CN102286627A - Simultaneous identification method for mycobacterium bovis and human mycobacterium tuberculosis and reagent kit thereof - Google Patents
Simultaneous identification method for mycobacterium bovis and human mycobacterium tuberculosis and reagent kit thereof Download PDFInfo
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- CN102286627A CN102286627A CN201110264464XA CN201110264464A CN102286627A CN 102286627 A CN102286627 A CN 102286627A CN 201110264464X A CN201110264464X A CN 201110264464XA CN 201110264464 A CN201110264464 A CN 201110264464A CN 102286627 A CN102286627 A CN 102286627A
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Abstract
The invention relates to the field of biological detection, in particular to a simultaneous identification method for mycobacterium bovis and human mycobacterium tuberculosis and a reagent kit of the simultaneous identification method. The invention designs more than one forward primer and two different reverse primers (the reverse primers respectively aim at the mycobacterium bovis and the human mycobacterium tuberculosis) by utilizing the genome differences between the human mycobacterium tuberculosis and the mycobacterium bovis, Taqman probes are respectively designed according to amplification target sections, in addition, the two probes carry out different fluorescence labeling, the goal of simultaneously detecting the mycobacterium bovis and the human mycobacterium tuberculosis in the same reaction tube is realized, in addition, the identification is carried out through different fluorescence passages, the results can be directly judged and read on software during the amplification without electrophoresis, the aerosol pollution is avoided, and greater application prospects are realized clinically.
Description
Technical field
The present invention relates to field of biological detection, authentication method and test kit thereof when being specifically related to a kind of Mycobacterium bovis and bacillus tuberculosis typus humanus.
Technical background
Tuberculosis is the chronic infectious disease that a kind of people and multiple animal suffer from altogether.According to the WHO report, there are 2,000 ten thousand tuberculosis patients in the whole world, and 800～1,000 ten thousand newly-increased case is arranged every year, and the present tuberculosis patient of China increases case 100～1,300,000 every year newly about 6,000,000.Tuberculosis has become one of disease of serious crisis human health of 21 century.Occurring in nature exists the various human mycobacterium tuberculosis: people's tuberculosis (or human-like) mycobacterium, Mycobacterium bovis (or ox type) are waited other bacillus tuberculosis typus humanus.Ox type bacillus tuberculosis typus humanus not only can make the ox morbidity, all right infringer and multiple animal, milk cow is to propagate tuberculosis to give the most dangerous human animal, and the tuberculosis morbidity of milk cow is higher, contact for a long time with sick ox, or drink the milk that contains the bacillus tuberculosis typus humanus accidentally and all might infect tuberculosis, particularly drink raw milk's custom in addition in the part area of China, causing infecting probability lungy increases greatly.Ox bacillus tuberculosis typus humanus's the therapy and the difference of tuberculotherapy are that the ox bacillus tuberculosis typus humanus is innately to the pyrazinoic acid amide resistance of one of tuberculosis standard drug.Have document to point out, it is to be caused by Mycobacterium bovis that 5%～10% tuberculosis is arranged in the tuberculosis patient of China, therefore, detects for the discriminating of bacillus tuberculosis typus humanus and Mycobacterium bovis and to seem most important.
At present, clinically the difference of bacillus tuberculosis typus humanus and Mycobacterium bovis is identified and need be carried out pure culture to bacterial strain, adopt biochemical method to differentiate simultaneously, but bacillus tuberculosis typus humanus and Mycobacterium bovis are difficult to cultivate, even cultivate successful required time also about 8 weeks, and these discrimination methods are also unreliable, therefore press for a kind of quick discriminating detection method of research and development.PCR detects outstanding high specificity, the characteristics that susceptibility is high of showing in numerous methods, in also a large amount of in recent years evaluations that has been applied in the ox bacillus tuberculosis typus humanus, but can produce a large amount of aerosols owing to need that the amplified production open pipe is carried out electrophoresis, cause this method to produce a large amount of false positives, so further do not used clinically.And fluorescence real-time quantitative PCR then can both can effectively be avoided aerocolloidal method fast for we provide a kind of.
Real-time fluorescence quantitative PCR technology (Real-time quantitative Polymerase Chain Reaction is called for short Real Time PCR) is the nucleic acid quantification technology (HIGUCHIR that grows up on the qualitative PCR technical foundation, FOCKLERC, DOLLINGER G, et al.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions[J] .Biotechnology, 1993,11:1026; Han Junying, Ceng Ruiping. fluorescent quantitative PCR technique and application thereof [J]. foreign medical science genetics fascicle, 2000,23 (3): 177., be incorporated by reference in this text and examine, as narration in full).
In the PCR reaction system, add fluorophor, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, each circulation is become " as seen ", by Ct value and typical curve the initial concentration of the DNA in the sample (orcDNA) is carried out quantitative methods at last.Real-time fluorescence quantitative PCR is to determine in the sample the most responsive, the method the most accurately of DNA (or cDNA) copy number at present.Have sensitivity and specificity height, can realize multiple reaction, level of automation height, characteristics such as pollution-free, real-time and accurate, this technology is having great significance aspect clinical medicine check and the clinic study.At present, adopt fluorescence quantitative PCR detection technique to measure to multiple pathogenic agent such as gonococcus, chlamydia trachomatis, Ureaplasma urealyticum, human papillomavirus, hsv, hepatitis viroid, bacillus tuberculosis typus humanus, assays for parvovirus B 19, Epstein-Barr virus and human cytomegalic inclusion disease viruss.Compare with traditional detection method have highly sensitive, sampling less, advantage such as fast and convenient.
The present invention utilizes the genome difference between bacillus tuberculosis typus humanus and the Mycobacterium bovis, design 1 upstream primer and 2 different downstream primers (downstream primer is respectively at Mycobacterium bovis and bacillus tuberculosis typus humanus), target fragment according to amplification designs the Taqman probe respectively, and two probes are carried out different fluorescent marks, Mycobacterium bovis and bacillus tuberculosis typus humanus have been realized in same reaction tubes, detecting simultaneously, and differentiated by different fluorescence channels, in amplification time, is sentence read result on software directly, and swim without leakage of electricity, avoided the aerosol pollution, bigger application prospect has been arranged clinically.
Summary of the invention
Detect take time length and discriminating difficulty at Mycobacterium bovis in the prior art and bacillus tuberculosis typus humanus, and regular-PCR causes the shortcoming of aerosol pollution, rapid identification method and test kit thereof when the invention provides a kind of Mycobacterium bovis and bacillus tuberculosis typus humanus easily.
Concrete, a kind of method of identifying Mycobacterium bovis and bacillus tuberculosis typus humanus simultaneously provided by the invention, comprise that adopting following primer and Taqman probe to carry out real-time fluorescence quantitative PCR detects: 5 '-AGCGCAACACTCTTGGAG-3 ' is for differentiating Mycobacterium bovis and bacillus tuberculosis typus humanus's shared upstream primer; Bacillus tuberculosis typus humanus's downstream primer is 5 '-GCTCGACATGCTGAATGC-3 '; Bacillus tuberculosis typus humanus Taqman probe sequence is 5 '-CGAGTCGCCGTGGCTTCTCTT-3 '; The Mycobacterium bovis downstream primer is 5 '-CCCGTAGCGTTACTGAGAAA-3 '; Mycobacterium bovis Taqman probe sequence is 5 '-TTGACCAGCTAAGATATC CGGTACGCC-3 '.
Preferably, bacillus tuberculosis typus humanus Taqman probe 5 ' end flag F AM fluorophor, 3 ' end mark BHQ quenching group; Mycobacterium bovis Taqman probe 5 ' end mark JOE fluorophor, 3 ' end mark BHQ quenching group.
The present invention further provides the test kit that is used for aforesaid method, test kit comprises following primer and Taqman probe: 5 '-AGCGCAACACTCTTGGAG-3 ' is a shared upstream primer of differentiating Mycobacterium bovis and bacillus tuberculosis typus humanus; Bacillus tuberculosis typus humanus's downstream primer is 5 '-GCTCGACATGCTGAATGC-3 '; Bacillus tuberculosis typus humanus Taqman probe sequence is 5 '-CGAGTCGCCGTGGCTTCTCTT-3 '; The Mycobacterium bovis downstream primer is 5 '-CCCGTAGCGTTACTGAGAAA-3 '; Mycobacterium bovis Taqman probe sequence is 5 '-TTGACCAGCTAAGATATC CGGTACGCC-3 '.
The present invention further provides the application of aforesaid method and test kit in pure cultures of bacteria and the direct context of detection of clinical sample.
Beneficial effect: the present invention utilizes the genome difference between bacillus tuberculosis typus humanus and the Mycobacterium bovis, design 1 upstream primer and 2 different downstream primers (downstream primer is respectively at Mycobacterium bovis and bacillus tuberculosis typus humanus), target fragment according to amplification designs the Taqman probe respectively, and two probes are carried out different fluorescent marks, Mycobacterium bovis and bacillus tuberculosis typus humanus have been realized in same reaction tubes, detecting simultaneously, and differentiated by different fluorescence channels, in amplification time, is sentence read result on software directly, and swim without leakage of electricity, avoided the aerosol pollution, bigger application prospect has been arranged clinically.
Description of drawings
Fig. 1 primer probe design synoptic diagram
Fig. 2 Taqman probe technique principle of work synoptic diagram
Fig. 3 real-time quantitative PCR amplification curve
Fig. 4 is at the amplification curve of Mycobacterium bovis template
Fig. 5 is at the amplification curve of bacillus tuberculosis typus humanus's template
Fig. 6 is at the amplification curve of Mycobacterium bovis template and two kinds of hybrid templates of bacillus tuberculosis typus humanus's template
Fig. 7 Mycobacterium bovis and bacillus tuberculosis typus humanus's genomic dna dilution back amplification curve
Embodiment
The invention provides a kind of quick discriminating Mycobacterium bovis and bacillus tuberculosis typus humanus's method.Difference according to Mycobacterium bovis and tuberculosis group, design a upstream primer and two downstream primers, upstream primer is a general primer, downstream primer is respectively at Mycobacterium bovis and bacillus tuberculosis typus humanus, design two Taqman probes respectively at Mycobacterium bovis and bacillus tuberculosis typus humanus, probe 5 ' the end mark JOE (2 of Mycobacterium bovis, 7-dimethyl-4,5-two chloro-6-Fluoresceincarboxylic acids) fluorophor, 3 ' end mark BHQ (Black hole quenchers) quenching group, bacillus tuberculosis typus humanus's probe 5 ' end flag F AM (6-Fluoresceincarboxylic acid) fluorophor, 3 ' end mark BHQ quenching group.When only having bacillus tuberculosis typus humanus DNA in the sample, the bacillus tuberculosis typus humanus's system in the reaction system can increase, and has fluorescent signal to occur at the FAM passage, and real-time amplification curve is arranged; When only having Mycobacterium bovis DNA in the sample, the Mycobacterium bovis system can increase in the reaction system, has fluorescent signal to occur at the JOE passage, and real-time amplification curve is arranged; When having the DNA of two kinds of bacterium simultaneously, two individual system can be worked (concentration ratio is in 1: 102) simultaneously, all real-time amplification curve can occur at two passages, thereby realize the discriminating to both.
If single passage real-time fluorescence PCR instrument can be in charge of operation, two equal flag F AM fluorophors of probe 5 ' end.
Primer sequence provided by the present invention is as follows: upstream primer F1 is 5 '-AGCGCAACACTCTTGGAG-3 ' (SEQ ID NO:1), is general primer.Bacillus tuberculosis typus humanus's downstream primer R1 is 5 '-GCTCGACATGCTGAATGC-3 ' (SEQ ID NO:2), and bacillus tuberculosis typus humanus's specific probe P1 sequence is 5 '-CGAGTCGCCGTGGCTTCTCTT-3 ' (SEQ ID NO:3); Mycobacterium bovis downstream primer R2 is 5 '-CCCGTAGCGTTACTGAGAAA-3 ' (SEQ ID NO:4), and Mycobacterium bovis probe sequence P2 is 5 '-TTGACCAGCTAAGATATCCGGTACGCC-3 ' (SEQ ID NO:5).
Reaction system is: template 5 μ l, 10 * Taq enzyme buffer, 5 μ l, 2.5mM dNTP 4 μ l, F1 primer 2 μ l (concentration is 20pmol), R1 primer 1 μ l (concentration is 20pmol), R2 primer 1 μ l (concentration is 20pmol), P1 probe 1 μ l (concentration is 20pmol), P2 probe 1 μ l (concentration is 20pmol), Taq enzyme 2.5U, ddH
2O mends to 50 μ l.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 40s, totally 40 circulations; On quantitative real time PCR Instrument, increase, FAM and JOE passage are carried out signal collection.
Method therefor and reagent are ordinary method if no special instructions in the following experimental example, and it is synthetic that institute's synthetic primer probe work is given birth to the worker by Shanghai.
1, the design of primer probe is with synthetic
Compare according to Mycobacterium bovis that is provided in the Pub-Med gene pool and bacillus tuberculosis typus humanus's genome sequence, find that Mycobacterium bovis and bacillus tuberculosis typus humanus's genome homology on nucleotide level can reach 99.95%, but the genome of Mycobacterium bovis is little than bacillus tuberculosis typus humanus's genome, exist a large amount of disappearances, wherein a Zui Da disappearance is RD4, about 12.7Kb, we are primarily aimed at this section disappearance design primer probe greatly.
As shown in Figure 1, red area is the fragment of Mycobacterium bovis than the 12.7kb of bacillus tuberculosis typus humanus's disappearance, according to this disappearance, design upstream primer F1 is a general primer, with Mycobacterium bovis and bacillus tuberculosis typus humanus all can in conjunction with, design downstream primer R1 and probe P1 have guaranteed that like this bacillus tuberculosis typus humanus's amplification system can not carry out amplified reaction with Mycobacterium bovis on the Mycobacterium bovis deletion fragment; Probe P2 of Mycobacterium bovis and downstream primer R2, though can combine with the bacillus tuberculosis typus humanus, but owing to differ far (at interval＞12.7kb) with upstream primer, can not form amplification, guarantee that so also the amplification system of Mycobacterium bovis can not react with the bacillus tuberculosis typus humanus.
Synthetic and the mark of all primer probes is all given birth to the worker by Shanghai and is finished.
2, principle of work
The principle of work of TaqMan fluorescent probe is: add a specific fluorescent probe during pcr amplification when adding a pair of primer, this probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 '～3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, make the report fluorophor separate (as Fig. 2) with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously, form amplification curve (as Fig. 3).
Based on the above, quantitative fluorescent PCR can avoid aerosol to pollute, and carries out multiple reaction.
The fluorescence group that Mycobacterium bovis and bacillus tuberculosis typus humanus's differentiation depends on mark on the specific probe separately is different, can adopt different passages that fluorescent signal is collected.When only having the bacillus tuberculosis typus humanus in the template DNA, have only the FAM passage can receive fluorescent signal, form amplification curve; On the contrary, when only having Mycobacterium bovis in the template, have only the JOE passage can collect fluorescent signal, form amplification curve; When two kinds of templates all existed, two passages all can be received fluorescent signal, formed amplification curve.By judging in the template it is Mycobacterium bovis and bacillus tuberculosis typus humanus at the observed fluorescence curve of the fluorescent signal of different passages.
Embodiment
Experiment material: Mycobacterium bovis (AF2122/97), bacillus tuberculosis typus humanus (H
37R
V), Taq enzyme suit (Takara), primers F 1, R1, R2, probe P1, P2, ddH
2O
Laboratory apparatus: ABI 7500 (fluorescent PCR instrument)
Experimental technique:
Whole reaction system such as table 1:
| Sequence number | Component | Initial concentration | Quantity | Sequence number | Component | Initial concentration | Quantity | |
| 1 | Ultrapure water | / | 29.5μl | 8 | The Taq enzyme | 5U/L | 0.5 |
|
| 2 | 10×buffer | / | 5μl | 9 | dNTP | 25mM | 4μl | |
| 3 | | 10μM | 2μl | 10 | Template | ? | 5μl | |
| 4 | primer?R1 | 10μM | 1μl | 11 | Total | ? | |
|
| 5 | primer?R2 | 10μM | 1μl | 12 | ? | ? | ? | |
| 6 | ProbeP1 | 10μM | 1μl | 13 | ? | ? | ? | |
| 7 | ProbeP2 | 10μM | 1μl | 14 | ? | ? | ? |
If three kinds of processing: template A are the nucleic acid that extracts after the deactivation of Mycobacterium bovis pure growth; The nucleic acid of template B for extracting after the deactivation of bacillus tuberculosis typus humanus's pure growth; Template C is both mixtures.
Be configured according to above-mentioned reaction system, increase on the ABI7500PCR instrument, the reaction conditions of PCR is as follows: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 40s, totally 40 circulations; FAM and JOE passage are carried out signal collection (can be in charge of operation as the single passage instrument, two equal flag F AM of probe).
Fig. 4,5,6 is respectively the amplification curve at template A, B, C.
As can be seen from Figure 4, when only containing the Mycobacterium bovis genomic dna in template, only the passage corresponding Mycobacterium bovis has amplification curve, and does not have amplification curve to occur at bacillus tuberculosis typus humanus's passage.
As can be seen from Figure 5, when only containing bacillus tuberculosis typus humanus's genomic dna in template, only the passage corresponding bacillus tuberculosis typus humanus has amplification curve, and does not have amplification curve to occur at the passage of Mycobacterium bovis.
As can be seen from Figure 6, when in template, containing Mycobacterium bovis and bacillus tuberculosis typus humanus's genomic dna simultaneously, can detect fluorescent signal at two passages.
From top Fig. 4,5,6 as can be seen, two kinds of systems can well be carried out work in same reaction tubes, do not have cross reaction, and specificity is fine.
The interference phenomenon that may take place in view of double PCR is carried out gradient dilution to Mycobacterium bovis and bacillus tuberculosis typus humanus's genomic dna, is diluted to four gradients, joins respectively in the reaction system, how sees expanding effect, result such as Fig. 7.A, B form for bacillus tuberculosis typus humanus DNA dilution among the figure amplification curve and canonical plotting, the primer probe that visible double PCR reaction is introduced does not influence its work; Amplification curve and canonical plotting that C, D form for Mycobacterium bovis DNA dilution, the primer probe that visible double PCR reaction is introduced does not influence its work.
Because two shared upstream primers of reaction, and the shared situation of raw material that exists in the double PCR reaction may make to have competitive relation in the reaction process, so contrived experiment is proved under two kinds of simultaneous situations of template the working condition of whole system.Experiment material is the same, and amplification system is the same, and variation has taken place template, the Mycobacterium bovis genomic dna is diluted to 4 gradients, is respectively 1,2,3,4, bacillus tuberculosis typus humanus's genomic dna is diluted to 4 gradients, be respectively A, B, C, D, then template mixed, forming A (1-4) (is that gradient A mixes with gradient 1-4 respectively, down together), B (1-4), C (1-4), D (1-4) combination, the experiment of increasing, result such as table 2:
Two kinds of different concns templates of table 2 are mixed and are had system working condition down
| Sequence number | FAM channel C t value | JOE channel C t value | Sequence number | FAM channel C t value | JOE channel C t value |
| A1 | ?27.31 | ?27.79 | C1 | ?33.83 | ?30.62 |
| A2 | ?27.25 | ?30.85 | C2 | ?33.77 | ?33.99 |
| A3 | ?27.37 | ?38.02 | C3 | ?34.22 | ?37.82 |
| A4 | ?27.29 | ?ND | C4 | ?ND | ?27.45 |
| B1 | ?30.61 | ?27.53 | D1 | ?ND | ?27.66 |
| B2 | ?30.77 | ?30.98 | D2 | ?ND | ?30.57 |
| B3 | ?30.73 | ?34.34 | D3 | ?37.42 | ?33.34 |
| B4 | ?30.64 | ?ND | D4 | ?36.60 | ?37.02 |
As can be seen from the table: when both concentration differ 10
2Within the time, the two can not produce tangible interference; But when concentration difference reaches 10
3The time, may be owing to make the low side amplification of concentration be suppressed to the competition that has primer and raw material.
Conclusion: through a series of experimental verifications, this system can effectively be distinguished Mycobacterium bovis and bacillus tuberculosis typus humanus, and the strain isolated sample needs about 2h, and clinical sample 3～4h can finish detection; Amplification is finished simultaneously with detection, has avoided the aerosol pollution, and very big using value is arranged clinically.
Sequence table
Claims (5)
1. method of identifying Mycobacterium bovis and bacillus tuberculosis typus humanus simultaneously is characterized in that adopting following primer and Taqman probe to carry out real-time fluorescence quantitative PCR and detects:
5 '-AGCGCAACACTCTTGGAG-3 ' is for differentiating Mycobacterium bovis and bacillus tuberculosis typus humanus's shared upstream primer; Bacillus tuberculosis typus humanus's downstream primer is 5 '-GCTCGACATGCTGAATGC-3 '; Bacillus tuberculosis typus humanus Taqman probe sequence is 5 '-CGAGTCGCCGTGGCTTCTCTT-3 '; The Mycobacterium bovis downstream primer is 5 '-CCCGTAGCGTTACTGAGAAA-3 '; Mycobacterium bovis Taqman probe sequence is 5 '-TTGACCAGCTAAGATATCCGGTACGCC-3 '.
2. the method for claim 1 is characterized in that bacillus tuberculosis typus humanus Taqman probe 5 ' end flag F AM fluorophor, 3 ' end mark BHQ quenching group; Mycobacterium bovis Taqman probe 5 ' end mark JOE fluorophor, 3 ' end mark BHQ quenching group.
3. a test kit that is used for claim 1 or 2 described methods is characterized in that test kit comprises following primer and Taqman probe: 5 '-AGCGCAACACTCTTGGAG-3 ' is a shared upstream primer of differentiating Mycobacterium bovis and bacillus tuberculosis typus humanus; Bacillus tuberculosis typus humanus's downstream primer is 5 '-GCTCGACATGCTGAATGC-3 '; Bacillus tuberculosis typus humanus Taqman probe sequence is 5 '-CGAGTCGCCGTGGCTTCTCTT-3 ', 5 ' end flag F AM fluorophor, 3 ' end mark BHQ quenching group; The Mycobacterium bovis downstream primer is 5 '-CCCGTAGCGTTACTGAGAAA-3 '; Mycobacterium bovis Taqman probe sequence is 5 '-TTGACCAGCTAAGATATC CGGTACGCC-3 ', 5 ' end mark JOE fluorophor, 3 ' end mark BHQ quenching group.
As claimed in claim 1 or 2 method in the application of pure cultures of bacteria and the direct context of detection of clinical sample.
5. as the application of test kit as described in the claim 3 in pure cultures of bacteria and the direct context of detection of clinical sample.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981260A (en) * | 2014-05-06 | 2014-08-13 | 山东省农业科学院奶牛研究中心 | Detection method for mycobacterium bovis and mycobacterium tuberculosis in aerosol |
| CN105219882A (en) * | 2015-11-19 | 2016-01-06 | 北京利德曼生化股份有限公司 | Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method |
| CN109852713A (en) * | 2019-02-15 | 2019-06-07 | 安徽理工大学 | It is a kind of difference mycobacterium tuberculosis simultaneously identifier box and its identification method |
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| CN101168780A (en) * | 2007-11-06 | 2008-04-30 | 广东出入境检验检疫局检验检疫技术中心 | Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit |
| CN101333556A (en) * | 2008-06-05 | 2008-12-31 | 北京爱普益生物科技有限公司 | Gene chip for detecting bacterial infection for flocks and herds, preparing and detecting process and kit |
| EP2097533B1 (en) * | 2006-11-28 | 2010-08-25 | Proyecto de Biomedicina Cima, S.L. | Viral vector and uses thereof |
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| EP2097533B1 (en) * | 2006-11-28 | 2010-08-25 | Proyecto de Biomedicina Cima, S.L. | Viral vector and uses thereof |
| CN101168780A (en) * | 2007-11-06 | 2008-04-30 | 广东出入境检验检疫局检验检疫技术中心 | Zoonosis tuberculosis fluorescence PCR rapid diagnosis kit |
| CN101333556A (en) * | 2008-06-05 | 2008-12-31 | 北京爱普益生物科技有限公司 | Gene chip for detecting bacterial infection for flocks and herds, preparing and detecting process and kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981260A (en) * | 2014-05-06 | 2014-08-13 | 山东省农业科学院奶牛研究中心 | Detection method for mycobacterium bovis and mycobacterium tuberculosis in aerosol |
| CN103981260B (en) * | 2014-05-06 | 2016-03-09 | 山东省农业科学院奶牛研究中心 | A kind of method detecting Mycobacterium bovis and mycobacterium tuberculosis in aerosol |
| CN105219882A (en) * | 2015-11-19 | 2016-01-06 | 北京利德曼生化股份有限公司 | Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method |
| CN109852713A (en) * | 2019-02-15 | 2019-06-07 | 安徽理工大学 | It is a kind of difference mycobacterium tuberculosis simultaneously identifier box and its identification method |
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