefore, must synthesize the corresponding specific probe of mycobacterium of distinct group simultaneously, detects amplified production and just can obtain assay accurately.In addition, mycobacterium tuberculosis complex body (Mycobactrium tuberculosis complex, MTBC) comprise Mycobacterium bovis, mycobacterium tuberculosis, mycobacterium microti and mycobacterium africanum, the total gene of MTBC mainly contains PMT64 gene, 32kD protein gene and insertion sequences such as IS6110 and IS1081.Wherein the tumor-necrosis factor glycoproteins of IS6110 special the strongest, susceptibility is higher, mycobacterium tuberculosis complex body PCR such as can yet be regarded as Bacillus tuberculosis and mycobacterium bovis BCG detect the preferred genes of design of primers.Combine with total gene order of these MTBC and round pcr, progressively develop and multiple molecular biology method based on round pcr.As PCR finger-printing technique, multiple PCR method etc.But use the PCR detection that said gene is carried out, still can not differentiate Mycobacterium bovis and mycobacterium tuberculosis, and be merely able to differentiate mycobacterium tuberculosis mixture and non-tuberculous mycobacteria mixture.Along with to mycobacterium tuberculosis and deepening continuously of Mycobacterium bovis research and finishing of genom sequence, found to be present in specific marker gene in mycobacterium tuberculosis and the Mycobacterium bovis gradually.
The MPB70 antigen gene is peculiar by Bacillus tuberculosis bovis, and the primer that is designed by this gene order carries out PCR, the detection of Bacillus tuberculosis bovis is had the specificity and the susceptibility of height.But conventional is that goal gene can not detect all mycobacterium bovis BCGs with MPB70.
By detection validation 500bp gene fragment is that the bacillus tuberculosis bovis genome is peculiar, and segmental amplification characteristic of the 500bp in the mycobacterium tuberculosis var bovis genome and mycobacterium tuberculosis var bovis are cultivated and the biochemical test characteristic has 100% dependency.This shows that this gene can be used for the diagnosis and the discriminating of mycobacterium tuberculosis var bovis.
The mtp40 protein gene is the peculiar gene of Bacillus tuberculosis, is that target sequence carries out the PCR detection with this gene, the Bacillus tuberculosis DNA that can only increase, but also can not detect all Bacillus tuberculosis.
PncA (pyrazinamidase) gene is to be present in a kind of peculiar gene in Mycobacterium bovis and the mycobacterium tuberculosis, homology with height, the homologous sequence analysis revealed exists difference between Mycobacterium bovis and mycobacterium tuberculosis PncA (pyrazinamidase) gene.Therefore, can by the control of primer, reach the purpose that specificity is identified Mycobacterium bovis and mycobacterium tuberculosis according to this species diversity design primer with the different and high annealing temperature of dna profiling bonding force.Shah etc. (2002) have reported according to pncA (pyrazinamidase) gene design of Mycobacterium bovis (ox type) and mycobacterium tuberculosis (human-like) multiple PCR method of step amplification, differentiate Mycobacterium bovis (ox type) and mycobacterium tuberculosis (human-like) fast.This multiple PCR method can detect the dna segment of Mycobacterium bovis and mycobacterium tuberculosis PncA gene 185bp specifically, but because the target gene fragment of two kinds of amplifications is smaller, is unfavorable for electrophoresis detection and result's observation.Therefore, the present invention improves according to people's such as Shah result of study, and goal gene is expanded as 294bp, so not only still has the characteristic that can differentiate Mycobacterium bovis and mycobacterium tuberculosis, and helps the judgement of electrophoresis observation and positive findings.
Exist difference on the PncA of Mycobacterium bovis and mycobacterium tuberculosis (pyrazinamidase) gene, at the indoor design of PncA gene identical upstream primer and the downstream primer that contains differential gene, by the control of downstream primer with the different and high annealing temperature of dna profiling bonding force: the Bacillus tuberculosis bovis primer is merely able to amplify Mycobacterium bovis PncA gene fragment, Bacillus tuberculosis's primer is merely able to amplify bacillus tuberculosis typus humanus PncA gene fragment, two gene fragments are 294bp, thereby finish task of the present invention.
2, the conventional method of differentiating Mycobacterium bovis and bacillus tuberculosis typus humanus needs through processes such as microbial culture, biochemical identification, and the time is longer, general 2-3 month.The present invention has saved above-mentioned loaded down with trivial details operation, needs promptly can finish from extracting template DNA in 1 day to only finishing of PCR reaction, and is easy and simple to handle, quick.
Embodiment:
Embodiment 1:
PncA (pyrazinamidase) gene is to be present in a kind of peculiar gene in Mycobacterium bovis and the mycobacterium tuberculosis, has the homology of height, the homologous sequence analysis revealed, and Mycobacterium bovis and mycobacterium tuberculosis PncA (pyrazinamidase) gene exist difference.Design identical upstream primer and the downstream primer that contains differential gene, by the control of downstream primer with the different and high annealing temperature of dna profiling bonding force, specificity is identified Mycobacterium bovis and mycobacterium tuberculosis; Owing to do not have this gene in the anonymous mycobacteria, therefore can distinguish Mycobacterium bovis, mycobacterium tuberculosis and anonymous mycobacteria again again.
1, increase respectively Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and Mycobacterium phlei genomic dna of upstream primer MB-F of Mycobacterium bovis and downstream primer TB-R.
Concrete operation method:
Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and TB-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.Set up negative control simultaneously.
The result: having only to add has Mycobacterium bovis Vallee strain gene group DNA can amplify the fragment of 294bp, conforms to the actual design size.And mycobacterium tuberculosis H37vR bacterial strain, ox mycobacterium paratuberculosis, Mycobacterium phlei genome and negative control all do not amplify dna fragmentation.
2, increase respectively Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and the genomic DNA of Mycobacterium phlei of the upstream primer MB-F of mycobacterium tuberculosis and downstream primer MT-R.
Concrete operation method: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and MT-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.While is also set up negative control.
Result: have only to add the fragment that can amplify 294bp that mycobacterium tuberculosis H37vR strain gene group DNA is arranged, conform to the actual design size.And Mycobacterium bovis Vallee bacterial strain, ox mycobacterium paratuberculosis, Mycobacterium phlei genome and negative control all do not amplify dna fragmentation.
The method of a kind of quick discriminating Mycobacterium bovis and mycobacterium tuberculosis, adding has Mycobacterium bovis Vallee strain gene group DNA to amplify the fragment of 294bp, primer with Mycobacterium bovis can only go out goal gene from the DNA of Mycobacterium bovis, adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify the fragment of 294bp, primer with mycobacterium tuberculosis can only amplify goal gene from the DNA of mycobacterium tuberculosis, identical upstream primer and two discrepant downstream primers, two amplification gene fragments are 294bp, 67 ℃ annealing temperature.
The method of described quick discriminating Mycobacterium bovis and mycobacterium tuberculosis: the segmental method that described adding has Mycobacterium bovis Vallee strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and TB-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
The method of described quick discriminating Mycobacterium bovis and mycobacterium tuberculosis: the segmental method that described adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and MT-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
Embodiment 2:
The method of a kind of quick discriminating Mycobacterium bovis and mycobacterium tuberculosis is in the application of the direct context of detection of pure cultures of bacteria and clinical sample.
Claims (4)
1. method of differentiating fast Mycobacterium bovis and mycobacterium tuberculosis, it is characterized in that: adding has Mycobacterium bovis Vallee strain gene group DNA to amplify the fragment of 294bp, primer with Mycobacterium bovis can only go out goal gene from the DNA of Mycobacterium bovis, adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify the fragment of 294bp, primer with mycobacterium tuberculosis can only amplify goal gene from the DNA of mycobacterium tuberculosis, identical upstream primer and two discrepant downstream primers, two amplification gene fragments are 294bp, 67 ℃ annealing temperature.
2. the method for quick discriminating Mycobacterium bovis according to claim 1 and mycobacterium tuberculosis: the segmental method that described adding has Mycobacterium bovis Vallee strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and TB-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
3. the method for quick discriminating Mycobacterium bovis according to claim 1 and 2 and mycobacterium tuberculosis: the segmental method that described adding has mycobacterium tuberculosis H37vR strain gene group DNA to amplify 294bp is: Mycobacterium bovis Vallee bacterial strain, mycobacterium tuberculosis H37Rv bacterial strain, ox mycobacterium paratuberculosis and each 1ul of Mycobacterium phlei genomic dna, each 1ul of primer MB-F and MT-R, primer concentration is 20pMol, 10 * TagDNA polysaccharase Buffer5ul, 2.5mMdNTP3ul, TagDNA polysaccharase 0.5ul, supply with water, reaction system is 50ul, carries out pcr amplification then.Amplification PCR products is 294bp.The condition of PCR reaction is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 67 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 circulations, 72 ℃ were extended 10 minutes.Ward off at 1.5% agar and to carry out electrophoresis on the gel, 100V voltage is observations after 20 minutes.
4. differentiate of the application of the method for Mycobacterium bovis and mycobacterium tuberculosis fast for one kind in the direct context of detection of pure cultures of bacteria and clinical sample.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157722B (en) * | 2006-12-06 | 2010-10-20 | 中国农业大学 | Cattle mycobacterium Mce4E protein, preparation method and uses thereof |
| CN102399751A (en) * | 2011-09-13 | 2012-04-04 | 中国农业科学院哈尔滨兽医研究所 | Anti-mycobacterium tuberculosis monoclonal antibody, epitope recognized by anti-mycobacterium tuberculosis monoclonal antibody, and use thereof |
| CN102409102A (en) * | 2011-11-30 | 2012-04-11 | 中国农业大学 | PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis |
| CN101732732B (en) * | 2009-12-17 | 2012-10-31 | 中国兽医药品监察所 | Bovine type tuberculin standard substance and preparation method thereof |
| CN105907861A (en) * | 2016-05-05 | 2016-08-31 | 广州金域医学检验中心有限公司 | Method and primer for quick detection and classification of mycobacteria |
| CN108004253A (en) * | 2017-12-01 | 2018-05-08 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN108165565A (en) * | 2017-12-01 | 2018-06-15 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN110408630A (en) * | 2018-04-28 | 2019-11-05 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN110408631A (en) * | 2018-04-28 | 2019-11-05 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101157722B (en) * | 2006-12-06 | 2010-10-20 | 中国农业大学 | Cattle mycobacterium Mce4E protein, preparation method and uses thereof |
| CN101732732B (en) * | 2009-12-17 | 2012-10-31 | 中国兽医药品监察所 | Bovine type tuberculin standard substance and preparation method thereof |
| CN102399751A (en) * | 2011-09-13 | 2012-04-04 | 中国农业科学院哈尔滨兽医研究所 | Anti-mycobacterium tuberculosis monoclonal antibody, epitope recognized by anti-mycobacterium tuberculosis monoclonal antibody, and use thereof |
| CN102409102A (en) * | 2011-11-30 | 2012-04-11 | 中国农业大学 | PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis |
| CN102409102B (en) * | 2011-11-30 | 2013-07-10 | 中国农业大学 | PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis |
| CN105907861A (en) * | 2016-05-05 | 2016-08-31 | 广州金域医学检验中心有限公司 | Method and primer for quick detection and classification of mycobacteria |
| CN108004253A (en) * | 2017-12-01 | 2018-05-08 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN108165565A (en) * | 2017-12-01 | 2018-06-15 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN108004253B (en) * | 2017-12-01 | 2021-04-20 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and application thereof |
| CN110408630A (en) * | 2018-04-28 | 2019-11-05 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN110408631A (en) * | 2018-04-28 | 2019-11-05 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
| CN110408631B (en) * | 2018-04-28 | 2021-04-20 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and application thereof |
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