CN105112399A - Fluorescent constant-temperature amplification technique - Google Patents
Fluorescent constant-temperature amplification technique Download PDFInfo
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- CN105112399A CN105112399A CN201510504145.XA CN201510504145A CN105112399A CN 105112399 A CN105112399 A CN 105112399A CN 201510504145 A CN201510504145 A CN 201510504145A CN 105112399 A CN105112399 A CN 105112399A
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Abstract
The invention discloses a fluorescent constant-temperature amplification technique. In water used as solvent for a fluorescent constant-temperature amplification reagent used in the technique, Tris-buffer solution, magnesium acetate, dithiothreitol, betaine, Tween 20, DMSO, mycose, PEG, BSA, dNTPs, and proper amounts of fluorescent dye, polymerase, single strand DNA-binding protein and recombinase are added. A constant-temperature amplification system of the invention is dependent of high-energy molecules such as ATP and creatine phosphate; compared with existing constant-temperature amplification systems, the constant-temperature amplification system is simpler in composition, lower in cost and more convenient to use. The constant-temperature amplification system is capable of amplifying DNA and RNA sequences, is applicable to amplification of complex templates stably and quickly, capable of finishing an amplification reaction within 30min, has high amplification sensitivity and accuracy without rare occurrence of mispairing, and has good amplification effect. The constant-temperature amplification system is applicable to real-time quantification and has a wider range of application.
Description
Technical field
The present invention relates to a kind of fluorescence nucleic acid isothermal amplification technology, particularly a kind of fluorescence isothermal amplification technology not relying on high energy kinetomeres.
Background technology
Nineteen eighty-three, Mullis etc. have invented polymerase chain reaction (PCR) technology, and between more than 30 year, round pcr obtains tremendous development, due to its advantage in sensitivity and specificity, round pcr is applied to rapidly all respects of scientific research and clinical study.This round pcr being similar to the natural reproduction process of DNA, its specificity depends on the Oligonucleolide primers with the complementation of target sequence two ends.Mainly comprise three primitive reaction steps: the sex change of (1) template DNA: after heating certain hour, make template DNA double-strand or dissociate through the double-stranded DNA that pcr amplification is formed, becoming strand; (2) annealing (renaturation) of template DNA and primer: temperature is down to about 55 DEG C, and the complementary sequence of primer and template DNA strand matches and combines; (3) extension of primer: DNA profiling--primer binding substances 72 DEG C, under the effect of archaeal dna polymerase (as Taq DNA polymerase), take dNTP as reaction raw materials, target sequence is template, by base pair complementarity and semiconservative replication principle, synthesize a semiconservative replication chain that the is new and complementation of template DNA chain, recirculation sex change--annealing--extends three processes and just can obtain more " semiconservative replication chain ", and this new chain can become again the template of circulation next time.The exploitation of this technology greatly facilitates the progress of biological study.
PCR also has some restriction in technology application: first, round pcr needs expensive Laboratory Instruments, instrument will possess meticulous temperature control program and heated die plate simultaneously, thus realize the sex change of the DNA profiling chain at very high temperature, at lower temperature, primed probe annealing extends template, and such repeated temperature realizes the amplification of template amount after changing tens circulations.Because each cycling time is shorter, instrument quick and precisely must can be elevated temperature, and this just needs the heating module of silvery or gold system, strengthens the cost of instrument development.The second, polysaccharase useful in PCR amplification system must have the ability of withstand high temperatures, otherwise then must rejoin new enzyme in each circulation, and to realize the amplification of next circulation, as easy as rolling off a log polluting also increases cost like this.3rd, in PCR circulation, the time very short (several seconds to tens seconds) of each circulation primer annealing, this just required that primer must find the section of homology coupling in template fast, to realize extending.This just requires that PCR system must have excessive PCR primer.Excessive primer can with template mispairing, mistake cause amplification, primer dimer etc., so suppression PCR amplification, particularly in the situation that template amount is low, this situation can be made to aggravate.
Compared with traditional round pcr, isothermal amplification technology mainly utilizes the activity of recombinase, does not carry out nucleic and melting and anneal under constant temperature, (as 37 DEG C) to carry out the amplification of nucleic acid, does not therefore need expensive instrument, it also avoid the loss of high temperature to enzyme.Simultaneously isothermal amplification technology does not need to add excessive primer in system, reduces primer and template mispairing or generates the possibility of primer dimer.Isothermal amplification technology comes into one's own day by day.
Fluorescent PCR can be more convenient for quantitative analysis, is more and more subject to people's attention, and fluorescence isothermal amplification technology have also been obtained further development.
Common isothermal amplification technique has following several: roll ring constant-temperature amplification (RCA), loop-mediated isothermal amplification (LAMP), relies on helicase constant-temperature amplification (HDA) etc.Existing isothermal amplification technology, its reaction system more complicated, simultaneously most constant-temperature amplification system or depend on ATP, depend on creatine kinase and phosphocreatine, or other high energy kinetomereses, required reagent type is various, and amplification rate is limited to the supply of energy, this causes isothermal amplification technology cost compare high, limits its application.
Summary of the invention
The object of the present invention is to provide a kind of the nucleic acid fluorescent isothermal amplification reactions reagent and the method that do not rely on high energy kinetomeres.
The technical solution used in the present invention is:
A kind of fluorescence isothermal amplification reactions reagent, solvent is water, and it is composed as follows:
Tris-damping fluid 0mM--500mM
Magnesium acetate 3mM--50mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-5M
Tween200%-10%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--10mg/mL
dNTPs180uM--1000uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each;
Recombinase is the recombinase not relying on kinetomeres.
Preferably, fluorescence isothermal amplification reactions reagent is composed as follows:
Tris-damping fluid 10mM--100mM
Magnesium acetate 5mM--20mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-2M
Tween200%-1%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--1mg/mL
dNTPs200uM--500uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each.
Preferably, the pH of Tris-damping fluid that fluorescence isothermal amplification reactions reagent is used is 7 ~ 9.
The Tris-damping fluid that fluorescence isothermal amplification reactions reagent is used is selected from Tris-acetic acid, Tris-H
2sO
4,tris-HCl.
The fluorescence dye that fluorescence isothermal amplification reactions reagent is used is SYBRgreen Ι, SYTO-9, SYTO-12, SYTO-60, SYTO-62, SYTO-16, SYTO-82 or POPO-3, the one in TOTO-3, TO-PRO-3.
Preferably, the recombinase that fluorescence isothermal amplification reactions reagent is used is selected from E.coliRecT albumen; Lambda particles phage β albumen; Cereuisiae fermentum Sep Ι/STP β; Cereuisiae fermentum DPA albumen; Cereuisiae fermentum STP α albumen; Schizosaccharomyces pombe p
140/ Exo ∥ albumen and Rrp Ι, HPP-Ι, v-SEP, ICP8 albumen.
Preferably, reversed transcriptive enzyme is added with in fluorescence isothermal amplification reactions reagent, for detecting RNA.
A kind of nucleic acid fluorescent constant-temperature amplification method, comprises probe, primer, template and isothermal amplification reactions reagent mix, after constant-temperature amplification to desired amount, by the enzyme-deactivating of isothermal amplification reactions system, complete constant-temperature amplification, wherein, isothermal amplification reactions reagent is described above.
Preferably, the temperature of constant-temperature amplification is 37 ~ 42 DEG C.
The invention has the beneficial effects as follows:
Fluorescence constant-temperature amplification system of the present invention, does not rely on the high energy such as ATP, phosphocreatine kinetomeres, relatively existing constant-temperature amplification system, and its composition is more simple, and cost is more cheap, uses more convenient.
Constant-temperature amplification system of the present invention can realize amplification to DNA and RNA sequence, is applicable to the amplification of template complex, and amplification rate is fast and stable, amplified reaction can be completed within 30min, sensitivity and the accuracy of amplification are high, and not easily occur mispairing, expanding effect is good.
Accompanying drawing explanation
Fig. 1 is the fluorescence constant-temperature amplification result figure of embodiment 1;
Fig. 2 is the fluorescence constant-temperature amplification result figure of embodiment 2;
Fig. 3 is the fluorescence constant-temperature amplification result figure of embodiment 3;
Fig. 4 is the fluorescence constant-temperature amplification result figure of embodiment 4;
Fig. 5 is the fluorescence constant-temperature amplification result figure of embodiment 5;
Fig. 6 is the fluorescence constant-temperature amplification result figure of embodiment 6;
Fig. 7 is the fluorescence constant-temperature amplification result figure of embodiment 7;
Fig. 8 is the fluorescence constant-temperature amplification result figure of embodiment 8.
Embodiment
A kind of fluorescence isothermal amplification reactions reagent, solvent is water, and it is composed as follows:
Tris-damping fluid 0mM--500mM
Magnesium acetate 3mM--50mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-5M
Tween200%-10%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--10mg/mL
dNTPs180uM--1000uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each;
Recombinase is the recombinase not relying on kinetomeres.
Preferably, it is composed as follows:
Tris-damping fluid 10mM--100mM
Magnesium acetate 5mM--20mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-2M
Tween200%-1%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--1mg/mL
dNTPs200uM--500uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each.
The effect of Tris-damping fluid is to maintain certain pH, so that enzyme carries out amplified reaction at optimal pH or preferably under pH.Its concentration can adjust as required accordingly.As the further improvement of above-mentioned reaction reagent, the pH of the Tris-damping fluid used is 7 ~ 9, comprises Tris-acetic acid, Tris-H
2sO
4,tris-HCl.
Magnesium acetate is to provide certain ionic strength, and its consumption can carry out certain adjustment according to nucleic acid amplification situation.
As the further improvement of above-mentioned reaction reagent, in isothermal amplification reactions reagent, be also added with fluorescence dye.Fluorescence dye is SYBRgreen Ι, SYTO-9, SYTO-12, SYTO-60, SYTO-62, SYTO-16, SYTO-82 or POPO-3, the one in TOTO-3, TO-PRO-3.
The consumption of polysaccharase, single strand binding protein, recombinase can adjust its addition as required, or determines its consumption by preliminary experiment.Polysaccharase is polysaccharase conventional in isothermal amplification reactions, is preferably Bsu or klenow.
As the further improvement of above-mentioned reaction reagent, recombinase is selected from E.coliRecT albumen; Lambda particles phage β albumen; Cereuisiae fermentum Sep Ι/STP β; Cereuisiae fermentum DPA albumen; Cereuisiae fermentum STP α albumen; Schizosaccharomyces pombe p
140/ Exo ∥ albumen and Rrp Ι, HPP-Ι, v-SEP, ICP8 albumen.
As the further improvement of above-mentioned reaction reagent, in isothermal amplification reactions reagent, be added with reversed transcriptive enzyme, for detecting RNA.
A kind of nucleic acid fluorescent constant-temperature amplification method, comprises primer, template and fluorescence isothermal amplification reactions reagent mix, after constant-temperature amplification to desired amount, by the enzyme-deactivating of isothermal amplification reactions system, completes constant-temperature amplification
As the further improvement of aforesaid method, the temperature of constant-temperature amplification is 37 ~ 42 DEG C.
Below in conjunction with embodiment, further illustrate technical scheme of the present invention.
embodiment 1:
The concrete composition relying on recombinase isothermal amplification reactions reagent is as follows:
Tris-H
2SO
4（pH7.4）10mM
Ammonium sulfate 100mM
Magnesium acetate 14mM
Dithiothreitol (DTT) 5mM
Trimethyl-glycine 0.8M
DMSO5%
Trehalose 3%
PEG5%
BSA0.1mg/mL
dNTPs200uM
Polysaccharase Bsu5U
Single strand binding protein 250ng/ul
Recombinase (lambda particles phage β albumen) 120ng/ul.
embodiment 2:
The concrete composition relying on recombinase isothermal amplification reactions reagent is as follows:
Tris-acetic acid (pH8.0) 50mM
Magnesium acetate 14mM
Trimethyl-glycine 0.5M
Tween200.34%
DMSO5%
Dithiothreitol (DTT) 4mM
PEG8%
BSA0.1mg/mL
dNTPs200uM
Polysaccharase klenow50ng/ul
Single strand binding protein 350ng/ul
Recombinase (STP α) 100ng/ul.
embodiment 3:
The concrete composition relying on recombinase isothermal amplification reactions reagent is as follows:
Tris-HCl（pH8.3）50mM
KCl60mM
Magnesium acetate 10mM
Dithiothreitol (DTT) 2mM
Trimethyl-glycine 1M
Trehalose 5.5%
PEG5.5%
BSA0.1mg/mL
dNTPs200uM
Polysaccharase Bsu50U
Single strand binding protein 262ng/ul
Recombinase (E.coliRecT) 360ng/ul.
embodiment 4
With embodiment 1, difference is that recombinase is the cereuisiae fermentum DPA albumen of 180ng/ul, polysaccharase klenow50ng/ul
embodiment 5
With embodiment 2, difference is that recombinase is the ICP8 albumen of 362ng/ul.
embodiment 6
With embodiment 3, difference is that recombinase is the v-SEP albumen of 70ng/ul, polysaccharase klenow50ng/ul
embodiment 7
With embodiment 2, difference is that recombinase is the Rrp Ι albumen of 90ng/ul, and polysaccharase BSU is 20U.
embodiment 8
With embodiment 3, difference is that Tris-HClpH value is 8.8, and concentration is 50mM; KCl concentration is 100mM; Magnesium acetate concentration is 14mM.
Below in conjunction with concrete experiment, further illustrate the advantage of isothermal amplification technology of the present invention.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Forward primer: GGACCGAAAACGGTGTATATAAAAGATGTGAG(SEQIDNO:1)
Reverse primer: ATCAGGTAGCTTGTAGGGTCGCCGTGTTGGA(SEQIDNO:2)
Probe: TTGTCCATAGACTGATGTGAGAAACACACCACAATACTATGGCGCGC
UmUmUmGmAmGmGmAmUmCmCmAmAmCm-inverteddT（SEQIDNO：3）
The present embodiment with hela cell as sample, with human gene group DNA as negative control, 2ulhela cell is added in reaction tubes 1,2ul human gene group DNA is added as negative control in reaction tubes 2, in reaction tubes 1 and 2, add the dependence recombinase isothermal amplification reactions reagent (reaction system of embodiment 1) of 25ul simultaneously, respectively add the upstream and downstream primer of 200nM again, system sterilized water is supplied 30ul.
SYTO13：0.8uM
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 1.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 2, and in system, respectively add the forward primer of fluorescence dye SYTO13, HPV18 type, reverse primer and probe, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYTO13：0.8uM
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 2.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 3, and add fluorescence dye SYTO13, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYTO13：0.8uM
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 3.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 4, and add fluorescence dye SYBRGreen Ι, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYBRGreenΙ：1:10000
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 4.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 5, and add fluorescence dye SYBRGreen Ι, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYBRGreenΙ：1:10000
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 5.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 6, and add fluorescence dye SYBRGreen Ι, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYBRGreenΙ：1:10000
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 6.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 7, and add fluorescence dye SYBRGreen Ι, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYBRGreenΙ：1:10000
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 7.
the fluorescence constant-temperature amplification of HPV18 C-type virus C
Two pipe reagent are prepared respectively according to the reaction system of embodiment 8, and add fluorescence dye SYBRGreen Ι, HPV18 type forward primer, reverse primer and probe wherein, then, 2ulhela cell is added in reaction tubes 1, in reaction tubes 2, add 2ul human gene group DNA as negative control, system sterilized water is supplied 30ul.
SYBRGreenΙ：1:10000
Forward primer: 200nM
Reverse primer: 200nM
Probe: 200nM
Select quantitative real time PCR Instrument to carry out increasing and collecting fluorescence, concrete upper machine condition is: 39 DEG C, 60s, 20 circulations, fluorescence is collected in each circulation.Melting curve is arranged: 95 C, 15s; 25 C, 60s.; 95 C, 15s; 60 °, 15s.Result is as Fig. 8.
As can be seen from Fig. 1 ~ 8, positive template amplification corresponding to 8 reaction systems has melting curve, and curve peak figure is sharp-pointed, reacting positive.Negative template is at 75 C without melting curve, and amplification is negative.Illustrate that constant-temperature amplification result of the present invention is better, specificity is good, highly sensitive.
<110> Guangzhou two kinds of substance synthesis into another Technology Co., Ltd.
<120> fluorescence isothermal amplification technology
<130>
<160>3
<170>PatentInversion3.5
<210>1
<211>32
<212>DNA
<213> artificial sequence
<400>1
ggaccgaaaacggtgtatataaaagatgtgag32
<210>2
<211>31
<212>DNA
<213> artificial sequence
<400>2
atcaggtagcttgtagggtcgccgtgttgga31
<210>3
<211>75
<212>DNA
<213> artificial sequence
<400>3
ttgtccatagactgatgtgagaaacacaccacaatactatggcgcgcumumumgmamgmg60
mamumcmcmamamcm75
Claims (9)
1. a fluorescence isothermal amplification reactions reagent, solvent is water, and it is composed as follows:
Tris-damping fluid 0mM--500mM
Magnesium acetate 3mM--50mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-5M
Tween200%-10%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--10mg/mL
dNTPs180uM--1000uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each;
Recombinase is the recombinase not relying on kinetomeres.
2. fluorescence isothermal amplification reactions reagent according to claim 1, is characterized in that: it is composed as follows:
Tris-damping fluid 10mM--100mM
Magnesium acetate 5mM--20mM
Dithiothreitol (DTT) 0mM--10mM
Trimethyl-glycine 0M-2M
Tween200%-1%
DMSO0%-10%
Trehalose 0%-10%
PEG1%--20%
BSA0mg/mL--1mg/mL
dNTPs200uM--500uM
Fluorescence dye, polysaccharase, single strand binding protein, recombinase, in right amount each.
3. fluorescence isothermal amplification reactions reagent according to claim 1, is characterized in that: the pH of Tris-damping fluid is 7 ~ 9.
4. the fluorescence isothermal amplification reactions reagent according to claims 1 to 3 any one, is characterized in that: Tris-damping fluid is selected from Tris-acetic acid, Tris-H
2sO
4,tris-HCl.
5. the fluorescence isothermal amplification reactions reagent according to claims 1 to 3 any one, is characterized in that: fluorescence dye is SYBRgreen Ι, SYTO-9, SYTO-12, SYTO-60, SYTO-62, SYTO-16, one in SYTO-82 or POPO-3, TOTO-3, TO-PRO-3.
6. the fluorescence isothermal amplification reactions reagent according to claims 1 to 3 any one, is characterized in that: recombinase is selected from E.coliRecT albumen; Lambda particles phage β albumen; Cereuisiae fermentum Sep Ι/STP β; Cereuisiae fermentum DPA albumen; Cereuisiae fermentum STP α albumen; Schizosaccharomyces pombe p
140/ Exo ∥ albumen and Rrp Ι, HPP-Ι, v-SEP, ICP8 albumen.
7. the fluorescence isothermal amplification reactions reagent according to claims 1 to 3 any one, is characterized in that: be added with reversed transcriptive enzyme in fluorescence isothermal amplification reactions reagent, for detecting RNA.
8. a nucleic acid fluorescent constant-temperature amplification method, comprise probe, primer, template and isothermal amplification reactions reagent mix, after constant-temperature amplification to desired amount, by the enzyme-deactivating of isothermal amplification reactions system, complete constant-temperature amplification, wherein, isothermal amplification reactions reagent is as described in claim 1 ~ 7 any one.
9. according to claim 8 kind of nucleic acid fluorescent constant-temperature amplification method, is characterized in that: the temperature of constant-temperature amplification is 37 ~ 42 DEG C.
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