CN105483285A - Construction and application of guanine-based super-quenched molecular beacon - Google Patents
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Abstract
The invention provides construction and application of a guanine-based super-quenched molecular beacon. Particularly, the super-quenched molecular beacon which is simple in structure, easy to synthesize, high in stability and high in speed of reaction with a target is invented by utilizing double quenching effect, on certain organic fluorophores, of G basic groups and organic quenching groups. A method of utilizing the super-quenched molecular beacon to quantitatively detecting a target molecule is provided, and a method for virus detection is established by utilizing the super molecular beacon.
Description
Technical field
The present invention relates to biology and technical field of analytical chemistry, be specifically related to utilize two quenching effects of organic quencher and guanine to build a kind of super quenching molecules beacon sensor and application thereof.
Background technology
Molecular beacon (molecularbeacons) is a kind of oligonucleotide probe of the stem-ring double-tagging in hairpin structure.Classical molecular beacon is invented in 1996 first by Tyagi, is made up of stem, ring, organic fluorescence group and organic quencher four part.Since molecular beacon is used for detection of nucleic acids by Tyagi first in 1996, many researchers have synthesized different classes of molecular beacon on the basis of classical molecular beacon.Molecular beacon have simple to operate, selectivity good and need not with the advantage such as unreacted probe separates can detect in real time, now be widely used in the Real-Time Monitoring of polymerase chain reaction (PCR), the real-time analysis of transgenation, nucleic acid and protein detection, bacterium and virus analysis, the aspects such as the repercussion study of enzyme liberating monitoring and nucleic acid-protein.
Although molecular beacon all obtains and comparatively successfully applies in every field, existing molecular beacon, in the quantitative analysis of actual sample, also exists comparatively outstanding contradiction between fluorescence background and its structure.Classical molecular beacon structure is simple, synthesis easily, but exists higher fluorescence background, and the existence of fluorescence background seriously affects its sensitivity in quantitative analysis and quantitative limit.There is the problem of higher fluorescence background to solve classical molecular beacon, many researchers reduce its fluorescence background by the quencher changed in classical molecular beacon.Such as Maxwell and Mao etc. to substitute the quencher in classical molecular beacon as fluorescence quencher with golden nanometer particle, devise novel molecular beacon.In this molecular beacon, the average quencher efficiency of golden nanometer particle to fluorophor Fluoresceincarboxylic acid (FAM) reaches 98.68%; Yang etc. carry out quencher to a fluorophor by being assembled into a quenching molecules array with multiple quencher simultaneously, construct super quenching molecules beacon.They can reach 99.5% by the average quencher efficiency of 3 4-dimethylamine nitrogen benzide-4-carboxylic acid (DABCYL) molecular molecular array to FAM; Lu etc. utilize graphene oxide to substitute organic quencher in molecular beacon, significantly reduce the fluorescence background of molecular beacon, obtain very high signal to noise ratio.Although these molecular beacons improved significantly reduce fluorescence background, complex structure, and the structure of complexity brings new problem.One is that the molecular beacon preparation process that has is very complicated, and preparation time is long; Two is that the molecular beacon self stability that has is relatively poor, and the repeatability of detected result is bad; Three is owing to there being the existence of the nanoparticle of greater particle size in the molecular beacon had, greatly sterically hindered, slow with target compound speed of response.
Theoretically, a desirable molecular beacon should meet following condition: (1) fluorescence background is low; (2) good stability and structure are simple, and preparation process is easy; (3) fast with target compound speed of response.But current reported molecular beacon is all difficult to meet above condition simultaneously.
Guanine (G base) has good quenching effect to a lot of organic fluorescent dye.Its ultimate principle is that G base can be used as fluorescent energy acceptor in FRET (fluorescence resonance energy transfer), and the fluorescence of many organic fluorescent dyes can by the mode of Photo-induced electron transfer by its quencher.Such as Torimura etc. utilize nucleic acid hybridization test to have studied the quencher effect of G base pair organic fluorescence group, and result shows that the quencher efficiency of G base pair FAM can reach 86%, can reach 89% to the quencher efficiency of carboxyl tetramethylrhodamine (TAMRA).The present inventor utilizes the quenching effect of G base pair FAM and TAMRA to devise a kind of compound molecule beacon, and result shows that the quencher efficiency of G base pair FAM can reach 82.3%, can reach 81.6% to the quencher efficiency of TAMRA.This absolutely proves that G base pair FAM and TAMRA has good quencher effect really.
For existing molecular beacon Problems existing in quantitative analysis, in conjunction with the quenching effect of some fluorophor of G base pair, the present inventor utilizes G base and organic quencher to the dual quenching effect of some organic fluorescence group, invented that a kind of structure is simple, synthesis easily, good stability, the super quenching molecules beacon fast with target compound speed of response, and invented super quenching molecules beacon to be applied.
Summary of the invention
Object of the present invention is exactly the limitation for existing molecular beacon, provide that a kind of structure is simple, synthesis easily, the super quenching molecules beacon of good stability, and to apply it in the detection of nucleic acid and virus.
A first aspect of the present invention, there are provided the super quenching molecules beacon probe (Fig. 1) constructed by a kind of dual quenching effect based on G base and organic quencher.Super quenching molecules beacon probe 5 ' end mark can be carried out the fluorophor of dual quencher by G base and organic quencher, 3 ' end mark quencher; The part of probe ring is 13-45 base, can with target molecules generation specific reaction.Probe forms the hairpin structure of stem-ring under unbound state, fluorophor and organic quencher and G base close to each other, the fluorescence of fluorophor is by organic quencher and the quencher of G base, when probe and target molecule in conjunction with time, stem-ring structure is destroyed, fluorophor is away from organic quencher and G base, and the fluorescence of fluorophor recovers.Its Cleaning Principle as shown in Figure 1.
Preferably, described super quenching molecules beacon stem district is containing the nucleotide fragments of 4-8 base.More preferably, above-mentioned super quenching molecules beacon probe 5 ' end stem sequence is 5 '-CCCGCG-3 ', and 3 ' end stem sequence is 5 '-CGCGGG-3 '.
Preferably, the structure of described super quenching molecules beacon is specially: 5 '-fluorogene-(CH
2)
n-CCCGCG ring-shaped area CGCGGG-(CH
2)
mthe organic quencher-3 of – ', wherein, m, n be less than 12 optional natural number.
Preferably, described fluorophor by the quencher of G base institute, can be common are machine quencher institute quencher (as FAM, TAMRA, ROX and CY5 etc.) again.More preferably, described fluorophor is FAM or TAMRA.
Preferably, the fluorophor (as BHQ-1, BHQ-2, DABCYL etc.) of described quencher energy quenching molecules beacon probe 5 ' end mark.More preferably, described quencher is BHQ-1 or BHQ-2.
A second aspect of the present invention, there are provided a kind of method utilizing above-mentioned supramolecule beacon detection by quantitative target molecule.Described method comprises the steps: to be joined by target molecule in certain density super quenching molecules beacon, after hatching for some time at a certain temperature, then detects the fluorescent value of system.According to the size of fluorescent value, the fluorescent value of contrast standard solution, can obtain the concentration of target nucleic acid.
Preferably, described target molecule comprises nucleic acid and protein molecule.
A third aspect of the present invention, there are provided a kind of method utilizing above-mentioned super quenching molecules beacon detection virus.Described method comprises the steps: first to obtain the single-chain nucleic acid containing target dna by Asymmetric Polymerization polymerase chain reaction (asymmetric PCR), excess of imports quenching molecules beacon is added again in this system, after hatching for some time at a certain temperature, finally the fluorescent value of system is detected.The quantitative analysis step of virus is: first patients serum's normal human serum of known viruse copy number is mixed with a series of sample containing different copy number, carry out asymmetric PCR respectively, then in the sample of amplification, add excess of imports quenching molecules beacon respectively, respectively the fluorescence of each system is detected after reaction.
Beneficial effect of the present invention is:
(1) super quenching molecules beacon probe design: in existing molecular beacon, structure is simple, synthesis is easy to molecular beacon and there is higher fluorescence background; And the molecular beacon complex structure that fluorescence background is low, preparation time is long, and self stability is relatively poor, greatly sterically hindered, slow with target compound speed of response.Super quenching molecules beacon of the present invention is when designing, and the base on the Nucleotide be connected stem with quencher is designed to G base, utilizes the dual quenching effect of organic quencher and G base, builds super quenching molecules beacon.This super quenching molecules beacon had both maintained the simple structure of classical molecular beacon, significantly reduced again its fluorescence background (Fig. 2).
(2) the super quenching molecules beacon constructed by dual quenching effect that the present invention is based on organic quencher and G base is adopted, substantially increase target molecule (nucleic acid, protein) accuracy that detects and sensitivity, have synthesis easily, the feature such as detection speed is fast, testing cost is low.
(3) the super quenching molecules beacon in the present invention, changes putting in order of ring in molecular beacon, can realize the detection to multiple different target molecule.Not only can detect nucleic acid, also can detect protein equimolecular.Change the fluorophor in molecular beacon, the structure of quencher and ring, designs different molecular beacons simultaneously, also can realize detecting while multiple different target molecule.
(4) adopt the super quenching molecules beacon constructed by the present invention, in conjunction with PCR, the detection to virus can be realized.
Accompanying drawing explanation
Fig. 1: Cleaning Principle figure of the present invention
Fig. 2: with or without impact and the detection feasibility analysis of G base pair molecular beacon fluorescence background, a, has the molecular beacon of G base (super quenching molecules beacon, 15nM); B, does not have the molecular beacon (15nM) of G base; C, 15nM surpass quenching molecules beacon+7.5nM target nucleic acid; D, 15nM surpass quenching molecules beacon+15nM target nucleic acid
Fig. 3: utilize the typical curve that super quenching molecules beacon detects nucleic acid quantification, the concentration of target nucleic acid is (a → j × 10
-10m): 0,2,4,8,16,32,80,120,160,200.
Fig. 4: add template and do not add the PCR system fluorescence comparison diagram of template, a, 10nM surpass quenching molecules beacon; B, the PCR system+10nM not adding template surpasses quenching molecules beacon; C, the PCR system+10nM adding template surpasses quenching molecules beacon
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.
Embodiment 1: utilize super quenching molecules beacon to the detection by quantitative of nucleic acid
(1) according to target nucleic acid sequence (5 '-TTGTCCTGGCTACCGCTGGATTTGTCTGC-3 '), super quenching molecules beacon probe is designed.As previously mentioned, quencher is BHQ-1 to the structure of super quenching molecules beacon, and fluorophor is FAM.Be specially: 5 '-FAM-(CH
2)
6-CCCGCGGCAGACAAATCCAGCGGTAGCCAGGACAACGCGGG-(CH
2)
6-BHQ-1-3 ' (SEQIDNO.1).Structure without the molecular beacon of G base quencher is: 5 '-FAM-(CH
2)
6-ATATATGCAGACAAATCCAGCGGTAGCCAGGACAAATATAT-(CH
2)
6-BHQ-1-3 ' (SEQIDNO.2).
Target nucleic acid and super quenching molecules beacon synthesize by Shanghai Sheng Gong Bioisystech Co., Ltd (China) and carry out purifying by high performance liquid chromatography.
(2) feasibility analysis detected
Super quenching molecules beacon and the fluorescence without the molecular beacon of G base quencher are compared, result is as Fig. 2.Fig. 2 shows that the fluorescence background of super quenching molecules beacon is far below the molecular beacon without the quencher of G base, and this illustrates that the super quenching molecules beacon constructed by the present invention is successful.In super quenching molecules beacon, add target nucleic acid, the fluorescent signal of system significantly strengthens, and the concentration of target nucleic acid is different, and fluorescence intensity has significant difference.This illustrates and utilizes the detection of super quenching molecules beacon to target nucleic acid to be feasible.
(3) optimization of testing conditions
The data of all fluorescence spectrums and fluorescence intensity are all obtained by synchronous fluorimetry by RF-5301PC type fluorescence spectrophotometer (Shimadzu, Japan), and wavelength interval arranges 26nm, excite and launch slit width to be all set to 10nm.
Preferably, the concentration of described super quenching molecules beacon probe is 5-50nM, more preferably, and described super quenching molecules beacon probe 20nM.
Preferably, buffered soln used is Tris-HCl buffered soln, phosphate buffer solution, borate buffer solution or the extensive buffered soln of BR.More preferably, buffered soln used is 0.2M phosphate buffer solution.
Preferably, the pH of buffered soln used is 7.0-9.0.More preferably, the pH of buffered soln used is 8.2.
Preferably, the temperature that super quenching molecules beacon and target nucleic acid are hatched is 30-70 DEG C, and more preferably, the temperature that super quenching molecules beacon and target nucleic acid are hatched is 50 DEG C.
Preferably, the time that super quenching molecules beacon and target nucleic acid are hatched is 3-9min, and more preferably, the time that super quenching molecules beacon and target nucleic acid are hatched is 6min.
Preferably, the concentration adding sodium-chlor in buffered soln used is 20-100mM.More preferably, the concentration of sodium-chlor is 40mM.
(4) detection by quantitative of nucleic acid
Under optimal condition, target nucleic acid is detected.In super quenching molecules beacon, add the target nucleic acid of different concns, fully after reaction, measure its fluorescence intensity respectively with spectrophotometer.The fluorescence spectrum figure of Fig. 3 corresponding to the target nucleic acid of different concns.Be 2 × 10 in target nucleic acid concentration
-10-200 × 10
-10time between M, fluorescence intensity increases with the increase of the concentration of target dna, and the regression equation of its matching is Δ I=4.628C+2.0340 (R
2=0.9972).In linearity range, detect respectively the parallel sample of 13 same concentrations, its relative standard deviation (RSD) is 2.33%, and illustration method has good precision.Detecting of the method is limited to 8 × 10
-11m.
Embodiment 2: utilize super quenching molecules beacon to the detection of HBV virus
(1) select specific HBV target sequence (5 '-CCGCGTCGCAGAAGAT-3 ' (SEQIDNO.3)), according to target sequence design excess of export quenching molecules beacon, its structure is: 5 '-FAM-(CH
2)
6-CCCGCGATCTTCTGCGACGCGGCGCGGG-(CH
2)
6-BHQ-1-3 '.Target nucleic acid and super quenching molecules beacon synthesize by Shanghai Sheng Gong Bioisystech Co., Ltd (China) and carry out purifying by high performance liquid chromatography.
(2) sample preparation.Sample is obtained by asymmetric PCR.First design asymmetric PCR primer, carry out asymmetric PCR amplification and obtain target sequence, then add excess of imports quenching molecules beacon, reacted rear and its fluorescence intensity is detected.Upstream primer is 5 '-CACCATATGCCCCTTTCTTA-3 ' (SEQIDNO.4), and downstream primer is 5 '-GTTTGCCACCTTAAGAGTCC-3 ' (SEQIDNO.5).The condition of asymmetric PCR amplification is: 10 μ L templates (coming from the blood sample of HBV infection), PCR damping fluid 10 μ L, the MgCl of 10mmol/L
2the downstream primer 4 μ L of the upstream primer 4 μ L of the dNTPs4 μ L of solution 10 μ L, 5mmol/L, 20nM, 2nmol/L, 5UTaq polysaccharase, adding distil water to cumulative volume is 50 μ L.Response procedures is: after 95 DEG C of denaturation 3min, and by 95 DEG C of sex change 50s, 55 DEG C of renaturation 50s, 72 DEG C extend 50s, 40 circulations.Asymmetric PCR reaction terminates the molecular beacon solution 50 μ L adding 10nM after rear buffered soln dilutes reaction system, with distilled water diluting to 500 μ L, after 50 DEG C of reaction 6min, detects the fluorescence of system.
。
Claims (8)
1. the super quenching molecules beacon based on guanine, comprise arrange successively fluorophor, molecular beacon stem district, ring-shaped area and organic quencher, described ring-shaped area is the nucleotide fragments with target molecules specific combination, and described molecular beacon stem district is molecular beacon double stranded section.
2. super quenching molecules beacon according to claim 1, is characterized in that, described ring-shaped area is the nucleotide fragments containing 13-45 base.
3. super quenching molecules beacon according to claim 1, is characterized in that, the nucleotide fragments of 4-8 base is contained in described molecular beacon stem district, and 3 bases of wherein holding near 3 ' are all G base, is all C base near 5 ' 3 bases of holding.
4. super quenching molecules beacon according to claim 3 is CCCGCG near the 5 ' nucleotide fragments held, and is CGCGGG near the 3 ' nucleotide fragments held.
5. super quenching molecules beacon according to claim 4, the structure of described super quenching molecules beacon is specially: 5 '-fluorogene-(CH
2)
n-CCCGCG-ring-shaped area-CGCGGG-(CH
2)
mthe organic quencher-3 of – ', wherein, m, n be less than or equal to 12 optional natural number.
6. super quenching molecules beacon according to claim 5, described organic quencher has good quenching effect to fluorophor, and fluorophor also can by the quencher of G base institute simultaneously.
7. the method for the super quenching molecules beacon detection by quantitative target molecule utilizing one of claim 1-6 described.
8. a viral detection method, comprise the steps: that, first for the specific sequence fragment of viral nucleic acid, the super quenching molecules beacon of design primer and correspondence, after increasing by asymmetric PCR, add designed super quenching molecules beacon again, again the fluorescence of system is detected after reaction.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112852927A (en) * | 2021-02-02 | 2021-05-28 | 中国科学院合肥物质科学研究院 | Isothermal amplification system and method based on fluorescence self-inhibition probe |
| CN116004772A (en) * | 2022-11-28 | 2023-04-25 | 郑州轻工业大学 | Fluorescent start sensor for detecting BRCA2 and preparation method and application thereof |
| WO2023109031A1 (en) * | 2021-12-14 | 2023-06-22 | 广州市金圻睿生物科技有限责任公司 | Respiratory pathogen detection kit, and preparation method therefor and use thereof |
| WO2023109032A1 (en) * | 2021-12-14 | 2023-06-22 | 广州市金圻睿生物科技有限责任公司 | Multiple nucleic acid detection system, and preparation method therefor and use thereof |
| CN116445586A (en) * | 2023-06-13 | 2023-07-18 | 中国农业大学 | Biosensor based on fluorescence hybridization chain reaction |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852927A (en) * | 2021-02-02 | 2021-05-28 | 中国科学院合肥物质科学研究院 | Isothermal amplification system and method based on fluorescence self-inhibition probe |
| WO2023109031A1 (en) * | 2021-12-14 | 2023-06-22 | 广州市金圻睿生物科技有限责任公司 | Respiratory pathogen detection kit, and preparation method therefor and use thereof |
| WO2023109032A1 (en) * | 2021-12-14 | 2023-06-22 | 广州市金圻睿生物科技有限责任公司 | Multiple nucleic acid detection system, and preparation method therefor and use thereof |
| CN116004772A (en) * | 2022-11-28 | 2023-04-25 | 郑州轻工业大学 | Fluorescent start sensor for detecting BRCA2 and preparation method and application thereof |
| CN116445586A (en) * | 2023-06-13 | 2023-07-18 | 中国农业大学 | Biosensor based on fluorescence hybridization chain reaction |
| CN116445586B (en) * | 2023-06-13 | 2023-09-01 | 中国农业大学 | Biosensor based on fluorescence hybridization chain reaction |
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