阅读说明：本技术 两部分式中介探针 (Two-part intermediary probe ) 是由 马丁·特罗特 西蒙·瓦德勒 费利克斯·冯·施特滕 莉萨·贝赫勒 于 2017-12-15 设计创作，主要内容包括：本发明涉及用于检测至少一种靶分子的包括至少两个寡核苷酸的中介探针。根据本发明的中介探针的第一寡核苷酸包括探针区和中介体结合区,其中,探针区对靶分子和/或模板分子具有亲和力,并且中介体结合区对至少一种中介体具有亲和力。中介探针的至少一个另外的寡核苷酸是中介体,其经由中介体结合区与中介探针的第一寡核苷酸结合,并且对至少一种检测分子具有亲和力。在中介探针的第一寡核苷酸释放后,中介体通过与检测分子的相互作用触发可检测的信号。此外,本发明涉及包括至少一种根据本发明的中介探针和至少一种检测分子的系统,以及用于检测至少一种靶分子的方法。(The present invention relates to intermediary's probes including at least two oligonucleotides for detecting at least one target molecule.First oligonucleotides of intermediary's probe according to the present invention includes probe region and mediator combined area, wherein probe region has affinity to target molecule and/or template molecule, and mediator combined area has affinity at least one mediator.At least one other oligonucleotides of intermediary's probe is mediator, via mediator combined area in conjunction with the first oligonucleotides of intermediary probe, and has affinity at least one detection molecules.After the first oligonucleotides release of intermediary's probe, mediator triggers detectable signal by the interaction with detection molecules.In addition, the present invention relates to the system including at least one intermediary's probe and at least one detection molecules according to the present invention, and the method for detecting at least one target molecule.)

1. intermediary's probe for detecting at least one target molecule, wherein intermediary's probe includes at least two oligonucleotides, It is characterized in that

A) the first oligonucleotides includes probe region and mediator combined area, wherein

Probe region described in i has affinity to target molecule and/or template molecule, and

Mediator combined area described in ii has affinity at least one mediator, and

B) at least one other oligonucleotides is mediator, the mediator

I via the mediator combined area in conjunction with first oligonucleotides of intermediary's probe, and

Ii has affinity at least one detection molecules, wherein first widow of the mediator from intermediary's probe By triggering detectable signal with detection molecules interaction after nucleotide release.

2. intermediary's probe according to claim 1, which is characterized in that first oligonucleotides of intermediary's probe And/or mediator does not include the marker for generating signal.

3. intermediary's probe according to claim 1 or 2, which is characterized in that the first oligonucleotides of intermediary's probe and/ Or the mediator contain it is one or more for generating the markers of signal, preferably fluorescent molecule, Redox molecules, shine The unit of molecule or other generation signals.

4. it include at least one according to claim 1 to the system of intermediary's probe and at least one detection molecules described in one of 3, It is characterized in that, at least one detection molecules include one or more oligonucleotides and including at least one mediator At least one first area of interaction, and

A) second area, chemical group and/or chemistry including fluorescence acceptor or fluorogenic donor and/or for combining solid phase are protected Group and/or redox modification and/or the modification that shines are protected, and/or

B) third region, chemical group and/or chemistry including fluorogenic donor or fluorescence acceptor and/or for combining solid phase are protected Group and/or redox modification and/or the modification that shines are protected, or

C) at least one the fourth region, at least one described the fourth region with there is fluorogenic donor and/or fluorescence acceptor extremely The the first probe interaction of few one kind, and/or

D) at least one the 5th region, at least one described the 5th region with include fluorogenic donor and/or fluorescence acceptor at least A kind of second probe interaction.

5. the system according to preceding claims, which is characterized in that the detection molecules have hairpin structure.

6. a kind of method for detecting at least one target molecule, comprising the following steps:

A) it provides at least one according to claim 1 to intermediary's probe described in 3 and/or system according to claim 4 or 5 System,

B) make probe region and the template molecule and/or the target molecule of the first oligonucleotides of at least one intermediary's probe The combination of sequence,

C) the first oligonucleotides of amplification at least one intermediary's probe and/or template molecule and/or target molecule,

D) at least one mediator is discharged by least one accessory molecule,

E) optionally make the mediator of at least one release and the combination of at least one detection molecules, and

F) variation of signal is detected.

7. the method according to preceding claims, which is characterized in that at least one mediator is in conjunction with the detection point The first area of son is simultaneously extended through at least one accessory molecule come enzymatic, and the accessory molecule is preferably and in conjunction with mediator The end 3' combine, physically or chemically measurable variation thus occurs in the detection molecules.

8. method described in one of according to claim 6 or 7, which is characterized in that in the first oligonucleotides of intermediary's probe Probe region in conjunction with the sequence of template molecule and/or target molecule after, the end 3' of the first oligonucleotides of intermediary's probe Extended through accessory molecule enzymatic.

9. the method according to any one of claim 6 to 8, which is characterized in that the first few nucleosides of intermediary's probe The amplification of acid and/or template molecule and/or target molecule is carried out via isothermal or non-isothermal amplification method.

10. the method according to one of claim 6 to 9, which is characterized in that the detection molecules have at least one fluorescence Or the modification that shines, and with after at least one intermediary precursor reactant, the fluorescence or the modification that shines can be by means of auxiliary Molecule is cut away from the detection molecules, and/or removes the end 5' and/or the expansion hair of the hairpin structure of the detection molecules Clamping structure, and detect in the detection molecules variation of the fluorescence signal or luminous signal.

11. the method according to one of claim 6 to 10, which is characterized in that every kind of intermediary's probe uses several mediators And/or every kind of target molecule uses several intermediary's probes and/or several detection molecules.

12. the method according to any one of claim 6 to 11, which is characterized in that at least one accessory molecule tool There are DNA chain separation effect and/or assembly effect, wherein the accessory molecule is preferably strand displacement polymerase.

13. the method according to any one of claim 6 to 12, which is characterized in that the target molecule and/or template molecule It is the biomolecule selected from the group being made up of: DNA, RNA, peptide, protein, aptamer, and/or combination thereof.

14. the method according to any one of claim 6 to 13, which is characterized in that the accessory molecule is selected from by following The group of composition: polymerase, protein, nucleic acid, natural materials, enzyme, enzyme system, cell lysate, cellular component, spreads out at catalyst It is born from the derivative and/or synthetic molecules of cellular component.

15. the method according to any one of claim 6 to 14, which is characterized in that fluorescence, shines, quality, suction at phosphorescence Receipts, light scattering, the touching of conductivity, enzymatic activity and/or affinity, electrochemical potentials or signal, refractive index, surface plasma Hair, magnetic relaxation, magnetism, impedance or the measurable variation of capacitor are by fixed or revocable detection molecules and at least one Directly or indirectly interacting and occurring between mediator.

16. the method according to any one of claim 6 to 15, which is characterized in that at least one mediator is released Put is to expand at least one mediator by means of isothermal or non-isothermal amplification method to detect.

17. the method according to one of claim 6 to 16, which is characterized in that the mediator of at least one release is logical Cross sequencing detection.

18. the method according to one of claim 6 to 17, which is characterized in that the mediator of at least one release is logical It crosses hybridization to extend optionally after in conjunction with the detection molecules by accessory molecule in conjunction with the detection molecules, and then Carry out melting curve analysis.

Background technique

DNA cloning is particularly in the clinical diagnosis of disease research.DNA cloning is related to copying for a large amount of required target sequences of duplication Shellfish, so that initially a small amount of DNA becomes visible.

DNA cloning can carry out by various methods.PCR in addition to needing to carry out thermal cycle between about 60 DEG C to 95 DEG C Except, also use isothermal amplification method, such as LAMP (62 DEG C) or RPA (39 DEG C).It can be used for real-time tracking DNA there are many method The detection of amplification and amplified production.In particular, the detection method based on bioluminescence, chemiluminescence, turbidimetry and fluorescence can Detection and quantitative DNA to be checked.The total amount for the DNA that most of above methods can only expand in test sample, and cannot Distinguish different target sequences.Therefore, these methods are only applicable to so-called substance verifying.Based on fluorescence and shine, electrochemistry Other application possibility is opened with other detection methods.In addition to being embedded in the detection point with DNA chain non-specific interaction It is sub outer, also use the oligonucleotides of modification.The latter can be used for the analysis of target sequence specificity, and is embedded in detection molecules and often leads Cause the false positive detection of unspecific by-products.

In clinical analysis and in-vitro diagnosis, can in the reaction several target molecules of Parallel testing it is meaningful, because The reason of for for example different bacteriums or virus may be various diseases.Therefore, multiple analyte verifying is extremely important, thus below List some examples: for example, ABO Genotyping is not only related with blood group determination, but also with people's neutrophil cell spectrotype (HNA) generation is related, and blood and tissue infusion must be determined.Also use the skill based on immunoassays or nucleic acid Art has routinely carried out the HIV variant of blood donors sample and the parallel testing of Type B or hepatitis C virus.Pathogen it is special Property detection it needs to be determined that several genetic locis, so as to diagnosis derived from being carried out after short analysis time.

The determination of activity of different markers and crt gene allows to generate express spectra.For example, this can be used for identifying influence Cell division and cell differentiation and oncogene therefore closely related with cancer, or for (a according to the genotype of patient Propertyization medical treatment) prediction some drugs the effect of.It may also detect that the something lost often occurred in (antenatal) diagnosis of molecular biology Hereditary diseases, including cystic fibrosis (cystic fibrosis), phenylketonuria (metabolic disorder) and thalassemia (red blood cell drop Solution).In addition, joint-detection marker of inflammation such as Procalcitonin or cell factor are it can be concluded that about the knot for infecting severity By.

If in examples detailed above, diagnosis problem need to analyze several target molecules, genetic loci or other markers with And internal contrast or reference, then analysis every time is only allowed to determine the method usually not meaning of single parameter.On the other hand, such as Fruit carries out different single analyses in parallel to record several parameters, then this is uneconomic: sample solution must be separated into several Detect the reaction batch of different target molecules.It leads to the problem of as follows: by the way that sample solution is divided into n aliquot, individually The amount of substance in reaction is reduced to 1/n, therefore the sensitivity for detecting reaction accordingly reduces.

In order to avoid these disadvantages, develop the homogeneous or heterogeneous reaction method of the different target molecule of Parallel testing with Capture several parameters.Using the various oligonucleotides marked for detection, target molecule to be detected is specifically bound.Upper It states in the directly relying on property between the oligonucleotides of label and target molecule, problem appear to is that, if there is new experiment problem, For example, if to detect different virogene types, it is necessary to use new probe.This makes must for each new experiment problem The oligonucleotides of new label need to be developed for detecting.Due to the modification of the oligonucleotides needed for detecting, this is to be time-consuming and expensive 's.

As the alternative solution of Parallel testing difference target sequence in homogeneous reaction method, can in solid phase immobilization widow core Thuja acid is for detecting (heterogeneous detection).Depending on the signal location of stationary phase, the presence of certain target sequences can be inferred that.Mark Directly relying on property between the oligonucleotides and target molecule of note causes following problems again: the oligonucleotides of immobilization must adapt to Experiment problem.For each new experiment problem, it is necessary to by new oligonucleotide pair in solid phase.Due to complicated preparation Journey, this is very time-consuming.

Target sequence specific oligonucleotide with the marker for detection or the target sequence positioned at solid phase different location The unfavorable purposes of specific oligonucleotide results in the need for universal test method, is sequence-specific and is still to have cost Benefit.In universal method, the sequence of oligonucleotides (detection molecules) of signal is generated independent of target sequence to be detected. The oligonucleotides of the generation signal of identical optimization can be used for different target sequences.As a result, working time and therefore can be saved Labor cost is saved, because being not necessarily each detection reaction readjusts the oligonucleotides for generating signal.

The universal test method of sequence-specific is known, but they have the shortcomings that.Particularly, the enzyme used It is only mainly that non-isothermal amplification method is compatible with certain amplification methods.These include, for example, more according to Faltin in 2012 et al. Analyte reporting system and the general dual probe used according to Yang in 2008 et al..In the method being previously mentioned, there is this The enzyme of kind nuclease, such as polymerase are indispensable, although polymerase used in LAMP or RPA does not have this Kind nuclease.So-called strand displacement polymerase (stranddisplacement polymerases) is difficult to sequence spy Anisotropic general detection.In addition, there is the risk for generating false positive signal according to the program of Yang in 2008 et al..

2013079307 A1 of WO describe using including the system of intermediary's probe and general reporter molecule detect to A kind of universal method of few target molecule.The enzyme for by the mediator of cutting release needing that there is nuclease.In PCR, gather Synthase has this nuclease in most of forms, this is why this amplification method does not need additional enzyme. However, in isothermal amplification method, such as LAMP or RPA, or in PCDR, polymerase used does not have this enzymatically active nucleic acid Property.Therefore, mediator only could be discharged by cutting by the enzyme that addition shows nuclease.The disadvantage is that additional Other components in enzyme and reaction mixture interact, and therefore may influence the efficiency of detection reaction.In addition, to additional Enzyme reaction mixture of the increase in demand for detecting reaction cost.Changing in addition, being resulted in using additional enzyme Under conditions of optimizing detection react additional workload.

2016/0312271 A1 of US is described using including the system of cleavable probe and general detection molecules and is detected The universal method of at least one target molecule.In the program according to US 2016/0312271A1, it is similar to WO 2013079307 A1 triggers detection reaction by cutting oligonucleotides.Therefore, there is identical disadvantage, i.e. the enzyme with nuclease is It is required.

Also describe the art methods using primer comprising hairpin-forming sequences, covalently bound fluorogen or The probe of covalently bound fluorescent marker.In addition, the probe of the second fluorescent marker has been used, and it can be in conjunction with amplicon The interaction of one fluorogen.Second probe of hairpin-forming sequences and the target sequence specificity of target sequence specificity leads to fluorogen Not the shortcomings that attached universal sequence section, and therefore this method cannot be general.It therefore, must be individually for new detection reaction Optimize signal to generate.In addition, there are risks for the second additional probe, this is because and primer when using strand displacement polymerase In conjunction with the first probe can extend and therefore replace the second probe.

For this, it should be mentioned that produced using strand displacement polymerase to amplification in LAMP according to Tanner in 2012 et al. The sequence-specific detection that object carries out.By this detection method, fluorogenic donor and fluorescence acceptor and target sequence specificity Oligonucleotides combines, this is why this method is not general, and therefore having must for each new detection reaction The shortcomings that palpus optimizing detection.

In addition, describing the detection method based on molecular beacon comprising therefore primer sequence simultaneously has target sequence special The region of property.It has another disadvantage that due to the dependence to target sequence, because to be positioned at target sequence special for the marker for generating signal The oligonucleotides of property, this is why this detection method is not general.In addition, the closure of fluorescent yield and molecular beacon and Balance between open conformation depends on primer sequence.Therefore, necessary single optimization detection is reacted instead for each new detection It answers.

Also had the shortcomings that described in document using the general technology of strand displacement polymerase.For example, according to 2006 Li et al. people or 101328498 A of CN are using the molecular beacon with primer hybridization, due to the stabilization of the hairpin structure of molecular beacon Property, there is the risk (Li et al. people, 2007 years) for generating false positive signal in the case where lacking target molecule.In addition, the detection side Method is only used for the amplified reaction via PCR so far.The function of isothermal amplification method has not proved out, and hairpin structure is steady It is qualitative more obvious in lower temperature (LAMP, RPA), this it is not recommended that this method and isothermal duplication combined purposes.According to The use of the general fluorescence labeling primer of G.J.Nuovo in 1999 et al. further relates to following risk: the hybridization of universal primer can also It can lead to the generation of the false positive signal in non-specific amplification product.

Summary of the invention

The purpose is realized by independent claims.Advantageous embodiment is obtained by dependent claims.

According to the present invention, this skill is realized by providing for detecting the two-part intermediary probe of at least one target molecule Art purpose.

Intermediary's probe of the invention for detecting at least one target molecule includes at least two oligonucleotides, and it is special Sign is

A) the first oligonucleotides includes probe region and mediator combined area, wherein

The probe region o has affinity to target molecule and/or template molecule, and

O mediator combined area has affinity at least one mediator, and

B) at least one other oligonucleotides is mediator,

O via mediator combined area in conjunction with the first oligonucleotides of intermediary probe, and

O has affinity at least one detection molecules, wherein after being discharged from the first oligonucleotides of intermediary's probe, Mediator triggers detectable signal by the interaction with detection molecules.

Therefore, intermediary's probe according to the present invention includes that the first molecule or the first oligonucleotides and the second molecule or second are few Nucleotide, i.e. mediator；First molecule or the first few nucleosides include mediator combined area and probe region.The probe region of first molecule There is affinity to target molecule and/or template molecule, and mediator combined area is to a kind of mediator (mediator) or a variety of Mediator (mediators) has affinity.If probe region cannot directly interact with target molecule, template point is used Son.Therefore, template molecule is used as the medium between target molecule and probe region.

After probe region is in conjunction with target molecule and/or template molecule, mediator is set to by molecule from mediator combined area It changes, which preferably has the enzyme of DNA chain separation effect and certain forms with additional assembly effect, preferably strand displacement Polymerase.The interaction of mediator and detection molecules triggers detectable signal.Strand displacement polymerase has strand-displacement activity, And the chain complementary with amplification chain is replaced during amplification.

It is all beyond one's expectations, by dexterously utilizing the general applicability of intermediary's probe, can be used for detecting Different target molecules., it is surprising that several intermediary's probes according to the present invention can be used in a sample while examining Survey several target molecules.

Intermediary's probe according to the present invention can carry out any nucleic acid sequence of target molecule and/or template molecule general Sequence dependent detection.The detection molecules with fixed design can be used, fixed design is visited independent of target sequence or intermediary The probe region of needle.

, it is surprising that the release of mediator and it is subsequent can be with by the signal generation that interacts with detection molecules Applied to different amplification methods, and it is not limited to specific system.It is each in different amplification methods by dexterously utilizing Condition, above-mentioned mediator release can be easily adaptable to each system.

There are the prior art systems that the various oligonucleotide probes based on label or primer are used to detect target nucleic acid sequence. However, compared to it is described herein based on the system according to the present invention and intermediary's probe according to the present invention according to this hair Bright method, these methods are not the universal test methods that can be carried out independent of the different target-specific moleculars of target sequence.

The modification of signal, such as fluorogen and quencher are generated, the oligonucleotides for being usually applied to target sequence specificity (draws Object).In these cases, the molecule for generating signal cannot be used for different detection reactions, because it must be for every kind of target point Son is individually designed and optimizes.Therefore, the great advantages of the present invention compared with the existing technology are to generate the general detection molecules of signal General applicability, which is used together with intermediary's probe according to the present invention.These general reporter molecules Containing the molecule for generating signal, but without the segment of target sequence specificity.General reporter molecule can be used for detecting different Target molecule, without redesigning or optimizing reporter molecule.Only need to adjust two-part intermediary probe.According to the present invention Jie's probe also has simplified design of primers.It is contrasted with the system of the prior art, the oligonucleotides as primer need not contain Having can fluoresce or the molecule of connection molecule, also not contain the region of the second target sequence specificity.

During amplification procedure, the fluorogen label of primer and quencher label remainder is polymerize by strand displacement Enzyme is replaced from target molecule, so that signal is restored to its reset condition and cannot be guaranteed lasting signal intensity.In addition, being permitted The enzyme that the detection method of more prior arts needs to have nuclease, for example, polymerase, but in the case where LAMP or RPA, Polymerase used in the present invention does not have this nuclease., according to the invention it is preferred to nuclease is not needed, this It is why to use isothermal amplification method.However, the detection method of many prior arts cannot be with isothermal amplification method such as LAMP is used together.

It is contrasted with the program of the prior art, does not preferably divide intermediary's probe according to the method for the present invention.With the system System is contrasted, and intermediary's probe is two-part, allows to occur the release of mediator without total by displacement division Valence link.This advantageouslys allow for the real-time detection target molecule in isothermal amplification, wherein without using poly- with nuclease Synthase.In addition, can be with augmentation detection signal and the different mediators of probe using a variety of mediators by every kind of intermediary's probe It can produce different detection signals.According to the present invention, even if every kind of intermediary's probe uses several mediators, one kind is also only needed The specific binding site of target molecule.However, for the system or program of the known prior art, if to discharge a variety of intermediaries Body must then use several complete intermediary's probe systems.

The system of known contains the primer with target molecule specific sequence, and can mark with fluorogen Probe hybridization.However, being contrasted with these methods, the mediator of two-part intermediary according to the present invention probe is preferably not Load any marker for generating signal.If fluorogen is hybridized in conjunction with primer by individual probe, fluorogen still may be used It can be influenced by the target sequence specificity portion of primer.If there are guanine base in primer sequence, the fluorescence of fluorogen is produced Rate may be by the negative effect of guanine base.Therefore, the fluorescent yield of fluorogen is to special by target sequence in primer sequence Specific section has an impact/dependence to independent probe.In addition, in most cases, without using the inspection with universal sequence Survey molecule.

The primer of the prior art is also described, with hairpin-forming sequences and covalently or passes through what hybridization probe combined Fluorogen.Hairpin-forming sequences include the second target sequence specific regions.It is preferable, however, that intermediary's probe according to the present invention First part only include a target sequence specific regions or sequence.In addition, intermediary's probe according to the present invention does not preferably wrap Include hairpin-forming sequences and only one target sequence specific regions.Additionally, it is known that system need primer and it is usual two kinds it is additional Label probe, wherein at least one probe be target sequence specificity.Therefore, this primer/probe system is by with target Two or three molecular compositions of molecular specificity sequence.On the contrary, intermediary's probe of the invention preferably only has target with one The molecule of molecular specificity sequence.In addition, some form of intermediary's probe does not include marker.Mediator does not further preferably include target Molecular specificity sequence.

In view of the known prior art, the present invention is a kind of completely new and surprising development.The prior art does not disclose The detection system of the strand-displacement activity work of enzyme is similarly used.On the contrary, which depict use can not have under the conditions of PCR The detection method that the polymerase of strand-displacement activity carries out.

It is all beyond one's expectations, develops intermediary's probe, the system and program according to the present invention, using by with chain The activity displacement of the mediator of the enzyme of displacement effect.The method of many prior arts is based on the indispensable of polymerase used Nuclease.It is not contacted significantly between cutting and displacement reaction.In addition, for expert, to using polymerase Nuclease make it through the known method that substituted enzyme works in this way and be adjusted be it is unconspicuous or it is micro- not Sufficient road, because a large amount of reaction condition must be modified or be recombinated in different ways.

For expert, in the case where not using common detection principle, by known universal test method and use The known method combination of the target sequence specific primer and target sequence specific probe of hybridization is nor obvious.

The advantages of intermediary's probe according to the present invention described herein, is especially suitable for intermediary's probe, system according to the present invention System and preferred implementing form according to the method for the present invention.

Detection target molecule means either quantitatively or qualitatively to detect sample target to be studied in the context of the present invention Presence.According to the present invention, target molecule be in the sample its there are molecules to be detected.It is biomolecule, such as but unlimited In the combination of nucleic acid molecules, protein, peptide, glycan molecule, lipid or these molecules, such as glycosylated protein or other glycosylations Biomolecule.

Term nucleic acid in the sense of the present invention include but is not limited to DNA, RNA, PNA, ssDNA, dsDNA, RNA, mRNA, tRNA、lncRNA、ncRNA、microRNA、siRNA、rRNA、sgRNA、piRNA、rmRNA、snRNA、snoRNA、scaRNA、 GRNA, viral RNA or the RNA of modification such as LNA.Oligonucleotides in the sense of the present invention be include approximately up to 200 nucleosides The relatively short nucleic acid molecules of the length of acid.

Glycan molecule in the sense of the present invention carbohydrate or carbohydrate in particular, and including monosaccharide, disaccharides, oligosaccharides and Polysaccharide.Glycosylation describes a series of enzymatics or chemical reaction, wherein carbohydrate and protein, lipid or other glucosides Aglucon combines.In the case where lipid is as glycolipid, or in the case where protein is as glycoprotein or peptide glycan, gained is anti- Product is answered to be known as glucosides.

In the sense of the present invention, term " lipid " refer to it is complete or it is at least most of be water-insoluble (hydrophobic) object Matter, this is because their low polarity, they are very well dissolved in hydrophobic (or lipophilic) solvent.Lipid is in cell membrane Structural constituent, and also it is used as energy storage or signaling molecule in living organism.It is most of biology lipids be it is amphiphilic, i.e., They have lipophilicity hydrocarbon residue and polar hydrophilic head group, this is why their shapes in polar solvent such as water At micella or film.Lipid particularly includes fatty acid, triacylglyceride (fat and fat oil), wax, phosphatide, sphingolipid, rouge Polysaccharide and isoprenoid (steroids, carotenoid etc.).

Probe region is preferably complementary with the section of target molecule and/or template molecule.The spy of first oligonucleotides of intermediary's probe Needle area is in conjunction with target molecule and/or template molecule.In conjunction with being occurred via the probe region of intermediary's probe, because it is to target molecule And/or template molecule has affinity.Mediator combined area does not need to have template molecule any affinity and not need With complementary sequence fragment.

Mediator preferably has the region complementary with the section of detection molecules.For mediator in conjunction with detection molecules, triggering can The signal of detection.Detectable signal allows to obtain the existing conclusion about target molecule or template molecule.Template molecule itself Can be target molecule to be detected or it associated with target molecule can allow to generate via template molecule about target point Information existing for son.

Detection molecules according to the present invention are oligonucleotides, can interact and can lead to indirectly with target molecule Crossing processing (such as variation of fluorescence signal, electrochemical signals or quality) causes detection to be reacted.

If the reagent of detection method is used for, for example, primer or probe or intermediary's probe of the invention cannot be straight When connecing with target molecule interaction, template molecule is the nucleic acid molecules that can be used.Therefore, template molecule may be used as target molecule Mediator between primer or probe.Aptamer is typically used as template molecule.

Aptamer is oligonucleotides, this is because their architectural characteristic and other molecules or molecular complex interaction or In conjunction with.It can be protein, peptide, glycan molecule, lipid molecular, nucleic acid molecules or formed by these molecules by the molecule that aptamer combines Molecular complex.Aptamer can combine and different conformations is presented under unbound state, so that the different sequences of such as aptamer Column region can interact with complementary oligonucleotides such as primer or probe, this depends on conformation.

In the sense of the present invention interact refers to the interaction between each other of different interacting molecules.This can be, example Such as, the covalently or non-covalently key between two molecules, or by other one or more numerator mediated indirect keys, such as in molecule In compound.

In the context of the present invention, detectable signal refers to any kind of change that can physically or chemically measure Change.These variations include but is not limited to cutting, digestion, chain duplication, internal hybrid, phosphorylation, dephosphorylation, amidation, chemistry The combination or cutting of group, fluorescence, phosphorescence or variations in light.

In the preferred design of intermediary's probe according to the present invention, the first oligonucleotides and/or mediator of intermediary's probe It does not include the marker for generating signal.The decisive advantage of this unlabelled intermediary's probe is that it can be used for according to this In the method for invention, for detecting at least one target molecule without for new measurement optimization labour and cost.With it is existing Technology is compared, and intermediary's probe is made of oligonucleotides, can cost-effectively be synthesized, and is repaired without technical complicated Decorations, such as fluorogenic donor and/or fluorescence acceptor and blocking group.

Terms tag object refers preferably to the atom of any signal that may be used to provide detectable (can preferably quantify) or divides Son, and it can be with nucleic acid or protein or other biological molecule covalent or Non-covalent binding.Marker can provide can be with Pass through redox reaction, luminous, fluorescence, radioactivity, colorimetric method, gravimetric analysis, X-ray diffraction or absorption, magnetism, enzymatic activity The signal of equal detections.

In a preferred form, the first oligonucleotides of intermediary's probe and/or mediator includes one or more for producing Give birth to the marker of signal, the preferably unit of fluorescent molecule, Redox molecules, light emitting molecule or other generation signals.

In another preferred form of the invention, mediator includes one or more for generating the marker of signal, It is preferred that fluorescent molecule, Redox molecules, light emitting molecule or other generate signals molecule or other generate signals unit.Root According to the present invention, mediator can be in conjunction with detection molecules after replacing from intermediary's probe, and detection molecules also contain at least one use In the marker for generating signal, cause detectable signal intensity.

For example, the mediator in conjunction with intermediary's probe can mark specific wavelength λ₁The fluorogenic donor of place's transmitting/connect Receptor.Intermediary's probe displacement after, mediator can be marked in second wave length λ₂Locate fluorescence acceptor/donor of transmitting Detection molecules combine, second wave length λ₂With λ₁It is different.It is led via FRET mechanism from fluorogenic donor to the energy transfer of fluorescence acceptor The detectable increase of the radiation intensity of fluorescence acceptor is caused, this allows λ₂Transmitting be detected.Alternatively, chemistry can be used Luminous or bioluminescence donor molecule.Non-luminescent fluorescence acceptor can also be used in preferably design.It is different by using having The detection molecules of nucleotide quantity can distinguish different target molecules by melting curve analysis simultaneously in a sample.

By marking mediator, the generic features of described detection method will not lose because mediator be it is general, Molecule independent of target sequence.In other design, several probes or primer and label of every kind of target molecule can be used Mediator combine, wherein the sequence of mediator and label can be different.It is, for example, possible to use with the glimmering of different emission Photoinitiator dye.

The preferred form of intermediary's probe according to the present invention is characterized in that from the first oligonucleotides of intermediary's probe During mediator release betides the amplification of target molecule and/or template molecule in conjunction with intermediary's probe.

Term amplification or amplified reaction in the sense of the present invention refers to biomolecule, the amplification of preferred nucleic acid molecule.It borrows Help the referred to as enzymatic amplification of polymerase nucleic acid.In amplification, initiation sequence is known as amplicon, and product is known as expanding production Object.

In the preferred form of intermediary's probe according to the present invention, the first oligonucleotides has 1 to 200, preferably 20 to 80, Particularly preferred 35 to 65 nucleotide, and mediator has 1 to 60, preferably 10 to 50, particularly preferred 15 to 40 nucleotide.

The preferred form of intermediary's probe according to the present invention, target molecule and/or template molecule are selected from the group being made up of Biomolecule: nucleic acid, DNA, RNA, peptide, protein, aptamer, and/or combination thereof.

In a preferred embodiment of the invention, target molecule is template molecule.In other preferred forms, target molecule and mould Plate interaction of molecules.

In the preferred form of intermediary's probe according to the present invention, the end 3' of the first oligonucleotides of intermediary's probe is used as The starting point of amplified reaction, and therefore may be used as primer.This is a kind of decisive better than the scheme of known Advantage because intermediary's probe for new target molecule need not redesign and select completely the probe region in target molecule, and shows Some primers can easily be modified to make it possible in this way plays function as intermediary's probe according to the present invention Energy.

In another preferred form of intermediary's probe according to the present invention, the end 3' of the first oligonucleotides of intermediary's probe End is not used as the starting point of amplified reaction.In the case where target molecule is not present, corresponding intermediary's probe can form hair clip knot Structure makes it be in closed form, and mediator is in conjunction with the first oligonucleotides of intermediary probe.In the presence of target molecule, Intermediary's probe is in conjunction with target molecule or template molecule, and thus primer can be in conjunction with the first few nucleosides of the intermediary's probe opened now Acid.By being handled with suitable enzyme system, the primer of attachment can be extended, thus intermediary's probe displacement mediator.In release Mediator can be detected by means of the detection molecules of specificity.Preferred execution example is shown in embodiment 18.

In preferred design, the first oligonucleotides of intermediary's probe according to the present invention may include aptamer area, intermediary Body combined area and primer binding zone.Target molecule to be detected can be protein or peptide, such as, but not limited to, this.In target molecule In the case where being not present, primer can combine the primer binding zone of the first oligonucleotides of intermediary's probe, and by using conjunction The end 3' of suitable enzyme system extension primer, mediator are discharged from intermediary's probe.Specificity can be used in the mediator of release Detection molecules or method trigger detectable signal.In the presence of target molecule, the aptamer area of intermediary's probe and target molecule In conjunction with the primer attached with primer binding zone domain thus cannot be extended, and therefore intermediary's probe will not discharge mediator.If There are target molecules, then with there is no compared with target molecule, detect signal decline.

In addition, the present invention relates to include that at least one intermediary's probe according to the present invention and at least one detection molecules are System, it is characterised in that at least one detection molecules include one or more oligonucleotides and including at least one mediator phase At least one first area of interaction, and wherein at least one detection molecules include one or more oligonucleotides, and Including at least one first area to interact at least one mediator, and

A) second area, chemical group and/or change including fluorescence acceptor or fluorogenic donor and/or for combining solid phase Blocking group and/or redox modification and/or the modification that shines are learned, and/or

B) third region, chemical group and/or change including fluorogenic donor or fluorescence acceptor and/or for combining solid phase Blocking group and/or redox modification and/or the modification that shines are learned,

Or

C) at least one the fourth region, with the first probe of at least one with fluorogenic donor and/or fluorescence acceptor Interaction, and/or

D) at least one the 5th region, at least one the second probe phase for including fluorogenic donor and/or fluorescence acceptor Interaction

Or

E) it is made of two oligonucleotides, two in two oligonucleotides or only one have fluorescence acceptor or fluorescence Donor and/or for combine solid phase chemical group and/or chemical protecting group and/or redox modification and/or shine repair Decorations.

Detection molecules in the sense of the present invention are considered with the probe of at least one detection molecules interaction Part.In this case, detection molecules include more than one oligonucleotides.

The different zones of detection molecules described herein can be overlapped or identical in a preferred form of the invention.

In addition, the present invention relates to include that at least one intermediary's probe according to the present invention and at least one detection molecules are System, it is characterised in that at least one detection molecules include one or more oligonucleotides and including at least one mediator phase At least one first area of interaction, and wherein at least one detection molecules include one or more oligonucleotides and wrap At least one first area with the interaction of at least one mediator is included, and

A) second area, chemical group and/or change including fluorescence acceptor or fluorogenic donor and/or for combining solid phase Blocking group and/or redox modification and/or the modification that shines are learned, and/or

B) third region, chemical group and/or change including fluorogenic donor or fluorescence acceptor and/or for combining solid phase Blocking group and/or redox modification and/or the modification that shines are learned,

Or

C) at least one the fourth region, with the first probe of at least one with fluorogenic donor and/or fluorescence acceptor Interaction, and/or

D) at least one the 5th region, at least one the second probe phase for including fluorogenic donor and/or fluorescence acceptor Interaction.

In addition, the present invention relates to a kind of systems comprising at least one intermediary's probe according to the present invention and at least one Detection molecules, wherein at least one detection molecules be oligonucleotides and including at least one mediator interaction extremely A few first area, and

A) positioned at the second area of the end 5' of at least one detection molecules, with fluorescence acceptor or fluorogenic donor And/or chemical group and/or chemical protecting group and/or redox modification and/or luminous modification for combination solid phase, with And

B) third region, including fluorogenic donor or fluorescence acceptor and/or redox modification and/or shine modification and/ Or chemical group and/or chemical protecting group for combining solid phase,

Or

C) at least one the fourth region, with the first probe of at least one with fluorogenic donor and/or fluorescence acceptor Interaction, and/or

D) at least one the 5th region, with the second probe of at least one for including fluorogenic donor and/or fluorescence acceptor Interaction.

By dexterously utilizing the general applicability of intermediary's probe according to the present invention, this system can be used to detect not Same target molecule.Using several general intermediary's probes, several target molecules can be detected simultaneously in a sample.

Due to fluorogenic donor and receptor and general detection molecules, and non-target sequences specific molecular combines, therefore fluorescence produces Amount and baseband signal are not influenced by target molecule structure.Compared with prior art, the detection molecules of optimization can be used for different Analysis is without sacrificing fluorescence quantum yield or baseband signal.

Fluorescence acceptor or acceptor dye are the molecules that can absorb the energy from fluorogenic donor.Fluorescence acceptor can also To be described as quencher in the sense of the present invention.Among other things, absorption efficiency depend on fluorescence acceptor and fluorogenic donor it Between distance.Fluorescence acceptor can have λ by absorbing₁Photon be activated to transmitting λ₂Or fluorescence acceptor can be It is non-emissive and lead to fluorescent quenching.

Fluorogenic donor is the dye molecule or fluorogen that can be fluoresced.Can not have by the fluorogenic donor of radioactivation Have and transfers energy into fluorescence acceptor via dipole-dipole interaction in the case where radiating.Fluorogenic donor has been quenched in this Fluorescence signal.Alternatively, the fluorescence signal of fluorogenic donor to be detected can be influenced by static and dynamic quenching.

Fluorogen (or fluorescent dye, be similar to chromophore) is a kind of fluorescent chemicals, can be sent out again when light triggers Light.Rhodamine and derivative such as moral are preferably included with the fluorogen marked in the oligonucleotides for constructing label of the invention Ke Sasi is red, fluorescein and derivative such as 5- bromomethyl fluorescein, fluorescein, IAEDANS, 7-Me2N- coumarin-4-acetic acid Ester, 7-OH-4-CH3- cumarin -3- acetic acid esters, 7-NH₂- 4CH3- cumarin -3- acetic acid esters (AMCA), single bromine diamines, pyrene three Sulfonate such as Cascade Blue and 5- (bromomethyl)-N, N, N, 2,6- pentamethyl -1,7- dioxo -1H, 7H- pyrazolo [1,2-A] pyrazoles -3- methyl ammonium (monobromotrimethylammoniobimane), FAM, TET, CAL Fluor Gold 540、HEX、JOE、VIC、CAL Fluor Orange 560、Cy3、NED、Quasar 570、Oyster 556、TMR、CAL Fluor Red 590, ROX, LC red 610, CAL Fluor Red 610, texas Red, LC red 610, CAL Fluor Red 610、LC red 640、CAL Fluor Red 635、Cy5、LC red 670、Quasar 670、Oyster 645, green (the Oregon Green) 488 in LC red 705, Cy5.5, BODIPY FL, Oregon, rhodamine is green, Oregon is green 514、Cal Gold、BODIPY R6Gj、Yakima Yellow、JOE、HEX、Cal Orange、BODIPY TMR-X、 Red-the X of Quasar-570/Cy3, TAMRA, rhodamine, Redmond Red, 581/591 BODIPY, Cy3.5, Cal Red/ moral gram Sa Sihong, BODIPY TR-X, BODIPY 630/665-X, Pulsar-650, Quasar-670/Cy5.

" quenching " refers to any process for reducing the fluorescence intensity of predetermined substance.Quenching is Foster Resonance energy transfer (FRET) basis analyzed.FRET is a kind of dynamic quenching mechanism, because energy transfer occurs when donor is active. Quencher is such molecule: being quenched by the molecule via FRET when by light source activation by the fluorescence of Fluorophore emission.In structure It builds in the oligonucleotides or probe of label of the invention and preferably includes DDQ-1 with the quencher marked, Dabcyl, Eclipse, TAMRA、Iowa Black FQ、BHQ-1、QSY-7、BHQ-2、DDQ-II、Iowa Black RQ、QSY-21、BHQ-3、QSY- 35,BHQ-0,QSY-9,ElleQuencher,Iowa Black.Expert can be such as document [Johansson, M.K.Methods in Molecular Biology 335,17-29(2006)；Marras,S.A.Methods in Molecular Biology 335,3-16 (2006)] described in the suitable reporter molecule quencher pair of selection.

In the preferred form of the system according to the present invention, detection molecules have hairpin structure.Here, hairpin structure can be with It is formed by the end 5' of detection molecules and the internal sequence section Complementary hybridization of detection molecules, and the end 3' includes unpaired Sequence section.

In addition, according to the present invention, detection molecules can include at least one fluorescence in the end 5' and/or hairpin structure Modification or redox modification or the modification that shines.

Hairpin structure in the sense of the present invention refers to the linear kernel with the sequence fragment by internal base pairing alignment The secondary structure of acid molecule or oligonucleotides.These structures betide two regions of same molecule --- usually there is palindrome core Nucleotide sequence --- when forming double helix, terminated in end with unpaired ring.

After forming hairpin structure, positioned at the fluorogenic donor or fluorescence acceptor of the end 5' (second area) and third region Fluorogenic donor or fluorescence acceptor can interact, lead to the inhibition of fluorescence signal (FRET).As second and third area The substitution of the fluorogenic donor of detection molecules and the modification of fluorescence acceptor in domain, can be used other modifications for generating signal, such as But be not limited to Redox molecules, Chemiluminescence Resonance energy transfer (CRET) to insertion molecule.

After release, mediator is diffusely present in reaction solution and can be with the 3' that is positioned at hair clip shape detection molecules The first area of the detection molecules of the unpaired sequence section of end interacts.Detection molecules can be fixed in solid phase or deposit It is in solution.The extension of mediator in conjunction with detection molecules can be influenced by suitable accessory molecule, (such as strand displacement is poly- Synthase), wherein with the internal sequence section Complementary hybridizations of detection molecules and therefore the of the detection molecules of hairpin structure is formed Two regions (end 5') are replaced by the end 3' of the extension of polymerase or mediator respectively.This is increased by the displacement end 5' The distance between fluorescence acceptor and fluorogenic donor, and restored the fluorescence signal of the fluorogenic donor of previous inhibition.Alternatively, position Redox molecules in the end detection molecules 5' have changed relative to the end 3' of detection molecules or the efficiency change of CRET Or the insertion of molecule changes, this is because the formation of the dual structure of mediator or its extension products and detection molecules.Pass through The end 5' of substitution crossing, eliminates the formation of secondary structure or hairpin structure.In this case, mediator can pass through institute State the end 5' that accessory molecule complementally extends up to detection molecules.

The preferred design of system according to the invention, detection molecules have the structure of molecular beacon and including at least one Mediator combined area.Molecular beacon is the double labelling detection molecules of a kind of special chain end with self-complementary, in its day Hairpin structure is formed under right state.Molecular beacon can load marker such as fluorogenic donor and fluorescence acceptor in chain end, Thus marker can interact with each other in a preferred form.Hairpin structure keeps fluorogenic donor and fluorescence acceptor close each other It is close, to inhibit fluorescence signal.It is spatially separated fluorogenic donor and fluorescence acceptor with hybridizing for mediator, this may As a part of amplified reaction, amplified reaction mediator extends and generates fluorescence signal.The detection molecules of molecular beacon form Better than carry inner marker detection molecules the advantages of be end mark (such as fluorescent marker) synthesis cost it is lower.

In another is preferably designed, the present invention relates to a kind of systems comprising during at least one is according to the present invention Jie's probe and at least one detection molecules, it is characterised in that at least one detection molecules include two oligonucleotides, wherein

- the first oligonucleotides includes the first area with the interaction of at least one mediator, and has fluorescence acceptor Or fluorogenic donor and/or for combine solid phase chemical group and/or chemical protecting group and/or redox modification and/or Shine the second area modified, and

- the second oligonucleotides includes third region, which has fluorogenic donor or fluorescence acceptor and/or be used for It modifies and/or shines in conjunction with the chemical group and/or chemical protecting group of solid phase and/or redox and modify,

Wherein, the first and second oligonucleotides have the region hybridized each other.

According to the design, the labeled oligonucleotide that detection molecules can be hybridized by two is formed, two of them oligonucleotides Two labels can be individually that be attached at least one fluorescence acceptor or fluorogenic donor of oligonucleotides end, wherein dimerization Fluorescence signal in body approaches decaying by the space of two labels or inhibits.One or two oligonucleotides also has intermediary Body combined area.By the way that mediator is attached to mediator combined area and subsequent extension, the nucleotide of label is separated and is marked The end 5' and 3' of note is spatially separated from each other, so that detectable signal increases.The advantages of structure, is this end It is very low with the synthesis cost of the oligonucleotides of single fluorescent marker.The preferred variants of this design of detection molecules are shown in Figure 19 Out.

The preferred design of system according to the invention, detection molecules include being located at the 6th region of the end detection molecules 3', With for combining the chemical group and/or chemical protecting group of solid phase.

Blocking group in the sense of the present invention, which refers to, to be introduced into molecule with temporary protection particular functional group and therefore prevents The substituent group of undesirable reaction.On molecule carry out required reaction elsewhere after, the blocking group can be separated again.

In preferred design, detection molecules are freely present in solution.In another is preferably designed, detection molecules It is integrated to solid phase.It will test molecule in the reaction vessel for being suitable for each detection method to be fixed in solid phase.Can will needed for Sample and reagent be added reaction vessel in and then mixture can be incubated under suitable conditions.Sample can be by DNA, RNA and/or peptide or protein matter composition.If there is target molecule, mediator is replaced or is discharged and can be with by intermediary's probe It is diffused into fixed detection molecules in the reactive mixture.

In a preferred form of the invention, after mediator release and in conjunction with detection molecules, pass through the electrification in solid phase Detect signal intensity.Detection molecules are fixed on while being represented on the electrode of solid phase.The mediator of release can be with inspection The mediator combined area hybridization of molecule is surveyed, and is for example extended through polymerase.After success extends, Redox molecules are embeddable In the dimer of detection molecules and the mediator of extension, and generate detectable electrochemical signals.

In addition, it is also possible to which sufficiently long mediator hybridizes with detection molecules and do not extend.Redox molecules can be with In the dimer of insertion detection molecules and mediator and generate electrochemical signals.The variant of the design is shown in FIG. 20.

In other form, mediator and/or detection molecules are marked with one or more Redox molecules.In if Mediator is marked with Redox molecules, then the combination of the mediator and fixed detection molecules that discharge leads to signal intensity, this is Since the space of redox modification and electrode surface is close.The variant of this form of the invention is shown in FIG. 21.

Each target and/or template molecule discharge multiple mediators may be advantageous with obtaining stronger signal.Every kind Target molecule discharges several mediators can realize for example, by mediator to be attached on several different primers.

In other form, for example, the mediator for being marked with redox modification can be after hybridizing with detection molecules Extended through polymerase, it is possible thereby to realize the advantageous stabilisation (Figure 22) of double-strand.

In other forms, the mediator for being marked with redox modification can be in conjunction with the detection point removed from electrode surface The chain end of son.Mediator can extend at longer detection molecules or mediator had it is similar to detection molecules Length and do not extend.Electric charge transfer between Redox molecules and electrode occurs via the conductivity of DNA, wherein passing through It forms double-strand and increases conductivity.The variant of this design is shown in FIG. 23.

In other form, detection molecules can load Redox molecules and mediator can be it is unmarked. The intermediary of release, which is embodied in, in conjunction with detection molecules and can extend, as shown in Figure 24.Alternatively, the mediator being sufficiently long It can be in conjunction with detection molecules.The formation of double-strand increases the conductivity of DNA, this is because possible intramolecular and intermolecular electricity Lotus metastasis.Therefore, it can detecte the signal intensity at electrode.

Electrochemical Detection can be by measuring various parameters such as impedance variations, cyclic voltammetry, square wave voltammetry or electricity Hold variation to carry out.

In a preferred form of the invention, mediator release and combine detection molecules after signal intensity detection via complete Internal reflection fluorescence microscopy (TIRF) carries out.In this design, detection molecules are fixed in the solid phase above TIRF lighting device. It is penetrated into sample volume by the evanescent field that total reflection is formed and is triggered it and be positioned at detection molecules and/or mediator and/or spy Fluorescent molecule at needle or in insertion dimer, it is possible thereby to detect the variation of fluorescence signal.

In other are preferably designed, the mediator of release and the combination of detection molecules can pass through surface plasma resonance Spectroscopic methodology detection.Mediator by the release of mediator and is then integrated to fixed detection molecules on the surface, Ke Yijian Measure the variation of refractive index in sample.Detection molecules can be directly anchored on the metal surface that plasma is activation, or For example, within/on being directly anchored to be positioned directly in the film on metal surface.

The combination of detection molecules and solid phase or the fixed release for being also beneficial to prove mediator by weight measurement and with inspection Survey the combination of molecule.For example, will test, molecule is fixed on the surface of the carrier, and weight can be measured with oscillator quartz.It therefore can To detect the weight change as caused by the combination of mediator and detection molecules.

Alternatively, detection molecules can be fixed on magnetic-particle.This makes it possible to detect target by magnetic relaxation mensuration Molecule.In magnetic relaxation measurement, magnetic particle is magnetized by short magnetic field impulse and detects the time deterioration of induction magnetic moment.Intermediary Body it is in combination via the detection molecules being fixed on particle and extend particle hydrodynamic drag it is bigger, i.e., relative to The hydrodynamic drag for the particle for not having mediator in combination.Therefore mediator is in combination and optionally extends Compared with no mediator particle in combination, their induction magnetic moment more slowly reduces particle.Therefore, the induction of the particle The relaxation time of magnetic moment is different from each other, so as to detect the release of mediator.By by magnetic relaxation method and melting curve analysis It combines, different target molecules can be detected simultaneously in a sample.

According to the present invention, mediator and/or detection molecules can mark fluorescent molecule, redox point The molecule of son, light emitting molecule or other generation signals.

According to the present invention, detection molecules can be single stranded nucleic acid molecule or nucleic acid derivative.The probe of several labels can be with Hybridize with this detection molecules.After discharging mediator, it is in conjunction with detection molecules and extends.This is released hybridizes with detection molecules Label probe, cause from probe mark issue detectable signal intensity.For example, since fluorogenic donor and fluorescence receive Body is spatially separating, and the release for being marked with the probe of fluorogenic donor and fluorescence acceptor causes fluorescence to increase.Single-stranded detection molecules It can be linear or cricoid, it can be homogeneous in the solution or be fixed in solid phase and can have several intermediaries Body binding site.This form of the invention is preferred for isothermal amplification method and target molecule is being not present with the probe for ensuring to mark In the case where in conjunction with detection molecules, and will not occur lead due to the high thermal energy (such as passing through PCR) of generation with detection molecules The dissociation of cause.The example of form of the invention depicted herein is shown in embodiment 9.

If detection molecules are cricoid and insert several mediator binding sites, quickly detection reaction can be By occurring in combination with positioned at the several mediators of different loci in good dynamic range.The cyclic structure of detection molecules allows The additional increase for realizing sensitivity, because the hybridization and extension of the mediator in detection molecules release the label of all combinations Probe, but regardless of mediator site in combination.Receive with the different fluorogenic donors and fluorescence emitted at different wave length The probe of body can be in conjunction with detection molecules.By combining different fluorescent dyes, different fluorescent dyes can also be with difference Concentration use (it is determined by the quantity of the probe of the label of every kind of detection molecules), so as to increase multiple degree.It can Certain concentration ratios to be assigned to determining detection molecules.

In preferred design, the system according to the present invention further includes at least one binding molecule, at least one first and/ Or at least one second probe can be in conjunction at least one binding molecule after discharging from detection molecules.Binding molecule can be used for Prevent the probe (for example, being marked with those of fluorogenic donor or fluorescence acceptor) discharged again in conjunction with detection molecules.This hair Bright respective instance is shown in FIG. 7.

In certain forms, detection molecules are made of several oligonucleotides, wherein unlabelled oligonucleotides and shorter The oligonucleotide hybridization of fluorescent marker.Unlabelled oligonucleotides can be with the oligonucleotide hybridization of several fluorescent markers.Fluorescence Acceptor and/or fluorogenic donor are attached to shorter oligonucleotides.They are spatially close to each other with fluorogen and quencher Mode arrange.The unmarked mediator of release has more higher than the detection molecules of fluorescent marker with unlabelled detection molecules In conjunction with energy, and therefore replace the relatively short oligonucleotide for being for example marked with quencher.Adjustable combination can make mediator exist Preferentially combine primer without combining detection molecules under reaction condition.Respective instance of the invention is shown in FIG. 25.

In addition, the present invention relates to the methods for detecting at least one target molecule, comprising the following steps:

A) provide it is at least one according to claim 1 to any one of 3 intermediary's probe and/or according to claim 4 or 5 System,

B) make the probe region of the first oligonucleotides of at least one intermediary's probe and the sequence of template molecule and/or target molecule Combination,

C) the first oligonucleotides of at least one intermediary's probe of amplification and/or template molecule and/or target molecule,

D) at least one mediator is discharged by least one accessory molecule,

E) optionally, make the mediator of at least one release in conjunction at least one detection molecules, and

F) change in detection signal.

Amplification step according to the method for the present invention may include isothermal and/or non-isothermal amplification method.

In isothermal or isothermal duplication, each reaction is carried out under steady temperature (isothermal) by chain transfer polymerization enzyme.By Carried out at a constant temperature in isothermal duplication, thus its can also in the case where no any major technique equipment into Row.Strand displacement polymerase, for example, the Article 2 chain of the polymerization existing double-stranded DNA of enzyme replacement of the Φ 29DNA from bacteriophage Φ 29, And it is generated with mutually homotactic new chain using first chain to form Article 2 chain.

The method of DNA isothermal duplication includes but is not limited to multiple displacement amplification, isothermal assembling, recombinase polymeric enzymatic amplification (RPA), ring mediate isothermal duplication (LAMP), the amplification (NASBA) based on nucleic acid sequence, helicase dependent amplification (HDA), Nickase amplified reaction (NEAR), rolling circle amplification (RCA) and strand displacement amplification (SDA).

In non-isothermal amplification method, heat-stabilised poly synthase is used, because temperature changes during reaction.Thermal cycler It can be used for this purpose.The example of non-isothermal amplification method is polymerase chain reaction (PCR), real-time PCR and polymerase chain displacement It reacts (PCDR).

An important advantage according to the method for the present invention is mediator release and subsequent signal generation, such as in The interaction of mediator and detection molecules can be applied to different amplification methods and be not limited to specific amplification system.It is logical It crosses dexterously using each condition in different amplification methods, above-mentioned mediator release can be easily adaptable to each system.

In a preferred form of the invention, mediator is separated from intermediary's probe by using the nuclease of enzyme And discharge, but pass through displacement release.In such a situation it is preferred to covalent bond not cut, because mediator and intermediary are visited First oligonucleotides of needle is not covalently bound.Preferably, release mediator is without cutting oligonucleotides.Preferably, at this It is not needed in inventive method using the enzyme with nuclease.

In preferred form according to the procedure of the present invention, the first area of mediator combination detection molecules, the wherein knot Conjunction can be indirect or direct interaction.By this interaction of the first area of mediator and detection molecules, Physically or chemically measurable variation of detection molecules can occur.

In preferred form according to the procedure of the present invention, the first area of at least one mediator combination detection molecules is simultaneously Extended through at least one accessory molecule enzymatic, which preferably in conjunction with the end 3' for combining mediator, thus occurs Physically or chemically measurable variation in detection molecules.Detection molecules these variation include but is not limited to cutting, digestion, Chain duplication, internal hybrid, phosphorylation, dephosphorylation, amidation, the combination of chemical group or cutting, fluorescence, phosphorescence or the change that shines Change.

It ties the first area of another preferred form of program according to the present invention, at least one mediator and detection molecules It closes, this leads to physically or chemically measurable variation of detection molecules.In order to generate measurable variation, mediator is not needed Enzymatic extend.

Intermediary's probe the first oligonucleotides probe region in conjunction with the sequence of template molecule and/or target molecule after, in The end 3' of first oligonucleotides of Jie's probe preferentially passes through accessory molecule enzymatic and extends.

Preferred design according to the method for the present invention is characterized in that the first oligonucleotides and/or template point of intermediary's probe The amplification of son and/or target molecule is carried out by isothermal or non-isothermal amplification method.

, according to the invention it is preferred to which PCR, PCDR or real-time PCR are as non-isothermal amplification method.LAMP or RPA is preferred Isothermal amplification method.

In non-isothermal amplified reaction, such as PCR or PCDR, one or more primers used can be modified, so that its It is intermediary's probe.Sample and reagent are placed in suitably in the reaction vessel that can wherein expand and Incubation mixtures. During this process, the signal intensity of such as fluorescence signal is detected in the reaction vessel.

In other kind form according to the method for the present invention, detection molecules, which have at least one fluorescence or shine, is repaired Decorations, and after through accessory molecule and at least one intermediary's precursor reactant, fluorescence or the modification that shines removed from detection molecules and/or Remove the end 5' and/or the expansion hairpin structure of the hairpin structure of detection molecules, and detect in detection molecules fluorescence or The variation of luminous signal.

In preferred form according to the procedure of the present invention, using several mediators of every kind of intermediary's probe and/or every kind of target Several intermediary's probes of molecule and/or several detection molecules.

This makes composite multiple analysis in experimental method while can detect multiple analytes.Multiple analysis makes it possible to Detect several different target molecules and/or template molecule in reaction mixture.In order to increase according to the method for the present invention more The degree of weight, can be used the different intermediary's probe of n kind to detect the different target molecule of n kind.Every kind of target molecule to be detected can To be assigned at least one intermediary's probe, probe region and target molecule or the template molecule specificity phase of at least one intermediary's probe Interaction.The mediator of mediator combined area and corresponding intermediary's probe is not affine or mutual with corresponding target molecule or template molecule It mends.However, corresponding mediator represents the specificity interaction gametophyte of determining detection molecules.Therefore, every kind of target molecule It is assigned at least one detection molecules indirectly, this is specified by intermediary's probe.The detection of different target molecules needs different detection point Son.

Since the probe region of intermediary's probe and mediator combined area can be freely combined independently of one another, in any spy In the case where needle area, by connecting and synthesizing matched mediator combined area with mediator, detection molecules can also and other Target molecule is associated.Therefore, target molecule is allowed to design independent of detection molecules according to the method for the present invention.Therefore, pass through Standardized detection molecules group can detect different target molecules in a sample, to by adjusting intermediary's probe and make The corresponding target molecule of reactive adaptation can cost-effectively be made with suitable accessory molecule (such as primer or aptamer).

According to the present invention, as a part of single intermediary's probe and in identical first oligonucleotides of intermediary probe Several different mediators that mediator combined area combines can be in conjunction with several different detection molecules.In dexterously combining Several mediators of Jie's probe can be greatly increased from different detection molecules, multiple degree according to the procedure of the present invention. Prerequisite is that several different detection molecules generate different signals, which is differentiable and preferably may be used To detect in parallel or simultaneously.

For example, using n kind detection molecules and two mediators by every intermediary's probe and target molecule, can detecte The different target molecule of kind.For giving the different detection molecules of quantity, binomial coefficient can be used for calculating detectable target point The quantity of son.Since target molecule can not only be identified by generating two different signals, such as there are two different waves for tool Two long fluorescence signals, and can be identified by generating individual signals, the value of binomial coefficient, which must increase n, to be counted Calculate the maximum value of the quantity of detectable target molecule.By four different detection molecules, can detecte different to 10 kinds Target molecule, and only 5 detection molecules can distinguish 15 kinds of target molecules.Corresponding example is shown in fig. 5.

Alternatively, several intermediary's probes can be used in every kind of target molecule, one of intermediary's probe can only include in one kind Mediator.

It can be used for the preferred shape of present procedure in conjunction with one or more intermediary's probes of identical target molecule or template molecule In formula, wherein a kind of these mediators of intermediary's probe or a variety of mediators can be in combination with one or more detection molecules.It is logical The mediator combined area in dexterously combine detection molecule is crossed, for example, three kinds of targets can be distinguished using only two kinds of detection molecules Molecule.By using n kind detection molecules and every kind of at least two mediator of detection molecules, it can detecte " 2ⁿ- 1 " a different target Molecule.This form according to the present invention, detection molecules respectively include at least two different mediator combined areas, wherein with extremely At least two mediators of few two different target sequence connections are respectively only in conjunction with a kind of specific detector molecules.Every kind of target molecule produces Raw signal specific.According to the present invention, connect with three or more target sequences the third or more mediator can tie At least two detection molecules are closed, and therefore trigger at least two different signals.The mediator discharged for two is tied simultaneously The sufficiently high probability of identical detection molecules is closed, the concentration of the mediator of release should be the magnitude of detection molecules concentration.Phase The example answered is shown in figure 5B.Further, it is also possible to using the detection molecules that can combine more than two difference mediator, and Several different intermediary's probes can combine identical target molecule.

Use several intermediary's probes by every kind of target molecule or template molecule, can be discharged when detecting target molecule it is several not Same mediator.It is, for example, possible to use several intermediary's probes, respectively selectively combine common target molecule or template point Son, wherein the mediator of mediator or these intermediary's probes has different sequences.Not homotactic several different mediators can To react wherein detecting only by being triggered in conjunction with several mediators in conjunction with one and identical detection molecules.It can pass through The interaction between the mediator of release is flexibly utilized by control signal and generate reaction, and to increase detection method Specificity.Specificity refers to that being correctly classified as the negative ratio of event or the missing of target molecule is also categorized as feminine gender Probability.For example, can be interacted in detection molecules in this way by two kinds of mediators that different intermediary's probes discharge, So that the signal intensity of detection molecules only just occurs when two kinds of mediators are all in conjunction with detection molecules.Corresponding example is being schemed It is shown in 5C.

Preferred design according to the method for the present invention is characterized in that target molecule and/or template molecule are that choosing is freely composed Group biomolecule: DNA, RNA, peptide, protein, aptamer, and/or combination thereof.

Compared with prior art, major advantage according to the method for the present invention is, can be with single step and in list Parallel testing different molecular and molecular classification, such as protein and nucleic acid in one reaction method, so as to create the group of sample The DNA-RNA- protein spectrum of conjunction.

Detection method according to the present invention can be used for for example detecting the specific RNA molecule as target molecule, wherein passing through Reverse transcription (RT) passes through another suitable enzyme system for rna transcription into cDNA, and has then expanded cDNA, wherein CDNA is used as template molecule.

According to the present invention, target molecule SPECIFIC APTAMER may be used as template molecule for detecting target molecule.Target to be detected Molecule can be protein or peptide, such as, but not limited to, this.Aptamer binding target molecule simultaneously changes its structure, so that in phase interaction Aptamer can be attached to rear aptamer specificity intermediary's probe and primer.It can make to be attached to by being handled with suitable enzyme system The primer extension of aptamer leads to the amplification of the aptamer sequence except the protein binding domains of aptamer.It is according to the present invention Intermediary's probe can interact with aptamer or the aptamer sequence of amplification.For example, by by intermediary's probe and linear amplification product In conjunction with can open probe and replace mediator from intermediary's probe using other primers.The detection point of specificity can be used Son or suitable detection method detect the mediator of release.According to the present invention, the aptamer of target molecule specificity may be used as examining The template molecule of target molecule is surveyed, which includes the target molecule combined area that flank is primer binding zone.In addition, having used according to this At least one intermediary's probe of invention, one of the primer binding zone of probe region specific binding aptamers.Target molecule is being not present In the case where, the target molecule combined area of aptamer can be expanded using intermediary's probe and other primers, discharge intermediary from intermediary's probe Body and change in detection signal.There are target molecule, aptamer binding target molecule, and due between aptamer and target molecule Combination, the primer of intermediary's probe or the first oligonucleotides cannot be extended, and mediator is not released.If there is target point It is sub then with there is no compared with target molecule, detect signal decline.The method according to this invention is able to carry out index detection reaction.

Another form according to the method for the present invention, accessory molecule are selected from the group being made up of: polymerase, RNA polymerization It is enzyme, archaeal dna polymerase, ligase, ribozyme, catalyst, protein, nucleic acid, natural products, enzyme, enzyme system, cell lysate, thin Born of the same parents' component, derivative and/or synthetic molecules derived from cellular component.Accessory molecule preferably come from nucleic acid amplification system and/ Or the molecule of limitation enzyme system.

Ligase is the enzyme for connecting DNA chain.They form ester bond between phosphate residue and sugared deoxyribose.It it is known that Ligase, which also has, extends the ability of nucleic acid molecules in the end 3' of nucleic acid.

Ribozyme is the RNA molecule with catalytic activity, can be reacted as enzyme with catalytic chemistry.Known certain ribozyme classes Being similar to polymerase can extend and amplifier nucleic acid molecule.Ribozyme can also be catalyzed other reactions, and the combination of such as peptide bond and RNA divide The montage of son.

Preferred form according to the method for the present invention, at least one accessory molecule have DNA chain separating effect and/or polymerization effect Fruit, accessory molecule are preferably strand displacement polymerase.

, it is surprising that additional enzyme is not needed, such as with core when using the polymerase with strand-displacement activity The enzyme of phytase activity, this is major advantage compared with the existing technology.

In the method according to the invention, different accessory molecules can be used for different process steps.It can be by auxiliary The process steps for helping molecule to carry out include but is not limited to the first oligonucleotides for expanding intermediary's probe according to the present invention, expand target Molecule and/or template molecule, cutting, the mediator of release or displacement from intermediary's probe, the mediator after combining detection molecules Enzymatic extend and detection molecules change.

In preferred form according to the method for the present invention, fluorescence, phosphorescence, shine, quality, absorption, light scattering, conductivity, Enzymatic activity and/or affinity, electrochemical potential or signal, refractive index, the triggering of surface plasma or magnetic relaxation can measure Variation be between detection molecules and at least one mediator by immobilization or on-fixed it is direct or indirect mutually Effect and occur.

In preferred design according to the method for the present invention, fluorescence, shines, quality, absorption, the scattering of light, conductance at phosphorescence Rate, enzymatic activity and/or affinity, electrochemical potential or signal, refractive index, the triggering of surface plasma, magnetic relaxation, magnetism, Impedance or measurable variation of capacitor be by immobilization or the detection molecules of on-fixed and at least one mediator it Between directly or indirectly interact and occur.

Preferred design according to the present invention be characterized in that the release of at least one mediator be by by means of isothermal or Non-isothermal amplification method expands at least one mediator to detect.The mediator of release can be for example in corresponding amplification enzyme In the presence of trigger rolling circle amplification.Therefore, target molecule can be identified by detecting the amplified production of rolling circle amplification.For example, rolling ring The amplified production of amplification can detect with carrying out sequence-specific via probe or via pH value variation, gel electrophoresis or colorimetric method.

According to the preferred form of program according to the present invention, the mediator of at least one release is detected by sequencing.By right Free mediator is sequenced, and can identify any amount of target molecule in sample simultaneously.

Sequencing is the measurement to the nucleotide sequence in nucleic acid molecules especially DNA.Sequencing approach in meaning of the present invention Including but not limited to Maxam and Gilbert method, Sanger dideoxy, pyrosequencing, sequencing by hybridization, ionic semiconductor DNA sequencing system, bridge-type synthesis order-checking, double alkali yl sequencing, paired end sequencing and nano-pore sequencing.

For example, (NGS) can be sequenced by the next generation to prove to detect.One example of NGS method is nano-pore sequencing, Potential change on pore membrane can be measured as molecule (such as nucleic acid) flows through film wherein, and therefore can measure nucleic acid Sequence.Release while sequencing can be used for detecting any amount of mediator, every kind of mediator all indicate that there are specific targets Molecule.Compared with conventional method (such as fluorescence measurement), multiple degree is dramatically increased.Sequencing approach is not limited to nano-pore survey Sequence.

In preferred form according to the method for the present invention, the mediator of at least one release passes through hybridization and detection molecules In conjunction with optionally extending after by accessory molecule in conjunction with detection molecules, and then carry out melting curve analysis.This allows to lead to It crosses using different detection molecules and realizes the additional increase of multiple degree, detection molecules are for example marked with different signal point Son.Preferred form according to the present invention, sequence-specific or sequence-nonspecific probe and mediator and/or detection molecules are mutual Effect, sequence-specific or sequence-nonspecific probe are former fluorescence and/or chromogen label or fluorescent dye.

In preferred form according to the method for the present invention, at least one target molecule is RNA, and RNA is transcribed into CDNA, and cDNA is used as template molecule.Primer with sequence overhang can be used for RNA it is inverse write reaction/reverse transcription at CDNA, and the probe region of intermediary's probe can be in conjunction with the area for including at least both section of cDNA and the section of sequence overhang Domain.

Another preferred form according to the method for the present invention, at least one target molecule is peptide or protein matter, and template is divided Son is aptamer, wherein aptamer and peptide or protein matter knot merga pass are by aptamer so that in being located at aptamer in conjunction with target molecule The binding site of the probe region of Jie's probe becomes prone to reach.

According to the present invention, sequence-specific or sequence-nonspecific probe, fluorescent dye or Redox molecules can be with At least one mediator and/or detection molecules interaction.

In addition, the present invention relates to according to the present invention and/or the system of method is for detecting one of mixture or a variety of The purposes of similar or different biomolecule.In this case, detection molecules according to the present invention can be in the end region 3' Domain has a chemical protecting group, the blocking group with intermediary's precursor reactant and 3' terminal OH groups generate after by auxiliary point Son is separated from detection molecules.

In addition, the present invention relates to kits comprising at least one detection molecules, and optionally at least a kind of present invention meaning Mediator, polymerase and dNTP in justice.

Detailed description of the invention

Hereinafter, the present invention will be explained by means of the example of attached drawing and implementation, but not limited to this, as follows:

Fig. 1: the schematic diagram of embodiment of the present invention intermediary probe structure.

Fig. 2: the schematic sequence that mediator is replaced during expanding there are strand displacement polymerase.

Fig. 3: the schematic diagram of possible detection molecules.

Fig. 4: the schematic diagram that enzymatic mediator extends.

Fig. 5: when every kind of target molecule is using several mediators and/or several intermediary's probes and/or several detection molecules Arrange possibility.

Fig. 6: the structure of the detection molecules with molecular beacon structure.

Fig. 7: the linear or cyclic annular detection molecules with the hybridization probe that fluorogenic donor and fluorescence acceptor mark.

Fig. 8: the Electrochemical Detection in solid phase.

Fig. 9: the schematic sequence that mediator is replaced during expanding there are strand displacement polymerase.

The mechanism that mediator release and subsequent signal generate during Figure 10: LAMP.

Figure 11: using intermediary's probe according to the present invention and detection molecules for detecting Escherichia coli (E.coli) The normalization fluorogram of the LAMP of (W3110, complete genome group).

Figure 12: the normalization of intermediary's probe of the present invention and the RT-LAMP for detecting HIV-1RNA of detection molecules is used Fluorogram.

Figure 13: not as the structure for intermediary's probe that primer works.

Figure 14: the detection method of target molecule is detected by the aptamer of target molecule specificity.

Figure 15: the detection of the invention of target molecule is detected by also comprising intermediary's probe in aptamer area and primer binding zone Method.

Figure 16: the invention of target molecule is detected by being used as primer and being able to carry out intermediary's probe of index detection reaction Detection method.

Figure 17: immobilization of the detection molecules in solid phase.

Figure 18: the immobilization of the detection molecules of label on the electrode.

Figure 19: the detection molecules that the hybridization oligonucleotide marked by two forms.

Figure 20-Figure 24: the Electrochemical Detection in solid phase.

Figure 25: the detection molecules being made of several oligonucleotides.

Figure 26: the normalization fluorogram of the LAMP of detection haemophilus ducreyi (H.ducreyi).

Figure 27: the normalization fluorogram of the LAMP of detection Spirochaeta pallida (T.pallidum).

Figure 28: the normalization fluorogram of the RT-LAMP of HTLV-1 is detected.

Figure 29: the normalization fluorogram of the RT-LAMP of TMV is detected.

Figure 30: the normalization fluorogram of the PCDR of detection 100pg G3PDH segment.

Figure 31: the combination of mediator and magnetic or magnetisable nano particle.

Figure 32: the evidence of the Electrochemical Detection of the mediator of electroactive label.

Fig. 1 shows the schematic diagram of the possibility structure of intermediary's probe of preferred form of the present invention.

What mediator was replaced during expanding in the case where Fig. 2 shows there is the strand displacement polymerase from intermediary's probe shows Meaning property sequence, the primer that should be used as from intermediary's probe in DNA cloning.

Fig. 3 (A) shows the linear expression of possible detection molecules.(B) indicate that 3'- is fixed under the formation of secondary structure The detection molecules of change.The reverse complementary sequence segment that interaction generates the secondary structure of detection molecules is shown as black region, Mediator binding sequence is shown as diagonal stripes region.

Fig. 4 shows the schematic diagram of enzymatic mediator extension.I) detection molecules are drifted in solution or are fixed in solid phase, And determining secondary structure is presented at reaction conditions.Two suitable fluorescent decoration F and Q are interacted with each other, and thus inhibit F Fluorescence signal.Ii) mediator can interact with detection molecules in determining position (mediator combined area, region 5), Iii)-iv), and to be extended through strand displacement polymerase by enzymatic.The region 1 of detection molecules and fluorescence acceptor molecule Q It replaces together, to restore the fluorescence intensity of fluorogenic donor F.Vi) after the displacement in region 1, mediator can further extend.

Fig. 5 (A-C) is shown when every kind of target molecule uses several mediators and/or several intermediary's probes and/or several inspections Survey several possible arrangements when molecule.(A) using a variety of mediators of every kind of intermediary's probe or every kind of target molecule it is a variety of in Jie's probe increases the quantity of detectable target molecule.When every kind of target molecule uses several mediators, as detection molecules number The maximum quantity of detectable target molecule of function can be calculated by the quantity of binomial coefficient and detection molecules.(B) it uses Detection molecules with multiple mediator combined areas increase the quantity of detectable target molecule.(C) every kind of target molecule uses more Kind mediator and the specificity for increasing detection reaction using the interaction between mediator.Two kinds of mediators discharge permission They are interacted in a manner of mutual and are interacted simultaneously with detection molecules, and one extended in mediator simultaneously causes detection Reaction.The interaction of single mediator not will lead to detection reaction.

Fig. 6 shows the structure of the detection molecules corresponding to molecular beacon structure.Fluorescence acceptor and fluorogenic donor are attached In the end 5' and 3', and mediator combined area is positioned in ring.

Fig. 7 shows the linear or cyclic annular detection molecules of the probe hybridization of fluorogenic donor and fluorescence acceptor label.Pass through Mediator is set to hybridize and extend with detection molecules, the probe and change in detection signal of release mark.It is marked with fluorescence in order to prevent The release probe of donor or fluorescence acceptor is recombined with detection molecules, binding molecule can be used, the probe of label is existed It can be in connection after release.

Fig. 8 shows a kind of form of the invention, wherein Electrochemical Detection is used for solid phase.Detection molecules are fixed on electrode On.After mediator hybridizes and extends at the detection molecules, Redox molecules present in solution can be embedded in detection molecules and In the dimer for extending mediator, it is possible thereby to detect the variation of electrochemical signals.

Fig. 9 is shown to be shown in the presence of what mediator during expanding in the case where the strand displacement polymerase from intermediary's probe was replaced Meaning property sequence, intermediary's probe are used as the primer in DNA cloning.Mediator and detection molecules, which respectively mark, to be had, such as Fruit mediator is hybridized by FRET energy transfer with detection molecules, then can be detected the fluorescence intensity at a wavelength and increase. When using the detection molecules with different number nucleotide, melting curve analysis can be used to distinguish different target molecules.

Figure 10 shows the mechanism that mediator release and subsequent signal generate during LAMP.Detection molecules and intermediary are visited It is abbreviated as Medc in mediator combined area in needle (corresponding to the sequence complementary with mediator).In this form of the invention, in Jie's probe is used as Loop primer simultaneously；Therefore, intermediary's probe is by the ring primer and miscellaneous with it that is extended through Medc (ring-type _ Medc) The mediator (Med) of friendship forms.After the LAMP step of most initial, dumbbell shaped amplified production is formd, intermediary's probe can be with Combination.By extending intermediary's probe and reconnecting to primer thereon, mediator is replaced by strand displacement polymerase.So The mediator discharged afterwards can be by generating detectable signal with detection molecules interaction.

Figure 11 is shown using intermediary's probe according to the present invention and detection molecules for detecting e. coli dna The normalization fluorogram of the LAMP of (W3110, complete genome group).The figure shows the correlations between amount of DNA and fluorescence process. Fluorescence intensity in 0min is normalized to initial value.DNA copy number is expressed as (such as 10cp pairs of copy number of each reaction It should be in 10 copies of the reaction that each total volume is 10 μ l).The each reaction of negative control includes 0 copy (NTC, no template Control).

Figure 12 shows the RT- for being used to detect HIV-1RNA using intermediary's probe according to the present invention and detection molecules The normalization fluorogram of LAMP.Fluorescence intensity in 0min is normalized to initial value.RNA copy number is expressed as each reaction Copy number (it is reaction 3400 of 10 μ l copies that 3400cp, which corresponds to each total volume).The each reaction of negative control includes 0 A copy (NTC, no template control).

Figure 13 shows the design for being not used as intermediary's probe of amplification starting point.

Figure 14 shows detection method according to the present invention, detects target molecule by target molecule SPECIFIC APTAMER.

Figure 15 shows detection method according to the present invention, by additionally visiting containing aptamer area and the intermediary of primer binding zone Needle detects target molecule.Amplification intermediary's probe in the case where target molecule is not present, and primer in the presence of target molecule The target molecule that is combined of extension block.Therefore, detectable signal will not be triggered in the presence of target molecule, and Target molecule produces detectable signal in the case where being not present.

Figure 16 shows detection method according to the present invention, by being used as primer and being able to carry out index detection reaction Intermediary probe detects target molecule.In the case where target molecule is not present, linear aptamer is amplified and mediator is during amplification Release, and the target molecule that the extension of primer is combined in the presence of target molecule blocks.Therefore, the feelings existing for target molecule Detectable signal will not be triggered under condition, and detectable signal is produced in the case where target molecule is not present.

Figure 17 shows the designs of detection method according to the present invention, wherein detection molecules are solid in suitable reaction vessels It is scheduled in solid phase.

Figure 18 shows test format according to the method for the present invention, wherein detection molecules are fixed on the electrode.Pass through intermediary Hybridization and extension of the body at detection molecules, the Redox molecules in conjunction with detection molecules spatially divide with electrode surface From to generate the variation of signal.Scheme a and b show the possibility knot of the mediator in two different zones of detection molecules Coincidence point.

Figure 19 shows the detection molecules that the hybridization oligonucleotide marked by two forms.Fluorescence acceptor and fluorogenic donor It is attached in the end 5' and 3' respectively；In addition, one in two oligonucleotides has mediator combined area.

Figure 20 is shown a possibility that carrying out Electrochemical Detection in solid phase.Detection molecules are fixed on the electrode.Mediator After hybridizing at detection molecules, Redox molecules present in solution can be embedded in the dimer of detection molecules and mediator In, it is possible thereby to detect the variation of electrochemical signals.

Figure 21 shows the Electrochemical Detection carried out in solid phase using the mediator of label.Detection molecules are fixed on electrode On.By hybridizing the mediator of label with detection molecules, the variation of electrochemical signals can detecte.

Figure 22 shows the Electrochemical Detection in solid phase.Detection molecules are fixed on the electrode.Pass through the mark in detection molecules The hybridization and extension of the mediator of note, can detecte the variation of electrochemical signals.

Figure 23 is shown a possibility that carrying out Electrochemical Detection in solid phase.Detection molecules are fixed on the electrode.Pass through inspection The hybridization and extension for surveying the mediator of the label on molecule, can detecte the variation of electrochemical signals.Redox molecules and electricity Charge transmission between pole is as caused by the formation of double-strand.

Figure 24 shows the Electrochemical Detection in solid phase.Detection molecules are fixed on the electrode.Pass through the detection molecules of label On mediator hybridization and extension, can detecte the variation of electrochemical signals.Charge between Redox molecules and electrode Transmission is as caused by the formation of double-strand.

Figure 25: detection molecules are made of several oligonucleotides, wherein unlabelled oligonucleotides and shorter fluorescent marker Oligonucleotide hybridization.Fluorescence acceptor and/or fluorogenic donor are attached to shorter oligonucleotides.They are with fluorogen and quenching Agent mode spatially close to each other arranges.The mediator of release and unlabelled detection molecules have higher combination energy, And therefore replace the shorter oligonucleotides for being for example marked with quencher.

Figure 26 is shown using intermediary's probe according to the present invention and detection molecules for detecting haemophilus ducreyi LAMP normalization fluorogram.Fluorescence intensity in 0min is normalized to initial value.Negative control (NTC, no template pair According to) haemophilus ducreyi DNA is free of, positive control is mixed with the haemophilus ducreyi DNA of purifying.

Figure 27 is shown using intermediary's probe according to the present invention and detection molecules for detecting Spirochaeta pallida The normalization fluorogram of LAMP.Fluorescence intensity in 0min is normalized to initial value.Negative control (NTC, no template pair According to) Spirochaeta pallida DNA is free of, positive control is mixed with the Spirochaeta pallida DNA of purifying.

Figure 28 shows the RT-LAMP for being used to detect HTLV-1 using intermediary's probe according to the present invention and detection molecules Normalization fluorogram.Fluorescence intensity in 0min is normalized to initial value.Negative control (NTC, no template control) is no Containing HTLV-1RNA, positive control is mixed with the HTLV-1RNA of purifying.

Figure 29 shows the RT-LAMP for detecting TMV using intermediary's probe according to the present invention and detection molecules Normalize fluorogram.Fluorescence intensity in 0min is normalized to initial value.Negative control (NTC, no template control) is free of TMV RNA, positive control are mixed with the TMV RNA of purifying.

Figure 30 is shown using intermediary's probe according to the present invention and detection molecules for detecting 100pg mouse G3PDH The normalization fluorogram of the PCDR of DNA.The fluorescence intensity recycled at 0 is normalized to initial value.

Figure 31 shows the mediator in conjunction with magnetic or magnetisable nano particle.It is discharged in the presence of target molecule Afterwards, mediator can detect variation magnetic on solid phase surface in conjunction with detection molecules.

Figure 32 shows the functional of the Electrochemical Detection of the mediator of electroactive label and proves.In positive control (large intestine The 60 of bacillus DNA, 000 copy) in, mediator is replaced during LAMP reacts, and then can be hybridized with detection molecules. Therefore, at the electrode surface, this causes in the electrochemical analysis (side of being here for (being herein methylene blue) the mediator accumulation of label Wave voltammetry) at -0.39V characteristic peak formation.On the contrary, no peak value at NTC, this shows that there is no in significant Mediator release.

Design embodiment:

In the following explanation, several mediators can be with the first oligonucleotides of intermediary's probe according to the present invention or specific Mediator can be set to increase the mediator in sample in primer combination and/or several different primers and/or intermediary's probe Concentration.

Embodiment 1: intermediary's probe

Invention design embodiment includes intermediary's probe for detecting at least one target molecule, wherein intermediary's probe includes At least two oligonucleotides.First oligonucleotides has mediator combined area and probe region.Mediator combined area is positioned at few core The end 5' of thuja acid, and probe region is located at the end 3' of oligonucleotides.Second or other several oligonucleotides, an intermediary Body (mediator) or multiple mediators (mediators), chemistry, biology and/or physically with the first oligonucleotides Mediator combined area combines.Mediator can be made of DNA, RNA, PNA or the RNA of modification such as LNA.First oligonucleotides Probe region has affinity to target molecule and/or template molecule, and mediator combined area is to a mediator (mediator) Or multiple mediators (mediators) have affinity (Fig. 1).One mediator (mediator) or multiple mediators (mediators) there is affinity at least one detection molecules.