CN110195117A - Detect the method and its kit of mycobacteria - Google Patents
Detect the method and its kit of mycobacteria Download PDFInfo
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- CN110195117A CN110195117A CN201810161343.4A CN201810161343A CN110195117A CN 110195117 A CN110195117 A CN 110195117A CN 201810161343 A CN201810161343 A CN 201810161343A CN 110195117 A CN110195117 A CN 110195117A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The present invention provides a kind of method and its kit for detecting mycobacteria.The method comprises the steps of a) offer sample；B) primer pair is provided, selected from as SEQ ID NO:1, SEQ ID NO:2, with sequence of the SEQ ID NO:1 with about 45% to about 99% similarity, with group composed by sequence of the SEQ ID NO:2 with about 60% to about 99% similarity, the complementary strand of SEQ ID NO:1 and the complementary strand of SEQ ID NO:2；C) polymerase chain reaction is carried out to obtain product using primer pair and sample；And analyze product d) to detect the presence of mycobacteria.The present invention also provides a kind of kits for detecting mycobacteria, include the aforementioned primer pair being previously mentioned.
Description
Technical field
The present invention relates to a kind of detection method and its kit, especially a kind of method and its reagent for detecting mycobacteria
Box.
Background technique
Mycobacteria can be divided into mycobacterium tuberculosis group (Mycobacterium tuberculosis complex, MTBC)
With non-tuberculous mycobacteria group.More lungy cause a disease belongs to originally in the bacterium of mycobacterium tuberculosis group (MTBC), such as Africa
Mycobacteria (M.africanum), Mycobacterium bovis (M.bovis), sheep mycobacteria (M.caprae), cassette mycobacteria
(M.canettii), mycobacterium microti (M.microti), sea dog mycobacteria (M.pinnipedii) and tuberculosis branch bar
Bacterium (M.tuberculosis).These originals of causing a disease will cause the tuberculosis of mankind or animal, wherein again with mycobacterium africanum
(M.africanum), Mycobacterium bovis (M.bovis) and mycobacterium tuberculosis (M.tuberculosis) are the clinically mankind
The main bacteria seed of pulmonary tuberculosis.
Tuberculosis can occur in any organ or tissue, such as lungs, lymph node, meninx, pleura, kidney, bone, skin
Skin, alimentary canal and urogenital tract etc..If giving antituberculosis drugs treat appropriate in early days, tuberculosis almost can be with percentage
Hundred recovery from illness.If but do not treated in early stage, there are about the lethalities of half in 3 years.So needing a kind of inspection at present
Survey method can clinically early detection go out whether by mycobacterium tuberculosis group cause of disease infection, for disease cured
The promotion of rate has its importance.
Summary of the invention
One aspect of the invention is a kind of method for detecting mycobacteria, a) offer sample is comprised the steps of；B) it mentions
For primer pair, primer pair, which is selected from by SEQ ID NO:1, SEQ ID NO:2, with SEQ ID NO:1, has about 45% to about 99%
The sequence of similarity has the sequence of about 60% to about 99% similarity, the complementary strand of SEQ ID NO:1 with SEQ ID NO:2
And group composed by the complementary strand of SEQ ID NO:2；C) polymerase chain reaction is carried out to obtain using primer pair and sample
Product；And analyze product d) to detect the presence of mycobacteria.
According to certain embodiments of the present invention, step a) offer sample includes: providing sample includes mycobacterium tuberculosis
Group (Mycobacterium tuberculosis complex, MTBC).
According to certain embodiments of the present invention, sample be blood, sputum, bronchial alveolar rinse liquid, urine, excrement or
A combination thereof.
According to certain embodiments of the present invention, step c) carries out polymerase chain reaction using primer pair and sample to obtain
Obtaining product includes the partial order for carrying out polymerase chain reaction and making IS6110 sequence in primer pair amplifies mycobacterium tuberculosis group
Column are to obtain product, and wherein this partial sequence is SEQ ID NO:3.
According to certain embodiments of the present invention, it further includes: oligonucleotide probe is provided.
According to certain embodiments of the present invention, step c) carries out polymerase chain reaction using primer pair and sample to obtain
Before obtaining product, comprising carrying out hybridization reaction to sample using oligonucleotide probe.
According to certain embodiments of the present invention, polymerase chain reaction is real-time quantitative polymerase chain reaction.
Another aspect of the invention is a kind of kits for detecting mycobacteria, include a primer pair.Above-mentioned primer pair choosing
Free SEQ ID NO:1, SEQ ID NO:2, sequence and SEQ with SEQ ID NO:1 with about 45% to about 99% similarity
ID NO:2 has that the sequence of about 60% to about 99% similarity, the complementary strand of SEQ ID NO:1 and SEQ ID NO:2's is mutual
Mend group composed by chain.
According to certain embodiments of the present invention, primer pair is SEQ ID NO:1 and SEQ ID NO:2.
According to certain embodiments of the present invention, sample is further included, wherein sample is blood, sputum, broncho-pulmonary
Steep flushing liquor, urine, excrement or combinations thereof.
According to certain embodiments of the present invention, target gene is further included, wherein target gene is tuberculosis branch bar
The IS6110 sequence of flora (Mycobacterium tuberculosis complex, MTBC).
According to certain embodiments of the present invention, template is further included, has about 100 base-pairs to about 250
The length of base-pair.
According to certain embodiments of the present invention, template is SEQ ID NO:3.
Detailed description of the invention
Content of this disclosure is better understood with when the following detailed description is read in conjunction with the drawings.But it should be noted that according to ability
The standing procedure in domain, various features are not drawn on drafting.In fact, the size of various features can quilt in order to clearly discuss
Arbitrarily enlarged or diminution.
Fig. 1 is some embodiments according to the present invention, shows the Partial Fragment of IS6110 sequence and the position of primer pair design
It sets.
Fig. 2 is some embodiments according to the present invention, is tested in the case of different templates amount, with primer pair SEQ ID
The amplification curve diagram of NO:1 and SEQ ID NO:2 progress real-time quantitative polymerase chain reaction.
Fig. 3 is to show the canonical plotting of its different templates initial amount according to Fig. 2.
Fig. 4 is some embodiments according to the present invention, when template quantity is 10 copy number, with primer pair SEQ ID NO:1
And SEQ ID NO:2 carries out the amplification curve diagram of real-time quantitative polymerase chain reaction.
Fig. 5 is some embodiments according to the present invention, is derived on the basis of SEQ ID NO:1 and SEQ ID NO:2
Similar primer pair, carry out real-time quantitative polymerase chain reaction amplification curve diagram.
Fig. 6 is some embodiments according to the present invention, is detected with primer pair SEQ ID NO:1 and SEQ ID NO:2
Electrophoresis result figure.
Specific embodiment
For keep the description of present disclosure more detailed with it is complete, below for embodiments of the present invention and specific implementation
Example proposes illustrative description, but this not implements or use the unique forms of the specific embodiment of the invention.As disclosed below
Each embodiment, beneficial in the case of can be combined with each other or replace, can also add others embodiments in one embodiment, and nothing
Further it need to record or illustrate.In the following description, many specific details be will be described in detail so that reader can fully understand
Embodiment below.However, the embodiment of the present invention can be practiced in the case where these specific details useless.
Herein, unless being particularly limited in interior text for article, otherwise " one " and "the" can refer to single or
It is multiple.It will be further appreciated that "comprising" used herein, " comprising ", " having " and similar vocabulary, indicate that it is remembered
Feature, region, integer, step, operation, component and/or the component of load, but it is not excluded for its described or additional one or more
Other feature, region, integer, step, operation, component, component, and/or group wherein.
The embodiment of the present invention provides a kind of method for detecting mycobacteria, comprises the steps of and a) provides sample and b)
Primer pair is provided.Primer pair is selected from by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:1 with about 45% to about
The sequence of 99% similarity, have with SEQ ID NO:2 the sequence of about 60% to about 99% similarity, SEQ ID NO:1 it is mutual
Mend group composed by the complementary strand of chain and SEQ ID NO:2.Then, c) utilize aforementioned primer pair and sample progress polymerase chain
Formula is reacted to obtain product.Finally, analyzing product d) to detect the presence of mycobacteria.
Sample may include the sample of various separate sources, such as blood, sputum, bronchial alveolar rinse liquid, urine, excrement
Or combinations thereof.In some embodiments, detecting sample provided in the method for mycobacteria includes mycobacterium tuberculosis group
(Mycobacterium tuberculosis complex,MTBC)。
The selection of primer pair is as mentioned before, however it is not limited to SEQ ID NO:1 and SEQ ID NO:2 disclosed herein.
Primer pair is in the choice other than it may include the complementary strand of SEQ ID NO:1 and the complementary strand of SEQ ID NO:2, SEQ ID
Sequence shown in NO:1 and SEQ ID NO:2 can also allow for variation to a certain degree.That is, having with SEQ ID NO:1
The sequence of about 45% to about 99% similarity and the sequence application with SEQ ID NO:2 with about 60% to about 99% similarity
Also there is identical effect in present embodiment.For example, the selection of primer pair may include the degenerate sequence of SEQ ID NO:1
And the degenerate sequence of SEQ ID NO:2." degenerate sequence " described herein refers in the middle part of oligonucleotide sequence disclosed herein
Pyrene thuja acid is replaced other nucleotide.In other words, the degenerate sequence of SEQ ID NO:1 means in SEQ ID NO:1 sequence
In the case of column length is constant, the degree of variation that its oligonucleotides has about 1% to about 55% may be allowed.And SEQ ID NO:2
Degenerate sequence mean in the case of SEQ ID NO:2 sequence length is constant, may be allowed its oligonucleotides with about 1% to
About 40% degree of variation.In further embodiments, the selection of primer pair also may include SEQ ID NO:1 derived sequence and
The derived sequence of SEQ ID NO:2." derived sequence " described herein refers in oligonucleotide sequence disclosed herein at 3 ' ends
Or 5 ' end may be trimmed, and still retain part or full sequence.In other words, the derived sequence of SEQ ID NO:1 means
In the case of SEQ ID NO:1 sequence length can be increased and decreased, the change that its oligonucleotides has about 1% to about 55% may be allowed
Off course degree.And the derived sequence of SEQ ID NO:2 means in the case of SEQ ID NO:2 sequence length can be increased and decreased,
It may be allowed the degree of variation that its oligonucleotides has about 1% to about 40%.In other embodiments, primer pair be selected from
Sequence of the SEQ ID NO:1 with about 80% to about 99% similarity and have about 80% to about 99% with SEQ ID NO:2
Group composed by the sequence of similarity.
In one embodiment, carry out polymerase chain reaction using primer pair and sample to obtain product, comprising into
Row polymerase chain reaction makes the partial sequence of IS6110 sequence in primer pair amplifies mycobacterium tuberculosis group to obtain product,
Wherein this partial sequence is SEQ ID NO:3.Polymerase chain reaction (Polymerase chain reaction, PCR) is one
Kind Protocols in Molecular Biology.Utilize the specific DNA of primer pair amplifies (DNA) segment with oligonucleotide sequence.
It should be understood that sequence disclosed herein can be used for the various technologies based on polymerase chain reaction.Implement at one
In example, polymerase chain reaction may include, but are not limited to reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative polymerase
Chain reaction (real-time PCR), nested-polymerase chain reaction (nested PCR), the reaction of multipair primed polymerase chain
(multiplex PCR) and hot asymmetric interlaced polymerase chain reaction (thermal asymmetric interlaced
PCR,TAIL-PCR)。
In addition, the fluorescing system of real-time quantitative polymerase chain reaction can be divided into " sonde-type " and " non-sonde-type ".Therefore
Embodiments of the present invention can further include offer oligonucleotide probe.In one embodiment, according to real time aggregation
Enzyme chain reaction is the fluorescing system of sonde-type, carry out polymerase chain reaction using primer pair and sample with obtain product it
Before, it further includes and hybridization reaction (hybridization) is carried out to sample using oligonucleotide probe, so that oligonucleotide probe knot
It closes on target sequence.
The embodiment of the present invention also provides a kind of kit (kit) for detecting mycobacteria, includes a primer pair.It is above-mentioned to draw
Object is to selected from by SEQ ID NO:1, SEQ ID NO:2, the sequence with SEQ ID NO:1 with about 45% to about 99% similarity
Column have the sequence of about 60% to about 99% similarity, the complementary strand and SEQ ID of SEQ ID NO:1 with SEQ ID NO:2
Group composed by the complementary strand of NO:2.In some embodiments, primer pair is SEQ ID NO:1 and SEQ ID NO:2.One
In a little embodiments, primer pair be with the SEQ ID NO:1 sequence with about 45% to about 99% similarity and with SEQ ID
NO:2 has the sequence of about 60% to about 99% similarity.In some embodiments, primer pair is the complementation of SEQ ID NO:1
The complementary strand of chain and SEQ ID NO:2.
In some embodiments, the kit for detecting mycobacteria can further include sample.The source of sample can be
Blood, sputum, bronchial alveolar rinse liquid, urine, excrement or combinations thereof.For example, the kit of this detection mycobacteria
It can be applied to each medical institutions, detected by the body fluid or excreta of acquisition individual (such as: people).In one embodiment
In, sample does not include the IS6110 sequence of mycobacteria.For example, this sample can also be one section of artificial synthesized sequence and can
In conjunction with the primer pair in present embodiment, to be expanded to this section of artificial synthesized sequence.
In some embodiments, the kit for detecting mycobacteria can further include target gene, and this target
Gene refers to the IS6110 sequence of mycobacterium tuberculosis group (Mycobacterium tuberculosis complex, MTBC).
There are also some embodiments, the kit for detecting mycobacteria can further include template, have about 100 base-pairs to about
The length of 250 base-pairs.For example, this template can be the partial sequence in IS6110 sequence, such as SEQ ID NO:3 institute
The sequence shown, the length with 141 base-pairs.But in other embodiments, template does not include shown in SEQ ID NO:3
Sequence, and for about 100 base-pairs to the artificial synthesized sequence of about 250 base pairs lengths, can also be with this implementation
Primer pair in mode combines to be amplified.It in some embodiments, can be directly by sequence structure shown in SEQ ID NO:3
When building into different carriers, also, being expanded using this carrier with SEQ ID NO:3 as template, specificity is high and examines
It is splendid to survey efficiency.
Further to confirm that various embodiments of the invention can be used for detecting the presence of mycobacteria, following examination is carried out
It tests.It should be noted that following embodiments are merely provided as demonstration purpose, it is not intended to limit the present invention.
Primer and probe design
The IS6110 sequence of mycobacterium tuberculosis group has height retention.Therefore this test utilizes Photographing On-line program such as
Primer 3 and GenScript Real-time PCR Primer Design is directed to the IS6110 sequence of mycobacterium tuberculosis group
(GenBank:LC005482) design of primer and probe is carried out.
The information about accession number No.LC005482 is provided according to GenBank database, Fig. 1 shows IS6110 sequence
Part positive strand sequence.Primer pair is SEQ ID NO.1 and SEQ ID NO.2 in this test.Nucleosides shown in SEQ ID NO.1
Acid sequence is designed (as shown in right arrow box) for the position of the 929-949 base-pair.Shown in SEQ ID NO.2
Nucleotide sequence be to be designed (as shown in left arrow box) for the position of the 1048-1069 base-pair.And few core
Thuja acid probe is designed for the segment between the 929th base-pair and the 1069th base-pair.Accordingly, using drawing
The object product amplifiable to SEQ ID NO:1-2 has the length of 141 base-pairs.
The sensitivity analysis of primer pair
According to sequence shown in aforementioned GenBank database accession number LC005482, it is cloned in pUC57 carrier
(Protech CO., Ltd, GenBank:Y14837.1), to obtain the standard plasmid with IS6110 sequence (hereinafter referred to as
IS6110 standard plasmid).
According to commercially available real-time quantitative polymerase chain reaction kit (QuantiNova Probe PCR Kit, Qiagen)
Operational manual, reaction mixture include the real-time quantitative polymerase chain reaction examination of template (IS6110 standard plasmid), 15 μ L
Agent (QuantiNova probe master mix), concentration be 1000nM primer pair (the SEQ ID NO:1 of 667nM and
The SEQ ID NO:2 of 333nM) and 27nM oligonucleotide probe, be configured to total volume be 30 μ L reaction mixture.In real time
Quantitative polyase chain reaction condition be 95 DEG C denaturation (denature) 5 seconds, 60 DEG C of annealing/amplification (annealing/
Amplification) 10 seconds, and by reaction mixture in real-time quantitative Polymerized human serum albumin (CFX-96, BioRad) into
45 circular responses of row.
It should be noted that being to carry out real-time quantitative polymerase chain reaction with different template quantities in this test, to survey
Try the sensitivity of primer pair SEQ ID NO.1 and SEQ ID NO.2.According to the formula of previous reaction mixed liquor, eight groups of differences are prepared
The reaction mixture of template quantity, is respectively provided with 101、102、103、104、105、106、107And 108Copy number (copies)
IS6110 standard plasmid.First it is directed to 102、103、104、105、106、107And 108The group of copy number carry out as it is above-mentioned it is real-time calmly
Polymerase chain reaction is measured, every group repeats test three times.It is respectively real-time quantitative polymerase chain reaction referring to Fig. 2 to Fig. 3
Resulting amplification curve diagram and canonical plotting afterwards.As shown in Fig. 2, horizontal axis is reaction cycle number (cycles), the longitudinal axis is fluorescence
Intensity (Δ Rn).Thus amplification curve diagram is it is found that 102To 108The positive trend of rising is presented in the fluorescent value of copy number.Such as following table
Shown in one, 102、103、104、105、106、107And 108The Cq value (quantification cycle) of copy number is respectively
33.19,30.08,26.75,23.35,19.99,16.72 and 13.88.
Table one
| Copy number | 102 | 103 | 104 | 105 | 106 | 107 | 108 |
| Cq value | 33.19 | 30.08 | 26.75 | 23.35 | 19.99 | 16.72 | 13.88 |
With continued reference to Fig. 3, i.e., the result value expanded Fig. 2 is made into canonical plotting.Horizontal axis is template copy numbers.It is vertical
Axis is threshold recurring number, as Cq value.The mark drawn according to real-time quantitative Polymerized human serum albumin (CFX-96, BioRad)
Directrix curve, the dynamic range (dynamic range) with confidence level are related coefficient (R2) it is greater than 0.98；Amplification efficiency
(E) between 90% to 110%.If aforementioned amplification efficiency is lower than 90%, it may indicate that design of primers is bad and cause annealing effect poor,
Therefore amplification efficiency is poor.If amplification efficiency is higher than 110%, it may indicate that primer has excessive nonspecific annealing to generate, cause
Amplification efficiency is excessively high.As shown in figure 3, according to aforementioned 102To 108Result value of the template quantity of copy number in 2 figures, can make
The straight line that slope is -3.265, related coefficient is 0.999 and amplification efficiency is 102.414%.Therefore primer pair SEQ ID
The sensitivity of NO.1 and SEQ ID NO.2 meets dynamic range.
In order to test primer pair sensitivity detectable limit, further choose 101The template quantity of copy number carries out real-time
Quantitative polyase chain reaction repeats 22 tests.As shown in figure 4, for resulting expansion after real-time quantitative polymerase chain reaction
Increase curve graph.Horizontal axis is reaction cycle number (cycles), and the longitudinal axis is Relative fluorescence units (relative fluorescence
unit,RFU).Thus amplification curve diagram is it is found that 101The positive trend of rising is presented in the fluorescent value of copy number.According to the expansion of Fig. 4
Increase as a result, its numerical value is summarized in following table two.
Table two
As shown in Table 2, when template quantity is 101When copy number, threshold threshold value is set as 40 recurring numbers (cutoff value=40),
It is not only detected once (NA), therefore positive rate is about 96%.Its Cq value model of remaining 21 groups of repetition group that detected
It is trapped among between 34.55 to 38.1, average out to 36.37.The above are confidence intervals for positive rate 95%, it may thus be appreciated that drawing using this test
Object pair, the lowest detection limit of template quantity is up to 101Copy number.
In addition, for the further specificity of test SEQ ID NO:1 and SEQ ID NO:2.This test is with SEQ ID
Based on sequence shown in NO:1 and SEQ ID NO:2, similar primer pair is designed.Specifically, this similar primer pair
Forward primer be 50% to be similar to SEQ ID NO:1, and reverse primer is 66% to be similar to SEQ ID NO:2.Then, with this
Similar primer pair carries out real-time quantitative polymerase chain reaction.Detection mode is for example aforementioned to be mentioned, and seldom does repetition herein.Reference
Fig. 5, for the amplification curve diagram for carrying out real-time quantitative polymerase chain reaction using similar primer pair.Thus amplification curve diagram can
Know, 102To 108The positive trend of rising is presented in the fluorescent value of copy number.According to the amplification of Fig. 5, its numerical value is summarized in down
Table three.
Table three
| Copy number | 102 | 103 | 104 | 105 | 106 | 107 | 108 |
| Cq value | 35.85 | 31.52 | 28.15 | 24.82 | 21.35 | 17.31 | 14.98 |
As shown in Table 3, even if SEQ ID NO:1 and SEQ ID NO:2 has variation to a certain degree, when template quantity is low
To 102When copy number, Cq value remains at 40 recurring numbers or less (i.e. 35.85), is only slightly higher than SEQ ID NO:1 and SEQ ID
Resulting 33.19 when NO:2 detects same template amount.That is, having no too many differences in the sensitivity of detection.
Clinical trial (A)
This test is further used for clinically detecting.Clinical sample is the sputum that pulmonary tuberculosis makes a definite diagnosis sufferer.Extract tuberculosis
The DNA of the sputum of patient.It is grasped according to commercially available polymerase chain reaction kit (Dr.Q Taq archaeal dna polymerase, BioFuture)
Book is explained, using 0.2 μM of SEQ ID NO:1 and SEQ ID NO:2 as primer, the sputum DNA of tuberculosis patient is as template
Carry out polymerase chain reaction.PCR condition be 95 DEG C effect 2 minutes after, 95 DEG C be denaturalized 5 seconds, 60 DEG C annealing/amplification 10 seconds, into
25 circular responses of row.Then product resulting after polymerase chain reaction is analyzed with gel electrophoresis.As shown in fig. 6,
Swimming lane 1 is molecular weight markers. Lanes (marker ladder).Swimming lane 2 shows the aforementioned aggregated enzyme chain of the sputum DNA for making a definite diagnosis sufferer
Amplified production obtained after formula reaction.Swimming lane 3 to swimming lane 5 is control group, and display is uninfected by the sputum DNA of individual lungy
Product obtained after aggregated enzyme chain reaction.According to aforementioned design of primers it is found that amplified production size is expected to 141bp,
And the amplified production of swimming lane 2 is single band, and size is between 200bp and 100bp.That is, SEQ ID NO:1
And SEQ ID NO:2 is generated without other non-specific amplified productions, and amplified production size also complies with expection, can be applied to
In clinical detection.
Clinical trial (B)
It can be after applying clinical sample, then by SEQ ID NO:1 through aforementioned authentication SEQ ID NO:1 and SEQ ID NO:2
And SEQ ID NO:2 is compared with commercially available primer pair kit.In other words, in this test, embodiment is to utilize this reality
SEQ ID NO:1 and SEQ the ID NO:2 for applying mode carries out real-time quantitative polymerase chain reaction, and comparative example is using commercially available
Primer pair kit carry out real-time quantitative polymerase chain reaction.As shown in following table four, sample 1, sample 2 and sample 3 divide
Not from three different tuberculosis patients, and in terms of Cq value, embodiment is 33.21 to 34.90, comparative example 42.70
To 45.3.It can less than comparative example about 7.8 to 12.09 circulations that is, detect required recurring number using embodiment.
In the case of same template amount, the sensitivity of embodiment is substantially better than comparative example.
Table four
The feature of the several embodiments of foregoing general description is so that those skilled in the art are better understood content of this disclosure.
It will be understood by a person skilled in the art that easily present disclosure can be used as design or modify for realizing identical purpose and/or
Reach the other methods of the same advantage of the embodiment introduced herein and the basis of structure.Those skilled in the art should also recognize
It arrives, the building of such equivalent, and can be in the spirit without prejudice to present disclosure without prejudice to the spirit and scope of present disclosure
And various changes can be made in this in the case where scope, substitution and change.
Sequence table
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<120>method and its kit of mycobacteria are detected
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gataacgtct ttcaggtcga g 141
Claims (13)
1. a kind of method for detecting mycobacteria, comprises the steps of
A) one sample is provided；
B) pair of primers pair is provided, which is selected from has by SEQ ID NO:1, SEQ ID NO:2, with SEQ ID NO:1
The sequence of about 45% to about 99% similarity, sequence, the SEQ ID with SEQ ID NO:2 with about 60% to about 99% similarity
Group composed by the complementary strand of NO:1 and the complementary strand of SEQ ID NO:2；
C) polymerase chain reaction is carried out to obtain a product using the primer pair and the sample；And
D) product is analyzed to detect the presence of mycobacteria.
2. the method for detection mycobacteria as described in claim 1, it includes to provide a packet that wherein step a), which provides the sample,
The sample of group containing mycobacterium tuberculosis (Mycobacterium tuberculosis complex, MTBC).
3. the method for detection mycobacteria as claimed in claim 2, wherein the sample is blood, sputum, bronchovesicular punching
Washing lotion, urine, excrement or combinations thereof.
4. the method for detection mycobacteria as claimed in claim 2, polymerize wherein carrying out this with the sample using the primer pair
Enzyme chain reaction makes primer pair amplifies mycobacterium tuberculosis group to obtain the product, comprising carrying out polymerase chain reaction
A part of sequence of middle IS6110 sequence is to obtain the product, and wherein the partial sequence is SEQ ID NO:3.
5. the method for detection mycobacteria as described in claim 1, further includes and provides an oligonucleotide probe.
6. the method for detection mycobacteria as claimed in claim 5, polymerize wherein carrying out this with the sample using the primer pair
Before enzyme chain reaction is to obtain the product, comprising carrying out hybridization reaction to the sample using the oligonucleotide probe.
7. the method for detection mycobacteria as claimed in claim 6, wherein the polymerase chain reaction is real-time quantitative polymerization
Enzyme chain reaction.
8. a kind of kit for detecting mycobacteria, it includes pair of primers pair, which is selected from by SEQ ID NO:1, SEQ
ID NO:2, with sequence of the SEQ ID NO:1 with about 45% to about 99% similarity, with SEQ ID NO:2 with about 60% to
Group composed by the complementary strand of the sequence of about 99% similarity, the complementary strand of SEQ ID NO:1 and SEQ ID NO:2.
9. the kit of detection mycobacteria as claimed in claim 8, wherein the primer pair is SEQ ID NO:1 and SEQ ID
NO:2.
10. the kit of detection mycobacteria as claimed in claim 8, further includes a sample, wherein the sample is
Blood, sputum, bronchial alveolar rinse liquid, urine, excrement or combinations thereof.
11. the kit of detection mycobacteria as claimed in claim 8, further includes a target gene, wherein the mesh
Mark gene is the IS6110 sequence of mycobacterium tuberculosis group (Mycobacterium tuberculosis complex, MTBC).
12. the kit of detection mycobacteria as claimed in claim 8, further includes a template, there are about 100
Base-pair to about 250 base-pairs length.
13. the kit of detection mycobacteria as claimed in claim 12, wherein the template is SEQ ID NO:3.
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