CN103773756A - Preparation method for specific nanogold DNA probe - Google Patents
Preparation method for specific nanogold DNA probe Download PDFInfo
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- CN103773756A CN103773756A CN201310495123.2A CN201310495123A CN103773756A CN 103773756 A CN103773756 A CN 103773756A CN 201310495123 A CN201310495123 A CN 201310495123A CN 103773756 A CN103773756 A CN 103773756A
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Abstract
The invention discloses a preparation method for a specific nanogold DNA probe. The method comprises the following steps: preparing 13-nm nanogold particles AuNPs and connecting the specific probe to the 13-nm nanogold particles AuNPs, wherein the specific probe comprises the complementary sequences of 16SrRNA conserved sequences, two sections of complementary hairpin sequences of complementary sequences mingled with the 16SrRNA conserved sequences, complementary sequences mingled with the 16SrRNA conserved sequences, fluorescent molecular FAM of two sections of complementary hairpin sequences, and quenching groups BHQ1 of fluorescent molecular FAM. When being used for detecting urine samples, the specific nanogold DNA probe prepared through the preparation method has the advantages of obvious, quick, and sensitive effect and strong specificity over a traditional bacteria detection method for urine samples.
Description
Technical field
The present invention relates to detection of nucleic acids field, particularly relate to a kind of preparation method of specific nano gold DNA probe.
Background technology
Bacterial infection is general the property disease in the whole world, such as pneumonia, pulmonary tuberculosis, urinary tract infections etc. disease is all to be caused by bacterium, the mankind's normal work and study has been brought to great puzzlement.Wherein bacillary urinary tract infections is second largest general bacterial infection disease of tissue system.Each year approximately has 7,000,000 sufferers, exceedes 1,000,000 people and needs hospital care.The expense that is used for the treatment of every year this disease is huge, this has all caused great impact to national Economic development, social productive forces, and there is the detection of a big chunk expense for bacterium, but the topmost shortcoming of traditional Bacteria Detection of urine specimen is exactly length consuming time, approximately need about two days to obtaining detected result from sample collection, cultivating cloning bacteria is main time-consuming process.Clinically, because do not obtain in time Bacteria Detection result, only administration by rule of thumb, is everlasting and occurs using clinically untoward reaction and the bacterial drug resistance of the improper generation of medicine.At present, the molecular detection technology growing up and the application of round pcr, there is certain advantage, but all need the target sequence that increases, convenient and swift not (1, Journal ofclinical microbiology2009,47 (8), 2405-10.2, Journal of clinical microbiology2009,47 (7), 2067-78.).Immunoblot assay, needed step more loaded down with trivial details (3, Genome biology2007,8 (5), R93.).
Summary of the invention
Technical problem to be solved by this invention is, overcomes the shortcoming of prior art, provides quick, convenient, the accurate and detecting step of a kind of energy simply to detect the preparation method of the carrier of Urinary Tract Pathogens: A.In order to solve above technical problem, the invention provides a kind of preparation method of specific nano gold DNA probe, for the preparation of the specific nano gold DNA probe that can detect Urinary Tract Pathogens: A.This preparation method comprises preparation 13nm nm gold particles AuNPs and specific probe is connected to upper two steps of 13nm nm gold particles AuNPs.
The step of preparation 13nm nm gold particles AuNPs comprises: step 1-1 gets 9.8ml ddH2O heated and boiled; Step 1-2 adds the 50mM HAuCl4 of 200 μ l precoolings and follows and rock; Step 1-3 adds the 38.8mM bis-hydration citrate three sodiums of 1ml precooling and follows and rock; Step 1-4 boils 10min and follows and rock; Step 1-5 is at the ultrasonic 30min of room temperature.
The step that specific probe is connected on 13nm nm gold particles AuNPs comprises: the DNA probe that step 2-1 gets 30 μ l20 μ M joins in 30 μ l50mM tri-(2-propyloic) the phosphine TCEP of fresh preparation, and gets 10 μ l0.5M NaOH adjusting PH to 5; After step 2-21h, add 10nM13nm AuNPs100 μ l; After step 2-31h, add 50mM PBS33 μ l, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards; Step 2-4 at room temperature connects 12h, adds afterwards 40 μ l50mM TCEP; After step 2-51h, add 0.01%SDS and 8 μ l3M NaCl, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards; Step 2-6, after 6h, adds 8 μ l3M NaCl again, and ultrasonic 10s under room temperature heats 10min afterwards at 50 ℃; Step 2-7 repeating step 2-6; Step 2-8 at room temperature leaves standstill and connects 48h.
The technical scheme that the present invention further limits is:
The preparation method of aforesaid specific nano gold DNA probe, specific probe comprise 16SrRNA conserved sequence complementary sequence, clip two sections of complementary complementary hairpins of the complementary sequence of 16SrRNA conserved sequence, clip the complementary sequence of 16SrRNA conserved sequence and the fluorescence molecule FAM of two sections of complementary hairpins and quenching group BHQ1 thereof.
The preparation method of aforesaid specific nano gold DNA probe, hairpin is: 5 '-ACCGAGC-3 ' and 5 '-GCTCGGT-3 '.
The preparation method of aforesaid specific nano gold DNA probe, specific probe also comprises catenation sequence 5 '-GAGGTC-3 ', and is connected with 13nm nm gold particles AuNPs by catenation sequence 5 '-GAG GTC-3 '.
The preparation method of aforesaid specific nano gold DNA probe, specific probe is 5 '-/FAM/ACCG AGC CATCGT TTA CGG CGT GGA CTA CCA GGG GCT CGGT/BHQ1/GAG GTC-3 ', and wherein 5 '-CAT CGTTTA CGG CGT GGA CTA CCA GGG-3 ' is the complementary sequence of 16S rRNA conserved sequence.
The specific nano of preparation gold DNA probe in this way, for detection of Urinary Tract Pathogens: A, under state of nature due to two sections of complementary hairpin specific bindings, the specific probe of specific nano gold DNA probe in the form of a ring, fluorescence molecule FAM and quenching group BHQ1 thereof are mutually close, make fluorescence molecule FAM cancellation, whole specific nano gold DNA probe.In the time that temperature raises, the ring texture of specific nano gold DNA probe is opened, now run into 16SrRNA conserved sequence, in the time that temperature declines again the complementary sequence of 16SrRNA conserved sequence can with 16SrRNA conserved sequence specific binding, the fluorescence molecule FAM of specific nano gold DNA probe and quenching group BHQ1 thereof mutually away from, specific nano gold DNA probe sends fluorescence, reaches the object of detection.
The invention has the beneficial effects as follows: the preparation method of (1) specific nano gold of the present invention DNA probe simply should go, and partly preparing link has realized commercialization, is convenient to produce in batches and drop into practice; (2) the specific nano gold DNA probe that uses the inventive method to prepare is easy to use, on RTPCR instrument, can realize detection; (3) the specific nano gold DNA probe that utilizes the inventive method to prepare, in the time that Urinary Tract Pathogens: A is detected, compared to traditional method of detecting bacterium of urine specimen, has more the advantage of remarkable, quick, sensitive and high specificity.
accompanying drawing explanation
Fig. 1 is specific probe structural representation of the present invention
Fig. 2 is detection principle schematic of the present invention
Fig. 3 is single DNA probe in detecting intestinal bacteria ultimate value image of the present invention
Fig. 4 is that nanometer gold DNA probe of the present invention detects intestinal bacteria ultimate value image
Embodiment
Embodiment 1
The present embodiment provides a kind of preparation method of specific nano gold DNA probe, for detection of Urinary Tract Pathogens: A.The structure of this specific nano gold DNA probe is: on 13nm nm gold particles AuNPs, in conjunction with more than one specific probe, this specific probe is 5 '-/FAM/ACCG AGC CAT CGT TTA CGG CGT GGA CTA CCA GGG GCTCGGT/BHQ1/GAG GTC-3 '.Wherein FAM is a kind of fluorescence molecule, and BHQ1 is the quenching group of fluorescence molecule FAM, and the two is not luminous near rear FAM fluorescence molecule, luminous away from rear FAM fluorescence molecule.As shown in Figure 1, in figure with complete black circular signal FAM fluorescence molecule, with complete black rectangle signal BHQ1 quenching group, 5 '-ACCG AGC-3 ' and 5 '-GCT CGGT-3 ' is two sections of complementary hair fastener sequences, its effect be make this specific probe in the time target sequence not detected in the form of a ring, FAM fluorescence molecule and quenching group BHQ1 is adjacent to each other does not fluoresce.5 '-CAT CGT TTA CGG CGT GGA CTA CCAGGG-3 ' is the complementary sequence of 16SrRNA conserved sequence, and 16SrRNA gene is the corresponding DNA sequence dna of rRNA of encoding on bacterium, is present in the germy genome of institute.5 '-GAG GTC-3 ' is catenation sequence, and specific probe is connected on the nm gold particles AuNPs of 13nm by it.
The detection principle of the above-mentioned gold of the specific nano for detection of Urinary Tract Pathogens: A DNA probe as shown in Figure 2, when opening the specific nano gold DNA probe generation specific binding of ring texture after the 16SrRNA conserved sequence on bacterium and heating, while returning normal temperature, also cannot form ring texture, FAM fluorescence molecule is luminous.If do not have 16SrRNA conserved sequence to participate in, after the heating of specific nano gold DNA probe, ring texture is opened, and returns under normal temperature and forms again ring texture, and FAM fluorescence molecule is not luminous.
After the specific probe of the above-mentioned gold of the specific nano for detection of Urinary Tract Pathogens: A DNA probe designs, transfer to the company of market operation to produce, thus in the time of this specific nano gold DNA probe of preparation, first, preparation 13nm nm gold particles AuNPs.Preparing required vessel all needs repeated ultrasonic to clean, and thoroughly removes metal ion.Concrete preparation process: (1) gets 9.8ml ddH
2(object is to remove O to O heated and boiled
2); (2) add the 50mM HAuCl of 200 μ l precoolings
4(in adition process, follow and rock); (3) add the 38.8mM bis-hydration citrate three sodiums (follow and rock in adition process) of 1ml precooling; (4) boil 10min (follow and rock in boiling part); (5) at the ultrasonic 30min of room temperature.By at the lower gold particle pattern of observing preparation of transmission electron microscope (Transmission electron microscope, TEM), can judge the about 13nm of nm gold particles size of preparation.Secondly, specific probe is connected on 13nm nm gold particles AuNPs.Concrete connection procedure: the DNA probe that (1) gets 30 μ l20 μ M joins in 30 μ l50mM tri-(2-propyloic) phosphines (TCEP) of fresh preparation, and get 10 μ l0.5MNaOH adjusting PH to 5; (2) after 1h, add 10nM13nmAuNPs100 μ l; (3) after 1h, add 50mM PBS33 μ l, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards; (4) at room temperature connect 12h, add afterwards 40 μ l50mM TCEP; (5), after 1h, add 0.01%SDS and 8 μ l3M NaCl, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards; (6) after 6h, again add 8 μ l3M NaCl, ultrasonic 10s under room temperature heats 10min afterwards at 50 ℃; (7) repeating step (6); (8) at room temperature leave standstill and connect 48h.
Intestinal bacteria account for 80% of urinary tract infection pathogenic bacteria, for verifying beneficial effect of the present invention, cultivate bacillus coli DH 5 alpha, and collect bacterium liquid, detect, to verify susceptibility and the specificity of this probe.The Escherichia coli bacteria liquid collection 1ml that gets cultivation is centrifugal, adds 10 μ l lysates and 10 μ l0.5M NaOH ruptured cells, discharges 16SrRNA, adds probe, preparation RTPCR20 μ l reaction system, and wherein PBS final concentration is 10mM.Testing process is carried out in RTPCR instrument, 80 ℃ of setting programs, and 3min, every 20s reduces by 1 ℃, is reduced to 40 ℃ from 80 ℃.Analysis of fluorescence value, nanometer gold DNA probe is higher than self Tm value, and ring texture is all opened, and along with the decline of temperature, in the situation that there is no target sequence, probe forms ring texture again, and fluorophor is quenched, and does not have fluorescent signal to occur; When occurring target sequence, target sequence is attached on probe, probe no longer forms ring texture, fluorophor is away from quenching group, in RTPCR instrument, fluorescence can be detected, fluorescent value is carried out to statistical study, judge whether to exist specific sequence with this, whole experimentation can be controlled in 1h.In conjunction with Fig. 3 and Fig. 4, DNA probe and nanometer gold DNA probe are detected to bacterium liquid result and contrast, the DNA probe sensitivity that shows to connect nanometer gold is 10 of independent DNA probe
3doubly, proves that the Nano-Au probe that uses the method to prepare really has fast compared with traditional detection method, the feature of highly sensitive and high specificity.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (6)
1. a preparation method for specific nano gold DNA probe, is characterized in that: comprise preparation 13nm nm gold particles AuNPs and specific probe is connected to upper two steps of 13nm nm gold particles AuNPs;
The step of described preparation 13nm nm gold particles AuNPs comprises:
Step 1-1 gets 9.8ml ddH2O heated and boiled;
Step 1-2 adds the 50mM HAuCl4 of 200 μ l precoolings and follows and rock;
Step 1-3 adds the 38.8mM bis-hydration citrate three sodiums of 1ml precooling and follows and rock;
Step 1-4 boils 10min and follows and rock;
Step 1-5 is at the ultrasonic 30min of room temperature;
The described step that specific probe is connected on 13nm nm gold particles AuNPs comprises:
The DNA probe that step 2-1 gets 30 μ l20 μ M joins in 30 μ l50mM tri-(2-propyloic) the phosphine TCEP of fresh preparation, and gets 10 μ l0.5M NaOH adjusting PH to 5;
After step 2-21h, add 10nM13nm AuNPs100 μ l;
After step 2-31h, add 50mM PBS33 μ l, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards;
Step 2-4 at room temperature connects 12h, adds afterwards 40 μ l50mM TCEP;
After step 2-51h, add 0.01%SDS and 8 μ l3M NaCl, and ultrasonic 10s at room temperature, at 50 ℃, heat 10min afterwards;
Step 2-6, after 6h, adds 8 μ l3M NaCl again, and ultrasonic 10s under room temperature heats 10min afterwards at 50 ℃;
Step 2-7 repeating step 2-6;
Step 2-8 at room temperature leaves standstill and connects 48h.
2. the preparation method of specific nano gold DNA probe according to claim 1, it is characterized in that: described specific probe comprise 16SrRNA conserved sequence complementary sequence, clip two sections of complementary complementary hairpins of the complementary sequence of 16SrRNA conserved sequence, clip the complementary sequence of 16SrRNA conserved sequence and the fluorescence molecule FAM of two sections of complementary hairpins and quenching group BHQ1 thereof.
3. the preparation method of specific nano gold DNA probe according to claim 2, is characterized in that: described hairpin is: 5 '-ACCGAGC-3 ' and 5 '-GCTCGGT-3 '.
4. the preparation method of specific nano gold DNA probe according to claim 3, is characterized in that: specific probe also comprises catenation sequence, and is connected with the nm gold particles AuNPs of 13nm by catenation sequence.
5. the preparation method of specific nano gold DNA probe according to claim 4, is characterized in that: catenation sequence is 5 '-GAG GTC-3 '.
6. according to the preparation method of arbitrary described specific nano gold DNA probe in claim 1 to 5, it is characterized in that: described specific probe is 5 '-/FAM/ACCG AGC CAT CGT TTA CGG CGT GGA CTA CCA GGG GCT CGGT/BHQ1/GAG GTC-3 ', and wherein 5 '-CAT CGT TTA CGG CGT GGA CTA CCA GGG-3 ' is the complementary sequence of 16SrRNA conserved sequence.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106119344A (en) * | 2016-06-20 | 2016-11-16 | 清华大学 | A kind of combined with fluorescent intensity and the nano-probe of fluorescence polarization detection DNA |
| CN108148810A (en) * | 2017-12-14 | 2018-06-12 | 临沂大学 | A kind of aptamer and the RNA films of luminol-gold nanoparticle functionalization and its preparation method and application |
| CN110296961A (en) * | 2018-03-22 | 2019-10-01 | 中国科学院上海高等研究院 | The building and application of controllable Nano-Au probe based on double block D NA |
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| CN102080131A (en) * | 2010-12-10 | 2011-06-01 | 郑州大学 | Hairpin type DNA modified gold colloid nano particles and synthesizing method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119344A (en) * | 2016-06-20 | 2016-11-16 | 清华大学 | A kind of combined with fluorescent intensity and the nano-probe of fluorescence polarization detection DNA |
| CN106119344B (en) * | 2016-06-20 | 2020-06-09 | 清华大学 | Nano probe for detecting DNA by combining fluorescence intensity and fluorescence polarization |
| CN108148810A (en) * | 2017-12-14 | 2018-06-12 | 临沂大学 | A kind of aptamer and the RNA films of luminol-gold nanoparticle functionalization and its preparation method and application |
| CN108148810B (en) * | 2017-12-14 | 2020-06-12 | 临沂大学 | Aptamer and luminol-gold nanoparticle functionalized RNA membrane and preparation method and application thereof |
| CN110296961A (en) * | 2018-03-22 | 2019-10-01 | 中国科学院上海高等研究院 | The building and application of controllable Nano-Au probe based on double block D NA |
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Effective date of registration: 20220926 Address after: Room 602, No. 66, Guangzhou Road, Development Zone, Nantong City, Jiangsu Province, 226000 Patentee after: Jiangsu Senxinda Biotechnology Co.,Ltd. Address before: Weigang Xuanwu District of Nanjing Jiangsu province 210095 No. 1 Patentee before: NANJING AGRICULTURAL University |