CN101033485B - Method of carrying parallel real-time quantitative detection to varies nucleic acid molecule - Google Patents
Method of carrying parallel real-time quantitative detection to varies nucleic acid molecule Download PDFInfo
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Abstract
The invention provides a method for parallel detecting and real-time quantitative analyzing a variety of nucleic acid molecule. In the invention, it uses the evanescent field excitation caused by total optical reflection or optical planar waveguide, or fluorescence resonance energy conversion to stimulate fluorescence group, but it almost does not excite a large number of uncombined goal molecules or fluorescent groups in the reaction solution.
Description
Technical field
The present invention relates to the detection of nucleic acid, gene diagnosis, gene sequencing, the analysis of gene type and polymorphum, and the biological technical fields such as analysis of genetic expression.Particularly at the nucleic acid detection technique of real-time quantitative polymerase chain reaction of gene test (Real time quality PCR) and array.
Background technology
Since 1985, since the Kary Mullis invention polymerase chain reaction, polymerase chain reaction technology became one of technological revolution with strongest influence power since the molecular biology development quickly.It is a kind of sophisticated that it makes that detection of nucleic acids becomes, highly sensitive, and detection technique fast, and in the very fast clinical detection that is widely applied to molecular biology research and microbial pathogen body, comprise the detection of fungi, bacterium, virus etc.Therefore Kary Mullis has also obtained the Nobel prize in 1993.
But, need loaded down with trivial details DNA electrophoresis detection behind the polymerase chain reaction, and be easy to generate sample crossed contamination each other thus.Make the polymerase chain reaction very high, and the error rate that detects is very high to technological operation personnel and Laboratory Request.The polymerase chain reaction has finally received challenge in the application of clinical detection.
To the middle and later periods of the nineties; The real-time quantitative polymerase chain reaction that on polymerase chain reaction technology and fluorescence technique basis, grows up (Real time quality PCR) technology; Realized the leap of PCR from qualitative to quantitative; Deducted the DNA electrophoresis detection, made more easy and simple to handle, and time of having saved the DNA electrophoresis detection.Simultaneously also make the probability of crossed contamination reduce, improved the accuracy that detects, and can quantize nucleic acid molecule to be detected more accurately.This technological detection linear range can reach the 7-9 one magnitude, and sensitivity can detect low nucleic acid molecule to several copies, and particularity can be less than 3% deviation.It with its high specificity, highly sensitive, good reproducibility, quantitatively accurately, advantages such as fast, the totally-enclosed reaction of speed become the important tool in the molecular biology research, and beginning is accepted by clinical detection.
The detection of Clinical microorganism be the real-time quantitative polymerase chain reaction technology now in the most important field of clinical detection, it has shortened detection time greatly than traditional Clinical microorganism detection technique, has improved the sensitivity that detects, and operation is easier.At present, be mainly used in some common virus, like HIV, HBV, the clinical detection of HCV etc., or the difficult bacterium of cultivating are in the detection like tubercule bacillus.At life science, particularly in the quantitative analysis of genetic expression, the real-time quantitative polymerase chain reaction technology also has a very wide range of applications.The real-time quantitative polymerase chain reaction technology also is used in environmental monitoring simultaneously, and public health detects, and biological weapon detection etc. analytically.
But a lot of detection of nucleic acids need detect with quantitative nucleic acid molecule a kind of incessantly.Carry out the parallel detection and the quantitative analysis of multiple nucleic acid and the real-time quantitative polymerase chain reaction technology is exactly difficult.A lot of biotech companies comprise ABI, Invitrogen etc., or the biotechnology research chamber is all at parallel detection of nucleic acid and the quantitative analysis tech of attempting to develop many flux.The real-time quantitative polymerase chain reaction technology of these multiple nucleic acid; Or through coming the different probe molecule of mark to detect different nucleic acid molecules, or in a plurality of reacting pipes, carry out a plurality of real-time quantitatives polymerase chain reaction respectively with multiple different spectrographic fluorescence molecules.And these technology or require the fluorescence molecule of multiple different optical bands or need highdensity reacting pipe, make original just relatively expensive real-time quantitative polymerase chain reaction technology, the growth at double of its reagent and instrument cost.And their detection flux has received the restriction of fluorescence molecule kind and reacting pipe volume.
Therefore, press in this area and provide improved, simply can be simultaneously carry out the method that real-time quantitative detects multiple nucleic acid molecule.
Summary of the invention
The present inventor has set up and can carry out the method that real-time quantitative detects to multiple nucleic acid molecule simultaneously through further investigation.
Particularly, first aspect present invention provides the existence of multiple nucleic acid molecule in a kind of replicate(determination) sample or the method for amount, and this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided, the stationkeeping that said carrier surface is different multiple nucleic acid probe molecules, said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased, when amplified reaction, with fluorophor mark amplified production;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) produce evanscent field at solid phase carrier and liquid surface place with excitation light source, make with the said amplified production of said probe molecule bonded on the fluorophor generation fluorescent signal that is excited;
G) detect the position and the intensity of said fluorescent signal, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
The present invention provides the existence of multiple nucleic acid molecule in a kind of replicate(determination) sample or the method for amount on the other hand, and this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided; The stationkeeping that said carrier surface is different multiple nucleic acid probe molecules; Said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample, is connected with first fluorophor on the said nucleic acid probe molecules;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased; When amplified reaction; With the second fluorophor mark amplified production; The PLE of the emission spectrum of wherein said first fluorophor and said second fluorophor is overlapping, or the PLE of the emission spectrum of said second fluorophor and said first fluorophor is overlapping;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) with one in said first fluorophor of optical excitation and said second fluorophor;
G) detect another signal emitted fluorescence position and intensity in said first fluorophor and said second fluorophor, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
Method of the present invention is brand-new; It can detect and quantitative technology by high-throughout nucleic acid molecule to hundreds and thousands of kinds through a reaction; It does not need multiple fluorophor mark; Do not need a plurality of reaction channels, thereby with detecting on the target molecule flux very big technical superiority is arranged at cost.
Description of drawings
Fig. 1 produces the schematic diagram of evanscent field for total reflection.
Fig. 2 has shown the variation of the relation of the evanscent field intensity and the degree of depth with incident angle (A) and specific refractory power (B).
Fig. 3 produces the schematic diagram of evanscent field for slab guide.
Fig. 4 is the FRET schematic diagram.
Fig. 5 is the change in fluorescence curve synoptic diagram of the real-time quantitative polymerase chain reaction of multiple nucleic acid.
Fig. 6 A has shown that nucleic acid probe molecules is connected on glass surface through chemical bond-linking.
Fig. 6 B has shown that nucleic acid probe molecules is embedded in metal oxide surface through the polymkeric substance connection.
Fig. 7 is for producing evanscent field carries out the parallel quantitative analysis method to multiple nucleic acid molecule outline figure through total reflection.
Fig. 8 has shown that evanscent field excites and be fixed on the fluorophor on the target nucleic acid of probe molecule hybridization of carrier surface.
Fig. 9 A shows, along with the carrying out of the temperature cycle of polymerase chain reaction, and the fluorescent signal in detected each probe points hybridization that is excited by evanscent field of CCD.
Fig. 9 B is the change in fluorescence curve of each probe points real-time quantitative polymerase chain reaction of being excited by evanscent field.
The Ct value that Fig. 9 C obtains for the real-time quantitative polymerase chain reaction through the evanscent field method, the start-up phase of quantitative four kinds of cut modes is to copy number.
Figure 10 is for producing evanscent field carries out the parallel quantitative analysis method to multiple nucleic acid molecule outline figure through slab guide.
Figure 11 is for carrying out the outline figure of parallel quantitative analysis method to multiple nucleic acid molecule through FRET.
Figure 12 excites through FRET for the target nucleic acid with the probe molecule hybridization that is fixed on carrier surface.
Figure 13 A is the carrying out along with the temperature cycle of polymerase chain reaction, the fluorescent signal that detected each probe points that excites through FRET of CCD is hybridized.
Figure 13 B is the change in fluorescence curve of each probe points real-time quantitative polymerase chain reaction of exciting through FRET.
The Ct value that Figure 13 C obtains for the real-time quantitative polymerase chain reaction through the FRET method, quantitatively the start-up phase of various bacterial genes to be detected is to copy number.
Preferable embodiment
First aspect present invention provides the existence of multiple nucleic acid molecule in a kind of replicate(determination) sample or the method for amount, and this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided, the stationkeeping that said carrier surface is different multiple nucleic acid probe molecules, said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased, when amplified reaction, with fluorophor mark amplified production;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) produce evanscent field at solid phase carrier and liquid surface place with excitation light source, make with the said amplified production of said probe molecule bonded on the fluorophor generation fluorescent signal that is excited;
G) detect the position and the intensity of said fluorescent signal, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
The sample that is applicable to the inventive method can be a various forms, as long as wherein have target nucleic acid molecules to be detected.For example can use well known or disclosed method (like digestion with restriction enzyme or mechanical means) obtains nucleic acid samples, like mixtures such as total DNA, genomic dna, cDNA, total RNA, mRNA.
Term used herein " nucleic acid ", " nucleic acid molecule " can exchange use, and it refers to the polymkeric substance of thymus nucleic acid or Yeast Nucleic Acid and strand or double chain form.Only if special qualification is arranged in addition, this term comprises the nucleic acid of the known analogue that contains natural nucleotide.This term " nucleic acid " can exchange with the mRNA of gene, cDNA and genes encoding and use.In addition, nucleic acid molecule of the present invention has also been contained nucleic acid fragment, nucleic acid subsequence, nucleic acid derivative and nucleic acid modifier.Described length nucleic acid can be different, and it can be the fragment of larger nucleic acid molecule or less part.Preferably, length nucleic acid to be detected is at least 30 base pairs, and better is that length is at least 50 base pairs, and for example 30-4000 is individual, 50-2000 is individual or 50-1000 base pair.Nucleic acid to be detected can be known or unknown sequence, can be strand or double chain form.Its nucleic acid to be amplified of can deriving from any source.
In a preferable embodiment of the present invention, method of the present invention can be used for the different connecting methods of the multiple exon of parallel detection and the multiple mRNA and the amount thereof that give expression to.In another preferable embodiment, method of the present invention can be used to identify the existence and the amount thereof of various bacteria in the sample.
Adopted a kind of solid phase carrier in the methods of the invention, the stationkeeping that said carrier surface is different multiple nucleic acid probe molecules, said nucleic acid probe molecules has specificity to the said multiple nucleic acid molecule in the sample.Term used herein " solid phase carrier " or " solid carrier " are meant that the excitation wavelength to fluorophor that nucleic acid can be fixed on it has the low solid surface that absorbs.For example; Materials such as glass, nylon membrane, quartz, pottery, metal, plastics, organic polymer can both be as solid carrier of the present invention; As long as this carrier can both detect tangible fluorescent signal low absorption of light to the fluorophor excitation wavelength section that will detect.Should be noted in the discussion above that also need not be transparent for carrier, as long as the low absorption of its light to certain wavelength period, promptly to only transparent the getting final product of this wave band, it possibly be opaque at its all band of visible light also.In addition, the specific refractory power of solid carrier need be greater than the specific refractory power of selected liquid phase, and preferable specific refractory power need be greater than 1.33 usually.In the present invention, preferred solid phase carrier is a glass.In preferable embodiment, these carriers should pass through chemical process and modify, thereby have reactive group, can with amino, aldehyde radical, phosphate on the nucleic acid or stable being connected of formation such as sulfydryl of modifying, enhancing is to the crystallized ability of nucleic acid probe molecules.
Term used herein " is fixed " and is meant nucleic acid is fixed on the solid support; Adhere to through chemical covalent attachment mode; Or through irreversible passive absorption, or through intermolecular avidity (for example utilizing biotinylated molecule joint to fix) in the surface that scribbles avidin.This strength of fixation must be enough under DNA sex change condition, can not be removed because of the washing of water or aqueous buffer solution.A kind of preferable fixing means is to adopt the bifunctional cross-linker to handle slide, to obtain having the surface of reactive crosslinked group (like amino, sulfo-or acrylic acid groups).Said linking agent can be; For example; 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), butanedioic anhydride, phenyl diisothiocyanic acid salt or maleic anhydride or Heterobifunctional group linking agent, as-maleimide phenylformic acid-N-hydroxy-succinamide ester (MBS), N-succinimide [4-iodoacetyl] benzaminic acid (SIAB), succinimide 4-[N-maleimide methyl] hexanaphthene-1-carboxylate (SMCC), N-y-maleimide butyryl oxygen-succinimide ester (GMBS), succinimide-4-[right-maleimide phenyl] butyrates (SMPB) etc.In addition can be referring to document: Nucleic Acids Research, 29:e69,2001.Analytical Biochemistry; 247:96-101,1997.Analytical Biochemistry, 266:23-30; 1999.Nucleic Acids Research, 30:4793-4802,2002.
The nucleic acid probe molecules that is fixed on the carrier surface can design according to the information of target gene, thus make its can with specific mode respectively with sample in plurality of target nucleic acid molecule or its amplified production specificity combine.The length of this probe can be carried out suitable selection according to demand by those skilled in the art, usually between 6-2000 Nucleotide (a preferable 10-50 Nucleotide, a better 15-30 Nucleotide).In addition, according to concrete purposes, possibly also need adopt the nucleic probe of modified.This nucleic probe can prepare with method well known to those skilled in the art.
Nucleic probe is being preferably the array that separates on the space on the solid phase carrier, it normally constitutes like this, and solid phase carrier (like slide) is divided into different zones, makes and only contains a kind of nucleic acid probe molecules in each zone.Through the DNA array technique, multiple dna probe molecule is arranged on the solid carrier.
The nucleic acid probe molecules that is used for detection of nucleic acids generally includes oligonucleotide, oligomerization thio nucleotides, PNAG3 (peptide nucleic acid), long-chain dna molecular, RNA molecule or their modifier.These nucleic acid probe molecules can be fixed on the solid carrier through modes such as chemical bond connection, physical adsorption or polymkeric substance embedding, etching or sticking couplet.Nucleic probe also can be connected on the solid phase carrier through joint or spacerarm.The dna probe molecule that is arranged on the solid carrier passes through the mutual pairing between base, combines with target molecule (like amplified production).Usually target molecule is by on the fluorophor mark; It can make the deoxynucleoside triphosphate (dNTP) of marks such as fluorophor be incorporated in the amplified production of target nucleic acid molecules through amplified reaction, or makes the method for fluorophor on the amplified production band through the primer of marks such as fluorophor.Through hybridizing the back wash-out, the target molecule that hybridization combines to get on fluorescent signal be detected.
Nucleic acid molecule can duplicate external through archaeal dna polymerase or RNA polymerase, transcribes the process of reverse transcription, thereby linearity or its copy number of the amplification of index.Road as is known to the person skilled in the art; Amplification procedure generally includes sex change; Annealing, synthetic three steps, i.e. unwind (sex change) of double chain acid molecule; The nucleic acid molecule that is unwind combines (annealing) with primer molecule, archaeal dna polymerase or RNA polymerase are synthetic complementary DNA or RNA molecule with it of template with the nucleic acid molecule that unwinds.
Can be used for nucleic acid amplification method of the present invention and include, but are not limited to, polymerase chain reaction (PCR), inverse transcription polymerase chain reaction (RT-PCR), random primer amplified reaction, composite polymeric PCR amplification etc.In a preferable embodiment, should adopt the polymerase chain reaction." polymerase chain reaction " or " PCR " refers to a kind of technology that is applicable to a small amount of particular segment of nucleic acid, RNA and/or DNA, and it has detailed description in for example United States Patent(USP) No. 4,683,195 (mandate on July 28th, 1987).Usually, need to obtain the sequence information of area-of-interest two ends or its extension, thus can the design oligonucleotides primer; These primers should be same or similar basically with the sequence of template opposite strand to be amplified.5 ' terminal nucleotide of two primers can overlap with the two ends of amplification material just.The polymerase chain reaction can be used to increase among the full gene group DNA, transcribe specific RNA sequence, specific dna sequence the cDNA of acquisition from whole-cell rna, phage or plasmid sequence etc.PCR used herein be considered to increase one of sample amplifying nucleic acid molecule but and not exclusive example, adopt known nucleic acid to increase in this method or produce the nucleic acid of particular segment as primer and nucleic acid polymerase.
In the polymerase chain reaction; Double-stranded dna molecular becomes single chain molecule in sex change more than 90 ℃, combines with primer molecule at 50-60 ℃ then, combines to go up archaeal dna polymerase simultaneously; Archaeal dna polymerase is a masterplate with this single strand dna; Deoxynucleoside triphosphate (dNTP) is a substrate, copies another complementary DNA with it, and the righttest synthesis temperature of archaeal dna polymerase in the polymerase chain reaction is 70-78 ℃ usually.Through duplicating, target dna molecule has increased one times.After reaction repeated n so repeatedly the circulation, target dna molecule has increased nearly 2
nDoubly.The example that specifically can be used for nucleic acid polymerase of the present invention is heat-stable DNA polymerase (like Taq, VENT, Pfu, Tli, Tfl archaeal dna polymerase) of archaeal dna polymerase (T4DNA polysaccharase), various thermophilric bacterias and the verivate (TaqGold of their genetic modifications; VENTexo, Pfu exo).Preferred archaeal dna polymerase is Taq or Tfl archaeal dna polymerase.Preferably, being used for ribonucleoside triphosphote molecule of the present invention is deoxyribonucleoside triphosphate, like dATP, dTTP, dCTP, dGTP, or ribonucleotide triphosphate, like ATP, UTP, CTP, GTP.Ribonucleoside triphosphote can be natural or non-natural forms.
Carry out in the process of amplified reaction at nucleic acid molecule, archaeal dna polymerase or RNA polymerase are that substrate synthesizes with deoxynucleoside triphosphate (dNTP) or ribonucleoside triphosphote (NTP), synthesize into new amplified production.Therefore, through at amplimer marked fluorophor or at substrate deoxynucleoside triphosphate (dNTP) or ribonucleoside triphosphote (NTP) marked fluorophor, just can make the target dna molecule of amplification will mark on required fluorophor.
The technology of mark fluorescent group is to know in the prior art.Common fluorophor includes, but are not limited to, and organic fluorescence divides subclass, like resorcinolphthalein, and TAMRA, Cy3, Cy5, Cy5.5, rhodamine, Alexa Fluor series etc.; The GFP class, like GFP, CFP, BFP, YFP etc.; Four kinds of time resolved fluorescence and quantum dots (Quantum dot) containing rare earth ion.
In the annealing process of amplified reaction; The amplified nucleic acid molecule product to be detected that previous round amplification obtains with the amplimer bonded simultaneously; Also can combine, thereby make the fluorophor on the target nucleic acid molecules amplified production in the combination be able to excited by specific mode of the present invention with the nucleic acid probe molecules on being fixed on carrier.Particularly, the fluorophor on the target nucleic acid molecules amplified production can excite by the interior evanscent field that total reflection or slab guide produced by light of suppressed by vector.
(i) evanscent field of the total reflection of light generation
As everyone knows, when light from high refractive index (n
1, like glass) medium (optically denser medium) shine low-refraction (n
2, liquid phase such as water) medium (optically thinner medium), and input angle is greater than critical angle (θ c, θ c=sin
-1(n
2/ n
1) work as n
2＜n
1) time, total reflection will take place in light in optically denser medium, thereby can not be refracted in the optically thinner medium.Yet when light generation total reflection, light wave can infiltrate in the optically thinner medium in the place of Strahlungseintritt with the form of EM field, and the intensity of the EM field of infiltration is along with the intensification of depth of penetration forms exponential attenuation (Fig. 1), and the degree of depth of its infiltration is:
d
p=(λ/2 π n
1) [1/ (sin
2θ-n
2 2/ n
1 2)]
1/2(λ, light wavelength in a vacuum; θ, incident angle)
Its degree of depth is usually between 100 to 300 nanometers.According to formula, it and input angle, lambda1-wavelength, specific refractory power relevant (Fig. 2).This EM field that is penetrated into optically thinner medium through total reflection just is called as evanscent field.Evanscent field is the same with other light sources, also can the fluorescence excitation group.If the optically denser medium surface is coated with noble metal film or particles such as the gold and silver, copper of nanometer scale (about 100-200 nanometer); The intensity of enhancing evanscent field fluorescence excitation that can be to a certain degree, enhancing degree is several times and do not wait to tens times, and the enhancing degree is relevant with factors such as metal species and thickness (referring to document: Analytical Biochemstry; 334:303-311; 2004.Journal of Fluorescence, 14:425-441,2004.Current Opinion in Biotechnology; 16:55-61,2005).Therefore; In a preferable embodiment of the present invention; Optically denser medium (being solid phase carrier) surface can be coated with the metallic membrane less than 1000 nanometer thickness (about usually 50-500 nanometer, better about 100-200 nanometer), and perhaps diameter is less than 1000 nanometers (about usually 50-500 nanometer; Better about 100-200 nanometer) metallic particles, wherein said metal is selected from gold and silver, copper or other precious metal.
Because the degree of depth of evanscent field infiltration is very little, has only the hundreds of nanometer, so evanscent field can only excite the fluorophor that is close in the dielectric layer 100-200 nanometer, and the fluorophor of the overwhelming majority in optically thinner medium can not excited.Evanscent field excited fluorescent strength of signal is equivalent to be attached to the amount of the target dna molecule of carrier surface.The concentration of target dna molecule is high more in the solution, and also many more with molecule on dna probe combines, the excited fluorescent signal is also strong more.
The (ii) evanscent field that produces of slab guide
Slab guide is generally the high refractive index film of 100-300 nanometer, for example tantalum pentoxide (Ta
2O
5), titanium oxide (TiO
2), zinc oxide (ZnO), zirconium white (ZrO
2), niobium oxides (Nb
2O
5), hafnia (HfO
2) wait metal oxide film.It is deposited on the solid substrate (like glass, transparent polymer etc.) of transparent low-refraction.After laser was coupled into this high refractive index film through methods such as prism or gratings, light transmitted in this flat film.Simultaneously, it will produce the evanscent field that the very strong intensification along with the degree of depth of permeating forms the exponential decay between high refractive index film and low-index layer.The degree of depth of evanscent field infiltration is:
d
p=(λ/2 π n
1) [1/ (sin
2θ-n
2 2/ n
1 2)]
1/2(λ, light wavelength in a vacuum; θ, incident angle)
This degree of depth is equally in about 100 nanometers (Fig. 3).After fixing nucleic acid probe molecules on this plane wave conducting shell; Evanscent field will excite the fluorescent probe molecule in the hybridization; If on the plane wave conducting shell, be coated with the noble metal film or the particle such as gold and silver, copper of nanometer scale (about 100-200 nanometer); The intensity of enhancing evanscent field fluorescence excitation that can be to a certain degree, enhancing degree are several times and do not wait to tens times that the enhancing degree is relevant with factors such as metal species and thickness.Same, because the evanscent field penetration depth that slab guide produces is at nano level, the fluorophors of the overwhelming majority in this optically thinner medium of reaction solution can not excited, thus the fluorescence intensity of the specific position on just can the detection plane ducting layer.According to the corresponding relation or the production standard curve of this fluorescence intensity and nucleic acid molecule concentration, just can detect the concentration of each target nucleic acid molecules in the solution.
The present invention provides the existence of multiple nucleic acid molecule in a kind of replicate(determination) sample or the method for amount on the other hand, and this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided; The stationkeeping that said carrier surface is different multiple nucleic acid probe molecules; Said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample, is connected with first fluorophor on the said nucleic acid probe molecules;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased; When amplified reaction; With the second fluorophor mark amplified production; The PLE of the emission spectrum of wherein said first fluorophor and said second fluorophor is overlapping, or the PLE of the emission spectrum of said second fluorophor and said first fluorophor is overlapping;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) with one in said first fluorophor of optical excitation and said second fluorophor;
G) detect another signal emitted fluorescence position and intensity in said first fluorophor and said second fluorophor, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
(Flourescent resonance energy transfer FRET) excites also can to pass through FRET with fluorophor on the probe molecule bonded target nucleic acid molecules amplified production.As shown in Figure 4; FRET is meant works as two kinds of different fluorescence chromophores close together spatially; And the PLE of the emission spectrum of wherein a kind of chromophore (donor) 401 and another kind of chromophore (acceptor) 407 has when overlapping; When donor is excited (shown in 402), acceptor 409 can be excited because of the transfer 408 of donor excitation energy.Theoretically, overlapping as long as two spectrum have, FRET will take place, just the efficient that shifts of energy is relevant with the factors such as distance of integration, fluorescence quantum efficiency and the donor acceptor of spectra overlapping.The distance R during (but reference literature: Proc Natl Acad Sci USA 58:719-726,1967) energy transferase 45 0%
0=[8.8x 10
23X κ
2X n
-4X QY
dX J (λ)]
1/6 
κ is the direction of polarization factor, and donor acceptor direction is to be generally 2/3 at random, and n is a specific refractory power, and the aqueous solution is generally 1.34, QY
dBe the fluorescence quantum efficiency of donor, J (λ) is the emmission spectrum and the acceptor excitation spectrum eclipsed integrated intensity of donor.Its visualize be exactly the fluorescence intensity that produces of donor will be low during than its exist singly many 410; And the acceptor emitted fluorescence strengthens 409 greatly, overlapping degree, donor and the dipolar relative orientation of acceptor transition of energy transfer efficiency and two chromophore's PLEs and emission spectrum, supplies the distance between acceptor that substantial connection is arranged.When FRET took place, intermolecular distance was less than 10nm.FRET fluorophor commonly used is to know to those skilled in the art, and it includes, but are not limited to, CFP-YFP; CFP-dsRED, BFP-GFP, GFP-dsRED, YFP-dsRED; Cy3-Cy5, Alexa488-Cy3, FITC-rhodamine (TRITC), YFP-TRITC; YFP-Cy3, Fluorescein-Tetramethylrhodamine, IAEDANS-Fluorescein, EDANS-Dabcyl; Fluorescein-QSY 7andQSY 9, Alexa Fluor 350-Alexa Fluor 488, Alexa Fluor 488-Alexa Fluor 546, Alexa Fluor488-Alexa Fluor 555; Alexa Fluor 488-Alexa Fluor 568, Alexa Fluor 546-Alexa Fluor568, Alexa Fluor 488-Alexa Fluor 594, Alexa Fluor 546-Alexa Fluor 594; Alexa Fluor555-Alexa Fluor 594, Alexa Fluor 488-Alexa Fluor 647, Alexa Fluor 546-Alexa Fluor647, Alexa Fluor 555-Alexa Fluor 647; Alexa Fluor 568-Alexa Fluor 647, Alexa Fluor594-Alexa Fluor 647, Alexa Fluor 350-QSY 35; Alexa Fluor 488-QSY 35, Alexa Fluor546-QSY 35, Alexa Fluor 350-dabcyl; Alexa Fluor 488-dabcyl, Alexa Fluor 546-dabcyl, Alexa Fluor 488-QSY 7and QSY 9; Alexa Fluor 546-QSY 7and QSY 9, Alexa Fluor555-QSY 7and QSY9, Alexa Fluor 568-QSY 7and QSY 9; Alexa Fluor 568-QSY 21, Alexa Fluor 594-QSY 21, Alexa Fluor 647-QSY 21 etc.Yet those skilled in the art can understand can also be with other FRET fluorophor to being used for method of the present invention.
Therefore, when utilizing the method detection of FRET, the probe molecule 404 that only need be fixed on the solid carrier connects upward fluorophor, and another kind of fluorophor on target nucleic acid 405 molecule markers of amplification.And these two kinds of fluorophors; When the PLE of the emission spectrum of wherein a kind of chromophore (donor) 401 and another kind of chromophore (acceptor) 407,409 has certain degree overlapping, optical excitation donor 402; And detection acceptor fluorescence 411; Have only the fluorescence of the nucleic acid molecule 406 in the hybridization just can be detected so, a large amount of in solution and be not excited 407 by the background fluorescence on the probe in the hybridization, or FRET owing to do not take place in the light that excites; The wavelength difference 403 of emission can be filtered and not be detected by filter.Like this, fluorescence signal intensity is equivalent to be attached to the amount of the target dna molecule of carrier surface.The concentration of target dna molecule is high more in the solution, and also many more with molecule on dna probe combines, the excited fluorescent signal is also strong more.
Fluorescent signal and intensity in above-mentioned two methods can detect with the instrument of routine, for example can adopt charge-coupled device (Charge-Coupled Device, CCD), PM (PMT), photodetector, perhaps test set such as light power meter.The preferred detection system of fluorescent mark is electric charge coupling appearance (CCD) photographic camera, and it can randomly be connected with multiplying arrangement (like microscope).
Usually, the above two kinds of methods of the present invention can be based on the optical table configuration of thinking roughly the same, and they comprise following configuration:
Excitation light source: comprise stable mercury lamp, xenon lamp, mercury xenon composite arc lamp source; To infrared band different wavelength of laser light source, comprise gas laser, semiconductor laser from ultraviolet.
Neutral density filter: excite light intensity in order to control.
Set of lenses: be used for integrating the band of light, the incident of control light.
The filter group: the fluorophor combining and configuring appropriate filter mirror group for selected comprises exciting emission, two look beam split etc.It can cut down the spectrum colour contamination, and raising will detect the signal/noise ratio of fluorescence.
Test set: it is characterized in that responding to a certain position light intensity, nucleus can be from highly sensitive PMT or cold CCD, and photodetector is perhaps selected in the light power meter.
In fact the present invention relates to a kind of real-time quantitative polymerase chain reaction technology; This technology adds fluorophor in the system of polymerase chain reaction; Utilize the accumulation of fluorescent signal; Monitor whole polymerase chain reaction process, the method for through the production standard curve amplified production of target nucleic acid molecules being carried out quantitative analysis at last in real time.The DNA copy number that in the process of polymerase chain reaction, produces is exponential manner to be increased, and along with the increase of reaction cycle number, final polymerase chain reaction no longer generates target nucleic acid amplified production (or be called touch plate) with exponential manner, thereby gets into plateau.In traditional P CR, gel electrophoresis commonly used separates and detects the final amplified production of polymerase chain reaction with fluorescent dye, with this end-point method quantitatively there is unreliable part in the polymerase chain reaction product.In the real-time quantitative polymerase chain reaction; Whole PCR reaction amplification procedure real-time monitoring and analysing amplified relevant fluorescent signal have continuously been carried out; Along with the carrying out in reaction times, the variation of the fluorescent signal that monitors can be depicted as a curve (Fig. 5).Early stage in the polymerase chain reaction; The level that produces fluorescent signal can not be distinguished with background significantly; The generation of fluorescent signal then gets into exponential phase, linear phase and final plateau; Certain that therefore can be in exponential phase in the polymerase chain reaction detects the amount of polymerase chain reaction product on a bit, and infers the content that template is initial thus.For the ease of the detection sample is compared; In the exponential phase of real-time quantitative polymerase chain reaction reaction; At first need set the thresholding of certain fluorescent signal, be considered to real signal if detect fluorescent signal above thresholding, it can be used for defining the thresholding cycle number (Ct) of sample.The implication of Ct value is: the cycle number that the fluorescent signal in each reaction tubes is experienced when reaching the thresholding of setting.Although when setting Ct value in the embodiment of the invention and beginning 3 times of signal value for amplified reaction, yet, should be appreciated that those skilled in the art can and confirm appropriate C t value according to the real needs selection.
The present invention's research shows that there is linear relationship in the logarithm of the Ct value of each template and the initial copy number of this template, and initial copy number is many more, and the Ct value is more little.Therefore, after the standard substance that utilize known initial copy number are made typical curve,, can calculate the initial copy number of this sample from typical curve as long as obtain the Ct value of unknown sample.
In the present invention; Amplification along with target dna molecule in the solution; The concentration of fluorescently-labeled target dna molecule is exponential manner to be increased, and with also increasing that the dna probe molecule that is fixed on carrier surface combines, the excited fluorescent signal also strengthens on the correspondent probe position.Therefore, can the real-time monitoring of multiple probe and analysing amplified relevant fluorescent signal have continuously been carried out in whole nucleic acid amplification reaction process.Can be depicted as many S type curves thereupon, draw the Ct value of a plurality of probe molecules, thereby judge and detect the kind and the copy number of target nucleic acid.This nucleic acid amplification reaction can comprise polymerase chain reaction (PCR); Inverse transcription polymerase chain reaction (RT-PCR); Random primer amplified reaction, or the relevant amplification technique in other polymerase chain reactions, and the reaction that includes these amplified reaction processes.It is characterized by the sex change and the annealed process that comprise nucleic acid, and can increase the reaction of specific polynucleotide chain.
For more detailed narration the present invention, specify as follows according to accompanying drawing.However, it should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Only if description is arranged in addition, the routine techniques that enforcement of the present invention will adopt those skilled in the art to know, these technology have complete description in following document: for example, Sambrook " molecular cloning experiment guide " the 2nd edition (1989); " dna clone " I and II volume (D.N.Glover edits 1985); " oligonucleotide is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames and S.J.Higgins edit .1984).Perhaps, can carry out according to the specification sheets that the reagent manufacturer is provided.
As embodiment 1, to being suitable for nucleic acid detection method of the present invention, be that example describes with parallel quantitative analysis to the multiple choices property splicing formal representation amount of decided gene (below be called target gene).
Eukaryotic genetic expression has the different connecting methods of a plurality of exons usually and gives expression to the form of multiple mRNA.In order to detect the multiple choices property splicing form expression amount separately of target gene; The present inventor designs the probe molecule that is directed to specific splicing form according to the selectivity splice sections of target gene, and various probe molecules are fixed on glass surface (Fig. 6 A) through chemical connection process.Certainly, they also can connect through chemical bond, physical adsorption, or mode such as polymkeric substance embedding is fixed on other high solid carriers of other transparent refractive index ratio reaction solns, like quartz, and polycarbonate, PS, Vilaterm, PMMA etc.
Simultaneously, can design the primer that can both increase that is directed to various splicing forms in the non-selective splice sections at the two ends of target gene selectivity splice sections.Through the step or the multiple splicing form of two one step RT-PCR method post transcription cloning target genes.
Be example specifically, design and synthesize following two primers with the expression amount that detects four kinds of selectivity connecting methods of Rab5A gene in the liver cell:
Cy3-5′-CATCAAGACATACAGTTTGGGTTAG-3′(SEQ?ID?NO：1)
Cy3-5′-CACAGTCCAGTGTGAGGGGAG-3′(SEQ?ID?NO：2)
Be directed to four kinds of selectivity connecting method Sp1, Sp2, Sp3, Sp4 design and synthesize following four specific specificity probes:
Rab5A-Sp1：5’-GTACCATTGGGGCTGCTTTTCTAA-3′-C
6-SH(SEQ?ID?NO：3)
Rab5A-Sp2：5’-CAACCTCTGCCTCATGGGTTCA-3′-C
6-SH(SEQ?ID?NO：4)
Rab5A-Sp3：5’-CATGAATTTCAAGAGAGTACCATTGGG-3′-C
6-SH(SEQ?IDNO：5)
Rab5A-Sp4：5’-CCTTTTCTTTCAGCTGCTTTTCTAACC-3′-C
6-SH(SEQ?ID?NO：6)
Then through said specific probe being fixed on glass surface (but concrete fixing means reference literature Analytical Biochemistry, 266:23-30, the method in 1999.).
For more detailed this invention of explanation, embodiment of the invention method and process are elaborated below in conjunction with Fig. 7 and Fig. 8.As shown in the figure, various nucleic probe 703,704,807 methods through the DNA array are fixed on K9 glass 709,809 surfaces.The glass of stationary probe and sample groove 701 driving fits form reaction cabin.From the tissue that will detect or cell behind the total RNA of separation and purification; Total RNA is mixed with the reaction solution 702 of post transcription cloning; Filled it up with the entire reaction cabin, reaction solution comprises the Tfl archaeal dna polymerase, the amplification buffer that the Tfl archaeal dna polymerase is corresponding; The amplimer (as above) 803 of Cy3 (801) mark, substrate dNTP.The entire reaction temperature receives the control of a heating and temperature control platform 705; Thereby carry out a plurality of cyclic amplifications; Comprise the process that (74 ℃) are extended in the segmental sex change repeatedly of target gene (94 ℃), annealing (55 ℃) and reaction, the target gene fragment 802 that amplification produces has been with Cy3 fluorophor 801 because of the primer of Cy3 mark.
In the annealing reaction process, the target gene fragment 802 of amplification and primer 803 bonded while also combine with the mutual pairing of fixed probe 807 through base.This binding signal is excited and is detected by the evanscent field 806 that total reflection produces.That is, after annealing process was carried out about one minute, Argon 706 was opened, and produced the laser of 514 nanometers.Laser is through a set of lenses 707, be integrated into wire after, through prism 708, advance in the slide 709, because input angle is greater than refraction critical angle (θ c=sin with the angle coupling of about 75 degree
-1(1.34/1.52)), laser produces total reflection repeatedly in slide, thereby produces evanscent field.
Some probe molecule is hybridization last 807 and 808 with it because corresponding splicing form is arranged target gene; Evanscent field excites the Cy3 fluorophor 805 on the target gene fragment in the hybridization; Make it produce the emission wavelength 804 about 552 nanometers; This emission wavelength is received by CCD (712) through behind the filter 711 of a centre wavelength 550 nanometers, can detect the locational fluorescence signal intensity of each probe points through CCD.The signal magnitude of each point differs on the array, corresponding to the concentration different (Fig. 9 A) of the various splicing forms of target gene.Along with a plurality of circulations of amplified reaction are carried out, the signal value of each point increases thereupon on the array, when certain any signal value reaches a thresholding, and when promptly amplified reaction begins 3 of signal value times, record cycle number Ct at this moment
i(i is the probe numbering) (Fig. 9 B).Ct according to each probe
iThe relative copy number of various selectivity splicing forms that can the quantitative objective gene.Because there is linear relationship in the logarithm of the Ct value of each template and the initial copy number of this template, and initial copy number is many more, the Ct value is more little, therefore, can extrapolate the initial relative copy number of target gene fragment (Fig. 9 C) according to the Ct value.
As embodiment 2,, be that example describes with quantitative analysis to several genes expression amount in the decided tissue to being suitable for nucleic acid detection method of the present invention.
From the tissue that will detect or cell, behind the separation and purification mRNA, be primer with oligonucleotide T7-Oligo (dT) 16, in ThermoScript II and respective reaction damping fluid, the mRNA reverse transcription becomes the cDNA of band T7 sequence.3 ' the end of the cDNA of reverse transcription is touched plate through the RNA ligase enzyme formation cDNA that links to each other with oligonucleotide T3 sequence.
Shown in figure 10, all etch the wide grating 908 of a treaty 0.5mm respectively, about 700 nanometers of grating cycle, the about 10-20 nanometer of the degree of depth at a thick sheet glass or the transparent plastic sheet 1010 surperficial two of about 1mm.At whole glass surface, comprise that the grating part all plates the tantalum pentoxide (Ta of the about 100-200 nanometer thickness of one deck again
2O
5) layer 1009, form the plane light wave conducting shell.The co-polymer that electrostatic interaction combines last layer poly (L-Methionin)-grafting-polyoxyethylene glycol (PLL-g-PEG) is passed through on surface at tantalum pentoxide, thereby its surface of activation (Fig. 6 B).
For more detailed this invention of explanation, embodiment of the invention method and process are described through Figure 10 and Fig. 8.As shown in the figure, (probe is the cDNA fragment, and length does not generally wait at 200-2000bp to design and synthesize corresponding probe fragment according to the various specific fragments that will detect the expression of gene sequence.Point out below the primer that with T7 and T3 primer, its sequence is respectively: T7,5 '-TAATACGACTCACTATAGGG-3 ' (SEQ ID NO:16); T3,5 '-ATTAACCCTCACTAAAG-3 ' (SEQ ID NO:17)).CDNA probe with the 3 ' terminal specific of mRNA is an example.With the method for cDNA probe 1003,1004,807, can be fixed on the tantalum pentoxide (Ta that is combined with the PLL-g-PEG co-polymer through the DNA array
2O
5) plane light wave conducting shell 1009,809 surfaces (Fig. 6 B), and in the middle of two stop positions.The sheet glass that is coated with tantalum pentoxide plane light wave conducting shell of stationary probe and sample groove 1001 driving fits form reaction cabin.The cDNA that connects is touched plate and polymerase chain reaction liquid 1002 is full of the entire reaction cabin, and reaction solution comprises the archaeal dna polymerase of polymerase chain reaction, like the Taq enzyme, and corresponding reaction buffer, the T7 of Cy5 (801) mark, T3 primer 803, substrate dNTP.The entire reaction temperature receives the control of a heating and temperature control platform 1005; Thereby carry out a plurality of cyclic amplifications; Comprise the process that (74 ℃) are extended in the segmental sex change repeatedly of expressing gene (94 ℃), annealing (55 ℃) and reaction, the expressing gene fragment 802 that amplification produces has been with Cy5 fluorophor 801 because of the primer of Cy5 mark.
In the annealing reaction process, the expressing gene fragment 802 of amplification and primer 803 bonded while also combine with the mutual pairing of fixed probe 807 through base.This binding signal is excited and is detected by the evanscent field 806 that planar optical waveguide produces.Promptly after reaction (74 ℃) process of extending was carried out about three minutes, helium-neon laser 906 was opened, and produces the laser of 633 nanometers.Laser is through a set of lenses 1007, be integrated into wire after, shine grating 1008 with about 15 degree angles, advance 1009 layers of tantalum pentoxides through coupling behind the grating, form slab guide, and the evanscent field that produces on the tantalum pentoxide surface.
Some cDNA probe molecule hybridizes last 807 with it because target gene has than high expression level; Evanscent field excites the Cy5 fluorophor 805 on the target gene fragment in the hybridization; Make it produce the emission wavelength 804 of about 665 nanometers; This emission wavelength is received by CCD (912), thereby can detect the locational fluorescence signal intensity of each probe points through behind the filter 1011 of one 670 nanometers.The signal magnitude of each point differs on the array, different corresponding to the expression amount of expressing gene.Along with a plurality of circulations of amplified reaction are carried out, the signal value of each point increases thereupon on the array, when certain any signal value reaches a thresholding, and when promptly amplified reaction begins 2 of signal value times, record cycle number Ct at this moment
i(i is the probe numbering).Ct according to each probe
iAnd quantitative expression expression of gene amount.
As embodiment 3,, be that example describes to identify bacterial species and quantitative analysis to being suitable for nucleic acid detection method of the present invention.
Nucleic acid sequence analysis is the domestic method to Bacteria Identification.Usually, through analyzing the relatively conservative gene order of bacterium, like 16S-rRNA, 23S-rRNA, GyrB, genes such as rpoD, and detect the genus kind of identifying bacterium.To analyze the 16S-rRNA gene order is example.Section at two evolution conservatives of 16S-rRNA gene designs suitable primer, and it can be increased to the bacterium of broad-spectrum.Variable section is designed the specific probe of wanting bacterial detection to certain in the centre of two conservative sections again.
In this example, design and synthesize the universal primer sequence of following bacterial 16 S-rRNA:
5 '-rhodamine is red-X-GAAGAGTTTGATCMTGGCTC-3 ' (M=A+C) (SEQ ID NO:7)
5 '-rhodamine is red-X-ACTGCTGCCTCCCGTAGGAG-3 ' (SEQ ID NO:8)
And to the synthetic narrow spectrum dna probe of following seven kinds of pathogenic agent bacteriums:
Streptococcus aureus (Staphylococcus aureus):
SH-C
6-5’-CGGACGAGAAGCTTGCTTCTCTGATGTTAGCG-3′-FITC(SEQ?IDNO：9)
Monocyte hyperplasia Li Site bacterium (Listeria monocytogenes):
SH-C
6-5’-AACGGAGGAAGAGCTTGCTCTTCCAAAGTTAGTGG-3′-FITC(SEQID?NO：10)
Intestinal bacteria (Escherichia.Coli):
SH-C
6-5’-CAGGAAGCAGCTTGCTGCTTTGCTGACG-3′-FITC(SEQ?ID?NO：11)
Streptococcus pneumoniae (Streptococcus pneumoniae):
SH-C
6-5’-AGAACGCTGAAGGAGGAGCTTGCTTCTCTGGAT-3′-FITC(SEQ?IDNO：12)
Neisseria meningitidis (Neisseria meningitidis):
SH-C
6-5’-GCAGCACAGAGAAGCTTGCTTCTCGGGTG-3′-FITC(SEQ?ID?NO：13)
Faecium (Enterococcus faecium):
SH-C
6-5’-CTTTTTCCACCGGAGCTTGCTCCACCGGAAA-3′-FITC(SEQ?ID?NO：14)
Pseudomonas aeruginosa (Pseudomonas aeruginosa):
SH-C
6-5’-GAAGGGAGCTTGCTCCTGGATTCAGCGG-3′-FITC(SEQ?ID?NO：15)
For more detailed this invention of explanation, embodiment of the invention method and process are described through Figure 11 and Figure 12.Like figure, with above-mentioned various probe 1101,1102,1204 usefulness FITC fluorophors 1203; After on 1206 marks, connect () through chemistry and be fixed on glass carrier 1103,1207 surfaces of embodiment 1; Carrier can be a metal, and pottery is on any solid material such as polymer materials.The glass of stationary probe and sample groove 1105 driving fits form reaction cabin.Get testing sample, carry out the separation and purification of genomic dna.The genomic dna of separator well and polymerase chain reaction liquid 1104 are added and is full of the entire reaction cabin; Reaction solution comprises the Taq archaeal dna polymerase of polymerase chain reaction; And corresponding reaction buffer, rhodamine is red-the above primer 1209 of X fluorophor 1211 marks, and the dNTP substrate.The entire reaction temperature receives the control of a heating and temperature control platform 1111; Thereby carry out a plurality of cyclic amplifications; Comprise the segmental sex change repeatedly of target gene (94 ℃); The process that (74 ℃) are extended in annealing (60 ℃) and reaction, the target gene fragment 1210 that amplification produces because rhodamine red-primer of X mark and be with rhodamine red-X fluorophor 1211.
In annealing process, target gene fragment 1205 combines through the mutual pairing between the base with fixed probe 1204.After annealing process was carried out about one minute, Argon 1108 was opened, and produced the laser of 488 nanometers.Laser is radiated at solid carrier surface through a set of lenses 1107 after the focusing, simultaneously, and laser scanning whole probe fixed zone.The dna molecular 1205 of the bacterium that contains in the sample by rhodamine on the amplification label red-the X fluorophor after, combine with corresponding probe molecule 1204.The laser 1201 of 488 nanometers excites the FITC group 1203 on the probe molecule and produces the emission wavelength of 518 nanometers; It by the rhodamine on the DNA amplification in the combination red-X group 1202 absorbs, rhodamine is red-the X group produces the emission wavelength 1208 of 590 nanometers and take on a red color.If the bacterium that in sample, does not have, have in its corresponding probe molecule 1206 debonds rhodamine red-molecule of X group, after being excited, produce the blue light 1215 of 518 nanometers.In the solution in the hybridization contain rhodamine red-primer 1209 of X group 1211 and be not excited by the dna molecular of amplification 1210.CCD (1110; 1214) through a centre wavelength be the filter 1109 of 600 nanometers; Be detected as phase after 1213; Because the ruddiness 1208 of 590 nanometers sent of probe molecule 1204 in the hybridization can see through the filter 1213 of 600 nanometers, and the blue light 1215 of 518 nanometers of not sent by the probe molecule 1206 in the hybridization, the laser 1212 of 488 nanometers that are scattered all can not see through.Therefore, the locational strength of signal of each probe points of CCD imaging is the amount of the DNA in the hybridization, its amount corresponding to the corresponding bacteria DNA cloning (Figure 13 A).
Along with a plurality of circulations of amplified reaction are carried out, the signal value of each point increases thereupon on the array, when certain any signal value reaches a thresholding, and when promptly amplified reaction begins 3 of signal value times, record cycle number Ct at this moment
i(i is the probe numbering) (Figure 13 B).Ct according to each probe
iAnd the relative copy number of quantitative all types of target bacterial detection gene.Because there is linear relationship in the logarithm of the Ct value of every kind of template and the initial copy number of this template, and initial copy number is many more, the Ct value is more little, therefore, can extrapolate the initial relative copy number of target gene fragment (Figure 13 C) according to the Ct value.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, accompanying claims has covered all these changes within the scope of the present invention.It is for referencial use that this paper is all included in all publications, patent and the patented claim that this paper quotes in.
Sequence table
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Claims (10)
1. the existence of multiple nucleic acid molecule or the method for amount in the replicate(determination) sample, this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided, the stationkeeping that said carrier surface is different multiple nucleic acid probe molecules, said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased, when amplified reaction, with fluorophor mark amplified production;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) produce evanscent field at solid phase carrier and liquid surface place with excitation light source, make with the said amplified production of said probe molecule bonded on the fluorophor generation fluorescent signal that is excited;
G) detect the position and the intensity of said fluorescent signal, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
2. the method for claim 1 is characterized in that, said solid phase carrier is selected from glass, nylon membrane, quartz, pottery, metal, plastics or organic polymer.
3. the method for claim 1 is characterized in that, is coated with the metallic membrane less than 1000 nanometer thickness on the said solid phase carrier, and perhaps diameter is less than the metallic particles of 1000 nanometers, and wherein said metal is selected from gold and silver, copper.
4. the method for claim 1 is characterized in that, said evanscent field is the total reflection through the light that in said solid phase carrier, takes place, and perhaps produces through slab guide.
5. method as claimed in claim 4 is characterized in that, said slab guide is the metal oxide film on said solid phase carrier, and said MOX is selected from tantalum pentoxide, titanium oxide, zinc oxide, zirconium white, niobium oxides or hafnia.
6. the method for claim 1 is characterized in that, in said step d), has the deoxynucleoside triphosphate of fluorophor mark, the primer that the fluorophor mark is arranged or its combination to make amplified production by the fluorophor mark through use.
7. the method for claim 1 is characterized in that, said nucleic acid probe molecules is fixed on carrier surface through chemical bond connection, physical adsorption, polymkeric substance embedding, etching or the sticking mode that joins.
8. the method for claim 1 is characterized in that, said nucleic acid amplification reaction comprises nucleic acid denaturation, annealing and increases the step of specific polynucleotide chain.
9. the existence of multiple nucleic acid molecule or the method for amount in the replicate(determination) sample, this method may further comprise the steps:
A) sample is provided, comprises nucleic acid molecule to be detected in the said sample;
B) solid phase carrier is provided; The stationkeeping that said carrier surface is different multiple nucleic acid probe molecules; Said nucleic acid probe molecules has specificity to the nucleic acid molecule to be detected that possibly exist in the said sample, is connected with first fluorophor on the said nucleic acid probe molecules;
C) said nucleic acid probe molecules is contacted with said sample liquid;
D) nucleic acid molecule in the said sample is increased; When amplified reaction; With the second fluorophor mark amplified production; The PLE of the emission spectrum of wherein said first fluorophor and said second fluorophor is overlapping, or the PLE of the emission spectrum of said second fluorophor and said first fluorophor is overlapping;
E) said amplified production and said nucleic acid probe molecules are mutually combined;
F) with one in said first fluorophor of optical excitation and said second fluorophor;
G) detect another signal emitted fluorescence position and intensity in said first fluorophor and said second fluorophor, thereby confirm the existence or the amount of multiple nucleic acid molecule in the sample.
10. method as claimed in claim 9 is characterized in that, said first fluorophor and second fluorophor be when the 1-10 nanometer, and fluorophor can absorb the radiative energy of another fluorophor and be excited.
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| 刘力等.倏逝场与穿透光线.《湖南师范大学学报（自然科学版）》.1984,(第4期),第17-24页. * |
| 毕玉晶等.光纤倏逝波生物传感器及其应用.《生物技术通讯》.2004,第15卷(第6期),第652-655页. * |
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