CN102732610A - Visualized detection probes and application thereof in tuberculosis rifampicin resistance mutation detection - Google Patents
Visualized detection probes and application thereof in tuberculosis rifampicin resistance mutation detection Download PDFInfo
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Abstract
The invention discloses novel visualized fast and sensitive detection probes and applications thereof in mycobacterium tuberculosis rifampicin resistance mutation detections. The visualized probes provided by the invention comprise: a common anchoring probe A, insulation probes L and R, mutation detection probes B, and a common competition probe SIB. The mutation detection probes B and the probe SIB have a same target DNA complementary region, and only the base types of the mutation sites are different. Aims at rifampicin resistance mutation, a detection probe cluster is designed and synthesized according to all the mutation sites included in a mycobacterium tuberculosis rifampicin resistance rpoB genetic mutation core. The probe cluster comprises 8 probe sets, wherein each probe set comprises a specific mutation detection probe B. With the method provided by the invention, the designed probe sets and probe clusters have good distinguishing ability upon synthesized wild-type and mutant detection fragments, and can be used for accurately detecting specific mutation sites of 530-533 site amino acids of the mycobacterium tuberculosis rifampicin resistance rpoB gene.
Description
Technical field
The invention belongs to Pathogen Biology, molecular microbiology and nucleic acid chemistry field; The method of design and the sequence that relate to a kind of novel point mutation detection probes; With and application in mycobacterium tuberculosis Rifampin medicament-resistant mutation detects, this method can be intuitively visual detection mycobacterium tuberculosis Rifampin medicament-resistant mutation also fast.
Background technology
White plaque (the Tuberculosis that causes by mycobacterium tuberculosis; Be called for short TB) be a kind of ancient disease, in wide-scale distribution just a long time ago, since the 1950's in the world; Vazadrine, Rifampin etc. be the discovery of treatment tuberculosis medicine effectively; Improved the white plaque curative ratio greatly, and reduced tuberculosis palindromia rate, made white plaque become a kind of disease of curing and controlling.In some developed countries, white plaque is controlled basically.Yet owing to the ignorance of some countries to tuberculosis prevention and treatment work, control lungy can not obtain desired result in the last few years; A lot of local white plaque chemotherapy regimens are unreasonable, and mismanagement lacks monitoring, causes the generation and the propagation of mycobacterium tuberculosis Resistant strain, the particularly bacterial strain of anti-the multiple medicines, strengthens the difficulty of tuberculotherapy, the deterioration of aggravation white plaque epidemic situation; Add that the popular of AIDS and the population that economic globalization causes extensively flow, impel white plaque to stage a comeback, become the urgent public health problem in the whole world again.To the nineties in 20th century, the white plaque epidemic situation is out of control in many places in the world, and the population in the whole world 1/3rd infects mycobacterium tuberculosis, and annual New Development tuberculosis patient is about 800～1,000 ten thousand, has every year 3000000 people to die from white plaque approximately.China's epidemic situation lungy is severe equally, presents the situation of morbidity height, mortality ratio is high, resistant rate is high, yearly decline factor is low " three high is low ".Current China is one of the whole world high burden of 22 white plaque state, and the tuberculosis patient sum only is less than India, is whole world patient more than second country.In the face of such situation, researcher need develop new diagnostic techniques and medicine, solves the thorny problem in the tuberculosis prevention and treatment.
Mycobacterium tuberculosis (Mycobacterium tuberculosis) also often abbreviates tubercule bacillus or tubercule bacillus as, is pathogenic bacterium lungy.The mycobacterium tuberculosis resistance mainly causes owing to genome Chinese traditional medicine target gene random mutation.A line antitubercular agent that uses at present mainly comprises Rifampin and vazadrine; Wherein the target protein of Rifampin in bacterium is the β subunit of RNA polymerase; Rifampin combines with the β subunit of RNA polymerase, and it is synthetic to hinder mRNA, suppresses transcription; The blocking-up protein of bacteria is synthetic, reaches the bacteriostatic effect.Research shows that mycobacterium tuberculosis Rifampin resistance is usually by due to its RNA polymerase β subunit coding gene rpoB sudden change; These sudden changes most of (more than 90%) are positioned at the zone of a 81bp; Be referred to as RFP resistance determining area (Rifampicin Resistant Determination Region, RRDR).
The method of resistance mycobacterium tuberculosis detection at present mainly is a culture method, but the mycobacterium tuberculosis poor growth need 4～8 weeks just can provide the result, and positive rate is not high, is merely 30%～40%, causes a lot of tuberculosis patients to can not get timely diagnosis and treatment.And have dependency between transgenation and the mycobacterium tuberculosis resistance, so Many researchers develops the method that various sudden changes detect, and detects the tubercule bacillus resistance through detecting relevant transgenation.At present investigator's method of being used to detect the mycobacterium tuberculosis medicament-resistant mutation comprises molecular beacon and reverse probe hybridization, but these two kinds of methods operations are complicated, and needs special instrument.
Summary of the invention
The objective of the invention is to provides a kind of visual detection probes to above-mentioned deficiency, is used for the visual detection of transgenation.
Another object of the present invention is, a kind of visual detection probes that detects to the resistance mycobacterium tuberculosis is provided, and is used for the visual detection of white plaque Rifampin medicament-resistant mutation.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
A kind of visual detection probes, it comprises:
Grappling probe, its 3 ' end contain 3 sections GGG sequences participates in G-four serobilas and forms, or its 3 ' end contains 1 section GGG sequence and participates in G-four serobilas and form;
The competition probe, its complementary district is complementary fully with the wildtype target sequence;
The sudden change detection probes, its complementary district is complementary fully with corresponding mutant target sequence, and its 5 ' end has 1 section GGG sequence participates in G-four serobilas and form, or its 5 ' end has 3 sections GGG sequences participates in G-four serobilas and forms;
The downstream with sudden change detection probes complementary segment on the complementation district of said grappling probe and the target sequence are complementary, when said grappling probe is combined on the corresponding mutant target sequence with the sudden change detection probes, can form G-four serobilas between the two;
When target sequence PCR product is detected; For preventing that hairpin structure from influencing combining of probe and target nucleic acid; It also comprises a pair of insulated probe, and said insulated probe is combined in the downstream of grappling probe calmodulin binding domain CaM and the upper reaches of sudden change detection probes calmodulin binding domain CaM respectively.In reaction system, adding these two probes can be complementary in the side areas of target nucleic acid calmodulin binding domain CaM with probe (grappling probe and sudden change detection probes); Form stable two strands with the strand target nucleic acid; Thereby open the hairpin structure that target nucleic acid forms, the combining of help suddenling change detection probes and target nucleic acid.
Detect to the resistance mycobacterium tuberculosis; It is goal gene that the present invention selects mycobacterium tuberculosis RNA polymerase β subunit coding gene rpoB for use; Mainly concentrate on its Rifampin resistance determining area; This section zone is responsible for coding and is equivalent to 507 to 533 of e. coli rna polymerase beta subunits, totally 27 amino acid.Wherein mutation frequency is the highest is 531 Serine (ser), and this site mutation can cause the high-caliber tolerance of Rifampin, and (Huanghai Sea is flourish, the strange Ma Yu of gold; Chen Xi, Li Huiwen, Zhuan Yuhui. the Chinese mycobacterium tuberculosis of anti-Rifampin rpoB transgenation characteristics. Chinese tuberculosis and breathing magazine; 2001,24 (4): 231～235).
Particularly; All mutation types design relevant detection probes that the present invention is comprised according to rpoB transgenation core area 530-533 amino acids bunch and some probe groups; Probe bunch comprises a total grappling probe A; Article one, total competition probe SIB reaches a series of sudden change detection probes B according to the mutation type design.Probe bunch comprises some probe groups, respectively to corresponding mutation type in the surveyed area.Said grappling probe has the nucleotide sequence shown in the SEQ ID No.2, and said competition probe has the nucleotide sequence shown in the SEQ ID No.3, and said sudden change detection probes is one or more in the nucleotide sequence shown in SEQ ID No.4～11.Can comprise insulated probe L and probe R in addition, its nucleotide sequence is respectively shown in SEQ IDNo.12 and 13.
Use for convenient, the present invention also comprises the test kit that contains said above-mentioned visual detection probes.
The application of above-mentioned visual detection probes in mycobacterium tuberculosis Rifampin medicament-resistant mutation detects.
The present invention also provides a kind of method of visual detection mycobacterium tuberculosis Rifampin medicament-resistant mutation, and it comprises the steps:
A, design dna px detection probes system: according to synthetic each the described detection probes of corresponding claim 1～2 of rpoB transgenation core area design;
B, with probe and the target nucleotide complementary pairing of above-mentioned A, and add Hemin, through the catalytic coupling reaction of DNA px, judge whether to exist mycobacterium tuberculosis Rifampin medicament-resistant mutation.
The advantage and the effect of invention:
1, can be used for visualized tuberculosis mycobacterium Rifampin multidigit point medicament-resistant mutation and detect, comprise 8 kinds of mutation types; Probe bunch and the probe groups that comprises thereof all can well be distinguished wild-type sequence and mutant sequence, and all probe groups are to the discrimination of corresponding mutant sequence and wild-type sequence, and promptly probe produces the ratio of strength of signal to the two, all greater than 15 times; And probe bunch to the discrimination of mutant sequence and wild-type sequence all greater than 10 times; All results all only detect by an unaided eye and can distinguish mutant sequence and wild-type sequence clearly.
2, designed probe system of the present invention comprises two levels, can satisfy the different needs that detect, and whether the Rifampin resistance determining area that probe bunch can be used for rapid screening mycobacterium tuberculosis rpoB gene exists sudden change; Probe groups then can be used for detecting certain specific sudden change, particularly those and the relevant mutation type of height resistance.
3, designed probe group of the present invention and probe bunch can go out good discrimination to the PCR product detected representation with complicated secondary structure in clinical sample source, at present the gained result to the discrimination of mutant and wild-type rpoB gene PCR product greater than 7 times.
Description of drawings
Fig. 1. mycobacterium tuberculosis rpoB gene Rifampin (RFP) resistance determining area mutational site
Mycobacterium tuberculosis Rifampin resistance determining area, rpoB gene 507-533 amino acids, wherein underscore representes to be prone to the base of sudden change.
Asymmetric fractionation-the assembling of Fig. 2 .DNA px
With DNA superoxide enzyme sequence d (GGGTAGGGCGGGTTGGG); Be divided into two parts asymmetricly; A part comprises 3 sections GGG sequences; Another part only contains one section GGG sequence, and the 5 ' end or the 3 ' end of these two sections sequences merge with target nucleic acid adjacent area complementary sequence respectively, form probe A and probe B.When having target DNA-C to exist, the adjacent area of probe A and probe B and target nucleic acid is complementary, in them the DNA px form sequence each other near, be folded into G-four chain body structures, after Hemin combines, be formed with active DNA px.Can pass through the catalytic coupling reaction of DNA px like this, detect the existence of target nucleic acid C in the solution.
Fig. 3. probe bunch selectivity to wild-type sequence and corresponding mutant sequence
Probe bunch sees from photo the selectivity that wild-type sequence and corresponding mutant sequence all show, and probe bunch itself and wild-type test sample are colourless basically, and various mutants (2～9) sample then is a green clearly.Form is probe bunch experimental program and result, and the probe bunch signal to corresponding mutant sequence is to more than 10 times of wild-type signal.
Fig. 4. each probe groups is to the selectivity of wild-type sequence and corresponding mutant sequence
The selectivity that each probe groups all shows wild-type sequence and corresponding mutant sequence, each probe groups is colourless basically to wild-type sequence from the photo, and (1b～8b) sequence then is tangible green to corresponding mutant.Form is each probe groups experimental program and result, and all probe groups are to more than 15 times of wild-type signal to the signal of corresponding mutant sequence, wherein to common mutations type, and Ser531Leu, the selection multiple of TCG → TTG reaches 40 times.
Fig. 5. probe bunch detection to saltant Ser531Trp
Probe bunch detection to No. 1 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation, probe bunch is colourless basically to wild type strain PCR product and contrast, and saltant PCR product then presents tangible green.Probe bunch is to more than 7 times of wild-type signal to saltant product signal.No. 1 bacterial strain of detected result has sudden change in 530-533 amino acids zone, conforms to sequencing result.
Fig. 6. the 2nd probe groups is to the detection of saltant Ser531Trp
The 2nd probe groups is to the detection of No. 1 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation; Probe groups is colourless basically to wild type strain PCR product and contrast; The saltant product then presents tangible green, and probe groups is to more than 10 times of wild-type signal to saltant product signal.No. 1 bacterial strain of detected result is the sudden changes of 531 amino acids, and TCG sports TGG, conforms to sequencing result.
Fig. 7. probe bunch detection to saltant Ser531Leu
Probe bunch detection to No. 2 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation, probe bunch is colourless basically to wild type strain PCR product and contrast, and saltant PCR product then presents tangible green.Probe bunch is to more than 7 times of wild-type signal to saltant product signal.No. 2 bacterial strains of detected result have sudden change in 530-533 amino acids zone, conform to sequencing result.
Fig. 8. the 3rd probe groups is to the detection of saltant Ser531Leu
The 3rd probe groups is to the detection of No. 2 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation; Probe groups is colourless basically to wild type strain PCR product and contrast; The saltant product then presents tangible green, and probe groups is to more than 10 times of wild-type signal to saltant product signal.No. 2 bacterial strains of detected result are the sudden changes of 531 amino acids, and TCG sports TTG, conforms to sequencing result.
Fig. 9. probe bunch detection to saltant Leu533Pro
Probe bunch detection to No. 3 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation, probe bunch is colourless basically to wild type strain PCR product and contrast, and saltant PCR product then presents tangible green.Probe bunch is to more than 7 times of wild-type signal to saltant product signal.No. 3 bacterial strains of detected result have sudden change in 530-533 amino acids zone, conform to sequencing result.
Figure 10. the 7th probe groups is to the detection of saltant Leu533Pro
The 7th probe groups is to the detection of No. 3 Rifampin resistance mycobacterium tuberculosis ropB transgenation situation; Probe groups is colourless basically to wild type strain PCR product and contrast; The saltant product then presents tangible green, and probe groups is to more than 7 times of wild-type signal to saltant product signal.No. 3 bacterial strains of detected result are the sudden changes of 533 amino acids, and CTG sports CCG, conforms to sequencing result.
Embodiment
Following examples are used to further specify the present invention, but should not be construed as limitation of the present invention.Do not specify that experiment involved among the present invention all is well known to those skilled in the art routine operation if having.
1.DNA the design of superoxide enzyme mutant detection probes system
(1) rpoB transgenation core area sequence is as shown in Figure 1
The present invention comprises 530 8 kinds of mutation types to 533 amino acids (underscore mark among Fig. 1), specifically sudden change and relevant detection probe such as following table:
| The mutational site | Mutation type | Corresponding detection probes |
| 531 | T→G | I-B531-1C |
| 531 | C→G | I-B531-2C |
| 531 | C→T | I-B531-2A |
| 532 | C→A | I-B532-2T |
| 532 | C→T | I-B532-2A |
| 533 | C→T | I-B533-1A |
| 533 | T→C | I-B533-2G |
| 531 | C→A，G→C | I-B531-2T3G |
(2) according to mycobacterium tuberculosis rpoB gene Rifampin (RFP) resistance determining area mutational site design relevant detection probe bunch, probe bunch comprises three parts: a total grappling probe A, its 3 ' end contain 3 sections GGG sequences to participate in G-four serobilas and forms; Article one, total competition probe SIB, its complementary district is complementary fully with the wildtype target target; According to a series of sudden change detection probes B of mutation type design, the complementation district of every probe B and the target of corresponding mutant are complementary fully, and 5 ' end has one section GGG sequence and participates in the formation of G-four serobilas for it.C is a gene fragment to be detected.
(4) the required detection target that reaches: probe bunch all can well be distinguished wild-type and corresponding mutant DNA sequence with each probe groups.
Following according to above principle designed probe group:
The 1st probe groups: A, SIB, I-B531-1C (TGGGTTGCCCCAGCGCCGCCAG)
The 2nd probe groups: A, SIB, I-B531-2C (TGGGTTGCCCCAGCGCCCACAG)
The 3rd probe groups: A, SIB, I-B531-2A (TGGGAGCCCCAGCGCCAACAG)
The 4th probe groups: A, SIB, I-B532-2T (TGGGTGCCCCAGCTCCGACAG)
The 5th probe groups: A, SIB, I-B532-2A (TGGGAGCCCCAGCACCGACAG)
The 6th probe groups: A, SIB, I-B533-1A (TGGGTGCCCCAACGCCGACAG)
The 7th probe groups: A, SIB, I-B533-2G (TGGGTGCCCCGGCGCCGACAG)
The 8th probe groups: A, SIB, I-B531-2T3G (TGGGTGCCCCAGCGCGTACAG)
Total grappling probe A:CTCACGTGACAGACCGCCGTGGGTTGGGTTGGG
Total competition probe SIB:CCAACTGCCCCAGCGCCGACAG
The direction of sudden change detection probes B and grappling probe A and target sequence is opposite, and they are combined in the downstream position of 533 amino acids, with target sequence be complete complementary.
Probe bunch comprises grappling probe A, and competition probe SIB and 8 kinds of sudden change detection probes B are about to above-mentioned 8 groups of probes and mix to constituting probe bunch together.
In addition when detecting the PCR product, also comprise insulated probe L and the R that helps to open PCR product complicacy secondary structure in each probe groups and the probe bunch.Its sequence is following:
L：TCGGCGCTTGTGGGTCAACCCCGACAG
R：CGTCGCGGACCTCCAGCCCGGCACG
2, checking probe bunch and each probe groups are to the selectivity of wild-type sequence and corresponding mutant sequence
(1) oligonucleotide is quantitative
(resistivity is 10 to add the ultrapure water of 10 times of microlitre numbers according to the nmole number that marked on each oligonucleotide (probe A, B, SIB, fragment C to be measured) sample hose
18Ω), fully dissolving.Getting 10 μ L oligonucleotide mother liquors adds in the 990 μ l distilled waters; Promptly dilute 100 times, it is quantitative to be used for oligonucleotide, 95 ℃ of heating of volumetric soiutions 5 minutes; Take out and place cooled on ice rapidly; Centrifugal behind the mixing, destroy the secondary structure that possibly exist in the high density oligonucleotide mother liquor, guarantee that oligonucleotide is the strand state.Measure the ultraviolet absorption value of oligonucleotide volumetric soiutions at the 260nm place, the mmole specific absorbance according to each oligonucleotide obtains mother liquid concentration, as required each oligonucleotide mother liquor is diluted to 100 μ M or 50 μ M ,-20 ℃ of preservations.
(2) kinetic test of test sample
DNA superoxide enzyme catalysis H
2O
2Oxidation ABTS
2-The ABTS that is generated
-At the 414nm place maximum absorption is arranged, we judge the peroxidase activity of detection architecture through the monitoring detection architecture in the absorption of 414nm, and then infer that whether target nucleic acid exists or the type of relevant sudden change.The DNA peroxidase reaction is at damping fluid: 25mM HEPES-NH
4Carry out reaction substrate H among OH (pH8.0), 200mM NaCl, the 20mM KCl
2O
2And ABTS
2-Concentration be 2mM.Each sample contains the various oligonucleotide and the 50nM Hemin of prescribed concentration.
Concrete steps; (probe A, the B final concentration in detection architecture is 100nM to the various oligonucleotide of the about 760 μ L ultrapure waters of adding, 100 μ L10 * mixing salt solutions (2MNaCl200mM KCl), 100 μ L, 10 * H-Buffer (250mM HEPES-NH4OH (pH=8.0)) and prescribed concentration in each 1.5mL centrifuge tube; SIB is 300nM; The C fragment final concentration of synthetic is 100nM) (see the form among Fig. 3 and Fig. 4; " C531-1G " representes that first codon mutation of the 531st in ropB amino acid is the to be checked artificial fragment of G in the form, and other fragment to be checked also takes similarly to represent mode, and Bmix representes the mixture of 8 sudden change detection probes B); The mixed evenly back of sample is centrifugal, and 95 ℃ were heated 5 minutes, takes out and is cooled to room temperature; Add 1 μ L, 50 μ M Hemin solution in each sample, the Hemin final concentration is 50nM, and is centrifugal behind the mixing, and the room temperature placement was placed 3 hours in 1 hour or 4 ℃.
Detection reaction is at room temperature carried out, with the dynamic process of the spectrophotometric kinetics model monitoring reaction of Shimadzu UV-2550 type.414nm is the monitoring wavelength, and 1 second record 1 secondary data write down 3 minutes.Before the reaction, add 40 μ L 50mM ABTS solution in each sample, mixing is got 500 μ L samples in reference cell, gets 1 μ L 1M H again
2O
2Stick to the sample pool inwall, remaining 500 μ L samples edge is stained with H
2O
2The sample pool inwall inject, close the sample door, click START button, the opening entry data.
3. utilize probe groups and probe bunch that clinical isolating Rifampin resistance mycobacterium tuberculosis rpoB gene core zone sudden change situation is detected
(1) extract wild-type and resistance mycobacterium tuberculosis genomic dna, a use day root company bacterial genomes extraction test kit (catalog number (Cat.No.): DP302) step is following:
Get 5 milliliters of inoculums, centrifugal 1 minute of 12000rpm abandons most supernatant as far as possible.Add the resuspended thalline of 180 μ l N,O-Diacetylmuramidase damping fluids (buffer formulation: 20mM TrisHCl, pH8.0; 2mM Na2-EDTA; 1.2%Triton, 20mg/ml N,O-Diacetylmuramidase), 37 ℃ of water-baths 2 hours add 4 μ l RNaseA (100mg/ml); Mixing, normal temperature 10 minutes adds 20 μ l Proteinase K solution; 55 ℃ of water-baths 40 minutes add 220 μ l damping fluid GB, vibrate 15 seconds; 70 ℃ of water-baths 10 minutes, centrifugal 1 minute of 12000rpm gets supernatant and transfers to new EP pipe.Add 220 μ l absolute ethyl alcohols, all change mixture over to centrifugal post CB3, centrifugal 1 minute of 12000rpm abandons waste liquid; In CB3, add 500 μ l damping fluid GD, centrifugal 1 minute of 12000rpm abandons waste liquid; In CB3, add 700 μ l damping fluid PW, centrifugal 1 minute of 12000rpm abandons waste liquid; In CB3, add 500 μ l damping fluid PW, centrifugal 1 minute of 12000rpm abandons waste liquid.Centrifugal 2 minutes of empty centrifugal post 12000rpm, room temperature left standstill 3 minutes afterwards, changed adsorption column CB3 over to a clean centrifuge tube; In the middle of adsorption film, add 100 μ l distilled waters; Left standstill 2 minutes, centrifugal 1 minute of 12000rpm adds 100 μ l distilled waters once more in the middle of adsorption film; Left standstill centrifugal 1 minute of 12000rpm 2 minutes.Be bacterial genomes DNA in the centrifuge tube.
(2) to rpoB gene design primer, PCR obtains the double-stranded fragment of 588bp;
The PCR reaction system is following:
94℃5min，94℃1min，55℃1min，72℃1min，30cycles，72℃5min。
Primer sequence is following:
ropP1：GAGCGGATGACCACCCAGG
ropP2：CAGGAAGGGAATCATCGCGG
(3) be template with the 588bp two strands, PCR obtains the 158bp single-chain fragment
94℃5min，94℃1min，52℃1min，72℃1min，40cycles，72℃5min。
Primer sequence is following:
ropTR8：GTGCACGTCGCGGACCTCCA
ropTR9：TCGCCGCGATCAAGGAGT
(4) with above-mentioned coloration method wild-type and resistance mycobacterium tuberculosis rpoB gene strand PCR product are detected Rifampin resistance mycobacterium tuberculosis is numbered respectively 1,2, No. 3; When its PCR product is detected, influence probe A for preventing hairpin structure, B combines with target nucleic acid; The special insulated probe L that adds, R, in reaction system, add these two probes can with probe A; B is complementary in the side areas of target nucleic acid calmodulin binding domain CaM; Form stable two strands with the strand target nucleic acid, thereby open the hairpin structure that target nucleic acid forms, help combining of detection probes and target nucleic acid.Detection method is with embodiment 1, and insulated probe L, the final concentration of R are 100nM, and that reaction result writes down is the result of 180 seconds (sec).Its reaction system such as following table:
(5) send company's order-checking with the double-stranded PCR product of 588bp, confirm concrete mutational site, the result compares with probe in detecting.
Detected result of the present invention is shown in Fig. 5～10, and the equal naked eyes of the result of mutant are visible, and the result and the sequencing result that detect by the inventive method compare, and the result is in full accord.
The sequence table explanation: SEQ ID No.1 is the base sequence of mycobacterium tuberculosis Rifampin resistance determining area; The grappling probe of SEQ IDNo.2 mycobacterium tuberculosis Rifampin Drug Resistance Detection; SEQ ID No.3 is the competition probe; SEQ IDNo.4～11 are 8 different sudden change detection probes, SEQ ID No.12～13rd, and insulated probe L and R, SEQ ID No.14～17 are respectively primer ropP1&ropP2 and ropTR8&ropTR9.
Sequence table
< 110>Zhang Xiaolian
< 120>visual detection probes and the application in white plaque Rifampin medicament-resistant mutation detects
<130>
<160> 17
<170> PatentIn?version?3.5
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ctgtcggcgc?tg 72
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ccaactgccc?cagcgccgac?ag 22
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Claims (5)
1. visual detection probes, it comprises:
Grappling probe, its 3 ' end contain 3 sections GGG sequences participates in G-four serobilas and forms, or its 3 ' end contains 1 section GGG sequence and participates in G-four serobilas and form;
The competition probe, its complementary district is complementary fully with the wildtype target sequence;
The sudden change detection probes, its complementary district is complementary fully with corresponding mutant target sequence, and its 5 ' end has 1 section GGG sequence participates in G-four serobilas and form, or its 5 ' end has 3 sections GGG sequences participates in G-four serobilas and forms;
Insulated probe comprises probe L and probe R, can be combined in the downstream of grappling probe target area and the upper reaches and the strand target nucleic acid of sudden change detection probes target area respectively and form stable two strands;
The downstream with sudden change detection probes complementary segment on the complementation district of said grappling probe and the target sequence are complementary, when said grappling probe is combined on the corresponding mutant target sequence with the sudden change detection probes, can form G-four serobilas between the two.
2. visual detection probes according to claim 1; It is characterized in that; Said grappling probe has the nucleotide sequence shown in the SEQ ID No.2, and said competition probe has the nucleotide sequence shown in the SEQ ID No.3, and said sudden change detection probes is one or more in the nucleotide sequence shown in SEQ ID No.4 ~ 11; Said insulated probe comprises probe L and probe R, and its nucleotide sequence is respectively shown in SEQ ID No.12 and 13.
3. the test kit that contains claim 1 or 2 said visual detection probes.
4. claim 1 or the 2 said visual detection probes application in mycobacterium tuberculosis Rifampin medicament-resistant mutation detects.
5. the method for a visual detection mycobacterium tuberculosis Rifampin medicament-resistant mutation, it comprises the steps:
A, design dna px detection probes system: according to
RpoBSynthetic corresponding claim 1 of transgenation core area design or 2 described detection probes;
B, with probe and the target nucleotide complementary pairing of above-mentioned A, and add Hemin, through the catalytic coupling reaction of DNA px, judge whether to exist mycobacterium tuberculosis Rifampin medicament-resistant mutation.
Priority Applications (1)
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