CN101194024A - Amplification of nucleic acids with magnetic detection - Google Patents

Abstract

The present invention describes a method of amplifying nucleic acids and determining the amount of amplified nucleic acids using magnetic detection. The detection can be performed during the amplification process of the nucleic acid. During the detection, the amplified nucleic acid is bound to a sensor via a biological molecule.

Description

Nucleic acid amplification and magnetic detection

The present invention relates to DNA and RNA that the nucleotide sequence of quantitative amplification for example increases or the field that participates in the reagent of amplification procedure, and be particularly related to DNA and RNA that the nucleotide sequence that is used for quantitative amplification for example increases or compositions and methods and the device that participates in amplification procedure.The invention still further relates to biomolecules magnetic detection field, and be particularly related to the method and apparatus that is used for the biomolecules magnetic detection.

By using the biological chemistry amplification method can be very responsive and determine nucleic acid (RNA, DNA) specifically.Nucleic acid amplification and detection subsequently are complicated processes, and it generally needs a plurality of treatment steps.In order to detect biomaterial (for example microorganism), these steps generally include (selectivity) enrichment, separation/purification and evaluation.All doing very big effort aspect simplification process and the raising analytical performance (for example susceptibility, specificity and speed).For example, shown and to have detected the RNA/DNA amplified production sensitively and specifically with the combination of ligandin by Protocols in Molecular Biology and immunoassay.Take place for signal, other detection methods all use fluorescence, chemoluminescence or multiple (immunity) sensor detector to replace colorimetry.But most detection methods of being developed all are relative complex, costliness and/or need (high-precision) instrument.

Developed the extraction of simplification and the combination of amplification method and current immunity detecting (LFIA) detection method, its susceptibility than gel electrophoresis exceeds several orders of magnitude, and obtains the result in 5 to 15 minutes.Say that in principle LFIA is applicable to that multiple analyte detects, i.e. maximum 6 the special RNA/DNA sequences of screening in a test set.Method based on the ligands specific on deceiving micelle and being fixed in the Nitrocellulose film makes it possible to detect 5 to 30 different parameters in mini array apparatus.Can be by platform-type scanning and image analysis so that the number of results wordization.

Quantitatively the dynamicrange of aspect and observed value is a major issue in the biological chemistry amplification.This is particularly outstanding in exponential amplification rule such as PCR, and the exponential amplification method has very precipitous conversion between the state of saturation of the amplicon concentration of inferior detection level and biological chemistry TRAP.The answer that the result usually is or denys, rather than the accurate numerical value of target level.

The method of improvement is a PCR in real time, wherein can dynamically measure the concentration that (for example using molecule marker) goes out amplicon in exponential biological chemistry amplification procedure.In quantitative PCR in real time, utilize application specific probe design, process control and process monitoring to draw quantitative data.From taking place, signal specific derives primary target concentration the required time.Quantitatively the shortcoming of PCR in real time is (i) complicated mensuration process; The cost level of the costliness of (ii) each test and (iii) be difficult to carry out multichannel and measure.

In its widely used form of institute, aforesaid method (ELISA, LFIA, PCR in real time) all relates to optical detection.Optical detection for example has some shortcomings:

The background signal of-Gao (for example from substrate or device materials, from specimen material or from the autofluorescence of the biologic material in the test set) is particularly when using complicated biological sample.

-marker character depends on biological chemical environment (for example fluorescence efficiency), and it can make the quantitatively complicated of observed value.

Connecting each other easily between-box (cartridge) and the reader makes a mistake.

-from the device and the scattering of light of liquid sample.

The optical property of liquid is depended in-incident light and emission light/catoptrical absorption.

-need expensive fetch equipment sometimes.

There are other detection methods that are different from based on the detection of light.For example, studying the biological diagnosis purposes of the magneticsensor that detects magnetic nanoparticle, this is because it has the advantage of expecting below: ((biomaterial has low-down magnetic background to susceptibility to the high analyte performance, can obtain highstrung transmitter)), speed (giving the credit to magnetic actuates), specificity (power that gives the credit to is differentiated (force discrimination)), combine measured step (for example target extracts and detects, and the both uses magnetic-particle), and use easily that (simple and reliable electronics is interconnected, transmitter and reader all are compact and cheapness).

Carrying out preliminary trial aspect combined nucleic acid amplification and the magnetic detection.WO00/61803 discloses nucleic acid amplification and has been right after the combination that magnetic sensor chips thereafter detects.This method comprises by magnetic force and applies severity.Solution concentrates on strand displacement amplification method (SDA) especially.EP0781346 discloses a kind of method, and wherein magnetic detection method and pcr amplification hocket.Magnetic detection is dependent on the difference in migration between the DNA of magnetic mark primer and amplification.

At defective based on the detection of light, need based on other detection methods of utilizing magneticsensor and magnetic-particle fast, responsive, quantitative, HDR and real-time nucleic acid detection method.These methods should comprise the least possible step, and the maximum that promptly has Biochemical processes and detection is integrated.

The present invention relates to be used for amplification of nucleic acid and be the Method and kit for of the amount of RNA or DNA that RNA or DNA and mensuration increased or the reagent that participates in amplification procedure.In this method, determine the step of the amount of nucleic acid that increases or the reagent that participates in amplification procedure by magnetic detection enforcement, and in the amplification procedure of described RNA or DNA, implement described determining step at least 1 time.In the method for the invention, the method that combines with transmitter by one or more biomolecules by the nucleic acid that wherein increased or described reagent implements to determine the step of the amount of nucleic acid that is increased or the reagent that participates in amplification procedure.

The specific embodiment of the present invention relates to the Method and kit for that is used to implement described these methods, and one or more biomolecules of nucleic acid that wherein is used in conjunction with amplification is DNA, peptide nucleic acid(PNA) (PNA) or RNA, itself and amplification of nucleic acid or amplicon specific combination.Perhaps, biomolecules can be albumen, VITAMIN, lipid or carbohydrate or can be nucleic acid and proteic combination that described albumen guarantees that amplicon combines with sensor surface.

The specific embodiment of the present invention relates to the Method and kit for that is used to implement these methods, wherein with isothermal method or thermal cycling method amplification of nucleic acid.

Another embodiment of the present invention relates to the Method and kit for that is used to implement these methods, wherein the primer that is used to increase with the magnetic-particle mark (amplimer).Another embodiment of the invention relates to the Method and kit for that is used to implement these methods, and wherein the mode by the specific hybrid probe detects amplicon, and described probe is labeled (amplimer magnetic-particle mark of no use in this embodiment).

The present invention also provides and has been used for working sample and whether has for example oligonucleotide or participate in the test kit of the reagent of amplification procedure of nucleic acid, wherein said test kit comprises at least: a device with sensor surface, and wherein one or more biomolecules links to each other with described sensor surface; One or more oligonucleotide (DNA or RNA), the intermittent at least coupling of wherein at least a oligonucleotide and magnetic-particle; With one or more DNA or RNA polymerase.Test kit also randomly comprises having the active enzyme of breach or can comprise the enzyme RNAseH for example with ribonuclease activity.

According to an embodiment of test kit of the present invention, with magnetic-particle link coupled Nucleotide for example oligonucleotide be the primer that is used to increase, guarantee during increasing to implement the mark of amplicon in view of the above.

According to another embodiment of test kit of the present invention, with Nucleotide of magnetic-particle link coupled be hybridization probe, itself and amplicon specific hybrid, and by allowing that with combining of amplicon magneticsensor implements to detect to it.

Method and kit for of the present invention allows that the analytical performance with improvement carries out polynucleotide mensuration, for example Gai Liang speed, susceptibility and specificity.Method and kit for of the present invention allow with the improvement comfort carry out the polynucleotide concentration determination, for example higher steadiness, lower error rate, simpler connecting each other and lower cost.

In one aspect of the invention, in order to determine the nucleic acid concentration in the sample, united magnetic-particle circulation and nucleic acid amplification, its advantage is that the integration of magnetic circulation and nucleic acid amplification working cycle (for example temperature cycle and reagent circulation) reaches synchronously.

Method of the present invention can may further comprise the steps: step, nucleic acid amplification step and detection step before the magnetic amplification of optional nucleic acid extraction step, optional definite initial nucleic acid amount, and wherein use biomolecules (for example DNA or RNA) that the nucleic acid of amplification is combined with sensor surface or another object.

Method of the present invention comprises by detecting the nucleic acid of amplification or the reagent of participation amplification procedure in conjunction with itself and transmitter bonded biomolecules.Biomolecules can be a covalent attachment with combining of sensor surface.

According to an embodiment, present method comprises following step:

-use the nucleic acid in the primer amplification bulk solution (bulk solution) that links to each other with magnetic-particle and/or sensor chip surface;

-as near the nano particle the function mensuration magneticsensor of time (for example particle that combines with sensor surface by biomolecules); With

First body amplification procedure and second surperficial testing process take place so that allow in-applied magnetic particle circulation efficiently.

More specifically, when using PCR method, the characteristics of method of the present invention are that application of temperature circulation and magnetic force are actuated, and detect the amplification progress to guarantee nearly real-time mode ground.

In a word, the invention provides the method that has made up nucleic acid amplification and responsive and real-time magnetic detection.Native system all has its advantage at aspects such as detection in real time, speed, process control, process monitoring, multichannel analysis, compactedness, comfort and low expenses.

Fig. 1 has shown the alternative configuration of magneticsensor that is used to detect amplifying nucleic acid sequence of the real-time detection that is used for embodiments of the present invention.

Fig. 2 has shown the magneticsensor example with two step configurations that amplification and free primer purifying module and magnetic sensor chips subsequently detect of the nucleotide sequence that is used to detect amplification according to the embodiment of the present invention.After detecting, the nucleic acid that increases is reintroduced in the amplification module.

Fig. 3 has shown the magneticsensor example of real-time configuration that is used to detect amplifying nucleic acid sequence according to the embodiment of the present invention, it has one three room modules, be used for amplification, purifying and detection (after detecting, the nucleic acid of amplification is reintroduced in the amplification module) (A), or have an one room module, be used for amplification and detect (B).

Fig. 4 has shown the schematic representation of apparatus with reaction chamber and magnetic nanoparticle according to the embodiment of the present invention (not demonstrating entrance and exit).Particle is actuated and carries out the magnetic-particle working cycle synchronous with biological amplification procedure.

Fig. 5 has shown the figure as the sensor signal of the function of time.

Fig. 6 has shown the illustrative method according to an embodiment of the invention.Implement pcr amplification in the camera incubata on sensor chip.In each circulation, application of temperature and magnetic field specific combination of actuating all is so that determine the nearly real-time status of amplification procedure.

Amplimer refers to as the nucleotides of the primer of DNA or RNA polymerase DNA for example Or RNA oligonucleotides. Amplimer in the PCR reaction is generally known as forward primer and oppositely draws Thing. The mark amplimer refers to and magnetic particle or nano particle and/or biomolecule covalency company The amplimer that connects, the not limited example of described biomolecule is biotin and fluorescein.

Amplicon refers to the nucleic acid that obtains by amplification procedure.

Hybridization probe or hybridized primer refer to for detection of the DNA of amplification or RNA (i.e. amplification Son) nucleotides is DNA or RNA oligonucleotides for example. In specific implementations of the present invention, Hybridization probe can be covalently bound with magnetic particle or nano particle and/or biomolecule.

Sensor probe or sensor primer refer to sensor surface and (namely are positioned at magnetic detection system The device part of system near-end) directly or indirectly combination and can be in conjunction with the nucleotides of amplicon For example DNA or RNA oligonucleotides. Amplicon comprises in the amplification technique of RNA therein, Can be with the RNA sensor probe in conjunction with cloning RNA. The knot of sensor probe and sensor surface Closing can be covalency. Perhaps, sensor probe can be by one or more and biomolecule covalency The biomolecule (part, antibody) that connects links to each other with sensor surface.

Describe the present invention with reference to the specific embodiment and certain figures, but the present invention is not limited to this, It only is subject to the scope of claims. Any drawing reference numeral in claims should not done Be limitation of the present invention. Accompanying drawing described herein is illustrational, and nonrestrictive. In the accompanying drawings, for illustrational purpose, the size of some elements may be exaggerated. In this theory Used term " comprises " element or the step of not getting rid of other in bright book and claims. When the expression singular noun is used indefinite article or definite article for example " one (a or an) " or " being somebody's turn to do (the) " The time, it comprises plural noun, unless otherwise specified.

In addition, the term first, second, third, etc. are used to the district in specification and claims Element like the phase-splitting might not be used for describing the order continuous or time. Understand used Term can exchange in suitable situation, and embodiments of the present invention energy described herein Enough carry out to be different from said or illustrational other order.

In addition, term top, below, upper and lower etc. in specification and claims, be used as Descriptive purpose is not certain relative position that is used for describing. Understand that used term is suitable Can exchange in the situation, and embodiments of the present invention described herein can be to be different from These carrying out described or illustrational other order.

In one aspect, the present invention relates to method, wherein nucleic acid amplification with utilize magnetic detection to expanding The mensuration of the quantitative and qualitative analysis of the DNA that increases replaces mutually, and relates to the worker for these methods of enforcement Tool.

Method of the present invention may further comprise the steps:

-amplification process (substep or continuous),

In-the amplification procedure and/or one or more detecting step afterwards, wherein use DNA, PNA or The RNA sensor probe is incorporated into sensor surface or another object with the nucleic acid of amplification. Sensor The combination of probe and sensor surface can be covalent bond.

Randomly, method of the present invention also comprises, before amplification:

-nucleic acid extraction step, and/or

Step before the amplification of-magnetic is in order to determine the amount of initial nucleic acid.

According to an embodiment, by being incorporated into, probe guarantees in the amplicon to implement amplification Nucleic acid or the detection of amplicon. According to an embodiment, method of the present invention comprises the following step Suddenly:

-use the nucleic acid in the primer amplification bulk solution that links to each other with magnetic nanoparticle or particulate.

-amplification of nucleic acid is contacted with the sensor probe that links to each other with sensor surface. Primer and sensor The connection on surface can be covalently bound.

-neutralize/implement detection or mensuration afterwards at least 1 time at amplification procedure to magnetic nanoparticle or particulate.

Randomly, the circulation of applied magnetic particle is implemented (first) a large amount of amplification procedures and (second) surperficial testing process efficiently so that allow.

In a special embodiment of the present invention, application of temperature circulation and magnetic force are actuated, so that can implement the detection to the nearly real-time mode of amplification process.

As mentioned above, method of the present invention comprises the nucleic acid amplification step in the bulk liquid for example.Different amplification method known in the art can be integrated in the method for the present invention.The amplification scheme usually be divided into target type and probe type amplification (Hill, CS. (1996) Journal ofClinical Ligand Assay 19,43-52.).

" target " amplification utilizes the target nucleic acid molecule as the synthetic copy that generate required target sequence of template by each Nucleotide.Example is polymerase chain reaction (PCR), transcriptive intermediate amplification (TMA), nucleotide sequence dependent amplification (NASBA) and strand displacement amplification (SDA) (Saiki etal. (1988) Science 239,487-491; Compton, J. (1991) Nature 350,91-92; Walker et al. (1992) Nucl.Acids Res.20,1691-1696; McDonough et al. (1997) In:Nucleic acid amplification technologies, Eds.Lee, H., Morse, S.andOlsvik, O., Natick, MA:Biotechniques Books, 113-123.).The variant of this method is asymmetry PCR, has wherein only increased and sensor probe complementary specific DNA chain.In this method, add the primer of inequality, become the strand amplification procedure to guide amplification.A variant of this method be Linear-After-The-Exponential (LATE)-PCR (Pierce et al. (2005) PNAS, 102,24,8609-8614), wherein primer is to deliberately being designed so that with concentration inequality.Special embodiment of the present invention relates to and utilizes pcr amplification specific DNA or RNA.On the other hand, the amplification of " probe type " has generated the modification version of the initial probe that is placed into reaction.The example of this method be ligase chain reaction (LCR) (Laffler et al. (1993) Annalesde Biologie Clinique 50,821-826).

For PCR and LCR, with the double-stranded intermediate of thermal cycler sex change.Other schemes (for example TMA, NASBA and SDA) are isothermal, the general heating installation (for example water bath or thermostat container) that only needs a steady temperature.

Dubbing method for example TMA has some different with PCR/LCR.TMA can be with RNA or single stranded DNA directly as target spot.In theory, these methods are faster than PCR/LCR, and they can generate 1,000,000,000 times amplification in 15 minutes, and PCR/LCR needs 3 to 4 hours ability to generate similar amount.On the other hand, TMA may be more not special than PCR, because this process is to implement under lower temperature.Compensate this species diversity with specific probe.

The combination of nearest technology (EXPAR) thermally-stabilised breach enzyme of use and polysaccharase (Van Nesset al. (2003) Proc.Natl.Acad.Sci 100,4504-4509).This is isothermal molecular chain reaction, has wherein generated short oligonucleotide.This method is high responsive, and can obtain＞106 times of amplifications.Whether the steadiness of index method, speed and susceptibility exist in the rapid detection sample aspect a small amount of specific DNA sequences all is useful.

Above different amplification techniques generally all be known as amplification procedure, promptly lost its active process to the amplicon that has obtained aequum or to the enzyme of having exhausted amplimer and/or having participated in amplification from the sample that begins to increase.

For the thermal cycling amplification procedure, this process comprises a plurality of isolating amplification step.For isothermal process, amplification is the successive process.Particle and the another kind of probe that is fixed in sensor surface by magnetic circulation and probe mark, amplification material (for example being exactly the single stranded RNA oligonucleotide for NASBA) can with surface bonding so that make it possible to detect nearly qualitative or quantitative data in real time.Preferably, different with the probe and the amplimer sequence of sequence hybridization of amplification material.

Perhaps, by treatment temp or by physically separate template and amplimer can be divided into amplification step with this process.When method of the present invention is known as two-step approach, wherein in different unit, module or the indoor enforcement amplification (with optional purifying) of equipment therefor and the method that detects.Usually implement two-step approach of the present invention in device, described device comprises the camera incubata (Fig. 2) of wherein implementing the amplification module of amplification and comprising transmitter (chip) surface.Randomly, device comprises the 3rd chamber, wherein is purified into free primer.

According to the specific embodiment of the present invention, before (first) amplification, with RNA and/or DNA extraction step pretreatment sample.In the reference manual (for example Sambrook etal.1989) of molecular cloning, suitable scheme has been described.But the DNA of some types or RNA extraction test kit are commercializations to be obtained (Pharmacia, Dynal, Waters).These extracting method (using magnetic-particle usually) have been removed interfering compound for example polysaccharide and polyphenol.

Also can use simple extracting method, wherein extract rRNA, described rRNA has thousands of copies (Kohne et al.1984) In Thornsberry et al. (Ed) in individual cells: Legionella:Proceedings of the 2nd International Symposium, WashingtonD.C., American Society for Microbiology, 107-108).The parameter that sample is depended in specific pretreated application is complicacy and concentration for example.

In another optional step, the nucleic acid that extracts with magnetic-particle at first can be directly used in the detection of magnetic sensor chips, so that determine the raw-material amount in the preceding step of amplification.

Method of the present invention also comprises the detection step of utilizing magneticsensor.According to special embodiment of the present invention, biomolecules is used to the nucleic acid of amplification is attached to sensor surface.(and also randomly in amplification procedure finishes) implements to detect step 1 time at least in amplification procedure.In continuous amplification procedure, can implement to detect at the particular point in time of amplification procedure.For example among the PCR, after extending step, implement the detection step at the substep amplification procedure usually.Can after each amplification cycles of amplification procedure, implement to detect step.Need therein in the specific implementations of quantitative assay, implement to detect step, normally be recycled between about 25 circulations at about 15 in the logarithmic phase stage of PCR reaction.

Be used in conjunction with amplicon and transmitter or allow by described sensor detecting to molecule on biomolecules can be albumen, nucleic acid, also can be other biological compound for example carbohydrate or VITAMIN (biological example element).

According to the specific embodiment of the present invention, the mode by sensor probe directly or indirectly is connected amplicon with sensor surface, and described sensor probe is the oligonucleotide that is specific to amplicon.Randomly, this sensor probe and sensor surface are covalently bound.

All types of probes in this definition comprise that sensor probe or hybridization probe also can for example albumen or organic molecule link to each other with other molecules, perhaps directly link to each other with oligonucleotide, perhaps through linking to each other indirectly with adhering to oligonucleotide of magnetic-particle, described magnetic-particle combines with oligonucleotide.Expected the connector (for example can be reduced agent cracked chemical linkers or can by the albumen of enzymatic lysis or dna fragmentation) of cleavable for some application.Guarantee bonded biology between the biomolecules required for the present invention interact be for example DNA/DNA in conjunction with, DNA/RNA in conjunction with, antigen/antibody in conjunction with, ligand-receptor in conjunction with, substrate-enzyme in conjunction with, inhibition-enzyme in conjunction with, affine combination (biological example element-(streptavidin) avidin, Zinc-His-Tag, GST-GST conjugated protein etc.).

According to an embodiment, will be fixed in as the biomolecules of capture molecules on the sensor surface, for example antibody, special attraction albumen or polymer surfaces etc.These capture molecules for example by the label (sensor probe) that combines with primer specifically in conjunction with amplicon.The project 1 of Fig. 1 has shown can be used for various Acquisition Scheme of the present invention.For example, capture molecules 16 for example antibody can be incorporated on the surface of transmitter 10, and described transmitter 10 comprises the magnetic field generator 12 that is embedded in the base material 14 for example magnetic stripe or electro-magnet.Capture molecules 16 can with the sensor surface covalent attachment.Capture molecules 16 at least with magnetic field generator 12 near sensor surface combine.Base material 14 can be semiconductor substrate for example " biochip ".Antibody 16 directly or by the label that links to each other with the probe that is specific to amplicon combines with amplicon 18 specificitys that amplification step generates.Magnetic-particle 20 is by ligandin 22 or another mode direct mode (mixing during the amplification) or combine with amplicon 18 by hybridization probe for example.

In yet another embodiment of the present invention, transmitter-probe directly links to each other with sensor surface, be that transmitter institute fixed part is actually special oligonucleotide sequence 24, itself and the end hybridization that is positioned at the amplicon 18 that the magnetic-particle 20 shown in Fig. 1 project 2 links to each other.Magnetic-particle 20 is by ligandin 22 or another mode direct mode (mixing during the amplification) or combine with amplicon 18 by hybridization probe for example.

The used magnetic-particle 20 of the present invention is magnetic nanoparticle or particulate at 2nm in 5 micrometer ranges normally.The size of magnetic-particle 20 should be too not little, actuates particle conveniently to detect and to make it possible to applied magnetic field.Size should be too not big yet, because this may cause non-specific binding and deposition.Preferably, particle size is in the scope between 10nm and 1 micron.Grain pattern and shape can be arbitrary suitable structure and for example single magnetic core of shape, many magnetic core, sphere, bar-shaped etc.Can also use (biology) polymer-coated magnetic-particle 20, with enhanced stability and the functional group that is provided for connecting biomolecules as mentioned above.

Near detecting or measure, the used transmitter 10 of the present invention whether has magnetic particle or nano particle 20, for example in 100 mu m ranges, more preferably in 10 mu m ranges." according to a concrete embodiment, transmitter is more responsive more than 10 times than the nano particle of the surperficial 10 μ m of rang sensor to the nano particle in sensor surface 1 mu m range ".Transmitter can be to detect arbitrary electromagnetic sensor for example electrode, wire, coil, magnetoresistive transducer, magneto strictive sensor, Hall transmitter, plane Hall transmitter, SOUID, the magnetic resonance sensors etc. that whether have magnetic-particle.Transmitter 10 is magnetoresistive transducer huge magnetic impedance (GMR) transmitter for example preferably, so that reach high signal noise ratio when being lower than 1kA/m in magnetic field.Transmitter can randomly be incorporated in the chip.

Preferably on sensor surface or at the near-end of sensor surface, integrated multiple function as much as possible, for example temperature sensation, heating and magnetic detection.Cooling element (for example Peltier element) can be integrated in the box (cartridge) or can be the part of reading device.When needs rapid heating and cooling (for example PCR), the contact area of sample liquids and well heater or cooling element is preferably big as far as possible.

Shift required magneticstrength and the field gradient of magnetic-particle by liquid and depend on some parameters, for example interact for example cluster or the existence of chain formation and the resistance to flow in the medium of particulate susceptibility and magnetic moment, particulate homogeneity and concentration, particle-particle.Can produce magnetic field by combination field generation device (electric current wire and magneticsubstance) in reader, in the box (cartridge) and in the chip.In our system, select these parameters, make it possible between test period, carry out in the biotron particulate and transport repeatedly.

By the biology key (seeing for example WO2005010527) that can survey or rupture at generation power or moment of torsion between particle and other elements between magnetic-particle and the another kind of element (for example sensor surface).In reaction mixture, introduce the biomolecules of (for example primer of mark and probe) and generation (for example mark amplicon) magnetic mark, implement processing treatment then.In embodiment, by the applying a magnetic field fracture and/or the biomolecules of mixing the magnetic mark in this mixture.For example, as nearest (Luxton et al. (2004) Anal.Chem.76 detect described in the particulate immunoassay by plate coil, 1715.), in detecting, biological chemistry uses magnetic-particle surface/body circulation (surface/bulk cycling).

The present invention includes the magnetic-particle round-robin addition method that is used on the magnetoresistive transducer and make body and surface process more efficient methods, for example use the described special-purpose promoting method of additional reagent or WO2005010527.

Interacting with the biology of particulate or nano particle can increase the steepness of melting curve consumingly and strengthen the specificity that detects, and may be because synergistic effect.

Different devices all is applicable to the method for the present invention of implementing.Device can have formula of flowing through (flow-over) or the formula of flowing through (flow-through) design.For example DE040286 EPP (in November, 2004 submission) has described the formula packing of flowing through.Need avoid dead angle or unnecessary recirculation carefully, for example have the fluid edge that seamlessly transits and avoid with acute angle.In using the washing step of washing soln, the design of chamber is preferably guaranteed Gao Gengxin rate on the sensor surface by dwindling fluid depth on the sensor location.Box (cartridge) is preferably made by the material with low non-specific biomaterial bonding force, loses target material and/or reagent so that avoid because of box (cartridge) wall.

Fig. 2 for example understands an embodiment of the invention, and it is used amplification 30, purifying 32 and detects 34 unitary continuous arrangements.Amplification procedure separates with the sensor detecting step.Avoid the unwanted interaction between the ligands specific on free primer and the sensor surface, if desired, in the purifying module of forming by for example silicon materials 32, removed free primer still residual behind the amplification procedure and other reaction contaminant.According to specific embodiment, the liquid in the amplification chamber is recycled, and wherein the purifying module is designed to allow that amplicon passes through and it is indoor that every other material (for example free primer) is retained in amplification.

Fig. 3 A for example understands an embodiment of the invention, wherein uses the single device with three chamber 30-34, separates each conversion with controlled valve system, so that it is next indoor to allow that solution controllably and in time enters into.Three chamber 30-34 and sensor chip 10 have been realized the effect similar to module 30-34 and sensor chip 10 in the described construct of Fig. 1.But liquid can shift between the 30-34 of chamber back and forth at this.

Another preferred embodiment in, processing treatment and proofing unit are shown in Fig. 3 B, promptly implement amplification and detection or mensuration in single assembly 36, preferably near the single chamber sensor chip 10, it allows the formed amplicon of direct detection.More specifically, device comprises thermally-stabilised sensor surface (preferably having the fixed part that is attached thereto), and has the ability of implementing the amplification scheme under differing temps (for example PCR) required as used amplification method or steady temperature (for example NASBA).In addition, device is applicable to allows and removes competitive free primer (if existence), so that avoid taking place for example primer-primer complex and take into account the special sign of used magnetic particulate of nonspecific reaction.

Shown in Fig. 4 diagram, the present invention includes the magnetic-particle round-robin addition method that is used on the magnetoresistive transducer and make body and surface process more efficient methods, for example use the described special-purpose promoting method of additional reagent or WO2005010527.

Variation to processing treatment and proofing unit is contained within the scope of the present invention.Particularly, other possible device geometry comprise the high surface area material that uses as sensor surface.In addition, can use the effluent and the formula structure of flowing through.

The present invention is applicable to multiple application.Non-limited list of application is for example to detect microorganism (detection food spoilage and poisoning, and the gene of the detection toxin-encoding of agricultural-food-environmental area; Estimate the active gene (mRNA in " genome " research) of farm crop and fruit (quality indicator)); Detect GMO (food safety and evaluation); Estimate forgery/swindle (mensuration foreign DNA); Detect allergenicity product/pollution; The microorganism that detection brings environmental consequences is MRSA, legionella, listeria bacteria for example; Microorganism in the identification of cell culture.Application in the clinical diagnosis for example is: detect relevant microorganism such as infection, meningitis, respiratory tract disease, tuberculosis, hepatitis, AIDS; Microorganism in the identification of cell culture is for example with the microorganism of above-mentioned disease-related.

Illustrate the present invention with the following examples now.

Embodiment 1: the pcr amplification that utilizes the amplimer that does not have magnetic mark

After optional nucleic acid extraction step, according to of the amplification of PCR scheme with enforcement nucleic acid in mentioned in the above arbitrary processing treatment of two specific amplification primer solution and the proofing unit.Amplification procedure has generated amplicon 18, one bar chain and hybridization probe hybridization, described probe and magnetic nanoparticle or particulate 20 covalent attachment.Another part of this of amplicon chain is sensor probe (being connected with sensor chip surface) hybridization with another complementary probe.Described connection can be covalently bound.When hybridization probe and sensor probe and incomplementarity during, obtained minimum interference in the extension intrachain nucleic acid array hybridizing of the used amplimer of amplification procedure.In this embodiment, intermittently measure at specific time point tumor-necrosis factor glycoproteins (circulation) and be connected to the particulate of transmitter or the concentration of nanoparticle.

Step below 3 has been described PCR circulation commonly used:

The heated die plate double-stranded DNA, so that dissociate single chain, normally between 90 and 99 ℃ (sex change).

Reduce temperature, become free chain, normally between 45 to 65 ℃, depend on amplimer length and sequence (annealing) so that allow primer annealing.

Improve temperature, bring up to 72 ℃ usually.Extend primer (extension) along template strand.

In order to detect and measure amplimer for example DNA, RNA, the step below having implemented:

After finishing DNA cloning, improve temperature, with the double-stranded DNA that dissociates.

Utilize magnetic force that hybridization probe and magnetic particle or nano particle are attracted to sensor surface, the field gradient that described magnetic force is generated from magnetic field generator 12 from bulk solution.To form sandwich structure with these probes with hybridization probe and detection probes complementary DNA chain (by hybridization).Therefore, the DNA chain has been fixed to sensor surface and by magnetic-particle institute mark.

For example unconjugated hybridization probe on the sensor surface can be removed by applying magnetic force subsequently, but can not rupture DNA-DNA heterozygote in chain-hybridization probe-sensor probe mixture of maximum magnetic force will be made by magnetic field and/or field gradient with correct slope.Magnetic force numerical value commonly used is in the pN scope, the special key (as described in WO2005010527) that will rupture of the power in the nN scope.

The magneticstrength of the magnetic-particle that mensuration links to each other with hybridization probe, this numerical value is relevant with the amount of formed amplicon in the pcr amplification.This method allows that nearly PCR in real time detects.

After determination step, improve temperature once more, so that the probe amplification subchain of dissociating interacts.Nano particle can be redistributed in the bulk solution by reversing field direction, their magnetic force of passing through to be applied is actuated and is participated in biochemical reaction once more afterwards.During this time, with the sample sex change, and prepare to enter into next amplification cycles.

Fig. 5 has shown the figure as the sensor signal of the function of time.

For those skilled in the art, it is apparent that the modification to the specific step in the said process is possible.For example, implement to detect step after each amplification step, perhaps low frequency ground is implemented to detect step or is only just implemented to detect step in the PCR circulation of implementing initial number and after not implementing to detect step.

Said process is known as " magnetic-particle and temperature cycle ", because magnetic-particle and temperature all are recycled.This allows that particulate and nano particle participate in body and surface process effectively.In this embodiment, amplicon forms and generation occurs in bulk solution, and particle detection occurs in sensor surface.This is a test format at a kind of intermittence, and the time between the single detection is equal to the cycling time of PCR method.By shortening PCR cycling time, the nearly real-time status of amplification procedure becomes more accurate.With regard to present state of the art, each circulation of the most small-sized amplification needed for 15 to 30 seconds.

By in process, add the internal standard thing be known quantity with reference to template and be used for the primer special of comparison purpose, can be quantitative also so that should closely implement amplification.Preferably, isolating point on the sensor surface (statistics at random) is specific to this internal standard thing.By record amplification efficiency (for example amount in each timed interval), the original bulk that calculates unknown template DNA is possible.

Implement to detect in preferred setting, wherein the measurer of the hybridized primer of particulate or nano particle has low amplicon fraction of coverage, and promptly mean value is less than amplicon of each nano particle.This has guaranteed that the possibility that each particulate or nano particle surpass 1 amplicon is low-down, and the number of particulate on the transmitter or nano particle can be become target level in the primary sample by translation quantitatively and exactly.

According to the present invention, the array implement multichannel analysis of transmitter 10 that also can be by having different capture molecules.In some cases, can use identical primer, and in other situations, need different primers.

Embodiment 2: the pcr amplification that utilizes the amplimer with magnetic mark

After optional nucleic acid extraction step, according to the PCR scheme, with one of them primer and magnetic-particle covalent attachment and another primer by the amplification of the amplimer of magnetic mark enforcement nucleic acid.Fig. 6 illustrates present embodiment.Amplification procedure has caused wherein, and an extended chain is marked as magnetic particle or nano particle by the primer that links to each other.

In this embodiment, at specific time point, intermittently measure the particulate that links to each other with transmitter 10 or the concentration of nanoparticle tumor-necrosis factor glycoproteins (circulation).

Step and the additional aspects shown in the present embodiment of describing preferred design and method are substantially the same with embodiment 1 described method, except amplimer, only need an additional probe (sensor probe) that links to each other with the sensor chip surface covalency.This sensor probe can comprise the sequence identical with the amplimer oligonucleotide or can cover the sequence of amplimer oligonucleotide, as shown in Figure 6.Perhaps, sensor probe can with the sequence hybridization that is different from the amplimer binding sequence of DNA amplification.

Another possibility as top scheme: relevant with primary target concentration during the detection step with the concentration of transmitter bonded magnetic-particle.In other words, for given detection time and testing process time, the granule density that is attached on the transmitter of formed amplification subchain has indicated primary target concentration.Another method of measuring formed amplicon amount is the amount that detects at particular detection still residual amplimer in the time.If in this method, institute's bonded particulate concentration will reduce along with the increase of formed amplicon concentration in the amplification procedure in conjunction with particulate amplimer target.Provided an example of this detection at this:

One type amplimer and magnetic-particle covalent coupling.On transmitter, stationary probe, it has been chosen to combine with this amplimer.Therefore, the particle with amplimer of this covalent coupling can combine with transmitter with high-bonding-ratio at first.When amplification procedure makes progress, manyly will be extended into the total length amplicon in conjunction with the particulate amplimer.Therefore, particle and transmitter bonded possibility reduce, this be because the number of come-at-able amplimer on the particle still less and/or be because with the steric hindrance of particle bonded amplicon.

Embodiment 3: microarray applications of the present invention

The previous described method of embodiment makes it possible to detect the amplification genetic material by the temperature cycle in magnetic field circulation and the certain situation.All these methods all are highly suitable for microarray applications.The interaction strength and the complementary degree between probe and the amplicon that are fixed between the amplicon (in this case, it is connected with magnetic nanoparticle) of the probe of sensor surface of array and sample solution more or less are proportional.By on array, applying different power (the different magnetic force that for example depends on array position) or passing through the power of change as the function of time, the inferior point of dissimilar arrays be can identify, for example the continuous amplicon and the point of magnetic nanoparticle (hybridization for example non-specific or that highly degenerate) discharged under the weak power to having the very strong probe and the point of the bonding force between the amplicon (for example high complementarity).In one situation of back, as the result of the magnetic-particle that is fixed in these particular points, magnetic signal is still and can be recorded.

In microarray was provided with, wherein specific probe was fixed to the very little and separated portions of sensor surface, and special probe and the collision opportunity that has between the mark magnetic-particle of special complementary amplicon/part continuous or that combine are very little.Magnetic force circulation with the speed composition (velocity component) perpendicular to sensor surface will increase specificity combination in the small volume on the sensor surface to the concentration of (being the magnetic-particle of surface-probe and amplicon/ligand-labeled).But the motion that particle is parallel to sensor surface is limited (also being the result in magnetic field).Therefore, relevant with the result's that must generation can measure the amplification procedure right interaction of specific combination is the process of a poor efficiency in microarray is provided with.

In order to overcome this defective, provided another embodiment of microarray form, wherein off and on or by applying magnetic force and speed with composition parallel with sensor surface with vertical composition coordinated mode, so that flatly mobile particle on sensor surface, make particle more approaching, further increase the contact of collision property.

Except influencing the joint efficiency, can also use magnetic field to actuate, so that provide added value to heavily dividing to interact under the specified temp by adjusting temperature.This makes it possible to detect SNP or other variations of nucleotide sequence, therefore can detect gene difference (note: sequential analysis can be measured as checking) when not having sequential analysis.This also makes it possible to gene to last mediation downward modulation and carries out more responsive and describe more accurately, i.e. the key gene of being studied in microarray is tested relevant with the special physiological mathematic(al) parameter of lesser number.This also makes it possible to identify more accurately microorganism, pathogenic agent or other biological and learns material.

Claims (13)

1. one kind is used to utilize one or more amplimer cloning RNA or DNA nucleic acid and determines the RNA or the DNA nucleic acid of described amplification or participate in the method for amount of the reagent of amplification procedure, wherein implement to determine the step of the amount of the nucleic acid of described amplification or described reagent by magnetic detection, and described step is implemented in the amplification procedure of described RNA or DNA 1 time at least, it is characterized in that implementing by the following method determining the step of the amount of the RNA of described amplification or DNA or described reagent, in described method, the nucleic acid of described amplification or described reagent combine with transmitter by biomolecules.

2. the process of claim 1 wherein that described biomolecules is DNA.

3. the process of claim 1 wherein with isothermal method increase described RNA or DNA.

4. the process of claim 1 wherein with thermal cycling method increase described RNA or DNA.

5. the process of claim 1 wherein with a kind of amplimer of magnetic-particle mark.

6. the process of claim 1 wherein that the hybridization probe with magnetic mark detects described amplicon.

7. each method in the aforementioned claim also comprises the magnetic force circulation that has with the vertical speed composition of sensor surface.

8. be used for the test kit of the amount of the reagent determining the amount of sample amplifying nucleic acid or participate in amplification procedure, described test kit comprises at least:

-have the device of sensor surface, wherein one or more biomolecules links to each other with described sensor surface;

-oligonucleotide, wherein at least a oligonucleotide and magnetic-particle coupling mutually, and one or more DNA or RNA polymerase.

9. the test kit of claim 8 also comprises having the active enzyme of breach.

10. the test kit of claim 8 also comprises the enzyme with RNA enzymic activity.

11. the test kit of claim 8, wherein said at least a oligonucleotide is an amplimer.

12. the test kit of claim 8, wherein said at least a oligonucleotide is a hybridization probe.

13. each test kit in the claim 8 to 12, wherein sensor surface is the part of microarray.

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