CN101078028B - Twice bio-bar-code nucleic acid detecting technique - Google Patents
Twice bio-bar-code nucleic acid detecting technique Download PDFInfo
- Publication number
- CN101078028B CN101078028B CN2007100389293A CN200710038929A CN101078028B CN 101078028 B CN101078028 B CN 101078028B CN 2007100389293 A CN2007100389293 A CN 2007100389293A CN 200710038929 A CN200710038929 A CN 200710038929A CN 101078028 B CN101078028 B CN 101078028B
- Authority
- CN
- China
- Prior art keywords
- probe
- magnetic bead
- nucleic acid
- nano
- bar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kind of twice bio-bar-code nuclear acid technique, comprising steps of: (1)hybridizing magnetic beads probes and target DNA mixed with namo-gold probe; (2)isolating and washing magnetic beads, adding eluent, still insulation hybridization, magnetic separation and washing; (3) suspension and precipitation again, adding the second gold namo-probe, mixing and hybridizing; (4) isolating magnetic beads, washing, adding eluent, removing magnetic beads, detecting supemetant. It is easy automation, and the specificity and sensitivity are high, which can be applied in broad field.
Description
Technical field
The invention belongs to the biotinylated nucleic acid detection range, particularly relate to a kind of twice bio-bar-code nucleic acid detecting technique.
Background technology
Nucleic acid detection technique can be divided into two classes, and the one, rely on enzymatic reaction, comprise various index amplification techniques, based on the linear amplification technology of ligase enzyme with based on the intrusion method of special construction specificity restriction endonuclease based on polymerase chain reaction (PCR); The 2nd, non-dependence enzymatic reaction mainly detects by hybridization fixed target sequence.
First kind nucleic acid detection technique can increase to target sequence, but because the storage movement requirement strictness of enzyme, and needs professional's operation usually, and complex operation, and consuming time longer, price is relatively costly, and automatization has difficulties.
If adopt the method for hybridization, because target sequence is not had amplification in earlier stage, relative detection sensitivity can not show a candle to the detection method of enzymatic reaction, but operation is easier, is content with very little at the detection needs of various complex sites.
Nanometer gold is applied to proteinic detection at first.Because nm gold particles can produce the variation of optical property in the accumulative process, color is by red stain indigo plant, thus mark the nm gold particles of nucleic acid probe can detect nucleic acid (Science, 1997) in the solution by the method for hybridization.
Along with the maturation of labeling technique and staining technique, the nano gold mark probe mainly contains both direction: the one, at mark mark fluorescent molecule again on the probe of nanometer gold, after realizing that hybridization separates, can be directly used in detection; The 2nd, mark the probe of nanometer gold after hybridization separates, on chip, hybridize again and dye colour developing with silver.Before a kind of method easy and simple to handle fast, but owing to there not be the process of amplifying, the sensitivity of detection is limited, a kind of method in back is because the amplification process that has silver to dye, and the hybridizing method of use biochip, signal is more concentrated, makes its detection sensitivity reach 10
-15Mol, near the detection sensitivity of PCR, but complex operation, the condition strictness, consuming time longer.
Bio-bar-code amplification (BCA) technology that starts from 2004 then attempts to address the above problem, and reaches the purpose that rapid sensitive detects, and whole system comprises magnetic bead, nanometer gold and five kinds of probes.Mark target sequence part a kind of and to be measured complementary probe on the magnetic bead, be total to two kinds of probes of mark on the nanometer gold, a kind ofly be and the other end parts complementary probe of target sequence to be measured, another kind is to be used for the probe that signal amplifies, the ratio of these two groups of probes on the nanometer gold surface is 1: 100, and second kind of probe also has one section complete complementary dna fragmentation, as the amplifying signal that detects.In the process of hybridization, by target sequence two kinds of nano particles are coupled together, by Nano-Au probe absorption and the purifying of magnetic separator with hybridization, the amplifying signal that will be used to detect discharges again, detects by corresponding molecule means (as biochip).The main advantage of this method is to have adopted the signal amplification reaction of target sequence to be measured having been carried out non-enzymatic.
The BCA technology has the advantage of two class nucleic acid detection techniques, and the entire operation process has only hybridization, separation, washing and wash-out, and last fluoroscopic examination.Surface markers probe by nanometer gold does not rely on and enzymatic reaction, simultaneously target sequence is effectively increased.Entire operation can be finished in centrifuge tube, separation and purification is also based on the magnetic separation principle of nanometer magnetic bead, the entire operation process can realize automatization completely, and owing to there is not the preservation problem of bioactive macromolecule, can be widely used in various places, as remote districts, on-the-spot blood sample examination, battlefield etc.
2005, the research that continuation such as Mirkin have been done a nearly step to the BCA technology improved, and the probe that uses is reduced to two kinds, a kind ofly was marked on the magnetic bead complementaryly with the target sequence part, and another kind is marked on the nanometer gold complementary with target sequence the other end part.Be marked at probe on the nanometer gold after finishing hybridization, separation, washing, available elutriant elutes from the surface of Nano-Au probe.Because diameter is at the nm gold particles of 30nm, the surface on average can 200～300 probe molecules of mark, thereby the probe that elutes can be used as amplifying signal and is used for Molecular Detection, detection mode can be with tense marker the detection of fluorescence, the further amplifying signal that perhaps is used for chip detects.According to the difference of detection means, the entire operation time is respectively in 3 hours and 6 hours.According to the literature, be 3 hours the shortest detection time of BCA technology, but up to now, detection method does not have report with the detection that the signal probe that utilizes capillary electrophoresis to BCA carries out fast high-sensitive fast.
Summary of the invention
The object of the invention provides a kind of twice bio-bar-code nucleic acid detecting technique, and this method is simple to operate, and specificity and sensitivity are higher, are easy to automatization, have wide practical use.
A kind of twice bio-bar-code nucleic acid detecting technique of the present invention comprises the steps:
(1) magnetic bead probe, DNA to be measured and first group of Nano-Au probe mix hybridization;
(2) separate magnetic bead, washing adds elutriant, continues insulation hybridization, and magnetic separates, washing;
(3) suspension precipitation again adds second group of Nano-Au probe, mixes hybridization;
(4) separate magnetic bead, washing adds elutriant, and the degaussing pearl detects supernatant liquor.
Described magnetic bead particles size is 0.5～1.5 μ m;
Described nm gold particles is 30nm;
Described DNA concentration to be measured is 10fm-100pm;
Described magnetic bead probe is meant that an end is a biotin modification, and an end is the abiotic plain probe of modifying, and length is 30～50 bases;
Described first group of Nano-Au probe is meant that an end is a sulfydryl modification, and an end is the probe of non-sulfydryl modification;
Described second group of Nano-Au probe is meant that an end is a sulfydryl modification, and an end is the probe that fluorescence is modified;
Described hybridization temperature is 30-55 ℃, and preferred 55 ℃, hybridization time is 5-60 minute, preferred 15 minutes.
The magnetic bead probe length that the present invention uses is 30～50 bases, be divided into two parts, an end of biotin modification is connected with magnetic bead, and this part sequence is a stochastic sequence, not with other probe and target complement sequence to be checked, an end complementation of an end of abiotic plain modification and target sequence to be checked.Nano-Au probe has two groups, and an end of first group of Nano-Au probe is a sulfydryl modification, and this part base can to modify end complementary with magnetic bead abiotic plain, but not the sulfydryl modification end then with target complement sequence to be checked.Second group of Nano-Au probe one end is sulfydryl modification, and an end is that fluorescence is modified, and the sulfydryl modification end is a stochastic sequence, and not with other probe and target complement sequence to be checked, and fluorescence is modified the non-sulfydryl modification end complementation of end and first group of probe.
Beneficial effect of the present invention: (1) does not rely on enzymatic reaction, with target sequence amplification 10
4～10
5Doubly, amplification efficiency reacts near PCR, and owing to do not need the participation of bioactive macromolecule, the transportation of reagent is preserved relatively easy; (2) owing to the hybridization characteristic of Nano-Au probe itself, this method is than general hybridizing method specificity height, and background is relatively low; (3) entire operation is simple, and participation that need not the professional easily is automated detection.
Description of drawings
Fig. 1 is the synoptic diagram of twice BCA technology for detection nucleic acid.
M is a magnetic bead, and N is a nanometer gold, and 1 is magnetic bead probe, and 2 is determined nucleic acid, and 3 is first group of Nano-Au probe, and 4 is second group of Nano-Au probe, and has fluorescent mark.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
With an end is magnetic bead (0.5～1.5 μ m) probe (30～50 bases) of biotin modification, one end is that nanometer gold (30nm) probe of sulfydryl modification mixes with sample HBV viral DNA, hybridized 15 minutes for 55 ℃, handle rapidly with magnetic separator, with washings washing 4～5 times, add elutriant, reacted 5～10 minutes, continue insulation hybridization 20～30 minutes, handle with magnetic separator, wash repeatedly, adding second group of one end is the Nano-Au probe that fluorescence is modified for the sulfydryl modification the other end, hybridized 15 minutes for 55 ℃, handle rapidly with magnetic separator,, add elutriant with washings washing 4～5 times, reacted 5～10 minutes, handle with magnetic separator, remove magnetic bead, detect supernatant liquor.
Claims (4)
1. twice bio-bar-code nucleic acid detection method comprises the steps:
(1) magnetic bead probe, DNA to be measured and first group of Nano-Au probe mix hybridization;
(2) separate magnetic bead, washing adds elutriant, continues insulation hybridization, and magnetic separates, washing;
(3) suspension precipitation again adds second group of Nano-Au probe, mixes hybridization;
(4) separate magnetic bead, washing adds elutriant, and the degaussing pearl detects supernatant liquor;
Described magnetic bead probe length is 30～50 bases, be divided into two parts, an end of biotin modification is connected with magnetic bead, and this part sequence is a stochastic sequence, not with other probe and target complement sequence to be checked, an end complementation of an end of abiotic plain modification and target sequence to be checked; Nano-Au probe has two groups, and an end of first group of Nano-Au probe is a sulfydryl modification, and this part base can to modify end complementary with magnetic bead abiotic plain, but not the sulfydryl modification end then with target complement sequence to be checked; Second group of Nano-Au probe one end is sulfydryl modification, and an end is that fluorescence is modified, and the sulfydryl modification end is a stochastic sequence, and not with other probe and target complement sequence to be checked, and fluorescence is modified the non-sulfydryl modification end complementation of end and first group of probe.
2. a kind of twice bio-bar-code nucleic acid detection method according to claim 1, it is characterized in that: described magnetic bead particles size is 0.5～1.5 μ m, and the nm gold particles size is 30nm, DNA concentration to be measured is 10fm-100pm.
3. a kind of twice bio-bar-code nucleic acid detection method according to claim 1, it is characterized in that: described hybridization temperature is 30-55 ℃, the time is 5-60 minute.
4. a kind of twice bio-bar-code nucleic acid detection method according to claim 3, it is characterized in that: described hybridization temperature is 55 ℃, the time is 15 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100389293A CN101078028B (en) | 2007-03-30 | 2007-03-30 | Twice bio-bar-code nucleic acid detecting technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100389293A CN101078028B (en) | 2007-03-30 | 2007-03-30 | Twice bio-bar-code nucleic acid detecting technique |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101078028A CN101078028A (en) | 2007-11-28 |
| CN101078028B true CN101078028B (en) | 2011-08-31 |
Family
ID=38905693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100389293A Expired - Fee Related CN101078028B (en) | 2007-03-30 | 2007-03-30 | Twice bio-bar-code nucleic acid detecting technique |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101078028B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8372340B2 (en) | 2005-10-19 | 2013-02-12 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
| CN101250585B (en) * | 2008-03-28 | 2012-01-25 | 广州市搏克生物技术有限公司 | Method for detecting DNA, RNA and ultramicro-amount protein |
| CN102414557A (en) * | 2009-03-11 | 2012-04-11 | 新加坡科技研究局 | Electrical sensor for ultrasensitive nucleic acid detection |
| TWI744234B (en) | 2015-06-05 | 2021-11-01 | 瑞士商諾華公司 | Cell processing system and method of flow-through cell processing |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1978668A (en) * | 2006-12-18 | 2007-06-13 | 中国人民解放军第三军医大学第三附属医院 | Method for detecting fetus father DNA mononucleotide difference from plasma in pregnaut women |
-
2007
- 2007-03-30 CN CN2007100389293A patent/CN101078028B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1978668A (en) * | 2006-12-18 | 2007-06-13 | 中国人民解放军第三军医大学第三附属医院 | Method for detecting fetus father DNA mononucleotide difference from plasma in pregnaut women |
Non-Patent Citations (2)
| Title |
|---|
| Daniel Clarke.Bio-bar-code-based DNA detection.《DDT：TARGETS》.2004,第3卷(第4期),第129页. * |
| Jwa-Min Nam et al.Bio-Bar-Code-Based DNA Detection with PCR-like Sensitivity.《J.AM.CHEM.SOC》.2004,第126卷(第19期),5932-5933. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101078028A (en) | 2007-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11434538B2 (en) | Method of nucleic acid sequence detection | |
| US10227632B2 (en) | Rolling circle amplification method | |
| CN107574226B (en) | Gene detection probe and gene detection method | |
| JP2006518194A5 (en) | ||
| US20220106647A1 (en) | Solution-phase, trans-activated reporter systems for use in crispr-based nucleic acid sequence detections | |
| CN102604934B (en) | Method for amplifying and sequencing nucleic acid based on solid phase carrier | |
| CN101078028B (en) | Twice bio-bar-code nucleic acid detecting technique | |
| CN108660191A (en) | A kind of digitlization multiple nucleic acid detection method based on coding microball reactor | |
| US9518289B2 (en) | Detection of target nucleic acid sequences by exonucleolytic activity using single-labeled immobilized probes on solid phase | |
| WO2007037282A1 (en) | Method of forming autoaggregate on microparticle and method of detecting target analyte | |
| CN107523624B (en) | Multi-cross isothermal amplification method combining AUDG and self-avoiding molecule recognition system | |
| TWI699435B (en) | Methods of constructing circular template and detecting dna molecules | |
| Guo et al. | A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani | |
| US7794985B2 (en) | Methods and compositions for rapid amplification, capture and detection of nucleic acids and proteins | |
| JP5317221B2 (en) | Aptamer selection method and apparatus, and sensor method using aptamer | |
| JP6853523B2 (en) | PCR using helicase | |
| US20230313309A1 (en) | Methods for Nucleic Acid Sequence Detection | |
| CN106701899A (en) | Twice bio-bar-code nucleic acid detection technology | |
| CN113624980B (en) | Method and kit for detecting protein based on identification induction isothermal amplification technology | |
| US20240158842A1 (en) | Methods and uses for determining the efficiency of genetic-editing procedures | |
| CN109457013B (en) | Method for detecting nucleic acid by combining cross primer amplification with nano biosensor and AUDG (autonomous Underwater vehicle) | |
| CN107727618B (en) | Biochip and method for detecting mercury ions by using DNA sequence of specific cleavage site | |
| JPWO2009054320A1 (en) | Method for forming nucleic acid probe and probe polymer | |
| Shi et al. | Nucleic Acid Amplification Strategy-Based Colorimetric Assays | |
| Zhuang et al. | Research progress of loop-mediated isothermal amplification in the detection of Salmonella for food safety applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110831 Termination date: 20140330 |