CN101440403A - Method for detecting Vibrio cholerae O139 by using suspension chip technology - Google Patents
Method for detecting Vibrio cholerae O139 by using suspension chip technology Download PDFInfo
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- CN101440403A CN101440403A CNA2008101811959A CN200810181195A CN101440403A CN 101440403 A CN101440403 A CN 101440403A CN A2008101811959 A CNA2008101811959 A CN A2008101811959A CN 200810181195 A CN200810181195 A CN 200810181195A CN 101440403 A CN101440403 A CN 101440403A
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Abstract
The invention relates to a suspended chip and a method for detecting vibrio cholerae O139. The suspended chip comprises a microspheric carrier and an oligonucleotide probe fixed on the carrier, wherein the oligonucleotide probe is a DNA sequence in a vibrio cholerae O139-specific gene rfb screened from gene clusters of O-antigen of vibrio cholerae. A designed primer is used to proliferate and label the genomic DNA of the sample under test, hybridization is performed with the suspended chip, and then whether the sample under test contains vibrio cholerae O139 or not is judged. The suspended chip has high sensitivity, reaches the level of fg genomic DNA, satisfies the requirement of detection on clinical and environmental samples, and is characterized by high specificity, simple operation, low cost and easy generalization. The invention adopts a UNG-taq catalytic PCR system which dramatically decreases the frequency of false-positive outcome in PCR.
Description
Technical field
The present invention relates to a kind of molecular Biological Detection chip, concrete is that a kind of using suspending chip technology detects cholera vibrio O 139.
Background technology
The suspending chip technology is that biochip technology is combined a kind of new technology that produces with low cytometric analysis, be that a kind of multiple data obtains and analysis platform, full name is " multi-functional many indexs Synchronization Analysis system " (Flexible Multiple Analyte Profiling, xMap), be also referred to as Multi-Analyte SuspensionArray, organic combination fluorescence-encoded micro-beads, laser technology, micro-fluidic technologies, fast signal handle and data analysis system, promptly guaranteed signal quality, high-throughout molecular detection technology platform of new generation is provided again.The maximum difference of suspending chip and traditional gene chip is, traditional gene chip sample applying is on slide or nylon membrane, rely on coordinate setting to carry out addressing, and suspending chip is covalently bonded to detection probes on the different colour code microballoons, according on the colour code microballoon with fluorescein distinguish.
Suspension chip system mainly is made up of three parts: 1, about microsphere diameter 5.6 μ m, be made of polystyrene, the surface has about 10
8Individual carboxyl site can join with the peptide bond coupling with oligonucleotide probe and albumen; 2, in the fluorescein microballoon mark redness and fluorescent orange element, every kind is divided into 10 concentration gradients, and microballoon is divided into 100 kinds of microballoons that can be identified, and after microballoon is by 635nm laser excitation, can launch the fluorescence of 658nm and 712nm; 3, detector is because of microsphere diameter 5.6 μ m, and by fluorescent mark, adopts the low cytometric analysis principle to detect.
Principle: each colour code microballoon can pass through carboxyl, with specific amalyzing substances (oligonucleotide probe, antigen, antibody etc.) covalent attachment.The microballoon of mark bioprobe is carried out specific the combination with determinand, with the reporter molecules reaction that has the third fluorescein, in a reacting hole, can detect 100 kinds of various objectives molecules in the sample simultaneously again.After the reaction, adopt 96 orifice plates to carry out sample introduction.Detector picks up reaction solution the adding peristaltic pump automatically, pumps in the microcapillary, and under the effect of sheath fluid, the single sense channel that passes through of the microballoon of single distribution.Be provided with two laser probes in the sense channel, a probe can be launched the laser of 635nm wavelength, but two kinds of fluoresceins of excitation labeling microballoon, thus discern different colour code microballoons, carry out qualitative detection; Another laser probe can excite the fluorescein of reporter molecules, is with the reporter molecules fluorescence signal intensity by measuring microballoon, can carry out detection by quantitative.Therefore, suspending chip can carry out the qualitative and quantitative detection target molecule.In sample, contain molecules of interest, molecules of interest and specific microballoon with probe when being attached together, twice laser institute excited fluorescent all can be detected; If do not contain molecules of interest in the sample, then only can detect microballoon with fluorescence.By digital signal processor and the automatic statistical software of computer, analyze the wavelength and the strength of signal of twice laser institute fluorescence excitation, thereby judge several extremely tens of kinds of detection target compounds in the sample to be checked, and, carry out quantitative analysis detecting thing by detecting fluorescence signal intensity.Autopipette adds different samples in the peristaltic pump successively, separates with a bit of gas column between the sample, not only can distinguish different samples like this in continuous analysis process, can be also to prevent the pollution between sample.
Compare suspending chip tool distinct advantages with other protein and nucleic acid detection method:
1) carboxyl of applied range microsphere surface can join with nucleic acid probe and protein molecular coupling, can carry out qualitative, quantitative research to purpose nucleic acid and protein molecular;
2) the susceptibility height as reaction carriers, has increased the reactant contact area with microballoon; Each microsphere surface has about 10
8Individual carboxyl site, with covalent coupling connection oligonucleotide probe, antibody or antigen, the probe density height, the signal of generation is strong; Use fluoroscopic examination, amplified reaction signal, susceptibility improves greatly.
3) biological respinse that good reproducibility carried out carries out in liquid phase environment, be beneficial to biomolecules native conformations such as keeping nucleic acid, albumen, overcome the influence of traditional die steric effect, also be more conducive to the reaction of probe and analyte, improved the accuracy that detects;
4) signal to noise ratio is hanged down in microcapillary single microballoon is detected, and the problem that does not exist the background influence to detect need not wash-out and removes background;
5) high-throughput can carry out qualitative and quantitative analysis to the multiple various objectives molecule in the same sample simultaneously, has promptly improved detection information flux, has saved the sample consumption again;
6) speed of response and detection speed have rapidly and efficiently been improved greatly.Because the biological respinse that is carried out carries out in liquid phase environment, hybridization only needed to finish in 15 minutes, had improved speed of response; In 35-60 minutes, can reach the detection of 100 kinds of indexs respectively simultaneously to 96 different samples; Utilize 96 orifice plates and automatic sample handling system, can carry out the detection of n * 96 sample continuously.
7) the low preparation process of cost need not specific installation, and the reactive polypeptide that is dehydrated into that only carries out between carboxyl and amino gets final product, and is simple; Owing to be the fluid storage mode, once preparation can repeatedly be used, and on average each index detects cost and only needs 2.5-5.0 yuan;
8) be easy to stdn based on These characteristics, make this technology can accomplish stdn, be easy to the development and the popularization of test kit.At present, suspending chip is the biochip that the unique approval of U.S. FDA is used for clinical diagnosis (autoimmune disease detects and the human leucocyte antigen typing).
Cholera is a kind of deadly infectious disease, once causes in history to be very popular for 7 times, has caused massive losses for people's lives and properties.At present, the vibrio cholerae of having identified reaches more than 190 serotype, causes that wherein cholera is pandemic for serotype O1, O139; The cholera by the cholera vibrio O 139 initiation of India's outburst in 1992 involves countries in Asia rapidly, causes 110,000 people to infect altogether.The vibrio cholerae conventional sense is based on traditional cultural method, complex operation not only, length consuming time, and false negative height.Therefore set up a kind of quick, special, sensitive detecting method is significant to the cholera preventing and controlling.
The analysis of vibrio cholerae O-antigen gene coma information science is found one section region of variability of tool on O-antigen synthase gene rfb can be at this zone screening different serotypes specific sequence (Diaz MR, 2006).
Summary of the invention
Purpose of the present invention aims to provide the suspending chip that detects cholera vibrio O 139.The suspending chip of detection cholera vibrio O 139 provided by the present invention comprises the diagnose microballoons and the specificity oligonucleotide probe of microballoon coupling connection therewith, and wherein the oligonucleotide probe sequence is one section sequence of cholera vibrio O 139 specific gene rfb.The invention also discloses one section oligonucleotide probe sequence, this sequence is O139 rfb-Probe:5 '-NH
2-(CH
2)
12-GTGTATGTGGTACCTGGGTTG-3 ', shown in sequence in the sequence table 3, and 5 ' carry out NH
2-(CH
2)
12Modify.The present invention also comprises a pair of specific primer in addition, and upstream primer is: O139 rfb-F:5 '-ATGAAGAAGGATGACCGTATTG-3 ' (seeing sequence 1 in the sequence table); Downstream primer is: B-O139 rfb-R:5 '-ACGGAGAAAGTGAGTTTTGCC-3 ' (see sequence 2 in the sequence table, and carry out the Biotin mark).Above-mentioned primer can increase and comprise one section rfb gene order of above-mentioned probe sequence.
Another object of the present invention is to provide the preparation method of above-mentioned suspending chip, this method may further comprise the steps:
1) preparation oligonucleotide probe;
2) with the amido modified probe of pure water dilution;
3) with the microballoon vibration of deposit, transfer to then in the brown EP pipe;
4) centrifugal collection microballoon adds MES liquid and vibrates;
5) in the suspension microballoon, add above-mentioned probe, and vibration;
6) in microspheres solution, add the new 10mg/ml EDC for preparing, vibration; Carry out incubation then;
7) add the new 10mg/ml EDC that disposes once more in microspheres solution, incubation is carried out in vibration then;
8) in microspheres solution, add 0.02% Tween-20, centrifugal collection microballoon;
9) with the resuspended microballoon of 0.1%SDS, centrifugal collection microballoon; With the TE microballoon that suspends;
10) with the number of blood counting chamber calculating microballoon, 2-8 ℃, keep in Dark Place.
Another object of the present invention is the method that using suspending chip detects cholera vibrio O 139.
According to cholera vibrio O 139 rfb gene design and the synthetic Auele Specific Primer that is used for pcr amplification, shown in sequence in the sequence table 1 and 2, and primer is wherein carried out Biotin modify; Prepare sample genomic dna to be checked according to a conventional method as template, use above-mentioned Auele Specific Primer to carry out pcr amplification, make Biotin modification on the PCR product band simultaneously; With the suspending chip hybridization of PCR product and preparation, just hybridize with the probe that coupling is associated on the microballoon, the hybridization suspending chip is carried out streptavidin phycoerythrin mark, detect fluorescent signal by flow cytometer then.
Another object of the present invention provides a kind of PCR of overcoming false-positive method.UNG-Taq enzyme system that the present invention adopts has effectively been controlled the PCR false negative and has been produced.Concrete grammar is in the PCR reaction system, and the dTTP amount is reduced by half, and is substituted by dUTP, and adds the UNG (ura DNA glycosidase) of 0.05U.Be impregnated in a certain amount of dUTP in the PCR product, UNG can not be identified as template by interrupting the glycosidic link on the dUTP on the nucleic acid chains, make the nucleic acid that contains dUTP.37 ℃ of incubation 5min of elder generation before operation PCR program make UNG fully digest the PCR product, and during 94 ℃ of template sex change, the UNG inactivation carries out normal PCR reaction.
Description of drawings
Fig. 1 specificity test-results;
Embodiment:
For further specifying the application of the present invention in cholera vibrio O 139 detects, describe especially exemplified by following preferred embodiment, but application of the present invention is not limited to embodiment.
Embodiment one: primer design and synthetic
1) sequence obtains: by to bunch sequential analysis of vibrio cholerae O-antigen gene, choose O139 specific gene rfb as target sequence, and obtain this gene order from the GenBank public database, be numbered Y07786;
2) design primer: adopt Primer Premier 5.0 software design primers, correlation parameter is: 55.0 ℃-59.0 ℃ of Tm values, and GC value 40.0%-60.0%, PCR product size is 100bp-500bp, primer size 22 ± 3bp;
3) primer is selected: the primer of software output is carried out suitable manual regulation, increase or reduce several bases, carry out online Blast comparison at GenBank then, choose the high primer of specificity, and will be wherein one carries out 5 ' terminal Biotin mark.The upstream primer sequence is O139 rfb-F:5 '-ATGAAGAAGGATGACCGTATTG-3 ', and downstream primer is B-O139 rfb-R:5 '-Biotin-ACGGAGAAAGTGAGTTTTGCC-3 ', and the amplified fragments size is 450bp;
4) primer is synthetic: the synthetic upstream primer O139 rfb-F of worker, the downstream primer B-O139 rfb-R of the synthetic Biotin mark of Dalian TakaRa are given birth in Shanghai.
Embodiment two: the design of probe and synthetic
1) sequence obtains: at the non-guiding region designing probe of above-mentioned amplified fragments;
2) designing probe: adopt Primer Premier 5.0 software design probes, selected HybridizationProbes order, designing probe on the Anti-sense chain, parameter is with embodiment one;
3) probe is selected: the probe of software output is carried out suitable manual regulation, increase or reduce several bases, carry out online Blast comparison at GenBank then, choose the high probe of specificity, carry out NH at 5 ' end
2-(CH
2)
12Modify, selected probe sequence is O139 rfb-Probe:5 '-NH
2-(CH
2)
12-GTGTATGTGGTACCTGGGTTG-3 ';
4) probe is synthetic: Dalian TakaRa synthesizes above-mentioned probe.
The preparation method of embodiment three suspending chips
1. dilute amido modified probe to 1mM (lnanomole/ μ l) with pure water;
2. with the microballoon vibration 20sec that lays in;
3. getting 200 μ l microballoons transfers in the brown EP pipe;
4. collection microballoon, 8000g, centrifugal 1-2min;
5. abandon supernatant, add 50 μ l 0.1M MES (2-[N-Morpholino] ethanesulfonic acid, 2-(N-morpholino) ethyl sulfonic acid) liquid pH4.5, vibration 20sec;
6. the probe that in the suspension microballoon, adds 2 μ l 1mM, vibration 20sec;
7. prepare fresh 10mg/ml EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimidehydrochloride, 1-ethyl-3-3-dimethylaminopropyl carbodiimide), use dH
2O;
8. the 10mg/ml EDC that in microspheres solution, adds the new configuration of 2.5 μ l, vibration;
9. incubation 30min under the room temperature dark condition;
10. prepare fresh 10mg/ml EDC once more, use dH
2O;
11. in microspheres solution, add the 10mg/ml EDC of the new configuration of 2.5 μ l, vibration;
12. incubation 30min under the room temperature dark condition;
13. in microspheres solution, add 1.0mL0.02% Tween-20;
14.8000 the centrifugal 1-2min of * g collects microballoon;
15. abandon supernatant, with the resuspended microballoon of 1.0mL0.1%SDS;
16.8000 the centrifugal 1-2min of * g collects microballoon;
17. with 100 μ l TE, pH=8.0, the suspension microballoon,
18. calculate the number of microballoon with blood counting chamber
19. with 2-8 ℃ of microballoons, it is standby to keep in Dark Place.
Embodiment four: suspending chip rapid detection cholera vibrio O 139 method
1) TIANamp Bacteria DNA kit test kit extracts sample genomic dna to be checked.
2) pcr amplification purpose fragment, reaction conditions is as follows:
The PCR response procedures is: 94 ℃ of sex change 3min; Move 35 circulations: 94 ℃ of 30sec, 53 ℃ of 30sec, 72 ℃ of 40sec; At last, 72 ℃ are extended 3min.
3) hybridization: hybridization solution is formed: 33.3 μ l, 1.5 * TMAC (tetramethylammonium chloride, tetramethylammonium chloride), and the above-mentioned PCR product of 5 μ l adds 5000 microballoons that join with the probe coupling, and 1 * TE adds to 50 μ l with volume.Hybridization solution at 95 ℃ of sex change 4min, is hybridized more than the 15min for 52 ℃.
4) detect: the solution after the above-mentioned hybridization is all transferred in the 96 hole filter plates, microballoon is collected in vacuum filtration, with the resuspended microballoon of 2 μ g/ml S-R-PE (Streptavidin-R-phycoerythrin, streptavidin phycoerythrin), put 52 ℃ of 10min, Luminex detects.
Embodiment five: the specificity checking of suspending chip
Adopt embodiment four directions method, 45 strain bacterial strains are carried out the specificity checking, the result shows the cholera vibrio O 139 result that is positive, and other Non-cholera vibrio O139 is all negative, and specificity reaches 100%.
The bacterial strain specifying information is as follows:
Sequence table
＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL
＜120〉a kind of method for detecting Vibrio cholerae O 139 by using suspension chip technology
<140>
<141>
<160>3
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<400>1
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<400>2
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<400>3
Claims (8)
1. one kind is used for the suspending chip that cholera vibrio O 139 detects, and comprises the oligonucleotide probe of diagnose microballoons and this diagnose microballoons coupling connection, it is characterized in that this oligonucleotide probe is one section sequence on the cholera vibrio O 139 O-antigen synthase gene rfb.
2. be used for the suspending chip that cholera vibrio O 139 detects according to claim 1 is described, wherein the oligonucleotide probe sequence shown in sequence in the sequence table 3, its 5 ' carry out NH
2-(CH
2)
12Modify.
3. describedly be used for the suspending chips that cholera vibrio O 139 detects according to claim 1 or 2, it is characterized in that this chip can identify and detect cholera vibrio O 139.
4. a kind of suspending chip that detects cholera vibrio O 139 according to claim 1 and 2 is characterized in that comprising a pair of Auele Specific Primer, and primer sequence and carries out 5 ' Biotin to sequence 2 and modifies shown in sequence in the sequence table 1 and 2.
5. a kind of suspending chip that detects cholera vibrio O 139 according to claim 1 and 2, its preparation method comprises the steps: to prepare oligonucleotide probe; With the amido modified probe of pure water dilution; With the microballoon vibration of deposit, transfer to then in the brown EP pipe; Centrifugal collection microballoon adds MES liquid and vibrates; In the suspension microballoon, add above-mentioned probe, and vibration; The EDC solution that in microspheres solution, adds new preparation, vibration; Carry out incubation then; Add the 10mg/ml EDC of new configuration once more in microspheres solution, incubation is carried out in vibration then; In microspheres solution, add 0.02% Tween-20, centrifugal collection microballoon; With the resuspended microballoon of 0.1% SDS, centrifugal collection microballoon; With the TE microballoon that suspends; Number with blood counting chamber calculating microballoon, keeps in Dark Place by 2-8 ℃.
6. method that detects cholera vibrio O 139, its feature may further comprise the steps:
A), and primer is wherein carried out Biotin modify according to cholera vibrio O 139 rfb gene design and the synthetic Auele Specific Primer that is used for pcr amplification;
B) prepare sample genomic dna to be checked according to a conventional method, and use primer in the step a) to treat this genomic dna of sample and carry out pcr amplification, make simultaneously that Biotin modifies on the PCR product band;
C) suspending chip described in PCR product in the step b) and the claim 1 is hybridized;
D) hybridization suspending chip in the step c) is carried out streptavidin phycoerythrin mark, and it is detected fluorescent signal.
7. a kind of method that detects cholera vibrio O 139 according to claim 6 is characterized in that above-mentioned steps b) the PCR reaction system set up is as follows:
Form 1 PCR reaction system
The PCR response procedures is: 37 ℃ of incubation 5min; 94 ℃ of sex change 3min; Move 35 circulations: 94 ℃ of 30sec, 53 ℃ of 30sec, 72 ℃ of 40sec; At last, 72 ℃ are extended 3min.
8. a kind of method that detects cholera vibrio O 139 according to claim 6, wherein hybridization temperature is 52 ℃, hybridization time is greater than 15min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2528099C2 (en) * | 2013-06-25 | 2014-09-10 | Федеральное казенное учреждение здравоохранения "Российский научно-исследовательский противочумный институт "Микроб" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека ("РосНИПЧИ "Микроб") | Biological microchip for detection and multiparameter analysis of anticholeraic antibodies |
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| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
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2008
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| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2528099C2 (en) * | 2013-06-25 | 2014-09-10 | Федеральное казенное учреждение здравоохранения "Российский научно-исследовательский противочумный институт "Микроб" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека ("РосНИПЧИ "Микроб") | Biological microchip for detection and multiparameter analysis of anticholeraic antibodies |
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