CN107016258A - One kind is based on recombinase-mediated isothermal nucleic acid amplification（RAA）The method that method carries out fluorescent quantitation calculating - Google Patents
One kind is based on recombinase-mediated isothermal nucleic acid amplification（RAA）The method that method carries out fluorescent quantitation calculating Download PDFInfo
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Abstract
The invention discloses a kind of method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method, it is to be directed to RAA nucleic acid amplification technologies features, establish mathematical model and computational methods, quantitative detection amplification starting template amount can accurately be carried out, preferably application and popularization RAA nucleic acid amplification technologies, make the detection and scientific research in the fields such as the faster more accurate wider array of agricultural for being applied to modernization of RAA technologies, medical science, food security, inspection and quarantine, disease control preventing and treating, environmental microorganism identification.
Description
Technical field
The invention belongs to molecular Biological Detection field, and in particular to the method that one kind carries out fluorescent quantitation calculating based on recombinase-mediated isothermal nucleic acid amplification (RAA) method, and can act the amount of making to nucleic acid amplification with this method carries out accurate quantitative analysis.
Background technology
In biological study and molecule diagnosis, DNA cloning is a kind of basic and necessary technological means, and most common of which is to utilize PCR (English full name：Polymerase Chain Reaction, abbreviation PCR) carry out nucleic acid in vitro amplification, the technology is invented in nineteen eighty-three by the Dr.Mullis in American scientist PE (Perkin Elmer Perkins-Ai Ermo) heredity portion of company, mountain is upper across significance of times in theoretical and application in round pcr, therefore Mullis obtains the chemical Nobel Prize in 1993, has been widely used at present in fields such as the agriculturals and medical science and food industry of modernization.Pass through this technology, it is possible to achieve the efficient amplification of trace dna, minimal amount of specific nucleic acid squences molecule is expanded to the level that can be detected to instrument.
Round pcr three fundamental reaction steps of nucleic acid amplification point are denaturation, annealing (renaturation) respectively and are extended；Specially：
1. template DNA is denatured：Template DNA was heated to after 90-95 DEG C of certain time, made template DNA double-strand or expand the double-stranded DNA to be formed through PCR to untwist, and made single-stranded, so that it is combined with primer, was that lower whorl reaction is prepared；
2. the annealing (renaturation) of template DNA and primer：Template DNA is heated be denatured into it is single-stranded after, temperature is down to 55-60 DEG C, and the primer complementary series pairing single-stranded with template DNA is combined；
3. the extension of primer：DNA profiling-primer conjugate is in the presence of archaeal dna polymerase, in 70-75 DEG C, using dNTP as reaction raw materials, and target sequence is template, by base pairing and semi-conservative replication principle, synthesizes a new semi-conservative replication chain complementary with template DNA chain；
Complete three above process and be referred to as an amplification cycles, often complete a circulation, the nucleic acid quantity that can be doubled in theory, can be by micro nucleic acid amplification to the level that can be detected with instrument by constantly repeating the circulation of the above.In general, often completing a circulation needs 2-4 minutes, millions of times of target gene amplification amplification could be reached the level that can be detected within 2-3 hours.
PCR can carry out exponential amplification to specific nucleotide segment, no matter qualitative or quantitative analysis, analysis is all PCR end-products.But in many cases, people it is interested be without PCR signals amplify before starting template amount.For example wonder the expression quantity of the copy number or a certain specific gene of a certain animals and plants transgenosis in particular organization, fluorescent quantitative PCR technique arises at the historic moment under this demand, and fluorescent quantitation application field is also more extensive.
Quantitative fluorescent PCR (realtime fluorescence quantitative PCR, RTFQ PCR) is a kind of quantitative determination technology released by Applied Biosystems companies of the U.S. in 1996.
The principle of quantitative fluorescent PCR adds a specific fluorescence probe while being and pair of primers is added when PCR is expanded, the probe is an oligonucleotides, two ends one reporter fluorescence group of mark and a quenching fluorescence group respectively.When probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions；Just when starting, probe is combined on any one of DNA is single-stranded：When PCR is expanded, hold 5 prime excision enzyme activity that probe digestion is degraded in 5 ' ends -3 ' of Taq enzyme, separate reporter fluorescence group and quenching fluorescence group, so as to which fluorescence monitoring system can receive fluorescence signal, often expand a DNA, just there is a fluorescence molecule to be formed, realize accumulation and the PCR primer formation Complete Synchronization of fluorescence signal.
Principle quantitative fluorescent PCR based on more than is to set up a kind of mathematical modeling and a kind of computational methods to realize that the starting template amount in amplification process is calculated.
In fluorescence surely most PCR, within the exponential amplification phase, ideally, the fluorescence volume after n-th of circulation is only related to starting template amount, period and amplification efficiency.Relation between them can be expressed as：
Rn=R0×(1+Eo)n, wherein RnFor the fluorescence volume after n-th of circulation, R0For starting template amount, E0For amplification efficiency, n is period., can be by amplification efficiency E for specific amplification0Regard constant as.
Quantitative fluorescent PCR is exactly to be detected in real time by the progress to each circulation products fluorescence signal in pcr amplification reaction, often by a circulation, collect a fluorescence intensity signals, it can thus accomplish by monitoring fluorescence intensity change to realize the change of monitoring product amount, it is possible to obtain a fluorescent amplification curve figure.
In general, PCR fluorescent amplification curves are segmented into three phases：Fluorescence background stage, fluorescence signal exponential amplification stage and plateau.In the fluorescence background stage, the fluorescence signal of amplification is covered by fluorescence background, it is impossible to judge the change of product amount.And in plateau, oneself increase no longer exponentially of amplified production.There is no linear relationship between PCR end-product amount and starting template amount, so starting DNA copy number can not be calculated according to final PCR primer amount.Only in the fluorescence signal exponential amplification stage, there is linear relationship between the logarithm value and starting template amount of PCR primer amount, can select to carry out quantitative analysis in this stage.For convenience that is quantitative and comparing, two very important concepts are introduced in Real-Time Fluorescent Quantitative PCR Technique：Fluorescence threshold and CrValue.Fluorescence threshold is a value being manually set in fluorescent amplification curve, and it may be set on the meaning of fluorescence signal exponential amplification stage thousand position, but 10 times of the general standard deviation for by the default setting of fluorescence thresholding being the 3-15 fluorescence signal circulated.Fluorescence signal in each reaction tube reaches the period undergone during the thresholding of setting and is referred to as CTValue.
CTValue and the relation research of starting template shows, the C of each templateTThere is linear relationship in value and the logarithm of the starting copy number of the template, starting copy number is more, CTValue is smaller.Standard curve can be made using the standard items of known starting copy number, wherein abscissa represents the logarithm of starting copy number, and ordinate represents CTValue.Therefore, as long as obtaining the C of unknown sampleTValue, you can the starting copy number of the sample is calculated from standard curve.This principle based on more than, can calculate the initial copy number of testing sample template.
But, PCR process is to be iteratively repeated progress by denaturation-fundamental reaction step of annealing-extension three, and because PCR needs to be continually changing temperature to realize the amplification of nucleic acid, from being born, the limitation for relying on superior instrument and equipment can not be broken away from all the time；Moreover, PCR is in operation, it is necessary to which professional is operated, particularly analysis of the quantitative fluorescent PCR to follow-up data is also required to certain theory and technical merit, higher to personnel requirement；In addition, the general completion within a few hours of the time of PCR amplifications, is not also adapted to need the occasion of quick detection, in consideration of it, the nucleic acid amplification technologies of more convenient and quicker arise at the historic moment.
Recently for over ten years, DNA cloning technology in simulation microbial body is quietly risen, nucleic acid in vitro amplification is set to become simpler and conveniently, with fastest developing speed is isothermal amplification, and such as TMA technologies (nucleic acid amplification technologies of transcriptive intermediate), SDA technologies (strand displacement nucleic acid amplification technologies), LAMP (ring mediation nucleic acid amplification technologies), HDA (unwindase dependence isothermal amplification), RAA (recombinase-mediated isothermal amplification) can carry out nucleic acid amplification under isothermal conditions.
The present invention be directed to recombinase-mediated isothermal amplification (Recombinase Aided Amplification, abbreviation RAA) and the method calculated for the technology fluorescent quantitation of invention, it can be realized with this method and accurate quantitative analysis is carried out to nucleic acid amplification starting template amount.
Accomplish quantitative there is presently no a kind of isothermal amplification, this method has been invented in order to preferably apply and promote RAA nucleic acid amplification technologies, the faster more accurate wider array of agricultural for being applied to modernization of RAA technologies, medical science, food security, rifle is tested the fields such as quarantine, disease control preventing and treating.
RAA nucleic acid amplification technologies are that one kind utilizes RecQ albumen, UvsY albumen and UvsX albumen under isothermy (25-45 DEG C) (optimal 37 DEG C of temperature of amplification), template DNA double-strand is set to untwist, stablize single-stranded and make that chain replacement occurs between primer and template, simultaneously, the nucleic acid under the catalysis of archaeal dna polymerase, and under conditions of single strand binding protein SSB and condensing agent polyethylene glycol are present, constantly repeat this process and finally realize the efficient amplification of nucleic acid, the gene magnification that can just be achieved the goal within 5-30 minutes amplifies millions of times, reach the level that can be detected.
RAA nucleic acid amplification technologies can realize that under lower isothermy (25-45 DEG C) faster nucleic acid amplification, process is easier, it is possible to achieve live nucleic acid amplification detection, therefore have a wide range of applications field.
The present invention is directed to RAA technical characterstics, has invented mathematical modeling and computational methods, it is possible to achieve monitoring nucleic acid amplification situation and quantitatively detection amplification starting template amount in real time.
The content of the invention
It is an object of the invention to provide a kind of method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method, accurate quantitative analysis can be carried out to nucleic acid amplification starting template amount, can realize detection, the scientific research in the fields such as the faster more accurate wider agricultural for being more easily applied to modernization, medical science, food security, inspection and quarantine, disease control preventing and treating with this method.
RAA nucleic acid amplification technologies are that one kind utilizes RecQ albumen, UvsY albumen and UvsX albumen under isothermy (25-45 DEG C) (optimum temperature expands 37 DEG C of temperature), template DNA double-strand is set to untwist, stablize single-stranded and make that chain replacement occurs between primer and template, simultaneously, the nucleic acid under the catalysis of archaeal dna polymerase, and under conditions of single strand binding protein SSB and condensing agent polyethylene glycol are present, constantly repeat this process and finally realize the efficient amplification of nucleic acid, amplification procedure is related to proliferation time, this is and the significantly different part of round pcr (PCR amplifications are related with period).The gene magnification that RAA expands within 5-30 minutes with regard to that can reach day amplifies millions of times, reaches the level that can be detected with instrument.
The present invention is based on RAA nucleic acid amplification technologies features, in order to be able to carry out fluorescent quantitation amplification, and the specificity fluorescent probe for specific primer is added in RAA nucleic acid amplification reaction units, and increases an exonuclease, forms RAA amplified fluorescence reaction systems.
The fluorescence probe of addition is an oligonucleotides, two ends one reporter fluorescence group of mark and a quenching fluorescence group respectively, when probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions, when starting amplification, template DNA double-strand is untwisted in the presence of unwindase, probe combine DNA thousand anticipate one it is single-stranded on, exonuclease nonrecognition；After probe and target dna are combined, exonuclease, which recognizes and will release 3 ' end resistances, to break, separate reporter fluorescence group and quenching fluorescence group, so as to which fluorescence monitoring system can receive fluorescence signal, a DNA is often expanded, just has a fluorescence molecule to be formed, realizes fluorescence volume and RAA amplified production amount Complete Synchronizations, one-to-one relationship is formed, amplified production amount can just be extrapolated by measuring fluorescence volume.The method is defined as by fluorescent quantitation RAA based on this principle present invention.
Shown according to RAA amplification principle and many experiments, it is exponential amplification to determine RAA amplifications.
In fluorescent quantitation RAA, many experiments show, ideally, within the exponential amplification phase, and fluorescence volume is only related to starting template amount, amplification efficiency and proliferation time, and the relation between them can be expressed as：
Fn=F0×(1+E0)a, wherein FnFor the fluorescence volume after the 1st minute, F0For starting template amount, E0For amplification efficiency, a is the function with time correlation, a=(t × 60) ÷ b can be expressed as, b refers to that RAA amplified production amounts double the required time, and fluorescence volume and RAA amplified production amount Complete Synchronizations, forms one-to-one relationship, correspond to fluorescence volume and double the required time, for specific amplification, b is constant, amplification efficiency E0Also it is constant.
Fluorescent quantitation RAA using can detect the instrument of fluorescence by fluorescence volume determine come, fluorescence volume is a process constantly accumulated, it is also simultaneously one-to-one with RAA amplified productions, by the real-time detection to the product fluorescence signal of dust in RAA amplified reactions, the change of product amount is monitored by fluorescence intensity change；Intervals are set to collect a fluorescence intensity signals (typically selecting 5-30 seconds), the fluorescence intensity signals that each time interval is measured connect into curve, obtain fluorescent amplification curve figure；Abscissa represents that ordinate is represented with fluorescent value with the time.
In general, RAA fluorescent amplification curves are segmented into two stages：Fluorescence signal initial period, fluorescence signal exponential amplification stage.
In fluorescence signal initial period, the fluorescence signal of amplification is covered by fluorescence background, it is impossible to judge the change of product amount：Amplified production also no longer has exponent relation, and does not have linear relationship between RAA end-product amount and starting template amount, so this stage can not calculate starting DNA copy number.
Only in the fluorescence signal exponential amplification stage, there is exponential function relation between RAA product amounts and starting template amount, selection carries out quantitative analysis in this stage.
For quantitative convenience, the present invention introduces three basic concepts in real time fluorescent quantitative RAR technologies：Baseline value, fluorescence threshold and T values.
Baseline value refers to the fluorescence background stage in fluorescent amplification curve, has not been entered into the weighted average of exponential amplification phase fluorescence volume；
Fluorescence threshold refers to that fluorescent amplification curve enters the catastrophe point fluorescent value in exponential amplification stage, and the line by fluorescence threshold parallel to X-axis is defined as threshold line；In theory, fluorescence threshold is can be set taking human as any, and condition is within the fluorescence index amplification phase.
For convenience of unified, the specific adopted fluorescence threshold of the present invention is for N times of all fluorescent value standard variances within 0-4 minutes as fluorescence threshold, and N takes the integer more than or equal to 3.
T values refer to that the fluorescence signal of amplified production reaches the time required during the threshold value of setting.
Introduce after T value concepts, exponential function originally can be expressed as FT=F0×(1+Eo)T
It can be released by above formula：logFT=log [F0×(1+E0)T]
logF0=logFT-Tlog(1+E0) set up.
T values and the relation research of starting template show that the T values of each template and the logarithm of the starting copy number of place near the steps template have linear relationship, and starting copy number is more, and T values are smaller.For specific any fluoroscopic examination, the fluorescence volume lower limit that can be measured is stable, therefore can be by logFTRegard constant as, also can be by FTReplaced with threshold value.
For computational methods, the amplification curve of known starting copy number standard items can be made by the way that the instrument of fluorescence volume can be measured for specific target DNA, fluorescence threshold is set as stated above, the parallel lines that mistake threshold value makees X-axis obtain threshold line；And threshold line there must be joining with amplification curve, the vertical line that intersection point makees X-axis is crossed, corresponding T values are obtained.
Different concentration known gradient standard samples are set, graded series amplification curve is made by the method for the above, unified fluorescence threshold is set, threshold line is made and obtains a series of T values with the time corresponding to the joining of various concentrations gradient standard sample amplification curve
Proliferation time is represented with abscissa, ordinate represents the logarithm of starting copy number, marked in coordinate diagram using different T values as abscissa, using the logarithm of corresponding known standard items starting copy number as the point of ordinate, these point fitting fluorescent quantitation standard curves are crossed, and obtain according to the standard curve of fitting the linear function of the standard curve.
It is all constant that the linear function, which can be expressed as Y=-a*T+b wherein a, b,.
Therefore, for the sample of specific unknown starting template amount, as long as obtaining the T values of the sample of unknown starting template amount, you can calculate the starting copy number of the sample from standard curve；Method was that abscissa T value points make the vertical line of X-axis and hand over standard curve in point A, crosses A points and makees the parallel lines of X-axis and hand over ordinate in B points, can read ordinate data Y, data Y is the logarithm of starting copy number, so the starting copy number of the sample is represented by 10Y。
T values can also be substituted into the linear function Y=-a*T+b of standard curve, obtain Y value, Y value is the logarithm of starting copy number, and starting copy number is represented by 10Y；
T values can be tried to achieve by the following method：The sample amplification curve of unknown starting template amount is obtained by fluorescence detection equipment, identical threshold value when fluorescence threshold is arranged to making standard curve, cross threshold value and make the parallel lines of X-axis and the sample amplification curve intersection of unknown starting template amount, cross the vertical line that intersection point makees X-axis, T values can be obtained.
This principle based on more than, can accurately calculate the initial copy number of testing sample template.
Method provided by the present invention, can apply in RAA instrument devices, and realizing that mutation that RAA nucleic acid amplifications are analyzed from qualitative to quantitative is played well-behaved will act on.
Accurate quantification can be easily realized on the basis of the present invention, be conducive to RAA technologies widely to apply, can Ying Chuan in the field such as agricultural, medical diagnosis, food security, inspection and quarantine, disease control preventing and treating and Site Detection of nucleic acid molecules laboratory research, modernization.Really realize that quick, accurate, real-time, quantitative nucleic acid amplification rifle is surveyed.
Brief description of the drawings
The yellow fever virus plasmid DNA standard amplification curves of different copy numbers known to Fig. 1
Fig. 2 yellow fever virus plasmid DNA standard curves
The unknown copy number yellow fever virus RNA amplification curves of Fig. 3
Embodiment
The present invention is described in further detail with embodiment below, but present disclosure is not limited thereto.
Embodiment one：Further the present invention is illustrated by taking yellow fever virus unknown concentration RNA as an example.
The present embodiment is carried out according to the following steps：
1st, it is unknown to copy several sample sources and yellow fever virus RNA extractions
The Yellow Fever Vaccine,Live (Beijing Tiantan Bio-pharmaceuticals) that virus-like strain is provided by Zhejiang International Travel Healthcare Center, RNA is extracted and is used into hot extracts kit AM1836 (ambion, life technologies), extraction equipment is KINGFISHER Full automatic instrument for extracting nucleic acid (Thermo), and carrying out nucleic acid extraction by description above and operating method obtains yellow fever virus RNA.
2nd, the yellow hot fluorescence detection reagent kit sources of RAA
The yellow hot fluorescence detection reagent kits of RAA are provided by the strange day gene Chen Wu Science and Technology Ltd.s in Jiangsu, and the specification that operating process is strictly provided by the said firm is carried out, and the RAA amplification reaction systems in reagent are freeze-dried powder state.
3rd, fluorescence detection equipment
Detect that the detecting instrument of fluorescence signal is provided by Jiangsu Qi Tian genes bio tech ltd；
INSTRUMENT MODEL is RAA-F6100
4th, experimentation
Make standard curve：The yellow fever virus RNA of concentration known is diluted by different concentration, the standard sample of various concentrations gradient, respectively 10 is made in the yellow fever virus plasmid of the concentration known provided with Jiangsu Qi Tian genes bio tech ltd7、106、105、104、103、102Copy.
Receive RAA amplification reaction system reaction tubes in 7 hot fluorescence detection reagent kits of Huang, it is separately added into final concentration of 6% (w/v's), divide as reaction buffer to freeze in the polyethylene glycol that amount is 35000 and dissolved again as 49 μ l in system, the μ L of yellow fever virus RNA standard samples 1 and reverse transcriptase of the various concentrations gradient prepared are separately added into again, it is well mixed, in brief centrifugation, the instrument RAA-F6100 for being put into detectable FAM fluorescence, reacted 20 minutes under the conditions of 39 DEG C.(note：To ensure to test accuracy, the system for being not added with template is set as negative control).
This example took a fluorescence signal by 20 seconds, obtained the yellow fever virus RNA amplification curve of various concentrations gradient, as shown in Figure 1.
The present embodiment is in order to better illustrate problem, fluorescence threshold is taken as into 500, and (fluorescence threshold can be taking human as any setting, condition is within the fluorescence index amplification phase, industry can uniformly be set to N times of all fluorescent value standard variances within 0-4 minutes, N takes the integer more than or equal to 3), cross the parallel lines and 10 that threshold value makees X-axis7、106、105、104、103、102Copy amplification curve to intersect, cross the vertical line that intersection point makees X-axis, obtain T values, respectively T1、T2、T3、T4、T5、T6, obtain using the time as abscissa is made after T values, corresponding concentration known logarithm is ordinate, makees standard curve, (T is marked in coordinate diagram1、7)、(T2、6)、(T3、5)、(T4、4)、(T5、3)、(T6, 2) six points, cross these point fitting fluorescent quantitation standard curves, and obtain according to the standard curve of fitting the linear function of the standard curve.Standard curve is as shown in Figure 2.
Obtaining standard curve function is：Y=-0.5786*T+10.41
It is another to take RAA amplification reaction system reaction tubes in a yellow hot fluorescence detection reagent kit, add final concentration of 6% (w/v's), molecular weight dissolves lyophilized system as 49 μ l as reaction buffer for 35000 polyethylene glycol again, add the unknown μ L of copy number yellow fever virus RNA standard samples 1 prepared and reverse transcriptase, it is well mixed, brief centrifugation, be put into can Eurya plant survey FAM fluorescence instrument carry in RAA-F6100, under the conditions of 39 DEG C react 20 minutes.(note：To ensure to test accuracy, the system for being not added with template is set as negative control).
Take within 20 seconds a fluorescence signal, unknown copy number yellow fever virus RNA amplification curve is obtained, fluorescence threshold is equally taken as 500, the parallel lines that mistake thresholds make X-axis obtain threshold line, threshold line intersects at A points with unknown copy amplification curve, crosses intersection point A and makees the vertical line friendship X-axis of X-axis in TnPut and obtain T values, T values are 8, as shown in Figure 3.
It is by the T values obtained substitution calibration curve equation：Y=-0.5786*8+10.41
It is 5.7812 to obtain Y value, and it is 10 to obtain starting copy number57812
Starting copy number can also be obtained from standard curve, cross in fig. 2 T values for 8 point make X-axis vertical line hand over amplification line andThe parallel lines that point A, excessively A points make X-axis hand over ordinate in B points, and it is about 5.8 to read data, it can be deduced that starting copy number 1058。
It the foregoing is only the present invention and implement example, be not intended to limit the invention, within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (8)
1. the method that one kind carries out fluorescent quantitation calculating based on recombinase-mediated isothermal nucleic acid amplification (RAA) method, it is characterised in that：Based on RAA nucleic acid amplification technologies features, accurate quantitative analysis is carried out to nucleic acid amplification starting template amount with the method for fluorescent quantitation, often one DNA of amplification is realized in RAA courses of reaction, just there is a fluorescence molecule to be formed, realize fluorescence volume and RAA amplified production amount Complete Synchronizations, form one-to-one relationship, amplified production amount can just be extrapolated by measuring fluorescence volume, the method is defined as fluorescent quantitation RAA by the present invention, and determine that RAA amplifications are exponential amplification, within the exponential amplification phase, fluorescence volume and starting template amount, amplification efficiency is related to proliferation time, relation between them is expressed as：
Fn=F0×(1+E0)a, wherein FnFor the fluorescence volume after t minutes, F0For starting template amount, E0For amplification efficiency, a is the function with time correlation, is expressed as a=(t × 60) ÷ b, and b refers to that RAA amplified production amounts double the required time, and for specific amplification, b is constant, amplification efficiency E0Also it is constant.
2. the method according to claim 1 that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method, it is characterised in that：Fluorescent quantitation RAA can using detect the instrument of fluorescence by fluorescence volume determine come, fluorescence volume is a process constantly accumulated, it is also simultaneously one-to-one with RAA amplified productions, by the real-time detection of the product fluorescence signal to being produced in RAA amplified reactions, the change of product amount is monitored by fluorescence intensity change；Intervals are set to collect a fluorescence intensity signals (typically selecting 5-30 seconds), the fluorescence intensity signals that each time interval is measured connect into curve, so as to obtain a fluorescent amplification curve figure；Abscissa represents that ordinate is represented with fluorescent value with the time.
3. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2, it is characterised in that：RAA fluorescent amplification curves are divided into two stages：In the fluorescence background stage, in the fluorescence signal exponential amplification stage, in fluorescence signal initial period, the fluorescence signal of amplification is covered by fluorescence background, it is impossible to judge the change of product amount；Amplified production does not also have exponent relation, there is no linear relationship between RAA end-product amount and starting template amount, only in the fluorescence signal exponential amplification stage, there is exponential function relation between RAA product amounts and starting template amount, selection carries out quantitative analysis in this stage；
The method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2, it is characterised in that：For quantitative convenience, three concepts are defined：Baseline value, fluorescence threshold and T values；
Baseline value refers to the fluorescence background stage in fluorescent amplification curve, has not been entered into the weighted average of exponential amplification phase fluorescence volume；
Fluorescence threshold refers to that fluorescent amplification curve enters the catastrophe point fluorescent value in exponential amplification stage, and the line by fluorescence threshold parallel to X-axis is defined as threshold line；In theory, fluorescence threshold can be taking human as any setting, and condition is within the fluorescence index amplification phase；For convenience of unified, fluorescence threshold is defined for N times of all fluorescent value standard variances within 0-4 minutes as fluorescence threshold, N takes the integer more than or equal to 3；
T values refer to that the fluorescence signal of amplified production reaches the time required during the threshold value of setting.
4. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2,3, it is characterised in that：Introduce after T value concepts, exponential function originally can be expressed as FT=F0×(1+E0)T
It can be released by above formula：logFT=log [F0×(1+E0)T]
logF0=logFT-Tlog(1+E0) set up；
There is linear relationship in T values and the logarithm of the starting copy number of the template, starting copy number is more, and T values are smaller.
5. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2,3,4, it is characterised in that：The amplification curve of known starting copy number standard items is made by the way that the instrument of fluorescence volume can be measured for specific target DNA or RNA, and fluorescence threshold is set, the parallel lines that mistake threshold value makees X-axis obtain threshold line；And threshold line intersects with amplification curve, the vertical line that intersection point makees X-axis is crossed, corresponding T values are obtained.
6. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2,3,4,5, it is characterised in that：By the process described above by setting the standard samples of different gradient concentration knowns, make graded series amplification curve, unified fluorescence threshold is set, make threshold line and obtain a series of T values with the time corresponding to the joining of various concentrations gradient standard sample amplification curve, proliferation time is represented with abscissa, ordinate represents the logarithm of starting copy number, marked in coordinate diagram using different T values as abscissa, using the logarithm of corresponding known standard items starting copy number as the point of ordinate, cross these point fitting fluorescent quantitation standard curves, and the linear function Y=-a of the standard curve is obtained according to the standard curve of fitting*T+b wherein a, b are constants.
7. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2,3,4,5,6, it is characterised in that：For the sample of specific unknown starting template amount, as long as obtaining the T values of the sample of unknown starting template amount, you can calculate the starting copy number of the sample from standard curve；The vertical line that method makees X-axis to cross abscissa T value points in standard curve hands over standard curve in point A, cross A points and make the parallel lines friendship ordinate of X-axis in B points, it is exactly the logarithm of starting copy number that ordinate data Y, data Y, which can be read, and the starting copy number of the sample is represented by 10Y；
T values can also be substituted into the linear function Y=-a of standard curve*In T+b, Y value is obtained, Y value is the logarithm of starting copy number, and starting copy number is represented by 10Y。
8. the method that fluorescent quantitation calculating is carried out based on recombinase-mediated isothermal nucleic acid amplification (RAA) method according to claim 1,2,3,4,5,6,7, it is characterised in that：T values are tried to achieve using the following method：The sample amplification curve of unknown starting template amount is obtained by fluorescence detection equipment, identical threshold value when fluorescence threshold is arranged to making standard curve, cross threshold value and make the parallel lines of X-axis and the sample amplification curve intersection of unknown starting template amount, cross the vertical line that intersection point makees X-axis, T values can be obtained.
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