CN104450920A - MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle - Google Patents
MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle Download PDFInfo
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Abstract
The invention discloses a microRNA trace detection method based on an exponential order non-enzymatic amplification and electrochemical luminescence principle. A non-enzymatic amplification and hybrid chain type reaction system is adopted, and the specific sequences of DNA hairpin probes H1, H2, H3 and H4 are designed based on a detection target microRNA sequence; when an amplification system contains to-be-detected microRNA, the subsequent hybrid chain type reaction process is triggered by virtue of an H1+H2 double-chain composite structure, and is finished by H3 and H4 together; and moreover, an amplification product is captured by virtue of streptavidin magnetic bead capture, and an electrochemical luminescence signal is generated and detected by virtue of an electrochemical detection system. According to the method, an enzyme is not involved in the whole process, the principle is simple, and the detection cost is low; and the method has the advantages of constant temperature amplification, high sensitivity, simple operation, simplicity in popularization and the like. The method is applied to nucleic acid detection and can be combined with a protein aptamer related technology to be used for protein detection.
Description
Technical field
The invention belongs to nov nucleic acid chemical trace detection technique field, particularly a kind ofly strengthen non-enzymatic amplification platform and the microRNA trace detection method of combined with electrochemical principle of luminosity based on exponential signal.
Background technology
MicroRNA (miRNA) is the non-coding of a class length in 19 ~ 25 nucleotide range, endogenous Microrna, and it is distributed widely in eukaryotic cells, and participates in important life course.In recent years, along with the progress of molecular biology and related discipline, investigator has found hundreds of miRNA, and determines the major function of wherein part miRNA.This type of RNA mainly exercises gene regulating function, and after being transcribed into primary transcript by non-coding sequence, primary transcript is through the shearing of different nuclease, processing and producing.Person infers according to the study, and in Animal genome, the encoding gene of about 30% all receives the direct of miRNA or indirect adjustments and controls.Research shows, miRNA participates in numerous important biochemistry and regulates approach, mainly comprises growth course, virus defense, allelotaxis and formation, cell proliferation and apoptosis etc.
As the miRNA detection means of classics, Northern is hybridized (Northern blotting) method and has been achieved significant achievement, but it is consuming time, sensitivity is not high and need professional to operate, this greatly reduces the popularization of the method and universal.In recent years, investigator proposes based on methods such as capillary electrophoresis, nucleic acid amplification detection technique, nanowire signal amplification system, surface plasma resonance technologies.These methods have higher detection sensitivity, consuming time short, but required complex operation, belong to technology-intensive type, and need expensive plant and instrument as the point of suppon of these technology.Therefore, develop a kind of highly sensitive, high special and method that is economical, detection miRNA fast and safely has become one of focus of numerous investigators' concern in recent years.In recent years, along with popularizing of non-enzymatic amplification technology, novel miRNA amplification method also occurs thereupon, is significant for miRNA detection field.At present, Chinese patent 201310294770.7 discloses a kind of MicroRNA detection method based on non-enzymatic amplification and electrochemiluminescence principle, the method achieve constant temperature, the microRNA detection mode of non-enzymatic amplification, but the sensitivity of the method is lower.
Summary of the invention
In order to the shortcoming overcoming prior art is with not enough, the object of the present invention is to provide a kind of microRNA trace detection method based on exponential non-enzymatic amplification and electrochemiluminescence principle.This detection method is the microRNA trace detection method combined based on exponential signal enhancing non-enzymatic amplification platform and electrochemiluminescence principle.The method is highly sensitive, specificity good, accurate, quick, simple to operate, and whole amplification procedure does not need the participation of enzyme.Therefore, the present invention on the basis reducing testing cost, for microRNA detection technique system provides a kind of brand-new MicroRNA detection method.
Object of the present invention is achieved through the following technical solutions: a kind of microRNA trace detection method based on exponential non-enzymatic amplification and electrochemiluminescence principle, comprises the following steps:
(1) the hair fastener probe of non-enzymatic amplification system is designed for
Hair fastener probe H1 needed for non-enzymatic amplification principle and microRNA sequences Design non-enzymatic amplification system and hair fastener probe H2 sequence; Hair fastener probe H1 adopts double end labelling strategies, and it is amino that its 5 ' end and 3 ' section are all marked with 6 carbon, for follow-up connection electrochemiluminescence group; 3 ' end mark the vitamin H of H2, as the target spot of Streptavidin MagneSphere in magnetic capture-process; Wherein, H1 comprises bridge sequence (numbering 5,6 section), for connecting follow-up hybridization chain reaction process;
(2) the hair fastener probe of hybridization chain reaction system is designed for
On the basis of non-enzymatic amplification system, and combine hybridization chain reaction principle, devise hybridization chain reaction system, to obtain the amplification of signal that persistence strengthens; Design on the basis of bridge sequence for hybridizing the sequence of hair fastener probe H3 and H4 of chain reaction system, the identification 2* that this reaction system can be special, 5,6 sections; When non-enzymatic amplification system is triggered, the 2* section of the single-chain state of generation, and coordinate 5,6 sections namely to can be used as the amplification triggering device of hybridization chain reaction, to produce hybridization chain reaction amplified signal; When not having target microRNA, the 2* section of hair fastener probe H1 is the integral part of hair fastener stem, is duplex structure, and therefore, hair fastener probe H1, H2, H3 and H4 can coexist mutually, do not increase; Thus specificity obtains effective guarantee;
(3) electrochemiluminescence group mark
In all hair fastener probes, 3 ' end mark vitamin H of H2 probe, and 5 ' of H1, H3, H4 and 3 ' holds mark 6 carbon amino, in order to connect electrochemiluminescence group; The strategy why H1, H3, H4 probe adopts " two ends " to mark is to improve sensitivity;
Electrochemiluminescence group mark mainly comprises the following steps:
A, get 2.5OD hair fastener probe H1, H3, H4, centrifugal 5min, make the DNA being bonded at tube wall fall at the bottom of pipe, add 75 μ L 0.1M Sodium Tetraboratees respectively, lid upper tube cap immediately, vibrate 5min fully up and down;
B, centrifugal, and add the electrochemiluminescence group of 30 μ L 11.2mM, fully after mixing, lucifuge hatches 12 hours; Then, add the dehydrated alcohol of 1mL precooling, put into Ultralow Temperature Freezer 2 hours;
C, centrifugal, remove pale yellow solution, in DNA precipitation, add the washing of 1mL precooling volume fraction 80% aqueous ethanolic solution, put into Ultralow Temperature Freezer 1h;
D, centrifugal, remove solution, collecting precipitation, add the washing of 1mL precooling volume fraction 80% aqueous ethanolic solution, put into Ultralow Temperature Freezer 1h;
E, centrifugal, remove solution, collecting precipitation, add 20 μ L water dilutions, obtain H1, H3, H4 probe solution respectively, put-20 DEG C of preservations;
(4) hair fastener probe pre-treatment
In order to make hair fastener probe H1, H2, H3, H4 fully form hairpin structure, in H1, H3, H4 probe solution obtained in H2 and step (3), add phosphate buffered saline buffer (PBS) respectively, and PBS final concentration is set to 1 ×; Subsequently, be heated to after 95 DEG C, make its abundant sex change; Finally, gradient cooling, per minute falls 5 DEG C, be down to normal temperature till, obtain the pretreatment product of hair fastener probe H1, H2, H3, H4 respectively, product is stored in 4 DEG C or-20 DEG C of environment for subsequent use;
(5) strengthen non-enzymatic amplification detection system based on exponential signal to build
Get the pretreatment product of hair fastener probe H1, H2, H3, the H4 (final concentration is all 50nM) obtained in a certain amount of step (4), and add PBS damping fluid (final concentration 1 ×), add RNA enzyme inhibitors (final concentration 1U/ μ L) again, finally add DEPC water to 100 μ L; After abundant mixing, add microRNA to be measured, 37 DEG C of temperature are bathed 2 hours;
(6) product cleaning and separation
In step (5), add Streptavidin MagneSphere, after 37 DEG C of temperature bath 30min, be separated with magnetic separator, after removing solution, then the PBS damping fluid (final concentration 1 ×) adding 100 μ L dissolves magnetic bead, then carries out Magneto separate, and clean, repeatedly clean three times thus;
(7) signal detection
By the product in step (6), put into electrochemiluminescence automatic lmunoassays analyzer and detect electrochemical luminescence signals, and record data.
DNA hair fastener probe described in step (1) is for self forming specific double-strand " stem ring " structural DNA, and its sequence can change according to the sequence of microRNA to be measured and be changed accordingly; When there is target microRNA in detection system, the front end strand bilge construction of DNA hair fastener probe H1 is combined with target microRNA by base complementrity principle, make that the double-stranded stem structure of DNA hair fastener probe is unwind due to rigidity effect, structure changes, thus reaches the object of DNA hair fastener probe identification target microRNA.The single stranded portion formed after hair fastener probe H1 is opened can act on hair fastener probe H2 phase, makes H2 open hairpin structure, and makes H1 and H2 form complementary duplex structure, and is separated by target microRNA and H1, reaches the object of cyclic amplification; Thus, the 2* section of the single-chain state that H1 probe produces and this 5,6 sections as single-stranded structure, by the HCR process of triggering following;
In the present invention, involved DNA product is by the synthesis of prompt base (Shanghai) trade Co., Ltd in the English Weihe River.
Bridge sequence described in step (1) is CCTAATGGTGTGGC;
The temperature of the Ultralow Temperature Freezer described in step (3) is preferably less than-60 DEG C, is more preferably-80 DEG C;
Centrifugal condition optimization described in step (3) b is the centrifugal 5min of 12000rpm;
Centrifugal condition optimization described in step (3) c is the centrifugal 20min of 12000rpm;
Step (3) d and the centrifugal condition optimization described in step (3) e are 12000rpm, 4 DEG C of centrifugal 20min;
The program of the gradient cooling described in step (4) is 95 DEG C of 5min, 90 DEG C of 5min, 85 DEG C of 5min, 80 DEG C of 5min, 75 DEG C of 5min, 70 DEG C of 5min, 65 DEG C of 5min, 60 DEG C of 5min, 55 DEG C of 5min, 50 DEG C of 5min, 45 DEG C of 5min, 40 DEG C of 5min, 35 DEG C of 5min, 30 DEG C of 5min, 25 DEG C of 5min;
Phosphate buffered saline buffer (PBS) described in step (4) is provided by Sheng Gong biotechnology company limited, and concentration is 20 ×; This damping fluid is through DEPC process;
Described DEPC water is 0.1% (v/v) DEPC water through autoclave sterilization; DEPC is tetra-sodium diethyl phthalate;
RNA enzyme inhibitors described in step (5) is provided by precious biotechnology company limited; 37 DEG C of isoperibols can be maintained by PCR instrument or water-bath;
MicroRNA to be measured described in step (5) is preferably microRNA21, and its sequence is UAGCUUAUCAGACUGAUGUUGA;
The sequence of described hair fastener probe H1 is TCAACATCAGTCTGATAAGCTACCATGTGTAGATAGCTTATCAGACTCCTAATGGT GTGGC; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon;
The sequence of described hair fastener probe H2 is ATAAGCTATCTACACATGGTAGCTTATCAGACTCCATGTGTAGA; Its 3 ' end mark vitamin H (biotin);
The sequence of described hair fastener probe H3 is CCTAATGGTGTGGCCCATGTTTTGCCACACCATTAGGAGTCTG; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon;
The sequence of described hair fastener probe H4 is ACATGGGCCACACCATTAGGTTTCAGACTCCTAATGGTGTGGC; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon;
Streptavidin MagneSphere described in step (6) refers to the magnetic bead of Streptavidin bag quilt, purchased from NEWENGLAND BioLabs company, and its final concentration 0.2mg/mL;
Described electrochemiluminescence group is preferably tris (bipyridine) ruthenium; Described tris (bipyridine) ruthenium is open in Chinese patent 201310721893.4, the nucleic acid detection method that amplifies based on polymer electrochemical luminous signal;
Electrochemiluminescence automatic lmunoassays analyzer described in step (7) is preferably Roche Elecsys2010 electrochemiluminescence automatic lmunoassays analyzer;
Further, the described microRNA trace detection method based on exponential non-enzymatic amplification and electrochemiluminescence principle, when selected microRNA is the microRNA21 be closely related with human cancer, its sequence is: UAGCUUAUCAGACUGAUGUUGA, and concrete steps are as follows:
1. design dna hair fastener probe H1, H2, H3, H4 probe: first, according to the sequence (hair fastener probe H1 and H2) of microRNA21 sequences Design non-enzymatic amplification system, and according to bridge sequence and the sequence (hairpin probe H3 and H4) of hybridizing the follow-up hybridization chain reaction amplification system of chain reaction principle design;
2. H1, H3 and H4 probe mark electrochemiluminescence group, experimental procedure is as previously mentioned; After having marked, be first heated to 95 DEG C, make its abundant sex change, then gradient cooling again, per minute falls 5 DEG C till normal temperature, and product is stored in 4 DEG C of environment for subsequent use;
3. H2 is dissolved in 1 × phosphate buffered saline buffer (PBS), and final concentration is 10 μMs, and performs gradient cooling process;
4. strengthen the structure of non-enzymatic amplification detection system based on exponential signal: 100 μ L systems by hair fastener probe H1 (50nM), H2 (50nM), H3 (50nM), H4 (50nM), PBS damping fluid (final concentration 1 ×), RNA enzyme inhibitors (1U/ μ L); Finally, add DEPC volume of water and mend to 100 μ L, fully mix;
5. microRNA21 to be measured is added;
6. 37 DEG C of temperature are bathed 2 hours;
7. in reaction system, add Streptavidin MagneSphere (final concentration 0.2mg/mL), after 37 DEG C of temperature bath 30min, be separated with magnetic separator, after removing solution, the PBS damping fluid (final concentration 1 ×) adding 100 μ L again dissolves, be separated with magnetic separator again, repeatedly clean three times thus;
8. electrochemical luminescence signals detects, by step 7. in product, put into Roche Elecsys2010 electrochemiluminescence automatic lmunoassays analyzer and detect electrochemical luminescence signals, and record data.
Ultimate principle of the present invention is as shown in Figure 1: amplification procedure of the present invention is made up of non-enzymatic amplification and hybridization chain reaction, and " signal provides " step adopts electrochemiluminescence pattern.First, for detection target microRNA sequences Design specific DNA hair fastener probe H1, H2, H3, H4 sequence; When amplification system does not exist microRNA to be measured, amplified reaction can not be activated, and can not react between hair fastener probe, therefore, can not produce amplified production.When containing microRNA to be measured in amplification system, microRNA to be measured can be combined with hair fastener probe H1, H1 stem is caused to open, consequent part single-stranded structure acts on H2 phase, hair fastener probe H2 stem is caused to open, and form H1+H2 double-strand composite structure with H1, and by the hybridization chain reaction process of H1+H2 double-strand composite structure triggering following.Hybridization chain reaction process is completed jointly by H3 and H4.Catch through Streptavidin MagneSphere, amplified production is caught, and is produced by electrochemical detection system (Elecsys2010) and detected electrochemical luminescence signals.
The present invention has following advantage and effect relative to prior art:
(1) the present invention adopts non-enzymatic amplification and hybridization chain reaction system: whole process does not need the participation of enzyme, and principle is simple and testing cost is low.
(2) constant-temperature amplification: carry out under constant temperature, only needs 37 DEG C of reaction 2h just can obtain desirable experimental result.Reaction unit is simple, also can with water-bath heating.
(3) highly sensitive: the sensitivity of electrochemiluminescence assay reaches 1amol.Achieve the object of microRNA trace detection.
(4) simple to operate, be easy to universal.
(5) be applicable to detection of nucleic acids, also associated proteins aptamers correlation technique can be used for Protein Detection: this experimental principle can be used for the detection of DNA and RNA, as long as according to target sequence appropriate design hair fastener probe sequence.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the microRNA trace detection method strengthening non-enzymatic amplification platform based on exponential signal.
Fig. 2 is hairpin probe two-dirnentional structure figure; The two-dirnentional structure figure of the two-dirnentional structure figure of Fig. 2 A to be the two-dirnentional structure figure of hairpin probe H1, Fig. 2 B be hairpin probe H2, Fig. 2 C to be the two-dirnentional structure figure of hairpin probe H3, Fig. 2 D be hairpin probe H4.
Fig. 3 is non-enzymatic amplification system amplification efficiency checking electrophorogram; Wherein, swimming lane H1 is the electrophorogram of hairpin probe H1; Swimming lane H2 is the electrophorogram of hairpin probe H2; Swimming lane H3 is the electrophorogram of hairpin probe H3; Swimming lane H4 is the electrophorogram of hairpin probe H4; Swimming lane control group is hairpin probe H1, H2, H3, H4 amplification efficiency electrophorogram without microRNA21; Swimming lane experimental group is hairpin probe H1, H2, H3, H4 and adds the amplification efficiency electrophorogram of microRNA21.
Fig. 4 is the result figure of non-enzymatic amplification/electrochemiluminescence detection platform sensitivity.
Fig. 5 is non-enzymatic amplification/specific result figure of electrochemiluminescence detection platform.
Fig. 6 is the result figure of tumour cell detection sensitivity; A is HepG2 clone; B is A549 clone; C is MCF7 clone; D is HepG2 clone, A549 clone and the MCF7 clone result figure relative to the relative electrochemical luminous intensity of LO2 clone.
Fig. 7 is the result figure of tumor tissues test experience; A is liver cancer tissue, and B is cancerous lung tissue, and C is breast cancer tissue.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 exponential signal strengthens structure and the feasibility checking thereof of non-enzymatic amplification platform
Strengthen non-enzymatic amplification platform principle according to above-mentioned exponential signal, for hair fastener probe H1, H2, H3, the H4 needed for microRNA21 sequences Design non-enzymatic amplification system, its sequence is as shown in table 1, and its two-dirnentional structure as shown in Figure 2.5 ' of hair fastener probe H1, H3, H4 and 3 ' end to be marked with six carbon amino, the 3 ' end of corresponding H2 is marked with vitamin H.Hair fastener probe is by the synthesis of prompt base (Shanghai) trade Co., Ltd in the English Weihe River, and H1, H3, H4 need mark electrochemistry luminophore, and H2 TE buffer solution gets final product (final concentration 10 μMs is stored in-20 refrigerators).H1, H3, H4 labeling process is as follows: get 2.5OD hair fastener probe, centrifugal 5min, makes the DNA being bonded at tube wall fall at the bottom of pipe, and add 75 μ L 0.1M Sodium Tetraboratees, lid upper tube cap immediately, vibrate 5min fully up and down; Centrifugal (12000rpm, 5min) collects, and add the tris (bipyridine) ruthenium of 30 μ L 11.2mM, lucifuge hatches 12 hours; Add the dehydrated alcohol of 1mL precooling, put into Ultralow Temperature Freezer (-80 DEG C) 2 hours, centrifugal (12000rpm, 20min); Remove pale yellow solution, add 1mL precooling 80% washing with alcohol, put into Ultralow Temperature Freezer (-80 DEG C) 1h; Centrifugal (12000rpm, 4 DEG C, 20min); Remove solution, add 1mL precooling 80% washing with alcohol, put into Ultralow Temperature Freezer (-80 DEG C) 1h, centrifugal (12000rpm, 4 DEG C, 20min), remove solution, collecting precipitation, add 20 μ L water dilutions, obtain hair fastener probe H1, H3, H4 probe solution, put-20 DEG C of preservations.
Described tris (bipyridine) ruthenium repeats in crystallization synthesis method and application thereof open at the tris (bipyridine) ruthenium that Chinese patent 201410076390.0, pH value rely on;
Sequence used in table 1 embodiment
Fully hairpin structure is formed in order to make hair fastener probe, PBS damping fluid (PBS buffer Final concentration is 1 ×) is added respectively in H2 and H1, H3, H4 probe solution, first be heated to after 95 DEG C, make its abundant sex change, then gradient cooling (per minute falls 5 DEG C till normal temperature) again, obtain the pretreatment product of hair fastener probe H1, H2, H3, H4 respectively, product is stored in 4 DEG C of environment for subsequent use.The program of gradient cooling is 95 DEG C of 5min, 90 DEG C of 5min, 85 DEG C of 5min, 80 DEG C of 5min, 75 DEG C of 5min, 70 DEG C of 5min, 65 DEG C of 5min, 60 DEG C of 5min, 55 DEG C of 5min, 50 DEG C of 5min, 45 DEG C of 5min, 40 DEG C of 5min, 35 DEG C of 5min, 30 DEG C of 5min, 25 DEG C of 5min.
Whole amplification system is set to 100 μ L, get the pretreatment product of hair fastener probe H1, H2, H3, H4, final concentration is 50nM, add 5 μ L PBS (20 ×), PBS final concentration is made to be 1 ×, add RNA enzyme inhibitors (final concentration is 1U/ μ L), finally, add DEPC process water and mend to 100 μ L.Abundant mixing.Add microRNA21 to be measured, 37 DEG C of incubations are after 2 hours, and amplified production polyacrylamide gel electrophoresis detects to verify non-enzymatic amplification principle feasibility and amplification efficiency.As shown in Figure 3, as seen from Figure 3, the amplification of this system is stable, fully demonstrates the feasibility of non-enzymatic amplification system for experimental result.Experimental group in Fig. 3 only adds the microRNA of 10pmol.
Embodiment 2 exponential signal strengthens foundation and the sensitivity experiment thereof of non-enzymatic amplification platform
The foundation that exponential signal strengthens non-enzymatic amplification platform comprises the foundation of amplification system and electrochemical signals detects two portions, and the foundation of amplification system as described in Example 1.Electrochemical luminescence signals detects key step: the magnetic bead (final concentration 0.2mg/mL) adding Streptavidin bag quilt in non-enzymatic amplification system, after 37 DEG C of temperature bath 30min, be separated with magnetic separator, after removing solution, the PBS damping fluid (final concentration is 1 ×) adding 100 μ L again dissolves, be separated again, repeatedly clean three times thus.Finally, product is put into Roche Elecsys2010 electrochemiluminescence automatic lmunoassays analyzer and detect electrochemical luminescence signals.
In order to verify the sensitivity of non-enzymatic amplification/electrochemiluminescence detection platform, arranging amount that microRNA21 adds respectively is 100pmol, 10pmol, 1pmol, 100fmol, 10fmol, 1fmol, 100amol, 10amol, 1amol, 100zmol, detect electrochemical luminescence signals, experimental result as shown in Figure 4.As shown in Figure 4, the sensitivity of this detection platform can reach 1amol.Through linear analysis, the program obtains linear (R preferably
2=0.9918).
Embodiment 3 exponential signal strengthens non-enzymatic amplification platform specificity verification
For checking exponential signal strengthens non-enzymatic amplification platform specificity, add microRNA210 and microRNA214 (amount added is 10pmol respectively) respectively to this platform, and detect electrochemical luminescence signals, experimental result as shown in Figure 5.As shown in Figure 5, control group is blank, and electrochemical luminescence signals and the control group of microRNA210, microRNA214 experimental group maintain an equal level, and experimental group obtains stronger electrochemical luminescence signals.Therefore, this platform specificity is better.
Embodiment 4 tumour cell detection sensitivity
In order to verify the feasibility that this platform detects for tumour cell, we devise tumour cell test experience.In this experiment, we have chosen three kinds of tumor cell lines: hepatoma cell line (HepG2 clone), lung cancer cell line (A549 clone), breast cancer cell line (MCF7 clone).MicroRNA21 has been proved to be the phenomenon that there is high expression level in these three kinds of clones.And using the control group of microRNA21 expression level in people's normal liver tissue clone (LO2 clone) as normal expression level in cell.
In this programme, first we be extracted total serum IgE in cell (the RNAplus total RNA extraction reagent box that Total RNAs extraction test kit used provides for Takara company), and carried out cell counting before extraction.And be provided with cell concn: 10
0~ 10
7individual cell, to verify the sensitivity of the method.From experimental result (shown in Fig. 6), the present invention, in the test experience of HepG2 clone, reaches the sensitivity (see Fig. 6 A) of 10 cells.Meanwhile, in A549 (see Fig. 6 B) and MCF 7 clone (see Fig. 6 C), achieve the sensitivity of 10 cells, 100 cells respectively.Thus, the method obtains checking for the feasibility of cell detection.In addition, we also compare the microRNA21 expression amount in normal cell and tumour cell.In this, by 10
6the electrochemical luminescence signals intensity that in individual LO2 cell, microRNA21 produces is artificial is defined as unit 1, and the electrochemical luminescence signals intensity that in the electrochemical luminescence signals intensity produced by the tumour cell of equal amts and LO2 cell, microRNA21 produces is divided by, obtain the ratio of relative electrochemical luminous intensity, weigh the difference of expression amount in microRNA in different clone with this.As shown in Figure 6 D, in HepG2 clone, A549 clone, MCF7 clone, all there is the phenomenon of microRNA21 high expression level.
Described HepG2 clone, A549 clone, MCF7 clone and LO2 clone are all purchased from Han Bo bio tech ltd, Shanghai.
Embodiment 5 tumor tissues test experience
After demonstrating cell detection sensitivity, we have done tumor tissues test experience.The present invention have chosen three kinds of neoplasmic tissue sample (each 20 of liver cancer tissue sample, lung cancer tissue sample, breast cancer tissue samples, totally 60 samples, all carry out the tumour patient as making a definite diagnosis), and through Total RNAs extraction process, be extracted the total serum IgE of 60 samples.Through test experience, 51 samples obtain positive signal.As shown in Figure 7, A is liver cancer tissue to the result of tumor tissues test experience, and B is cancerous lung tissue, and C is breast cancer tissue; Thus, this platform have also been obtained further checking for the feasibility detecting tumor tissues.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1., based on a microRNA trace detection method for exponential non-enzymatic amplification and electrochemiluminescence principle, it is characterized in that comprising the following steps:
(1) the hair fastener probe of non-enzymatic amplification system is designed for
Hair fastener probe H1 needed for non-enzymatic amplification principle and microRNA sequences Design non-enzymatic amplification system and hair fastener probe H2 sequence; It is amino that the 5 ' end of hair fastener probe H1 and 3 ' section are all marked with 6 carbon; 3 ' end mark the vitamin H of H2; Wherein, H1 comprises bridge sequence;
(2) the hair fastener probe of hybridization chain reaction system is designed for
On the basis of non-enzymatic amplification system, and combining hybridization chain reaction principle, devising the sequence of hair fastener probe H3 for hybridizing chain reaction system and hair fastener probe H4; Wherein, hair fastener probe H3 and H4 5 ' end and 3 ' section be all marked with 6 carbon amino; Hair fastener probe H1, H2, H3 and H4 can coexist mutually, do not increase;
(3) electrochemiluminescence group mark
Electrochemiluminescence group mark mainly comprises the following steps:
A, get 2.5OD hair fastener probe H1, H3, H4, centrifugal 5min, make the DNA being bonded at tube wall fall at the bottom of pipe, add 75 μ L 0.1M Sodium Tetraboratees respectively, lid upper tube cap immediately, vibrate 5min fully up and down;
B, centrifugal, and add the electrochemiluminescence group of 30 μ L 11.2mM, fully after mixing, lucifuge hatches 12 hours; Then, add the dehydrated alcohol of 1mL precooling, put into Ultralow Temperature Freezer 2 hours;
C, centrifugal, remove pale yellow solution, in DNA precipitation, add the washing of 1mL precooling volume fraction 80% aqueous ethanolic solution, put into Ultralow Temperature Freezer 1h;
D, centrifugal, remove solution, collecting precipitation, add the washing of 1mL precooling volume fraction 80% aqueous ethanolic solution, put into Ultralow Temperature Freezer 1h;
E, centrifugal, remove solution, collecting precipitation, add 20 μ L water dilutions, obtain H1, H3, H4 probe solution respectively, put-20 DEG C of preservations;
(4) hair fastener probe pre-treatment
In order to make hair fastener probe H1, H2, H3, H4 fully form hairpin structure, in H1, H3, H4 probe solution obtained in H2 and step (3), add phosphate buffered saline buffer respectively, and PBS final concentration is set to 1 ×; Subsequently, be heated to after 95 DEG C, make its abundant sex change; Finally, gradient cooling, per minute falls 5 DEG C, be down to normal temperature till, obtain the pretreatment product of hair fastener probe H1, H2, H3, H4 respectively, product is stored in 4 DEG C or-20 DEG C of environment for subsequent use;
(5) strengthen non-enzymatic amplification detection system based on exponential signal to build
Get the pretreatment product of hair fastener probe H1, H2, H3, the H4 obtained in a certain amount of step (4), make its final concentration be all 50nM, and add PBS damping fluid, its final concentration is made to be 1 ×, add RNA enzyme inhibitors again, make its final concentration be 1U/ μ L, finally add DEPC water to 100 μ L; After abundant mixing, add microRNA to be measured, 37 DEG C of temperature are bathed 2 hours;
(6) product cleaning and separation
In step (5), add Streptavidin MagneSphere, after 37 DEG C of temperature bath 30min, be separated with magnetic separator, after removing solution, add the PBS damping fluid of 100 μ L again, make its final concentration be 1 ×, dissolve magnetic bead, carry out Magneto separate again, and clean, repeatedly clean three times thus;
(7) signal detection
By the product in step (6), put into electrochemiluminescence automatic lmunoassays analyzer and detect electrochemical luminescence signals, and record data;
The sequence of described hair fastener probe changes according to the sequence of microRNA to be measured and is changed accordingly.
2. microRNA trace detection method according to claim 1, is characterized in that:
Bridge sequence described in step (1) is CCTAATGGTGTGGC.
3. microRNA trace detection method according to claim 1 and 2, is characterized in that:
MicroRNA to be measured described in step (5) is microRNA21, and its sequence is UAGCUUAUCAGACUGAUGUUGA.
4. microRNA trace detection method according to claim 3, is characterized in that:
The sequence of described hair fastener probe H1 is TCAACATCAGTCTGATAAGCTACCATGTGTAGATAGCTTATCAGACTCCTAATGGT GTGGC; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon;
The sequence of described hair fastener probe H2 is ATAAGCTATCTACACATGGTAGCTTATCAGACTCCATGTGTAGA; Its 3 ' end mark vitamin H;
The sequence of described hair fastener probe H3 is CCTAATGGTGTGGCCCATGTTTTGCCACACCATTAGGAGTCTG; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon;
The sequence of described hair fastener probe H4 is ACATGGGCCACACCATTAGGTTTCAGACTCCTAATGGTGTGGC; It is amino that its 5 ' end and 3 ' section are all marked with 6 carbon.
5. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
The program of the gradient cooling described in step (4) is 95 DEG C of 5min, 90 DEG C of 5min, 85 DEG C of 5min, 80 DEG C of 5min, 75 DEG C of 5min, 70 DEG C of 5min, 65 DEG C of 5min, 60 DEG C of 5min, 55 DEG C of 5min, 50 DEG C of 5min, 45 DEG C of 5min, 40 DEG C of 5min, 35 DEG C of 5min, 30 DEG C of 5min, 25 DEG C of 5min.
6. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
Suddenly the Streptavidin MagneSphere described in (6) refers to the magnetic bead of Streptavidin bag quilt, its final concentration 0.2mg/mL.
7. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
Described electrochemiluminescence group is tris (bipyridine) ruthenium.
8. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
Centrifugal condition described in step (3) b is the centrifugal 5min of 12000rpm;
Centrifugal condition described in step (3) c is the centrifugal 20min of 12000rpm.
9. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
Step (3) d and the centrifugal condition described in step (3) e are 12000rpm, 4 DEG C of centrifugal 20min.
10. the microRNA trace detection method according to claim 1,2 or 4, is characterized in that:
Electrochemiluminescence automatic lmunoassays analyzer described in step (7) is Roche Elecsys2010 electrochemiluminescence automatic lmunoassays analyzer.
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