CN102286627B - 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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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 many animals are suffered from altogether.According to WHO report, there are 2,000 ten thousand tuberculosis patients in the whole world, has every year 800～1,000 ten thousand newly-increased case, and the current tuberculosis patient of China is in 6,000,000 left and right, increases every year case 100～1,300,000 newly.Tuberculosis has become one of disease of 21 century serious crisis human health.Occurring in nature exists various human mycobacterium tuberculosis: other bacillus tuberculosis typus humanuses such as people's tuberculosis (or human-like) mycobacterium, Mycobacterium bovis (or ox type).Ox type bacillus tuberculosis typus humanus not only can make ox morbidity, all right infringer and many animals, milk cow is to propagate the most dangerous animal of tuberculosis to the mankind, and the tuberculosis morbidity of milk cow is higher, with sick ox Long Term Contact, or accidentally drink the milk that contains bacillus tuberculosis typus humanus and all likely infect tuberculosis, particularly in the part area of China, drink in addition raw milk's custom, causing infecting probability lungy increases greatly.Ox bacillus tuberculosis typus humanus's therapy and the difference of tuberculotherapy are the pyrazinoic acid amide resistance of one of born Antituberculous standard drug of ox bacillus tuberculosis typus humanus.Have document to point out, in the tuberculosis patient of China, having 5%～10% tuberculosis is to be caused by Mycobacterium bovis, therefore, for the discriminating of bacillus tuberculosis typus humanus and Mycobacterium bovis, detects and seems most important.
At present, clinically the difference of bacillus tuberculosis typus humanus and Mycobacterium bovis is identified and need to 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 if cultivate successful required time also at about 8 weeks, and these discrimination methods are also unreliable, therefore in the urgent need to researching and developing a kind of detection method of differentiating fast.PCR detects in numerous methods outstanding aobvious high specificity, feature that susceptibility is high, in recent years also in a large amount of evaluation that has been applied in ox bacillus tuberculosis typus humanus, but can produce a large amount of aerosols owing to need amplified production open pipe being carried out to electrophoresis, cause the method to produce a large amount of false positives, so further do not applied clinically.And fluorescence real-time quantitative PCR can both can effectively have been avoided aerocolloidal method fast for we provide one.
Real-Time Fluorescent Quantitative PCR Technique (Real-time quantitative Polymerase Chain Reaction is called for short Real Time PCR) is the nucleic acid quantification technology (HIGUCHIR growing up in 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, Zeng 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 narrated in full).
In PCR reaction system, add fluorophor, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, make each circulation become " visible ", finally by Ct value and typical curve, the initial concentration of the DNA in sample (orcDNA) is carried out to quantitative method.Real-time fluorescence quantitative PCR is to determine that at present DNA in sample (or cDNA) copy number is the most responsive, method the most accurately.Have sensitivity and specificity high, can realize the features such as multiple reaction, level of automation are high, pollution-free, real-time and accurate, this technology is having great significance aspect clinical medicine check and clinic study.At present, adopt fluorescence quantitative PCR detection technique to measure multiple pathogens 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.Have compared with traditional detection method highly sensitive, sampling less, the advantage such as fast and convenient.
The present invention utilizes the genome difference between bacillus tuberculosis typus humanus and Mycobacterium bovis, design 1 downstream primer (downstream primer is respectively for Mycobacterium bovis and bacillus tuberculosis typus humanus) that upstream primer is different with 2, according to the target fragment of amplification, design respectively Taqman probe, and two probes are carried out to different fluorescent marks, realized and in same reaction tubes, detected Mycobacterium bovis and bacillus tuberculosis typus humanus simultaneously, and differentiated by different fluorescence channels, amplification time directly sentence read result on software, and swim without leakage of electricity, avoided Aerosol Pollution, there is clinically larger application prospect.
Summary of the invention
For Mycobacterium bovis in prior art and bacillus tuberculosis typus humanus, detect the long and discriminating difficulty that takes time, and regular-PCR easily 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.
Concrete, a kind of method of simultaneously identifying Mycobacterium bovis and bacillus tuberculosis typus humanus provided by the invention, comprises and adopts following primer and Taqman probe to carry out real-time fluorescence quantitative PCR detection: 5 '-AGCGCAACACTCTTGGAG-3 ' 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 '; 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 for aforesaid method, test kit comprises following primer and Taqman probe: 5 '-AGCGCAACACTCTTGGAG-3 ' is the 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 '; 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 aspect pure cultures of bacteria and clinical sample direct-detection of aforesaid method and test kit.
Beneficial effect: the present invention utilizes the genome difference between bacillus tuberculosis typus humanus and Mycobacterium bovis, design 1 downstream primer (downstream primer is respectively for Mycobacterium bovis and bacillus tuberculosis typus humanus) that upstream primer is different with 2, according to the target fragment of amplification, design respectively Taqman probe, and two probes are carried out to different fluorescent marks, realized and in same reaction tubes, detected Mycobacterium bovis and bacillus tuberculosis typus humanus simultaneously, and differentiated by different fluorescence channels, amplification time directly sentence read result on software, and swim without leakage of electricity, avoided Aerosol Pollution, there is clinically larger application prospect.
Accompanying drawing explanation
Fig. 1 primer probe design schematic diagram
Fig. 2 Taqman probe technique principle of work schematic diagram
Fig. 3 real-time quantitative PCR amplification curve
Fig. 4 is for the amplification curve of Mycobacterium bovis template
Fig. 5 is for the amplification curve of bacillus tuberculosis typus humanus's template
Fig. 6 is for the amplification curve of Mycobacterium bovis template and two kinds of hybrid templates of bacillus tuberculosis typus humanus's template
Amplification curve after Fig. 7 Mycobacterium bovis and the dilution of bacillus tuberculosis typus humanus's genomic dna
Embodiment
The invention provides a kind of quick discriminating Mycobacterium bovis and bacillus tuberculosis typus humanus's method.According to the difference of Mycobacterium bovis and tuberculosis group, design a upstream primer and two downstream primers, upstream primer is general primer, downstream primer is respectively for Mycobacterium bovis and bacillus tuberculosis typus humanus, design two Taqman probes respectively for Mycobacterium bovis and bacillus tuberculosis typus humanus, probe 5 ' the end mark JOE(2 of Mycobacterium bovis, 7-dimethyl-4, the chloro-6-Fluoresceincarboxylic acid of 5-bis-) 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 there is bacillus tuberculosis typus humanus DNA in sample, the bacillus tuberculosis typus humanus's system in reaction system can increase, and has fluorescent signal to occur, and have real-time amplification curve at FAM passage; When only there is Mycobacterium bovis DNA in sample, in reaction system, Mycobacterium bovis system can increase, and has fluorescent signal to occur, and have real-time amplification curve at JOE passage; When there is the DNA of two kinds of bacterium, two individual system can be worked (concentration ratio is in 1:102) simultaneously simultaneously, at two passages, all can occur real-time amplification curve, thereby realize the discriminating to both.
If single passage real-time fluorescence PCR instrument can be in charge of operation, two probes 5 ' are held equal flag F AM fluorophor.
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 buffer5 μ l, 2.5mM dNTP4 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.
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 to signal collection.
In following experimental example, method therefor and reagent are ordinary method if no special instructions, and the primer probe work of synthesized is synthetic by the raw work in Shanghai.
1, the design of primer probe is with synthetic
According to the Mycobacterium bovis providing in Pub-Med gene pool and bacillus tuberculosis typus humanus's genome sequence, compare, 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 compared with bacillus tuberculosis typus humanus's genome, exist a large amount of disappearances, wherein a maximum disappearance is RD4, about 12.7Kb, we are mainly for this section disappearance design primer probe greatly.
As shown in Figure 1, red area is that Mycobacterium bovis is compared with the fragment of the 12.7kb of bacillus tuberculosis typus humanus's disappearance, according to this disappearance, design upstream primer F1 is general primer, with Mycobacterium bovis and bacillus tuberculosis typus humanus all can be in conjunction with, on Mycobacterium bovis deletion fragment, design downstream primer R1 and probe P1, guaranteed that like this bacillus tuberculosis typus humanus's amplification system can not carry out amplified reaction with Mycobacterium bovis; The probe P2 of Mycobacterium bovis and downstream primer R2, although can carry out combination with bacillus tuberculosis typus humanus, but owing to differing (interval >12.7kb) far away with upstream primer, can not form amplification, so also guarantee that the amplification system of Mycobacterium bovis can not react with bacillus tuberculosis typus humanus.
Synthetic and the mark of all primer probes is all completed by the raw work in Shanghai.
2, principle of work
The principle of work of TaqMan fluorescent probe is: during pcr amplification, when adding pair of primers, add a specific fluorescent probe, this probe is an oligonucleotide, and two ends are a report fluorophor of mark and a cancellation fluorophor respectively.When probe is complete, the fluorescent signal of reporter group transmitting is quenched group and absorbs; During pcr amplification, 5 '～3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded by probe enzyme, make to report fluorophor Fen Li with cancellation fluorophor (as Fig. 2), thereby fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just have a fluorescence molecule to form, accumulation and the PCR product of having realized fluorescent signal form Complete Synchronization, form amplification curve (as Fig. 3).
Based on the above, quantitative fluorescent PCR can be avoided Aerosol Pollution, carries out multiple reaction.
The fluorescence group that Mycobacterium bovis and bacillus tuberculosis typus humanus's differentiation depends on mark on specific probe is separately different, can adopt different passages to collect fluorescent signal.When only there is bacillus tuberculosis typus humanus in template DNA, only have FAM passage can receive fluorescent signal, form amplification curve; On the contrary, when only there is Mycobacterium bovis in template, only have JOE passage can collect fluorescent signal, form amplification curve; When two kinds of templates all exist, two passages all can be received fluorescent signal, form amplification curve.In carrying out judge templet by the fluorescence curve of observing at the fluorescent signal of different passages, be Mycobacterium bovis and bacillus tuberculosis typus humanus.
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: ABI7500 (fluorescent PCR instrument)
Experimental technique:
Whole reaction system is as table 1:
| Sequence number | Component | Initial concentration | Quantity | Sequence number | Component | | Quantity | |
| 1 | Ultrapure water | / | 29.5μl | 8 | Taq enzyme | 5U/L | 0.5 |
|
| 2 | 10×buffer | / | 5μl | 9 | | 25mM | 4μl | |
| 3 | | 10μM | 2μl | 10 | Template | ? | |
|
| 4 | primer?R1 | 10μM | 1μl | 11 | Total | ? | |
|
| 5 | primer?R2 | | 1μl | 12 | ? | ? | ? | |
| 6 | ProbeP1 | 10μM | 1μl | 13 | ? | ? | ? | |
| 7 | | 10μM | 1μl | 14 | ? | ? | ? |
If three kinds of processing: template A are the nucleic acid extracting after the deactivation of Mycobacterium bovis pure growth; Template B is the nucleic acid extracting after the deactivation of bacillus tuberculosis typus humanus's pure growth; Template C is both mixtures.
According to above-mentioned reaction system, be configured, on ABI7500PCR instrument, increase, 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 to signal collection (as single passage instrument can be in charge of operation, two equal flag F AM of probe).
Fig. 4,5,6 is respectively the amplification curve for template A, B, C.
As can be seen from Figure 4, while only containing Mycobacterium bovis genomic dna in template, only at the passage of corresponding Mycobacterium bovis, there is amplification curve, and do not have amplification curve to occur for bacillus tuberculosis typus humanus's passage.
As can be seen from Figure 5, while only containing bacillus tuberculosis typus humanus's genomic dna in template, only at corresponding bacillus tuberculosis typus humanus's passage, there is amplification curve, and do not have amplification curve to occur for the passage of Mycobacterium bovis.
As can be seen from Figure 6,, while containing Mycobacterium bovis and bacillus tuberculosis typus humanus's genomic dna in template, at two passages, fluorescent signal can be detected simultaneously.
From Fig. 4,5,6 above, can find out, two kinds of systems can well be carried out work in same reaction tubes, there is no cross reaction, and specificity is fine.
In view of the interference phenomenon that double PCR may occur, Mycobacterium bovis and bacillus tuberculosis typus humanus's genomic dna is carried out to gradient dilution, be diluted to four gradients, join respectively in reaction system, how see expanding effect, result is as Fig. 7.In figure, A, B are amplification curve and the canonical plotting that bacillus tuberculosis typus humanus DNA dilution forms, and the primer probe that visible double PCR reaction is introduced does not affect its work; C, D are amplification curve and the canonical plotting that Mycobacterium bovis DNA dilution forms, and the primer probe that visible double PCR reaction is introduced does not affect its work.
Because two reactions share upstream primers, and the raw material existing in double PCR reaction shares situation, may make to exist in reaction process competitive relation, thus contrived experiment prove in two kinds of simultaneous situations of template, the working condition of whole system.Experiment material is the same, amplification system is the same, there is variation in template, Mycobacterium bovis genomic dna is diluted to 4 gradients, be 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, lower same), B (1-4), C (1-4), D (1-4) combination, the experiment of increasing, result is as table 2:
Two kinds of different concns templates of table 2 are mixed the lower system working condition of existence
| 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 time, the two can not produce obvious interference; But when concentration difference reaches 10
3time, may be owing to making side's amplification that concentration is low be suppressed to the competition of total primer and raw material.
Conclusion: through series of experiments checking, this system can effectively be distinguished Mycobacterium bovis and bacillus tuberculosis typus humanus, strain isolated sample needs 2h left and right, and clinical sample 3～4h can complete detection; Amplification completes with detection simultaneously, has avoided Aerosol Pollution, has clinically very large using value.
Sequence table
Claims (2)
1. in the time of for non-medical diagnosis on disease or methods for the treatment of, identify Mycobacterium bovis and bacillus tuberculosis typus humanus's a method, it is characterized in that adopting following primer and Taqman probe to carry out real-time fluorescence quantitative PCR detection:
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 '; Mycobacterium bovis downstream primer is 5 '-CCCGTAGCGTTACTGAGAAA-3 '; Mycobacterium bovis Taqman probe sequence is 5 '-TTGACCAGCTAAGATATCCGGTACGCC-3 ';
Wherein, 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.
2. for a test kit for method described in claim 1, it is characterized in that test kit comprises following primer and Taqman probe: 5 '-AGCGCAACACTCTTGGAG-3 ' is the 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; 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.
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