CN101407839B - Method for detecting Shigella flexneri by using suspension chip technology - Google Patents
Method for detecting Shigella flexneri by using suspension chip technology Download PDFInfo
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- CN101407839B CN101407839B CN 200810181198 CN200810181198A CN101407839B CN 101407839 B CN101407839 B CN 101407839B CN 200810181198 CN200810181198 CN 200810181198 CN 200810181198 A CN200810181198 A CN 200810181198A CN 101407839 B CN101407839 B CN 101407839B
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Abstract
The invention relates to a suspension chip used for detecting Sh.flexneri and a detection method thereof. The suspension chip comprises a microballoon vector and an oligonucleotide probe fixed on the vector, wherein, the oligonucleotide probe is one section of DNA sequence in the specific gene rfc of the Sh.flexneri screened from the O-antigen gene cluster of Shigella. After a designed primer is used for amplifying and marking the DNA of a sample gene group to be detected, the suspension chip is utilized for carrying out cross-breeding; and whether the sample to be detected has the Sh.flexneri or not can be detected according to the intensity of the cross-breeding fluorescence. The suspension chip has high detection sensitivity which can achieve the level of the DNA of the fg-grade gene group, can meet the demands for detecting clinic samples and environment samples and is characterized by high specificity, simple operation, low cost, and the like, and is easy to be popularized. A UNG-Taq enzyme PCR reaction system is adopted, and therefore the false positive result of the PCR is greatly reduced.
Description
Technical field
The present invention relates to a kind of molecular Biological Detection chip, be specifically related to a kind of suspending chip for the Shigellae detection.
Background technology
The suspending chip technology is that biochip technology is combined a kind of new technology that produces with low cytometric analysis, that a kind of multiple data obtains and analysis platform, complete " multi-functional many indexs Synchronization Analysis system " (Flexible Multiple Analyte Profiling by name, xMap), be also referred to as Multi-Analyte SuspensionArray, organic combination fluorescence-encoded micro-beads, laser technology, micro-fluidic technologies, fast signal process and data analysis system, namely 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 comprised of three parts: 1, about microsphere diameter 5.6 μ m, be made of polystyrene, surface band has an appointment 10
8Individual carboxyl site can be coupled with peptide bond 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.Microballoon and the determinand of mark bioprobe are carried out specific combination, again with the reaction of the reporter molecules of the third fluorescein, in a reacting hole, can detect 100 kinds of different molecules of interest in the sample simultaneously.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 identify different colour code microballoons, carry out qualitative detection; Another laser probe can excite the fluorescein of reporter molecules, by measure microballoon with the intensity of reporter molecules fluorescent signal, 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, the fluorescence that twice laser excites 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 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 by detecting fluorescence signal intensity, carry out quantitative analysis to detecting thing.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 particular advantages with other oroteins and nucleic acid detection method:
1) carboxyl of applied range microsphere surface can be coupled with nucleic acid probe and protein molecular, 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 band has an appointment 10
8Individual carboxyl site is coupled oligonucleotide probe, antibody or antigen with covalent, and probe density is high, and the signal of generation is strong; Use fluoroscopic examination, amplified reaction signal, susceptibility improves greatly.
3) biological respinse that carries out of good reproducibility carries out in liquid phase environment, be beneficial to biomolecules native conformations such as keeping nucleic acid, albumen, overcome the impact 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 impact to detect need not wash-out and removes background;
5) high-throughput can carry out qualitative and quantitative analysis to the multiple different molecules of interest in the same sample simultaneously, has namely improved detection information flux, has saved again the sample consumption;
6) speed of response and detection speed have rapidly and efficiently greatly been improved.Because the biological respinse that carries out carries out in liquid phase environment, hybridization only needed to finish in 15 minutes, had improved speed of response; In 35-60 minute, can reach simultaneously respectively to 96 different samples the detection of 100 kinds of indexs; Utilize 96 orifice plates and automatic sample handling system, can carry out continuously the detection of n * 96 sample.
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 testing cost only needs 2.5-5.0 unit;
8) be easy to stdn based on These characteristics, so that this technology can be accomplished stdn, be easy to 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).
Human extremely sensitive to Shigellae, only need be less than ten bacterium can cause people's infection, so its infectivity is strong, and hazardness is large.Show that according to domestic related data Shigellae ranks first in China infectious diarrhea pathogenic bacteria; American National is declared disease monitoring system and public health Test Information system data and is shown, the annual case that causes because of Shigellae is up to 448,240 examples, dead 70 examples; The WHO annual report points out that the bacterial diarrhea case reaches 1.5-2.5 hundred million people, dead 650,000 in Asian, African and Latin American various countries.The clear Shigellae is essential items for inspection in the food in State Administration for Quality Supervision and Inspection and Quarantine 2002 No. 25,26 commands.
Four serotypes of Shigellae tool, shigella flexneri, shigella dysenteriae, Shigella bogdii,
Shigella sonnei, the obvious region of different serotypes Shigellae sickness rate tool; Account for the 80%(Ma Hong of Shigellae sickness rate at China's shigella flexneri sickness rate, 2006), be the Main Pathogenic Bacteria group.At present, in the ripe detection technique of Shigellae, level detection can only be realized belonging to, the information on kind of the level can't be further obtained.And therefore the isotype Shigellae does not realize that in larger gaps of aspect tool such as minimum infective dose, pathogenic, resistance the detection of kind of level will provide important information for clinical treatment, provides strong technical support for foodstuff production, environmental monitoring simultaneously yet.
Shigella flexneri O-antigen gene cluster bioinformatic analysis is found, one section high special sequence of this gene cocooning tool, this sequence is positioned on the O-antigen pol gene, only with salmonella typhi less homology is arranged, and with other serotype Shigellae, also have a larger difference (Huo-Shu H.Houng, 1997).By to the analysis of this gene order on molecular biology, can realize detection, evaluation to shigella flexneri at molecular level.
Summary of the invention
Purpose of the present invention aims to provide a kind of suspending chip that detects shigella flexneri.Suspending chip provided by the present invention comprises diagnose microballoons and the specificity oligonucleotide probe that is coupled of microballoon therewith, and wherein sequence oligonucleotide probe is one section sequence of shigella flexneri O-antigen pol gene.The invention also discloses one section oligonucleotide probe sequence, this sequence is: S.f rfc-Probe:5'-NH
2-(CH
2)
12-GAACCAAATCCTCTTCCAAAC-3' shown in sequence in the sequence table 3, and carries out NH at 5'
2-(CH
2)
12Modify.Suspending chip of the present invention also comprises a pair of Auele Specific Primer in addition, and upstream primer is: B-S.f rfc-F:5'-CGGATCACGCAGTGAAGAT-3'(sees sequence 1 in the sequence table, carries out the Biotin mark); Downstream primer is: S.f rfc-R:5'-CCAATAACGCCTGTTTTCTGA-3'(sees sequence 2 in the sequence table).Above-mentioned primer can increase and comprise one section rfc gene order of above-mentioned probe sequence.
Another object of the present invention is to provide the preparation method of above-mentioned suspending chip, the 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, then transfer 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; Then carry out incubation;
7) again add the new 10mg/ml EDC that disposes in microspheres solution, then vibration carries out incubation;
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.
Using suspending chip detection shigella flexneri of the present invention carries out by the following method.
According to shigella flexneri rfc 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 according to a conventional method sample genomic dna to be checked as template, use above-mentioned Auele Specific Primer to carry out pcr amplification, make simultaneously Biotin modification on the PCR product band; With the suspending chip hybridization of PCR product and preparation, namely hybridize with the probe that is coupled on microballoon, the hybridization suspending chip is carried out streptavidin phycoerythrin mark, then detect fluorescent signal by flow cytometer.
Another object of the present invention provides the false-positive method of a kind of PCR of overcoming.The 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.
The suspending chip of invention can carry out Identification and detection to Shigella flexneri, have high specific, highly sensitive, fast, low-cost and easy-to is in characteristics such as popularizations.
Description of drawings
Fig. 1 specific test result;
Embodiment:
For further specifying the application of the present invention in shigella flexneri detects, describe especially exemplified by following preferred embodiment, but application of the present invention is not limited to embodiment.
Embodiment one: the design of primer and synthetic
1) sequence obtains: by to the sequential analysis of shigella flexneri O-antigen gene cluster, choose specific gene rfc as target sequence, and obtain this gene order from the GenBank public database, be numbered L07293.1;
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 selection: the primer of software output is carried out suitable manual regulation, increase or reduce several bases, then carry out online Blast comparison at GenBank, choose the high primer of specificity, and will be wherein carries out the terminal Biotin mark of 5'.The upstream primer sequence is sequence 1 in the B-S.f rfc-F:5'-Biotin-CGGATCACGCAGTGAAGAT-3'(sequence table), downstream primer is sequence 2 in the S.f rfc-R:5'-CCAATAACGCCTGTTTTCTGA-3'(sequence table), the amplified fragments size is 174bp;
4) primer is synthetic: the synthetic downstream primer S.f rfc-R of worker, the upstream primer B-S.f rfc-F of the synthetic Biotin mark of Dalian TakaRa are given birth in Shanghai.
Embodiment two: the design of probe and synthetic
5) sequence obtains: at the non-guiding region designing probe of above-mentioned amplified fragments;
6) 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;
7) probe is selected: the probe of software output is carried out suitable manual regulation, increase or reduce several bases, then carry out online Blast comparison at GenBank, choose the high probe of specificity, carry out NH at the 5' end
2-(CH
2)
12Modify, selected probe sequence is S.f rfc-Probe:5'-NH
2-(CH
2)
12 Sequence 3 in the-GAACCAAATCCTCTTCCAAAC-3'(sequence table);
8) probe is synthetic: Dalian TakaRa synthesizes above-mentioned probe.
The preparation method of embodiment three suspending chips
1. dilute amido modified probe to 1mM (1nanomole/ 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 pH 4.5, vibration 20sec;
6. the probe that adds 2 μ l 1mM in the suspension microballoon, vibration 20sec;
7. prepare fresh 10mg/ml EDC(1-ethyl-3-[3dimethylaminopropyl] carbodiimidehydrochloride, 1-ethyl-3-3-dimethylaminopropyl carbodiimide), use dH
2O;
8. in microspheres solution, add the new 10mg/ml EDC that disposes of 2.5 μ l, vibration;
9. incubation 30min under the room temperature dark condition;
10. again prepare fresh 10mg/ml EDC, use dH
2O;
11. in microspheres solution, add the new 10mg/ml EDC that disposes of 2.5 μ l, vibration;
12. incubation 30min under the room temperature dark condition;
13. in microspheres solution, add 1.0mL 0.02% Tween-20;
14.8000 the centrifugal 1-2min of * g collects microballoon;
15. abandon supernatant, with the resuspended microballoon of 1.0mL 0.1%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 microballoon 2-8 ℃, it is for subsequent use to keep in Dark Place.
Embodiment four: suspending chip rapid detection shigella flexneri 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:
Form 1PCR reaction system
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 forms: 33.3 μ l, 1.5 * TMAC(tetramethylammonium chloride, tetramethylammonium chloride), the above-mentioned PCR product of 5 μ l adds 5000 microballoons that are coupled with probe, 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
Specific test:
With suspending chip of the present invention different strains is carried out the specificity checking, its sensing range comprises following three class bacterial strains:
A) belong to aforesaid 1 strain shigella flexneri and the non-shigella flexneri of 4 strains;
B) with nearer intestinal bacteria, the Salmonellas of Shigellae sibship;
C) other relevant bacterial strain.
The bacterial strain specifying information is as follows:
For above-mentioned bacterial strains, detect by the method among the embodiment four, detected result shows the shigella flexneri result that is positive, and other non-shigella flexneri is all negative, and specificity reaches 100%.
Claims (3)
1. one kind is used for the suspending chip that shigella flexneri detects, and is comprised of diagnose microballoons and the oligonucleotide probe that is fixed on this diagnose microballoons, it is characterized in that: the sequence of this oligonucleotide probe shown in sequence in the sequence table 3, its 5 ' carry out NH
2-(CH
2)
12Modify.
2. one kind is used for the primer that shigella flexneri detects, and it is characterized in that: primer sequence and carries out 5 ' Biotin to sequence 1 and modifies shown in sequence in the sequence table 1 and 2.
3. the preparation method of the described suspending chip that detects for Shigellae of claim 1 comprises the steps: to prepare oligonucleotide probe; With the amido modified probe of pure water dilution; With the microballoon vibration of deposit, then transfer 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; Then carry out incubation; Again add the new 10mg/ml EDC that disposes in microspheres solution, then vibration carries out incubation; 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 ℃.
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| CN 200810181198 CN101407839B (en) | 2008-11-27 | 2008-11-27 | Method for detecting Shigella flexneri by using suspension chip technology |
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| CN 200810181198 CN101407839B (en) | 2008-11-27 | 2008-11-27 | Method for detecting Shigella flexneri by using suspension chip technology |
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| CN101407839B true CN101407839B (en) | 2013-03-20 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1986830A (en) * | 2005-12-21 | 2007-06-27 | 天津生物芯片技术有限责任公司 | Gene chip for serotype detection of shigella and its detection process and reagent kit |
-
2008
- 2008-11-27 CN CN 200810181198 patent/CN101407839B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1986830A (en) * | 2005-12-21 | 2007-06-27 | 天津生物芯片技术有限责任公司 | Gene chip for serotype detection of shigella and its detection process and reagent kit |
Non-Patent Citations (4)
| Title |
|---|
| 《Multiplex, Bead-Based Suspension Array for Molecular Determination of Common Salmonella Serogroups》;Collette Fitzgerald等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20070718;第45卷(第10期);第3323－3334页 * |
| 《Quantitative, Multiplexed Detection of Salmonella and Other Pathogens by Luminex R xMAP TM Suspension Array》;Sherry A.Dunbar等;《Methods in Molecular Biology》;20071231;第394卷;第1－19页 * |
| ColletteFitzgerald等.《Multiplex Bead-Based Suspension Array for Molecular Determination of Common Salmonella Serogroups》.《JOURNAL OF CLINICAL MICROBIOLOGY》.2007 |
| SherryA.Dunbar等.《Quantitative Multiplexed Detection of Salmonella and Other Pathogens by Luminex R xMAP TM Suspension Array》.《Methods in Molecular Biology》.2007 |
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