CN105063190A - Solid chip constant temperature detection method of MiRNA - Google Patents

Solid chip constant temperature detection method of MiRNA Download PDF

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CN105063190A
CN105063190A CN201510460239.1A CN201510460239A CN105063190A CN 105063190 A CN105063190 A CN 105063190A CN 201510460239 A CN201510460239 A CN 201510460239A CN 105063190 A CN105063190 A CN 105063190A
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primer
solid phase
target molecule
phase chip
constant temperature
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黄庆
府伟灵
黄君富
夏涵
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Third Military Medical University TMMU
First Affiliated Hospital of TMMU
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First Affiliated Hospital of TMMU
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses a solid chip constant temperature detection method of MiRNA. For an upstream primer and a downstream primer with target molecule specificity, anchoring sequence areas contain nicking agent recognition sequence positive-sense strand sequences, recognition sequence areas are complementary to the 3'-end sequences of the miRNA and the complementary chain thereof, 'cutting-extending-strand displacement' constant temperature linear amplifications which are independent and have interaction are mediated under the synergistic effect of a nicking agent and DNA polymer, and finally exponential amplification of miRNA is realized under steady temperature condition; and by fixing the upstream primer and/or the downstream primer labeled by fluorescence reporter molecules and cancellation molecules on a solid chip, the high throughput detection on various target molecules can be realized by detecting fluorescence signals. With the method, the design difficulty of the amplification detection method caused by the inherent attributes of miRNA is overcome, a liquid constant temperature index amplification and detection system based on the oligonucleotide primer is provided, and constant temperature index amplification and detection of a plurality of target molecules can be realized under the steady temperature condition.

Description

The solid phase chip Constant Temperature Detection method of Microrna
Technical field
The invention belongs to life science and biological technical field, is a kind of solid phase chip Constant Temperature Detection method that mode quantitative and qualitative analysis by solid phase chip constant temperature exponential amplification detects multiple Microrna (microRNA, miRNA).
Background technology
Compare with nucleic acid molecule such as genomic dna, mRNA and long segment non-coding RNAs (lncRNA), Microrna (microRNA, miRNA) molecule has its singularity, as: nucleic acid molecule fragment minimum (≤22nt), GC content distribution extensively (5 ~ 95%), cause melting temperature (Tm) (meltingtemperature, Tm) difference large, be difficult to the amplimer and the detection probes that design coupling; The ratio extremely low (≤0.01%) of shared total serum IgE, needs highly sensitive detection method; Family molecule difference little (can only have single base difference) is high to the requirement of detection specificity; The expression level very different of different miRNA (can reach 4 orders of magnitude; As: tens copy/cell to 5 × 10 5copy/cell), require to detect linear wide ranges, can effectively identify low abundance miRNA simultaneously; Target sequence is present in pri-miRNA and pre-miRNA simultaneously; Lack poly (A) tail being similar to mRNA, make it be difficult to carry out reverse transcription detection by routine techniques means.The above-mentioned feature of miRNA proposes the specific demand near harshness to its nucleic acid molecule detection technique and method.Such as, minimum molecule fragment makes Standard PCR PRIMER DESIGN STRATEGY implement, and the base resolution capability of single base difference to primer or probe requires high, and the greatest differences of GC content causes primer and probe to be difficult to be oriented in homogeneous annealing temperature.
Since Late Cambrian miRNA in 1993, the detection method of miRNA from NorthernBlot probe hybridization method gradually to high-flux sequence method (as: Solexa method and SoLiD method etc.), chip hybridization methods, fluorescence in situ hybridization (fluorescenceinsituhybridization, ISH), real-time fluorescence reverse transcription quantitative PCR method (real-timereversetranscriptionquantitativePCR, RT-qPCR), the development of constant-temperature amplification detection technique.Due to the above-mentioned particular feature of miRNA target molecule, existing often kind of detection technique and method characteristic different, have his own strong points, but also have obvious shortcoming, thus make often kind of method have its optimal environment for use.
For the microarray technology based on chip hybridization methods the most frequently used at present, high-flux sequence method and RT-qPCR.The detection flux of high-flux sequence and microarray is large, and high-flux sequence also can be used for finding new miRNA, but the quantification accuracy of these two kinds of methods is poor, expensive, is generally applied to the primary dcreening operation research of miRNA.In addition, the preparation of high throughput sequencing technologies library is complicated, and the huge and complicated literature data analysis reaching megabyte (terabytes, TB) needs special computer resource and relevant bioinformatics tools and means, therefore, often provided by the company of specialty.Because the fragment of miRNA is little, and Tm value difference is greatly different, microarray technology can not carry out the hybridization efficiency of balanced different probe simply by the length increasing and reduce probe, therefore, often need by some special technique means, such as use lock nucleic acid probe (lockednucleicacid, LNA).The RT-qPCR technology being representative with stem ring primer RT-qPCR, poly (T) joint RT-qPCR is the method verifying that genomic level miRNA express spectra result of study is the most frequently used and the most classical, but this technology all needs reverse transcription step, experimental data normalization method by many factors restriction affects greatly on detected result tolerance range, " thermally denature-renaturation-extension " thermal cycle conditions needs highly purified miRNA, in addition, amplification efficiency is subject to impact and the restriction of many factors, easily occurs non-specific amplification; The amplified reaction time is long, general needs several hours; Be limited to thermokinetics feature, accurately cannot detect the differential expression of less than 2 times.
Desirable miRNA detection method should have following characteristics: highly sensitive, can reach and detect a small amount of or micro-parent material; Specificity is good, can identify the miRNA only having single base difference; Easy and simple to handle, without the need to reagent and the equipment of costliness; Versatility is high, can be applied to multiple field such as tissue and cell in-situ detection, high throughput testing, accurate quantification detection of miRNA.But as mentioned above, be limited to the particular attribute of miRNA target molecule and the inherent limitation of prior art, the existing detection technique of miRNA often can only meet above-mentioned portion requirements.If a kind of techniques and methods that can meet the demand better newly can be developed, on the one hand, can be more widely used, such as, adopt a kind of techniques and methods just can realize the application in multiple field, thus effectively expand the depth of investigation and the range of non-coding RNA (non-codingRNA, ncRNA).
Isothermal amplification technology is the substantially constant nucleic acid amplification technologies of temperature.Relative to the alternating temperature amplification technique taking PCR as representative, isothermal amplification technology has following main advantage: temperature of reaction is single, low to the requirement degree of equipment; There is not temperature variation, amplification efficiency and amplified nucleic acid fragment length are all better than Standard PCR technology; Reaction times is short, at 1 hour, even can realize effective amplification of target molecule in several minutes, and sensitivity is suitable with round pcr with specificity, even better.Above-mentioned technical superiority makes isothermal amplification technology be widely applied in the every field of life science, and, be particularly suitable for the detection of miRNA.
At present, develop multiple nucleic acids isothermal amplification technology, wherein, representative technology mainly comprises: strand displacement amplification (stranddisplacementamplification, SDA), ring mediation nucleic acid isothermal amplification technology (loop-mediatedisothermalamplification, LAMP), rolling circle amplification (rollingcircleamplification, RCA), depend on the amplification (nucleicacidsequence-basedamplification of nucleotide sequence, NASBA), depend on the isothermal amplification technique (helicase-dependentisothermalDNAamplification of helicase, HAD), transcript mediated amplification (transcription-mediatedamplification, TMA), single primer isothermal duplication (singleprimerisothermalamplification, SPIA), signal mediate rna amplification technique (signalmediatedamplificationofRNAtechnology, SMART), constant temperature exponential amplification (exponentialamplificationreaction, EXPAR).But, due to the singularity of miRNA target molecule, only have part constant temperature technology to be suitable for the detection of miRNA, such as SDA, RCA and EXPAR.But be limited to the technical limitation of existing constant temperature technology, existing miRNA Constant Temperature Detection technology often can only detect single miRNA target molecule, cannot realize detection that is multiple or high-throughput miRNA.
No matter be alternating temperature or isothermal amplification technology, archaeal dna polymerase is all indispensable enzymes of its reaction system.Generally speaking, archaeal dna polymerase has one or more biological functions following usually: 5' → 3' amplification active (amplificationactivities), 5' → 3' exonuclease activity (exonucleaseactivities), 3' → 5' exonuclease activity, strand-displacement activity (stranddisplacementactivities).
No matter be alternating temperature or isothermal amplification technology, in primer extension process, when its 3'-extending end arrives at downstream DNA chain, namely there is another double-stranded DNA (doublestrandDNA formed with template molecule complementary pairing in its 3'-extending end, dsDNA), during region, if archaeal dna polymerase neither possesses 5' → 3' 5 prime excision enzyme activity, also strand-displacement activity is not possessed, so, the 3'-end extension of primer is then subject to the prevention of downstream DNA chain and cannot continues to extend.But, when archaeal dna polymerase has 5' → 3' 5 prime excision enzyme activity (as: Taq DNA polymerase), downstream DNA chain can be hydrolyzed into mononucleotide according to 5' → 3' direction by this activity, thus the extension of primer is continued, as the hydrolysis probes technology in real-time fluorescence PCR; Or when archaeal dna polymerase has strand-displacement activity (as: BstDNA polysaccharase), this activity can make the extension of primer be continued, meanwhile, downstream DNA chain is peeled off from dsDNA region, makes the single stranded nucleic acid molecule that downstream DNA chain becomes free.The above-mentioned strand-displacement activity of archaeal dna polymerase is widely used in multiple constant-temperature amplification system, as SDA, LAMP, RCA etc.
There is strand-displacement activity, lack the strand replacement reaction that the archaeal dna polymerase of 5' → 3' 5 prime excision enzyme activity is widely used in relying on double chain acid molecule breach.So-called double chain acid molecule breach refers to that a chain of double chain acid molecule keeps integrity, the phosphodiester bond rupture between certain two contiguous nucleotides of another chain, thus forms a breach.The nucleic acid molecule end of these breach both sides is that 3'-terminal hydroxy group (3'-hydroxygroup, 3'-OH) and 5'-hold phosphate group (5'-phosphategroup, 5'-PO4) respectively.Under the effect that above-mentioned archaeal dna polymerase exists, have nucleic acid molecule extension from indentation, there 3'-OH of 3'-OH, meanwhile, the old chain in downstream is peeled off by its nascent nucleic acid strand extending synthesis, makes downstream old chain be transformed into free single stranded nucleic acid molecule.
Breach restriction endonuclease (nickingendonuclease), is also breach enzyme (nickingenzyme), is the class specific type enzyme in II type restriction enzyme (restrictionendonuclease).Have been found that more than 280 plant breach enzyme at present, commercial nearly more than 20 plant, and its thermostability is to reaching 65 DEG C or higher.This fermentoid only cuts a chain in double chain acid molecule, causes a double chain acid molecule breach, and the nucleic acid molecule end of these breach both sides is 3'-OH and 5'-PO4 respectively.Breach endonuclease recognition sequence (nickingendonucleaserecognitionsequences, NERS) is called as by the nucleotide sequence of breach restriction endonuclease identification wholly or in part in double chain acid molecule.
A kind of enzyme (as: HincII) of specific type is also had in restriction enzyme, this fermentoid has the function of convenient restriction restriction endonuclease, can identify and at restriction endonuclease recognition sequence (restrictionendonucleaserecognitionsequences, the RERS) place of natural double chain acid molecule simultaneously enzyme cut two chains of double chain acid molecule.But, when the chain of in double chain acid molecule at least contains a derivatized nucleotide (as: time α sulfydryl-deoxynucleotide (α-thiodeoxynucleotide) in RERS sequence, this derivatized nucleotide can stop restriction enzyme to cut this nucleic acid molecule chain, therefore, the native nucleic acid molecule chain that another does not contain derivatized nucleotide can only be cut.This only have the double chain acid molecule that the RERS sequence of a chain contains the cutting of prevention restriction enzyme to be called as half modification RESR.Visible, can identify and cut half restriction enzyme modifying RERS that there is the function similar to breach restriction endonuclease, can for the preparation of double chain acid molecule breach.
The above-mentioned biologic activity that the restriction enzyme of RESR is modified in breach restriction endonuclease and identifiable design hemisect half is widely used in relying on the strand replacement reaction of double chain acid molecule breach, and, this strand replacement reaction principle is widely used in multiple isothermal amplification technology, as SDA, EXPAR etc.Such as, breach restriction endonuclease (or: the restriction enzyme of RESR is modified in identifiable design hemisect half) and there is strand-displacement activity archaeal dna polymerase acting in conjunction under, there is nucleic acid molecule extension from indentation, there 3'-OH of 3'-OH, meanwhile, the old chain in downstream is peeled off by its nascent nucleic acid strand extending synthesis.The breach be closed because of chain extension can repeat to produce under the effect of breach restriction endonuclease (or: the restriction enzyme of RESR is modified in identifiable design hemisect half), thus the process of " cutting-extension-strand displacement " can be repeated, and in the process, constantly peel off or discharge the single stranded nucleic acid molecule (WalkerGT identical with downstream old chain sequence with linear or exponential manner, etal.Stranddisplacementamplification--anisothermal, invitroDNAamplificationtechnique.NucleicAcidsRes.1992; 20 (7): 1691-6.WalkerGT, etal.Stranddisplacementamplification--anisothermal, invitroDNAamplificationtechnique.NucleicAcidsRes.1992; 20 (7): 1691-6.VanNessJ, etal.Isothermalreactionsfortheamplificationofoligonucleo tides.ProcNatlAcadSciUSA.2003; 100 (8): 4504-9.ShiC, etal.Exponentialstrand-displacementamplificationfordetec tionofmicroRNAs.AnalChem.2014; 86 (1): 336-9).
Lock nucleic acid (lockednucleidacid, LNA) a kind of special double-ring oligonucleotide derivative, 2'-O and 4'-C of the β-D-RIBOSE of its ribose forms Oxymethylene bridge, sulphur methylene bridge or amine methylene bridge by different shrink effects, and connect into annular, this annular bridge has locked the N configuration of furanose C3'-inner mold, reduce the snappiness of ribose structure, add the stability of phosphate backbone local structure.Because LNA and DNA/RNA structurally has identical phosphate backbone, therefore it has good recognition capability and powerful avidity to DNA, RNA.Compared with other oligonucleotide analogs, DNA/LNA inserted type oligonucleotide has good recognition capability to single nucleotide variation, is widely used in different kinds of molecules Biological Detection technology with DNA/LNA chimeric primers or probe geometries.
Solid phase chip (solidchip) is also biochip (biochips), solid phase chip is a kind of high-throughout nucleic acid molecule detection means, has been widely used at research field tools such as gene expression spectrum analysis, Study on gene polymorphism, genome copies number variations.But, when biochip applications detects in the nucleic acid target molecule comprising miRNA, often depend on merely the mode of nucleic acid hybridization, target molecule needs amplification in advance, and in amplification procedure, in amplified production, introduce detectable signal (as: fluorescent mark), the hybridization of amplified production or target molecule and solid phase chip the step such as needs repeatedly to wash and removes non-specific hybridization and background signal disturbs, there are the problems such as detecting step is loaded down with trivial details, length consuming time, constrain the application of solid phase chip in field of nucleic acid detection to a certain extent.
Therefore, if effectively integrate breach enzyme, there is the synergy advantage of strand-displacement activity archaeal dna polymerase and fluorescent label DNA/LNA chimeric primers, under isothermal condition, the exponential amplification of target molecule just can be realized by " cutting-extension-strand displacement " autonomous chain circulation, simultaneously, above-mentioned constant temperature exponential amplification methods is combined with solid phase chip, greatly can simplify existing solid phase chip detecting step, the detection of hundreds and thousands of kinds of miRNA target molecules can be completed in very short time, thus effectively solve miRNA prior art bottleneck, realize the clinical practice application that miRNA detects.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of constant temperature exponential amplification system being fixed on the primer mediation of solid phase chip, this system can realize the constant temperature exponential amplification of target molecule at solid phase chip by " cutting-extension-strand displacement " autonomous chain circulation, and, the detectable signal that this exponential amplification discharges by solid phase chip immobilized primer characterizes, quantitative and qualitative analysis detection is carried out to hundreds and thousands of kinds of miRNA target molecules in theory simultaneously, there is detected energy high, high specificity, responsive high, reproducible, sample consumption is few, consuming time short, the advantages such as step is easy.
In order to solve the problems of the technologies described above, the invention provides following technical scheme: the solid phase chip Constant Temperature Detection method of Microrna, reaction mixture comprises following reacted constituent:
1. there is the miRNA target molecule of 3'-terminal hydroxy group;
2. upstream primer and downstream primer;
3. archaeal dna polymerase;
4. the breach agent of upstream primer and downstream primer breach agent recognition sequence is identified;
5. triphosphate deoxy-nucleotide;
6. ion and the buffer system of above-mentioned archaeal dna polymerase and breach agent biologic activity is met;
Described upstream primer and downstream primer are the sense strand sequence containing jagged dose of recognition sequence, and its 5' → 3' base is all followed successively by the recognition sequence district of anchor series district, specific recognition miRNA target molecule sequence; The anchor series district of upstream primer and downstream primer and miRNA target molecule are without homology, and the recognition sequence district of upstream primer, downstream primer is complementary with the 3'-terminal sequence of miRNA target molecule and miRNA target molecule complementary strand respectively;
Described upstream primer and downstream primer have target molecule specificity, often kind of specific upstream primer of target molecule and/or downstream primer are fixed on solid phase chip by its 5'-end, further, the upstream primer and/or the downstream primer that are fixed on solid phase chip mark the fluorescent reporter molecule and quencher molecule with FRET (fluorescence resonance energy transfer) effect respectively in the both sides of its breach agent recognition sequence.
Adopt solid phase chip constant temperature exponential amplification and the detection method of the Microrna of technical solution of the present invention, breach agent (nickingagent, NA) be used to identify and cutting upstream primer, downstream primer breach agent recognition sequence (nickingagentrecognitionsequences, NARS).When the breach agent recognition sequence of primer is breach endonuclease recognition sequence (nickingendonucleaserecognitionsequences, NERS) time, breach agent used is the breach restriction endonuclease (nickingendonuclease, NE) identifying this breach endonuclease recognition sequence; When the breach agent recognition sequence of primer is half modification restriction endonuclease recognition sequence (restrictionendonucleaserecognitionsequences, RERS) time, breach agent used is the restriction enzyme (restrictionendonuclease, RE) identifying this half modification RERS; In the reactive mixture, thing to be detected is the various biological specimens containing miRNA target molecule in the present invention.
Triphosphate deoxy-nucleotide, generally includes dCTP, dGTP, dTTP or dATP, synthesizes at biological DNA, and plays raw material effect in the chain extension reaction of DNA mediation.
Principle of work of the present invention and beneficial effect are: upstream primer and downstream primer are the sense strand sequence containing jagged dose of recognition sequence, and its 5' → 3' base is followed successively by the recognition sequence district of anchor series district, specific recognition miRNA target molecule sequence.The recognition sequence district of upstream primer P1 and downstream primer P2 is complementary with the 3'-terminal sequence of miRNA target molecule and miRNA target molecule complementary strand (that is: miRNA*) respectively, therefore, upstream primer and downstream primer can identify miRNA target molecule or miRNA target molecule complementary strand respectively, and have single nucleotide variation recognition capability; The sequence in anchor series district and miRNA target molecule, without homology, can not produce non-specific hybridization with miRNA target molecule or miRNA target molecule complementary strand.In amplification procedure, upstream primer P1 and downstream primer P2 anchor series district can hold extension products to form stable heteroduplex with the 3'-of miRNA target molecule and miRNA target molecule complementary strand respectively, thus enable upstream primer P1 and downstream primer P2 respectively " grappling " hold extension products end at the 3'-of miRNA target molecule and miRNA target molecule complementary strand, and, " grappling " should can form breach agent recognition sequence double chain acid molecule in the anchor series district of primer, thus enable breach agent at the breach agent recognition sequence positive-sense strand cutting primer of primer, and under the synergy of archaeal dna polymerase, continuous generation nascent DNA chain, archaeal dna polymerase typically refers to RNA dependent form archaeal dna polymerase.Therefore, when the recognition sequence district of upstream primer and downstream primer identifies miRNA target molecule and miRNA target molecule complementary strand and after forming heteroduplex respectively, under the effect of archaeal dna polymerase, upstream primer, downstream primer, miRNA target molecule, miRNA target molecule complementary strand all produce 5' → 3' direction chain extension, and at upstream primer and miRNA target molecule, form two kinds of complete double chain acid molecules between downstream primer and miRNA target molecule complementary strand respectively, then, breach agent is at breach agent recognition sequence sense strand sequence place cutting upstream primer and downstream primer.Now, upstream primer and downstream primer are under the effect of archaeal dna polymerase, extend nascent DNA chain in indentation, there respectively, this nascent DNA chain and original miRNA target molecule or miRNA target molecule complementary strand are complementary respectively, namely new miRNA target molecule or miRNA target molecule complementary strand.These new miRNA target molecules or miRNA target molecule complementary strand constantly increase from indentation, there cutting-extension-strand displacement process under the synergy of breach agent and archaeal dna polymerase formation double chain acid molecule in cement out, trigger the upstream primer of a new round and the above-mentioned reaction of downstream primer, circulation like this, realizes the exponential amplification of miRNA target molecule under constant temperature.In above process, the anchor series district of upstream primer, downstream primer then enables said process continue to occur because of its " grappling " effect.
Further, the described specific upstream primer of often kind of target molecule and downstream primer are fixed on a specific site of solid-state chip simultaneously; Or, often kind of target molecule only have it to have specific site that specific upstream primer is fixed on solid-state chip; Or, often kind of target molecule only have it to have specific site that specific downstream primer is fixed on solid-state chip; Or the same specific site of solid phase chip fixes the specific upstream primer of one or more target molecules and another kind or more the specific downstream primer of other target molecule simultaneously.
Further, described solid phase chip comprises microarray or microballon, and based on the biochip with multiple micro-reacting hole that microfluid, micro-fluidic or micro-manufacturing process produce.Target molecule specific upstream primer of the present invention and/or downstream primer is fixed at the specific site on solid-phase matrix surface.Described solid-phase matrix comprises the sheet glass of multiple method process, silicon chip, ceramic plate, plastics, cellulose nitrate, nylon membrane or rubber etc.
Each specific site of solid phase chip or micro-reacting hole only fix upstream primer corresponding to a kind of miRNA target molecule and/or downstream primer, now, each specific site or micro-reacting hole only detect a kind of miRNA target molecule, with this understanding, be fixed on upstream primer corresponding to often kind of miRNA target molecule of solid phase chip and/or downstream primer mark has FRET (fluorescence resonance energy transfer) (fluorescenceresonanceenergytransfer, FRET) fluorescent reporter molecule acted on and quencher molecule, the primer not being fixed on solid phase chip does not then make above-mentioned mark, with this understanding, whether the target molecule specific upstream primer fixed by each specific site or micro-reacting hole and/or downstream primer discharge fluorescent signal judges whether there is target molecule in sample to be detected, the fluorescence signal intensity of the target molecule specific upstream primer fixed by each specific site or micro-reacting hole and/or downstream primer release judges the target molecule abundance in sample to be detected, or, each specific site of solid phase chip or micro-reacting hole fix upstream primer corresponding to two or more miRNA target molecule and/or downstream primer simultaneously, and, the upstream primer that the simultaneously fixing often kind of miRNA target molecule of each specific site or micro-reacting hole is corresponding and/or downstream primer mark have FRET (fluorescence resonance energy transfer) (fluorescenceresonanceenergytransfer, FRET) the different fluorescent reporter molecule acted on and quencher molecule, the primer not being fixed on solid phase chip does not then make above-mentioned mark, with this understanding, two or more miRNA target molecule can be detected by quantitative and qualitative analysis specific site or micro-reacting hole while.
The upstream primer that solid phase chip is fixed and/or downstream primer comprise can produce detection signal there is the fluorescent reporter molecule (donor molecule) and quencher molecule (acceptor molecule) composition that FRET (fluorescence resonance energy transfer) (fluorescenceresonanceenergytransfer, FRET) acts on.In FRET technology, energy donor (that is: fluorescent reporter molecule or donor molecule) has fluorescence, and energy acceptor (that is: quencher molecule or acceptor molecule) can be fluorescence or non-fluorescent material.Two kinds of marks (that is: fluorescent reporter molecule and quencher molecule) with FRET effect of the present invention (are produced the part of breach cleavage sites by the specific site of in primer, i.e. breach agent recognition sequence positive-sense strand) be separated, thus, under target molecule existent condition, breach agent can at the breach agent recognition sequence positive-sense strand cutting primer of primer, this cleavage reaction can cause fluorescent reporter molecule and quencher molecule to be separated from each other, lose FRET effect, thus make fluorescent reporter molecule discharge fluorescent signal, produce the detected fluorescent signal characterizing target molecule.Because the primer that often kind of solid phase chip is fixing corresponds to a kind of specific miRNA target molecule, therefore, the solid phase chip kind by discharging fluorescent signal determines the kind of the miRNA target molecule existed in thing to be detected.Fluorescent reporter molecule and quencher molecule are fluorescent substance, all can utilize known any material in the technical field of the invention, comprise: PE (Fluorescentalgaeofeggs, phycoerythin) (620), Cy2 tM(506), YO-PRO tM-1 (509), YOYO tM-1 (509), Calcein (517), FITC (518), FluorX tM(519), Alexa tM(520), Rhodamine110 (520), OregonGreen tM500 (522), OregonGreen tM488 (524), RiboGreen tM(525), RhodamineGreen tM(527), Rhodamine123 (529), MagnesiumGreen tM(531), CalciumGreen tM(533), TO-PRO tM-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPYTMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3TM (570), Alexa tM546 (570), TRITC (572), MagnesiumOrange tM(575), PhycoerythrinR & B (575), RhodaminePhalloidin (575), CalciumOrange tM(576), PyroninY (580), RhodamineB (580), TAMRA (582), RhodamineRedTM (590), Cy3.5TM (596), ROX (608), CalciumCrimson tM(615), AlexaTM594 (615), TexasRed (615), NileRed (628), YO-PROTM-3 (631), YOYOTM-3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO tM-3 (660), TOTO3 (660), DiDDilC (5) (665), Cy5 tM(670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), BiosearchBlue (447), CALFluorGold540 (544), CALFluorOrange560 (559), CALFluorRed590 (591), CALFluorRed610 (610), CALFluorRed635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar650 (566), Quasar570 (667), Quasar670 (705) and Quasar705 (610).The numeral of bracket is the maximum emission wavelength represented with nanometer unit.
Quencher molecule utilizes the known black quencher molecule of non-fluorescence can carrying out cancellation to the fluorescence of wide range of wavelength or specific wavelength in the technical field of the invention, comprises black hole quencher, (BHQ:blackholequencher; Comprise BHQ1, BHQ2, BHQ3), 4-[4-(dimethylamino) benzeneazo] phenylformic acid (DABCYL:4-[4-(Dimethylamino) phenylazo] benzoicacid).
In the FRET mark of primer, fluorescent reporter molecule comprises the donor of FRET, and quencher molecule comprises the acceptor of FRET, such as, fluorescein(e) dye (fluoresceindye) is reporter molecules, and rhodamine (rhodaminedye) is quencher molecule.
Fluorescent reporter molecule and quencher molecule are all positioned at the anchor series district of primer, and, lay respectively at the 5'-end of breach agent recognition sequence and the optional position of 3'-end; Or fluorescent reporter molecule and quencher molecule lay respectively at anchor series district and recognition sequence district, and, lay respectively at the 5'-end of breach agent recognition sequence and the optional position of 3'-end.The common trait of above-mentioned mark is, because primer is fixed on solid phase chip by its 5'-end, there is the condition of target molecule in reaction system under, the primer being fixed on solid phase chip discharges fluorescence in the cutting action of its breach agent recognition sequence positive-sense strand because of breach agent, this fluorescence is attached to solid phase chip because of the 5'-end fixed action of primer, thus judge to there is target molecule in detection system by the fluorescent signal that the primer that solid phase chip is fixed produces, realize the qualitative detection of target molecule.When under the condition that there is not target molecule corresponding to its immobilized primer in reaction system, the primer being fixed on solid phase chip can not be cut by breach agent, its fluorescent reporter molecule and quencher molecule do not produce fluorescent signal because of FRET effect, now, its solid phase chip also can not detect the detected fluorescent signal that primer produces, thus judges to there is not target molecule in detection system.Meanwhile, the detected fluorescence signal intensity of primer generation fixed due to solid phase chip and the Initial abundance positive correlation of target molecule, therefore, the fluorescence intensity of the primer release can fixed further by solid phase chip realizes the detection by quantitative of target molecule.
The principle of its work is: when there is not miRNA target molecule in reaction system, is fixed on the fluorescent reporter molecule of the primer of solid phase chip and quencher molecule because of FRET effect to make primer not produce fluorescent signal; When there is miRNA target molecule in reaction system, upstream primer, downstream primer, miRNA target molecule and the miRNA target molecule complementary strand produced thereof all can form complete double chain acid molecule under the effect of archaeal dna polymerase, thus enable nicking agent cutting be fixed on the upstream primer of solid phase chip and/or the breach agent recognition sequence positive-sense strand of downstream primer, now, the fluorescent reporter molecule and the quencher molecule that are fixed on solid phase chip lose FRET (fluorescence resonance energy transfer) effect and produce detectable fluorescent signal.When solid phase chip in reaction system has multiple specific site, and, often kind of specific site secures the specific upstream primer of target molecule and/or downstream primer, just same solid phase chip while, multiple target molecule can be detected by quantitative and qualitative analysis.Such as, according to the known technology of existing the technical field of the invention, at least can detect hundreds and thousands of kinds of miRNA target molecules in single micro-array chip simultaneously.
As depicted in figs. 1 and 2, the upstream primer of solid phase chip is fixed on and/or downstream primer comprises the fluorescent reporter molecule and quencher molecule with FRET effect by its 5'-end, and, fluorescent reporter molecule and quencher molecule lay respectively at 5'-end and 3'-end optional position (Fig. 1) of primer anchor series district breach agent recognition sequence, or, fluorescent reporter molecule and quencher molecule lay respectively at primer anchor series district and recognition sequence district, further, the 5'-end and the 3'-that lay respectively at breach agent recognition sequence hold optional position (Fig. 2).The upstream primer and/or the downstream primer that are not fixed on solid phase chip mark the fluorescent reporter molecule and the quencher molecule (Fig. 1, Fig. 2) that do not comprise FRET effect.When the primer being fixed on solid phase chip is cut by breach agent, detectable fluorescent signal can be produced, and, because its 5'-end is fixed on solid phase chip, therefore, solid phase chip is made to produce producible fluorescent signal, further, this fluorescent signal has target molecule specificity (Fig. 1, Fig. 2).
As shown in Figure 3,5' → 3' base sequence of upstream primer P1 and downstream primer P2 is the anchor series district at breach agent recognition sequence (nickingagentrecognitionsequences, NARS) sense strand sequence place, the recognition sequence district of specific recognition target miRNA successively.Above-mentioned primer has following three distinguishing feature (Fig. 1,2): the recognition sequence district of primer P1 and P2 is complementary with the 3'-terminal sequence of target miRNA and complementary strand (that is: miRNA*) thereof respectively, not only independent but also interactional " cutting-extension-strand displacement " linear amplification can be mediated respectively under the synergy of breach enzyme and archaeal dna polymerase, finally, the two coupling becomes an autonomous chain circulation, finally realizes the exponential amplification of target miRNA; The recognition sequence district specific position of the two all can introduce the LNA of proper amt, thus improve amplification temperature (reaching 55 ~ 65 DEG C), the Tm value (between 50 ~ 55 DEG C) of homogenization different GC content miRNA, strengthens the recognition capability of primer-template single base mismatch; Under the two can be fixed on solid phase chip condition simultaneously (Fig. 2), after primer to be combined with template specificity and to extend into double chain acid molecule, the cutting of breach agent will cause primer release fluorescence.Basic technique principle is as shown in Figure 3: target miRNA and primer P1 complementation combines, and because ripe miRNA and primer P1 all has 3'-OH, can extend into complete double chain acid molecule each other; Breach agent is at NARS sense strand sequence cutting primer P1, release fluorescent signal, simultaneously, the 5'-of this breach holds nucleic acid molecule, namely primer 5'-holds the 3'-terminal sequence of anchor series district place nucleic acid molecule because having 3'-OH, can extend further, and under the strand-displacement activity effect of archaeal dna polymerase, displace the nascent DNA chain (that is: miRNA*) with target miRNA complete complementary, thus form first " cutting-extension-strand displacement " linear amplification; The miRNA* that primer P1 discharges according to similarity principle, can trigger second " cutting-extension-strand displacement " linear amplification of primer P2 mediation again, and release and the identical nascent DNA chain (that is: miRNA) of target miRNA sequence; First with second linear amplification by the nascent DNA chain of separately release each other coupling become an autonomous chain circulation, and, often generate a nascent DNA chain, primer P1 or P2 all can be caused to discharge fluorescence, the fluorescence intensity of reaction system release and the proportional relation of initial amount of target miRNA; When the upstream primer that only fixing a kind of miRNA target molecule is corresponding on each specific site of solid phase chip and/or downstream primer, the upstream primer that often kind of miRNA target molecule is corresponding and/or downstream primer can mark identical fluorescent reporter molecule, now, each specific site only detects a kind of miRNA target molecule; Or, when each specific site of solid phase chip fixes upstream primer corresponding to two or more miRNA target molecule and/or downstream primer, with this understanding, the upstream primer that often kind of miRNA target molecule fixing of each specific site is corresponding and/or downstream primer mark different fluorescent reporter molecules, but the upstream primer that the miRNA target molecule fixed of different specific site is corresponding and/or downstream primer can mark identical fluorescent reporter molecule, with this understanding, solid phase chip can detect two or more different miRNA target molecule at each specific site.Because the fluorescent signal of primer release is all attached to solid phase chip, therefore, detect while the fluorescent signal kind discharged and abundance realize multiple target molecule by each specific site of solid phase chip.
Each specific site of solid phase chip only fixes upstream primer corresponding to miRNA target molecule (Fig. 4) or downstream primer (Fig. 5).It is similar with downstream primer (Fig. 3) that the principle of above-mentioned two kinds of cases release fluorescent signal and solid phase chip fix upstream primer simultaneously, do not affect the methodology parameters such as the sensitivity of detection method and specificity.
Further, the anchor series district 5'-of the described upstream primer being fixed on solid phase chip and/downstream primer holds and increases poly base, comprises poly born of the same parents gland pyrimidine bases (Poly (T n)) or poly adenine base (Poly (A n)), and be fixed on solid phase chip by the 5'-end of described poly base.Solid phase chip is fixed on to simplify above-mentioned primer, and, can minimize and (spacehindrance) is hindered to the space of enzyme effect (as: cutting action of breach agent), and increase the hybridization efficiency of primer and target molecule, improve constant-temperature amplification efficiency.Poly (T n) or Poly (A n) do not affect typical structure and the function thereof of primer of the present invention.
Further, target molecule specific upstream primer and/or downstream primer are directly or indirectly fixed on solid phase chip by its 5'-end, fixing mode comprises fixes with the combination of covalent linkage and non-co-part key, or, be coupled mode by physical adsorption and/or chemistry and fix, or, alkyl or the aryl compound with amido are fixed as connection peptides, or, there is the alkyl of thiol group or aryl compound as connection peptides immobilized primer, or, fixed by arm molecule.Its fixing means comprises known technique for fixing in the technical field of the invention, comprise and be coupled mode by physical adsorption and/or chemistry upstream primer and/or downstream primer are connected to solid phase chip, or, comprise there is amido alkyl or aryl compound as connection peptides immobilized primer, or, comprise there is thiol group alkyl or aryl compound as connection peptides immobilized primer, or, be included in solid phase chip and/or primer mark, modify or synthesize and have arm molecule, and by arm molecule therebetween, primer is fixed on solid phase chip by the interaction of arm molecule and other molecule.
Further, anchor series district, the recognition sequence district of the upstream primer that the miRNA target molecule of described multiple kind is corresponding and/or downstream primer all introduce derivatized nucleotide, and derivatized nucleotide comprises lock nucleic acid, peptide nucleic acid(PNA) or thio-modification base.
Further, containing the Nucleotide with miRNA target molecule complementary sequence 3'-end penultimate and/or the 3rd bit base mispairing in the upstream primer that the miRNA target molecule of described multiple kind is corresponding and/or the recognition sequence district of downstream primer;
Or, in described reaction mixture, add the bioactive molecules that can suppress non-specific amplification, comprise TaqMutS, RecA.
Further, described archaeal dna polymerase is the archaeal dna polymerase with strand-displacement activity; Or described archaeal dna polymerase does not have strand-displacement activity, and in described reaction mixture, add the bioactive molecules with strand-displacement activity.
Further, the range of reaction temperature of described constant temperature is 16-70 DEG C.
Further, the temperature of reaction of described constant temperature is 37 DEG C, 55 DEG C, 60 DEG C or 65 DEG C.
Further, the described reaction times is 10-80min.
Further, the described reaction times is 10min, 20min, 30min, 40min, 50min or 60min.
Further, present invention also offers the detection reagent or test kit that utilize the real-time constant temperature exponential amplification methods of solid phase chip of the present invention, its principal character and the reacted constituent comprised comprise: employ upstream primer of the present invention and downstream primer; Secure the solid phase chip of upstream primer and/or downstream primer; Thing to be detected is miRNA target molecule or has other small segment nucleic acid molecule of 3'-OH, or thing to be detected is containing miRNA target molecule or the various biological specimens with other small segment nucleic acid molecule of 3'-OH; Archaeal dna polymerase; Identify the breach agent of upstream primer and downstream primer breach agent recognition sequence; Triphosphate deoxy-nucleotide; Meet ion and the buffer system of above-mentioned archaeal dna polymerase and breach agent biologic activity function; Qualitative and/or the detection by quantitative of the high-throughput of multiple target molecule is realized by the detectable signal detecting the release of solid phase chip immobilized primer.
Explanation of nouns:
" derivatized nucleotide (derivatizednucleotide) " refers to other type Nucleotide outside natural nucleotide.
" non-specific amplification " refers to the amplification that non-target molecules causes.
" target molecule " refers to the material adopting the method for the invention directly or indirectly to detect.
" oligonucleotide (oligonucleotide; ODN) " refers to small molecules nucleic acid, by nucleotide residue (fragment), by phosphodiester bond (phosphodiester) or other chemical bond, (as: phosphorothioate bond (phosphorothioates) connects or is polymerized, molecular weight between nucleic acid and Nucleotide, and tends to Nucleotide.The number of the present invention to nucleotide residue there is no strict boundary.
" nascent DNA chain " refers to the DNA molecular that primer synthesizes at the effect downward-extension of archaeal dna polymerase.
" qualitative detection " refers to whether detect nucleic acid target molecule exists, or whether detection target molecule is present in thing to be detected.
" detection by quantitative " refers to the concentration detecting target molecule, or detects the concentration of thing target to be detected, such as, detects the copy number of thing target to be detected.
" breach " refers to that a nucleic acid molecule chain of double chain acid molecule keeps integrity, the phosphodiester bond rupture between certain two contiguous nucleotides of another nucleic acid molecule chain, thus forms a breach.The nucleic acid molecule end of these breach both sides is 3'-OH and 5'-PO4 respectively.
" 5'-of breach holds nucleic acid molecule " refers to the nucleic acid molecule chain of double chain acid molecule indentation, there with 3'-OH.
" cutting (nicking) " refers to a chain of cutting complete complementary double chain acid molecule, or a chain in the double-stranded region of cutting part double chain acid molecule, and cutting position is positioned at the specific position of NARS.The specific position that nucleic acid molecule is cut is called as in " breach site (nickingsite, NS) " in the present invention
" breach agent recognition sequence (nickingagentrecognitionsequences, NARS) " refer to wholly or in part in double chain acid molecule by the nucleotide sequence of breach agent identification.NARS of the present invention comprises NERS and partly modifies RERS.
" breach endonuclease recognition sequence (nickingendonucleaserecognitionsequences, NERS) " refer to wholly or in part in double chain acid molecule by the nucleotide sequence of breach restriction endonuclease identification.
" restriction endonuclease recognition sequence (restrictionendonucleaserecognitionsequences, RERS) " refers to the nucleotide sequence that identifies of enzyme (RE) earnestly of being limited property in double chain acid molecule wholly or in part.
" partly modify restriction endonuclease recognition sequence (hemimodifiedRERS) " and refer in the RERS sequence of a chain in double chain acid molecule wholly or in part at least containing a derivatized nucleotide (as: sulfydryl-deoxynucleotide (-thiodeoxynucleotide), and, this derivatized nucleotide can stop the chain of restriction enzyme cutting containing this derivatized nucleotide (that is: cannot cut the nucleic acid molecule chain containing above-mentioned derivatized nucleotide in its recognition sequence at restriction enzyme) that can identify this RERS, another chain is then cut at the specific position of its recognition sequence, thus make restriction enzyme have the biological function the same with breach restriction endonuclease, that is: a chain in double chain acid molecule wholly or in part is only cut.
" breach agent (nickingagent, NA) " refers to the NARS sequence of identifiable design double chain acid molecule wholly or in part, and, the restriction endonuclease of a nucleic acid molecule chain is only cut in the breach site of NARS sequence double-stranded region.Breach agent comprises (but being not limited to) breach restriction endonuclease (nickingendonuclease, NE; As: N.BstNBI), restriction enzyme (restrictionendonuclease, RE; As: HincII).For restriction enzyme, only have when double chain acid molecule contains half modification RERS wholly or in part, restriction enzyme is just used by as breach agent.
" breach restriction endonuclease (nickingendonuclease; NE) " refer to a kind of nucleotide sequence that can identify double chain acid molecule wholly or in part, and only relative to its recognition sequence, namely the specific position of NERS cuts the restriction endonuclease of a nucleic acid molecule chain.This function is different from restriction enzyme, restriction enzyme needs to have a derivatized nucleotide at least usually in the recognition sequence of double chain acid molecule wholly or in part, this derivatized nucleotide can stop restriction enzyme cutting to contain the nucleic acid molecule chain of this derivatized nucleotide, and breach enzyme identifies natural nucleotide usually, further, a chain in double chain acid molecule wholly or in part is only cut.
" NARS sense strand sequence (sequenceofthesensestrandoftheNARS) " refers to the NARS sequence can cut by breach agent in double chain acid molecule wholly or in part, and this sequence contains the breach site of the breach agent identifying this NARS.
" NARS antisense strand sequence (sequenceoftheantisensestrandoftheNARS) " refers to the NARS sequence can not cut by breach agent in double chain acid molecule wholly or in part, and this sequence does not identify the breach site of the breach agent of this NARS.
" NERS sense strand sequence (sequenceofthesensestrandoftheNERS) " refers to the NERS sequence can cut by breach restriction endonuclease in double chain acid molecule wholly or in part, and this sequence contains the breach site of the breach agent identifying this NERS.
" NERS antisense strand sequence (sequenceoftheantisensestrandoftheNERS) " refers to the NARS sequence can not cut by breach restriction endonuclease in double chain acid molecule wholly or in part, and this sequence does not identify the breach site of the breach agent of this NERS.
" half modify RERS sense strand sequence (sequenceofthesensestrandofthehemimodifiedRERS) " refers in double chain acid molecule wholly or in part can the RERS sequence of being limited property restriction endonuclease cutting, this sequence contains the breach site of the breach agent identifying this RERS, it is characterized in that RERS sequence is all natural nucleotides.
" half modify RERS antisense strand sequence (sequenceoftheantisensestrandofthehemimodifiedRERS) " refers in double chain acid molecule wholly or in part can not the RARS sequence of being limited property restriction endonuclease cutting, this sequence does not identify the breach site of the breach agent of this RERS, it is characterized in that in RERS sequence at least containing a derivatized nucleotide (as: sulfydryl-deoxynucleotide, further, this derivatized nucleotide can stop and can identify that the restriction enzyme of this RERS cuts this sequence.
" constant temperature (isothermalconditions) " refers in amplification procedure, temperature of reaction keeps substantially invariable reaction conditions, and (that is: temperature is identical, or top temperature and minimum temperature difference are no more than the narrow temperature variation range of 20 ° of C).
" fixing " of the present invention refers to that being coupled mode by physical adsorption and/or chemistry is connected to solid phase chip by Oligonucleolide primers.
" physical adsorption " of the present invention refers to that Oligonucleolide primers to be connected with solid phase chip by secondary key (such as: ionic linkage) and to fix, or with non covalent bond effect by Oligonucleolide primers directly or constant potential be adsorbed onto solid phase chip, or pass through electrostatic interaction by the decorative layer that the phosphate radical negative ion in primer of the present invention and solid phase chip are positively charged and fix.
" chemistry is coupled " of the present invention is by forming covalent linkage (such as: amido linkage, ester bond, ehter bond etc.) active group of Oligonucleolide primers and solid phase chip is interacted, thus Oligonucleolide primers is fixed to solid phase chip, such as: first activating pretreatment solid phase chip, introduce various required active group, as amino, carboxyl, sulfydryl, hydroxyl, halogen radical (comprises fluorine, chlorine, bromine, iodine etc.) etc., or derivatized nucleotide, it is made to bring suitable functional gene, with bi-functional reagents or coupling activator contact, Oligonucleolide primers is fixed to solid phase chip subsequently, conventional double functional group has glutaraldehyde (GA), p-nitrophenyl chloroformate (NPC), maleimide (MA), diisothio-cyanate etc.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, technical solution of the present invention is further illustrated:
Fig. 1 is the typical consequence exemplary plot one of primer of the present invention;
Fig. 2 is the typical consequence exemplary plot two of primer of the present invention;
Fig. 3 present invention uses the constant temperature exponential amplification principle schematic that solid phase chip secures fluorescent mark upstream primer and downstream primer simultaneously;
Fig. 4 present invention uses the constant temperature exponential amplification principle schematic that solid phase chip only secures fluorescent mark upstream primer;
Fig. 5 present invention uses the constant temperature exponential amplification principle schematic that solid phase chip only secures fluorescent mark downstream primer;
Fig. 6 is detected result example of the present invention.
Embodiment
In specific implementation process; for a person skilled in the art, under the prerequisite not departing from structure of the present invention, some distortion and improvement can also be made; these also should be considered as protection scope of the present invention, and these all can not affect effect of the invention process and practical applicability.
Embodiment one: each specific site of solid phase chip fixes upstream primer corresponding to a kind of miRNA target molecule and/or downstream primer, detects 6 kinds of miRNA target molecules in individual solid phase chip simultaneously
The present invention only using the detection of 6 kinds of miRNA target molecules as embodiment, so that Detection results of the present invention to be described.In the solid phase chip of primer is fixed, in order to increase its simplification, the 5'-in the anchor series district of the present invention of immobilized primer holds introducing 10 continuous print poly T base (Poly (T 10)), and by Poly (T 10) chemistry of the amino of 5'-end and the carboxyl of solid phase chip finishing is coupled and realizes fixing at solid phase chip of primer.The chemistry that the present embodiment adopts is coupled, the fluoroscopic examination of solid phase chip, solid phase chip is all place of the present invention technical fields known technology and method.
1. the Design and synthesis of target molecule Auele Specific Primer
SEQNo.1 and SEQNo.2 is upstream primer and the downstream primer of amplification miRNA target molecule miR-105 (SEQNo.3) respectively, SEQNo.4 and SEQNo.5 is upstream primer and the downstream primer of amplification miRNA target molecule miR-26a (SEQNo.6) respectively, SEQNo.7 and SEQNo.8 is upstream primer and the downstream primer of amplification miRNA target molecule miR-16 (SEQNo.9) respectively, SEQNo.10 and SEQNo.11 is upstream primer and the downstream primer of amplification miRNA target molecule miR-189 (SEQNo.12) respectively, SEQNo.13 and SEQNo.14 is upstream primer and the downstream primer of amplification miRNA target molecule miR-451 (SEQNo.15) respectively, SEQNo.16 and SEQNo.17 is upstream primer and the downstream primer of amplification miRNA target molecule miR-7e (SEQNo.18) respectively.
The consensus feature in each primer 5' → 3' direction is containing Nt.BstNBI otch restriction endonuclease NERS sense strand sequence (that is: 5'-successively gAGTC-the recognition sequence district of anchor series district 3'), specific recognition miRNA target molecule (SEQNo.3, SEQNo.6, SEQNo.9, SEQNo.12, SEQNo.15, SEQNo.18) and complementary strand thereof.Wherein, the recognition sequence district of upstream primer and downstream primer is complementary with the 3'-end sequence of miRNA target molecule and complementary strand thereof respectively, and is designed with lock nucleic acid (LNA at the portion in recognition sequence district; In sequence with+number base).For the primer being fixed on solid phase chip, comprise SEQNO.1, SEQNO.4, SEQNO.8, SEQNO.11, SEQNO.13, SEQNO.14, SEQNO.16, SEQNO.17, the 5'-end all in the anchor series district of above-mentioned primer introduces Poly (T 10), and at Poly (T 10) the end modified amino group of 5'-.
Above-mentioned all oligonucleotide molecules are synthesized by specialized company.
Upstream primer SEQNo.1 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)ACC+AC+A+GG+AG
Downstream primer SEQNo.2 (5' → 3' direction)
CCGATCTAGT GAGTCtgttcttTC+AA+ATGC+TCA
MiRNA-105SEQNo.3 (5' → 3' direction)
UCAAAUGCUCAGACUCCUGUGGU
Upstream primer SEQNo.4 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)AGCC+TA+TCC+TG
Downstream primer SEQNo.5 (5' → 3' direction)
CCGATCTAGT GAGTCtgttcttT+TCA+AGT+AAT
MiR-26aSEQNo.6 (5' → 3' direction)
UUCAAGUAAUCCAGGAUAGGCU
Upstream primer SEQNo.7 (5' → 3' direction)
CCGATCTAGT GAGTCtgttcttCGCC+AA+TA+TT
Downstream primer SEQNo.8 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)T+AGC+AGC+ACGT
MiR-16SEQNo.9 (5' → 3' direction)
UAGCAGCACGUAAAUAUUGGCG
Upstream primer SEQNo.10 (5' → 3' direction)
CCGATCTAGT GAGTCtgttcttAC+TGA+TA+TCAG
Downstream primer SEQNo.11 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)TGCC+TAC+TG+AG
MiR-189SEQNo.12 (5' → 3' direction)
UGCCUACUGAGCUGAUAUCAGU
Upstream primer SEQNo.13 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)A+ACTC+AGT+AATG
Downstream primer SEQNo.14 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)A+A+ACCGT+TACC
MiR-451SEQNo.15 (5' → 3' direction)
AAACCGUUACCAUUACUGAGUU
Upstream primer SEQNo.16 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)A+ACTA+TACA+ACC
Downstream primer SEQNo.17 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GAGTCtgttctt(BHQ1)TG+AGGT+AGG+AG
Let-7eSEQNo.18 (5' → 3' direction)
UGAGGUAGGAGGUUGUAUAGUU。
2. the upstream primer that different target molecule is corresponding and/or downstream primer are fixed on the specific site of solid phase chip
Adopt the technical field of the invention known technological method, as shown in Figure 6, by the Poly (T of upstream primer and/or downstream primer 10) 5'-terminal amino group and the chemistry of carboxylic group of solid phase chip be coupled, primer is fixed on the specific site on solid phase chip surface, wherein:
The specific site that the first row three that the upstream primer SEQNo.1 that miR-105 target molecule is corresponding is fixed on solid phase chip repeats, the object of three specific sites repeated improves the accuracy of detected result and repeatability;
The specific site that the secondary series that the upstream primer SEQNo.4 that miR-26a target molecule is corresponding is fixed on three repeats, the object of three specific sites repeated improves the accuracy of detected result and repeatability;
The specific site that the 3rd row three that the downstream primer SEQNo.8 that miR-16 target molecule is corresponding is fixed on repeat, the object of three specific sites repeated improves the accuracy of detected result and repeatability;
The specific site that the 4th row three that the downstream primer SEQNo.11 that miR-189 target molecule is corresponding is fixed on repeat, the object of three specific sites repeated improves the accuracy of detected result and repeatability;
The specific site that the 5th row three that the upstream primer SEQNo.13 that miR-451 target molecule is corresponding and downstream primer SEQNo.14 is fixed on simultaneously repeat, the object of three specific sites repeated improves the accuracy of detected result and repeatability;
The specific site that the 6th row three that the upstream primer SEQNo.16 that miR-7e target molecule is corresponding and downstream primer SEQNo.17 is fixed on simultaneously repeat, the object of three specific sites repeated improves the accuracy of detected result and repeatability.
Further, the upstream described above and/or the downstream primer that are fixed on solid phase chip are all marked with PE fluorescent reporter molecule and BHQ3 quencher molecule respectively in the base of the NERS sense strand sequence both sides at place, anchor series district.
3. the extraction of serum total serum IgE
Commercialization total RNA extraction reagent box is adopted to extract 10 routine human peripheral blood leukocytes total serum IgE.
4. the structure of solid phase chip constant temperature exponential amplification system
Reaction system amounts to 50 μ L, and this system comprises following component: 20mMTris-HCl, 10mM (NH 4) 2sO 4, 50mMKCl, 8mMMgSO 4, 0.1%Tween-20,100 μ g/mlBSA, 5%DMSO, 0.2 unit Vent r(exo-) archaeal dna polymerase (NEB), 1.6 unit Nt.BstNBI breach enzyme (NEB), 1.5 μ g heat-resisting mismatch binding albumen TaqMutS (NipponGene), 600 μMs of dNTPs (Promega), the upstream and/or the downstream primer that are not fixed on solid phase chip in step 1 and 2 are often kind of primer 50nM, in step 2, surface secures the solid phase chip of upstream and/or downstream primer, target molecule is that the miRNA target molecule of the 0.1zmol of synthesis (comprising: SEQNo.3, SEQNo.6, SEQNo.9, SEQNo.12, SEQNo.15, SEQNo.18), or 10 routine human peripheral blood leukocytes total serum IgE prepared by step 3.
4. the amplification of target molecule and fluoroscopic examination
The reaction system of above-mentioned preparation is placed in ViiA7 real-time fluorescence quantitative PCR, and reaction conditions is: 60 DEG C 40 minutes.After completion of the reaction, the micro-array chip scanner of Beijing Bo Ao company is adopted to detect the fluorescence of each specific site of solid phase chip.
5. detected result and analysis
5.1 when only having miR-105 target molecule in reaction system, only have three of first row specific sites repeated FAM fluorescent signal can be detected, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.2 when only having miR-26a target molecule in reaction system, only have three of secondary series specific sites repeated FAM fluorescent signal can be detected, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.3 when only having miR-16 target molecule in reaction system, only have tertial three specific sites repeated FAM fluorescent signal can be detected, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.4 when only having miR-189 target molecule in reaction system, three that only have the 4th the to arrange specific sites repeated can detect FAM fluorescent signal, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.5 when only having miR-451 target molecule in reaction system, three that only have the 5th the to arrange specific sites repeated can detect FAM fluorescent signal, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.6 when only having miR-7e target molecule in reaction system, three that only have the 6th the to arrange specific sites repeated can detect FAM fluorescent signal, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.7 when there is 6 kinds of target molecules described in the present embodiment in reaction system simultaneously, first row can detect FAM fluorescent signal to three specific sites repeated of the 6th row, and the intensity of fluorescent signal is directly proportional (Fig. 6) to the starting point concentration of each target molecule.
The detected result of 5.8 clinical samples shows, miR-105, miR-26a, miR-16, miR-189, miR-451, miR-7e detected result of 10 routine samples is 5.23 ~ 7.28 × 10 respectively 3, 1.27 ~ 4.38 × 10 4, 2.13 ~ 3.24 × 10 4, 6.23 ~ 8.23 × 10 3, 4.23 ~ 6.89 × 10 4.
5.9 relative to the existing solid phase chip detection method based on hybridization, the present embodiment is nucleic acid amplification and the detection method being detected as one, nucleic acid amplification can be completed in 40 minutes, amplified production can be directly used in relevant device and detect, without the need to follow-up hybridization step, totally only about 50 minutes consuming time, well below the existing similar detection method based on hybridization, further, its methodology such as sensitivity and specificity parameter and existing similar detection method have good dependency.
Embodiment two: each specific site of solid phase chip fixes upstream primer corresponding to two kinds of miRNA target molecules and/or downstream primer, detects 6 kinds of miRNA target molecules in a solid phase chip simultaneously
In the solid phase chip of primer is fixed, in order to increase its simplification, the 5'-in the anchor series district of the present invention of immobilized primer holds introducing 10 continuous print poly T base (Poly (T 10)), and by Poly (T 10) chemistry of the amino of 5'-end and the carboxyl of solid phase chip is coupled and realizes fixing at solid phase chip of primer.The chemistry that the present embodiment adopts is coupled, the fluoroscopic examination of solid phase chip, solid phase chip is all place of the present invention technical fields known technology and method.
1. the Design and synthesis of target molecule Auele Specific Primer
SEQNo.1 and SEQNo.2 is upstream primer and the downstream primer of amplification miRNA target molecule miR-105 (SEQNo.3) respectively, SEQNo.4 and SEQNo.5 is upstream primer and the downstream primer of amplification miRNA target molecule miR-26a (SEQNo.6) respectively, SEQNo.7 and SEQNo.8 is upstream primer and the downstream primer of amplification miRNA target molecule miR-16 (SEQNo.9) respectively, SEQNo.10 and SEQNo.11 is upstream primer and the downstream primer of amplification miRNA target molecule miR-189 (SEQNo.12) respectively, SEQNo.13 and SEQNo.14 is upstream primer and the downstream primer of amplification miRNA target molecule miR-451 (SEQNo.15) respectively, SEQNo.16 and SEQNo.17 is upstream primer and the downstream primer of amplification miRNA target molecule miR-7e (SEQNo.18) respectively.
The consensus feature in each primer 5' → 3' direction is containing Nt.AlwI otch restriction endonuclease NERS sense strand sequence (that is: 5'-successively gGATC-the recognition sequence district of anchor series district 3'), specific recognition miRNA target molecule (SEQNo.3, SEQNo.6, SEQNo.9, SEQNo.12, SEQNo.15, SEQNo.18) and complementary strand thereof.Wherein, the recognition sequence district of upstream primer and downstream primer is complementary with the 3'-end sequence of miRNA target molecule and complementary strand thereof respectively, and is designed with lock nucleic acid (LNA at the portion in the recognition sequence district of part primer; In sequence with+number base).For the immobilized primer of solid phase chip, comprise SEQNO.1, SEQNO.4, SEQNO.8, SEQNO.11, SEQNO.13, SEQNO.14, SEQNO.16, SEQNO.17, the 5'-end all in the anchor series district of above-mentioned primer introduces Poly (T 10), and at Poly (T 10) the end modified amino group of 5'-.
Above-mentioned all oligonucleotide molecules are synthesized by specialized company.
Upstream primer SEQNo.1 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GGATCtgttcttACC(BHQ1)ACAGGAG
Downstream primer SEQNo.2 (5' → 3' direction)
CCGATCTAGT GGATCtgttcttTCAAATGC+TCA
MiRNA-105SEQNo.3 (5' → 3' direction)
UCAAAUGCUCAGACUCCUGUGGU
Upstream primer SEQNo.4 (5' → 3' direction)
NH 2-tttttttttt(VIC)CCGATCTAGT GGATCtgttcttAGCCT(BHQ1)A+TCCTG
Downstream primer SEQNo.5 (5' → 3' direction)
CCGATCTAGT GGATCtgttcttTTCAAGTAAT
MiR-26aSEQNo.6 (5' → 3' direction)
UUCAAGUAAUCCAGGAUAGGCU
Upstream primer SEQNo.7 (5' → 3' direction)
CCGATCTAGT GGATCtgttcttCGCCAATATT
Downstream primer SEQNo.8 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GGATCtgttctt(BHQ1)TAGCAGCACGT
MiR-16SEQNo.9 (5' → 3' direction)
UAGCAGCACGUAAAUAUUGGCG
Upstream primer SEQNo.10 (5' → 3' direction)
CCGATCTAGT GGATCtgttcttACTGA+TATCAG
Downstream primer SEQNo.11 (5' → 3' direction)
NH 2-tttttttttt(VIC)CCGATCTAGT GGATCtgttcttTGCCT(BHQ1)ACTGAG
MiR-189SEQNo.12 (5' → 3' direction)
UGCCUACUGAGCUGAUAUCAGU
Upstream primer SEQNo.13 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GGATCtgttctt(BHQ1)AACTCAGTAATG
Downstream primer SEQNo.14 (5' → 3' direction)
NH 2-tttttttttt(FAM)CCGATCTAGT GGATCtgttctt(BHQ1)A+AACCGTTACC
MiR-451SEQNo.15 (5' → 3' direction)
AAACCGUUACCAUUACUGAGUU
Upstream primer SEQNo.16 (5' → 3' direction)
NH 2-tttttttttt(VIC)CCGATCTAGT GGATCtgttctt(BHQ1)AACTATACAACC
Downstream primer SEQNo.17 (5' → 3' direction)
NH 2-tttttttttt(VIC)CCGATCTAGT GGATCtgttcttTGAGGT(BHQ1)+AGGAG
Let-7eSEQNo.18 (5' → 3' direction)
UGAGGUAGGAGGUUGUAUAGUU。
2. the upstream primer that different target molecule is corresponding and/or downstream primer are fixed on solid phase chip
Adopt the technical field of the invention known technological method, utilize Micrometer-Nanometer Processing Technology, the solid phase chip of preparation containing 3 micro-reacting holes, by the Poly (T of upstream primer and/or downstream primer 10) 5'-terminal amino group and the chemistry of carboxylic group of the micro-reacting hole of solid phase chip be coupled, primer is fixed on micro-reacting hole of solid phase chip, wherein:
The upstream primer SEQNo.1 that miR-105 target molecule is corresponding and upstream primer SEQNo.4 corresponding to miR-26a target molecule is fixed on first micro-reacting hole of solid phase chip simultaneously, primer flag F AM and the VIC fluorescent reporter molecule respectively of the two;
The downstream primer SEQNo.8 that miR-16 target molecule is corresponding and downstream primer SEQNo.11 corresponding to miR-189 target molecule is fixed on second micro-reacting hole of solid phase chip simultaneously, primer flag F AM and the VIC fluorescent reporter molecule respectively of the two;
The upstream primer SEQNo.13 that miR-451 target molecule is corresponding and upstream primer SEQNo.16 corresponding to downstream primer SEQNo.14, miR-7e target molecule and downstream primer SEQNo.17 is fixed on the 3rd micro-reacting hole of solid phase chip all simultaneously, primer flag F AM and the VIC fluorescent reporter molecule respectively of the two.
3. the extraction of serum total serum IgE
Commercialization total RNA extraction reagent box is adopted to extract 10 routine human peripheral blood leukocytes total serum IgE.
4. the structure of solid phase chip constant temperature exponential amplification system
Reaction system amounts to 50 μ L, and this system comprises following component: 50mMNaCl, 10mMTris-HCl, 10mMMgCl 2, 100 μ g/mlBSA, 0.5 unit K lenow (exo-) archaeal dna polymerase (NEB), 2 unit Nt.AlwI breach enzyme (NEB), 600 μMs of dNTPs (Promega), the upstream and/or the downstream primer that are not fixed on solid phase chip in step 1 and 2 are often kind of primer 50nM, the solid phase chip of upstream and/or downstream primer is secured in step 2, target molecule is that the miRNA target molecule of the 0.1zmol of synthesis (comprising: SEQNo.3, SEQNo.6, SEQNo.9, SEQNo.12, SEQNo.15, SEQNo.18), or 10 routine human peripheral blood leukocytes total serum IgE prepared by step 3.
4. the amplification of target molecule and fluoroscopic examination
The reaction system of above-mentioned preparation is placed in ViiA7 real-time fluorescence quantitative PCR, and reaction conditions is: 37 DEG C 30 minutes.After completion of the reaction, the micro-array chip scanner of Beijing Bo Ao company is adopted to detect fluorescent species and the intensity of each micro-reacting hole of solid phase chip.
5. detected result and analysis
5.1 when only having miR-105 target molecule in reaction system, only have the detected FAM fluorescent signal of first of solid phase chip micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.2 when only having miR-26a target molecule in reaction system, only have the detected VIC fluorescent signal of first of solid phase chip micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.3 when only having miR-16 target molecule in reaction system, only have the detected FAM fluorescent signal of second of solid phase chip micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.4 when only having miR-189 target molecule in reaction system, only have the detected VIC fluorescent signal of second of solid phase chip micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.5 when only having miR-451 target molecule in reaction system, only have the detected FAM fluorescent signal of the 3rd of solid phase chip the micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.6 when only having miR-7e target molecule in reaction system, only have the detected VIC fluorescent signal of the 3rd of solid phase chip the micro-reacting hole immobilized primer release, and the intensity of fluorescent signal is directly proportional to the starting point concentration of this target molecule.
5.7 when there is 6 kinds of target molecules described in the present embodiment in reaction system simultaneously, each of solid phase chip all can detect to the 3rd micro-reacting hole FAM and the VIC fluorescent signal that immobilized primer discharges, and the intensity of fluorescent signal is directly proportional to the starting point concentration of each target molecule.
The detected result of 5.8 clinical samples shows, miR-105, miR-26a, miR-16, miR-189, miR-451, miR-7e detected result of 10 routine samples is 5.23 ~ 7.28 × 10 respectively 3, 1.27 ~ 4.38 × 10 4, 2.13 ~ 3.24 × 10 4, 6.23 ~ 8.23 × 10 3, 4.23 ~ 6.89 × 10 4.
5.9 relative to the existing solid phase chip detection method based on hybridization, the present embodiment is nucleic acid amplification and the detection method being detected as one, nucleic acid amplification can be completed in 30 minutes, amplified production can be directly used in relevant device and detect, without the need to follow-up hybridization step, totally only about 40 minutes consuming time, well below the existing similar detection method based on hybridization, further, its methodology such as sensitivity and specificity parameter and existing similar detection method have good dependency.

Claims (14)

1. the solid phase chip Constant Temperature Detection method of Microrna, it is characterized in that: comprise reaction mixture, reaction mixture comprises following reacted constituent:
1. there is the miRNA target molecule of 3'-terminal hydroxy group;
2. upstream primer and downstream primer;
3. archaeal dna polymerase;
4. the breach agent of upstream primer and downstream primer breach agent recognition sequence is identified;
5. triphosphate deoxy-nucleotide;
6. ion and the buffer system of above-mentioned archaeal dna polymerase and breach agent biologic activity is met;
Described upstream primer and downstream primer are the sense strand sequence containing jagged dose of recognition sequence, and its 5' → 3' base is all followed successively by the recognition sequence district of anchor series district, specific recognition miRNA target molecule sequence; The anchor series district of upstream primer and downstream primer and miRNA target molecule are without homology, and the recognition sequence district of upstream primer, downstream primer is complementary with the 3'-terminal sequence of miRNA target molecule and miRNA target molecule complementary strand respectively;
Described upstream primer and downstream primer have target molecule specificity, often kind of specific upstream primer of target molecule and/or downstream primer are fixed on solid phase chip by its 5'-end, further, the upstream primer and/or the downstream primer that are fixed on solid phase chip mark the fluorescent reporter molecule and quencher molecule with FRET (fluorescence resonance energy transfer) effect respectively in the both sides of its breach agent recognition sequence.
2. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, is characterized in that: the described specific upstream primer of often kind of target molecule and downstream primer are fixed on a specific site of solid-state chip simultaneously;
Or, often kind of target molecule only have it to have specific site that specific upstream primer is fixed on solid-state chip;
Or, often kind of target molecule only have it to have specific site that specific downstream primer is fixed on solid-state chip;
Or the same specific site of solid phase chip fixes the specific upstream primer of one or more target molecules and another kind or more the specific downstream primer of other target molecule simultaneously.
3. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, it is characterized in that: described solid phase chip comprises microarray or microballon, and based on the biochip with multiple micro-reacting hole that microfluid, micro-fluidic or micro-manufacturing process produce.
4. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, it is characterized in that: the anchor series district 5'-of the described upstream primer being fixed on solid phase chip and/downstream primer holds and increases poly base, comprises poly born of the same parents gland pyrimidine bases (Poly (T n)) or poly adenine base (Poly (A n)), and be fixed on solid phase chip by the 5'-end of described poly base.
5. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, it is characterized in that: target molecule specific upstream primer and/or downstream primer are directly or indirectly fixed on solid phase chip by its 5'-end, fixing mode comprises fixes with the combination of covalent linkage and non-co-part key, or, be coupled mode by physical adsorption and/or chemistry to fix, or, alkyl or the aryl compound with amido are fixed as connection peptides, or, there is the alkyl of thiol group or aryl compound as connection peptides immobilized primer, or, fixed by arm molecule.
6. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, is characterized in that: the specific upstream primer and/or the downstream primer that are fixed on the different target molecules of different solid phase chip mark identical fluorescent reporter molecule and the quencher molecule with FRET (fluorescence resonance energy transfer) effect;
Or the specific upstream primer and/or the downstream primer that are fixed on the different target molecules of identical solid phase chip mark different fluorescent reporter molecule and the quencher molecule with FRET (fluorescence resonance energy transfer) effect.
7. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, it is characterized in that: the specific upstream primer of described miRNA target molecule and/or the anchor series district of downstream primer and/or recognition sequence district introduce derivatized nucleotide, derivatized nucleotide comprises lock nucleic acid, peptide nucleic acid(PNA) or thio-modification base.
8. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, is characterized in that: containing the Nucleotide with miRNA target molecule and complementary sequence 3'-end penultimate and/or the 3rd bit base mispairing in the specific upstream primer of described miRNA target molecule and/or the recognition sequence district of downstream primer;
Or, in described reaction mixture, add the bioactive molecules that can suppress non-specific amplification, comprise TaqMutS, RecA.
9. the solid phase chip Constant Temperature Detection method of Microrna as claimed in claim 1, is characterized in that: described archaeal dna polymerase is the archaeal dna polymerase with strand-displacement activity;
Or described archaeal dna polymerase does not have strand-displacement activity, and in described reaction mixture, add the bioactive molecules with strand-displacement activity.
10. the solid phase chip Constant Temperature Detection method of Microrna as in one of claimed in any of claims 1 to 9, is characterized in that: the range of reaction temperature of described constant temperature is 37-70 DEG C.
The solid phase chip Constant Temperature Detection method of 11. Micrornas as claimed in claim 10, is characterized in that: the temperature of reaction of described constant temperature is 37 DEG C, 55 DEG C, 60 DEG C or 65 DEG C.
The solid phase chip Constant Temperature Detection method of 12. Micrornas as in one of claimed in any of claims 1 to 9, is characterized in that: the described reaction times is 10-80min.
The solid phase chip Constant Temperature Detection method of 13. Micrornas as claimed in claim 12, is characterized in that: the described reaction times is 10min, 20min, 30min, 40min, 50min or 60min.
14. detection reagent and the test kits that utilize the solid phase chip Constant Temperature Detection method of Microrna as described in any one in claim 1 to 9,11,13.
CN201510460239.1A 2015-07-30 2015-07-30 Solid chip constant temperature detection method of MiRNA Pending CN105063190A (en)

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Application publication date: 20151118