CN109536587A - A kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof - Google Patents
A kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof Download PDFInfo
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- CN109536587A CN109536587A CN201811651557.6A CN201811651557A CN109536587A CN 109536587 A CN109536587 A CN 109536587A CN 201811651557 A CN201811651557 A CN 201811651557A CN 109536587 A CN109536587 A CN 109536587A
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- 238000003753 real-time PCR Methods 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 15
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 239000004615 ingredient Substances 0.000 claims description 6
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Chinese gallotannin Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 238000009004 PCR Kit Methods 0.000 claims description 2
- -1 dNTPs Chemical compound 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 18
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 18
- 230000035945 sensitivity Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 6
- 239000012807 PCR reagent Substances 0.000 abstract description 4
- 239000000654 additive Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 51
- 230000007812 deficiency Effects 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
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- 239000003814 drug Substances 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
- 206010056740 Genital discharge Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The present invention provides the present invention wherein on the one hand to develop a kind of efficient multiple fluorescence quantitative PCR reaction solution and kit, preparation method and fluorescence quantifying PCR method.On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, which includes basis buffer and protein protection liquid.By the detection to reagent sensitivity and amplification capability, it is found that the multiple fluorescence quantitative PCR reagent that this research provides has sensitivity more higher than conventional reagents and amplification capability.Present invention optimizes multiple fluorescence quantitative PCR reaction solutions to improve sensitivity, specificity and the stability of reaction especially by some specific additives, such as a certain proportion of glycerol, BSA and DTT are added.
Description
Technical field
The invention belongs to molecules and cell biology, in particular to high-efficiency multiple fluorescence quantitative PCR detection.
Background technique
Quantitative fluorescent PCR (realtime fluores-cence quantitative PCR) is 1996 by the U.S.
A kind of novel quantitative experimental technique that Applied Biosystems company releases can be not only used for widely
The research of mRNA expression, the measurement of single nucleotide polymorphism (SNPs), pathogen measurement, tumor research and immune component
The medical treatment aspect such as analysis can be also used for new drug development, curative effect of medication research and new diagnostic and the exploitation of testing reagent etc.
Drug research field.Combination dye of the most frequently used SYBR Green I of dye method quantitative fluorescent PCR as DNA, this is a kind of DNA
Ditch unsaturation combination dye, can produce fluorescence when in conjunction with DNA, and it is free when do not generate fluorescence, one DNA double of every formation
Chain just has a certain number of dyestuffs and combines up, quantifies from the accumulation of signal to product.Although this method has
It is cheap, the wide advantage of applicability, but its poor specificity, it cannot distinguish between amplified production and non-specific amplification, it can not
Different target product is carried out respectively quantitatively, these deficiencies make the utilization of dye method quantitative fluorescent PCR by certain limit
System.And the quantitative fluorescent PCR of sonde method then compensates for these deficiencies completely, multiple fluorescence quantitative PCR is even more to realize simultaneously to more
A target product is quantified.Since multiplex amplification has Preference, and interfering with each other property is stronger between each product, therefore average probe
The quantitative fluorescent PCR reagent of method can not be used for multiple fluorescence quantitative PCR, so the optimization to multiple fluorescence quantitative reagent is just shown
It obtains very necessary.
Summary of the invention
In order to solve the deficiency of above-mentioned existing various methods, on the one hand the present invention wherein develops a kind of efficient multiple glimmering
Fluorescent Quantitative PCR reaction solution and kit, preparation method and fluorescence quantifying PCR method.By to reagent sensitivity and amplification capability
Detection, find this research provide multiple fluorescence quantitative PCR reagent have sensitivity more higher than conventional reagents and amplification property
Energy.
Present invention optimizes multiple fluorescence quantitative PCR reaction solutions, especially by some specific additives of addition, such as one
Glycerol, BSA and DTT of certainty ratio etc. improve sensitivity, specificity and the stability of reaction.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, this is efficient multiple
Fluorescence quantitative PCR reaction solution includes basis buffer and protein protection liquid.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, above-mentioned albumen quality guarantee
Protecting liquid includes glycerol, BSA and DTT.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, the basis buffering
The ingredient of liquid includes Tris-HCl, dNTPs, KCl, (NH4)2SO4、MgCl2。
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, above-mentioned multi-fluorescence
Quantitative PCR reaction solution is by Tris-HCl solution, dNTPs solution, KCl solution, (NH4)2SO4Solution, MgCl2Solution, BSA are molten
Liquid, glycerite and DTT solution composition.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, the multi-fluorescence is fixed
Measure PCR reaction solution include 200~700mM PH 8.3Tris-HCl, 0.3~1.5mM dNTPs, 25~75mM KCl, 75~
200mM(NH4)2SO4, 12~20mM MgCl2Solution and 8%~25% glycerol.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR reaction solution, the multi-fluorescence is fixed
Measure PCR reaction solution can also by 50~150 μ l 2M Tris-HCl PH, 8.3 solution, 5~20 μ l 25mM dNTPs solution,
5~15 μ l 2M KCl solution, 15~40 μ l 2M (NH4)2SO4Solution, 10~20 μ l 0.5M MgCl2Solution, 10~30 μ l
The BSA solution that mass fraction is 5%, the glycerite and 3~10 μ l 1M DTT solution that 70~200 μ l mass fractions are 50%
Composition.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR kit, the multi-fluorescence is fixed
Amount PCR kit wraps any one the multiple fluorescence quantitative PCR reaction solution stated.
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR kit, above-mentioned multi-fluorescence
Quantitative PCR kit also includes primer pair, probe, thermal starting archaeal dna polymerase.
On the one hand of the invention wherein provides plasmid construction information, carrier is pET30 (a), is inserted into aim sequence such as SEQ
ID NO.1。
5’-TCAGTGGTGGTGGTGGTGGTGCTCGAGCTGGTTCTTGACAAAGCTTTTGAGGTCCTCAAGAAAGG
TGAT ATATTCCTGGTGCTCCAGGGCTGTGCTGAGCTCCACCAACTCATCCGCAAAAGTGTTATCCACCCCTCGGT
CCGCAA GGAAATCCATTAGGTGGTCATACAAGGCCCAGTCCAGGGAATCTGTGTTGAGTGTATAGTTTGTATCCC
TCCACTCA GAGTCACCAGTGGCCTGAAAGCTAACTTCCTTGATAGAGAAAATATCACTCTCGGCCTCATCTTCGT
GTCCAATCTC ATCCTCAGGATAGTGACAGTCCAGTACAAGGGTCTTCTTGCCATCAGTCTTTGTAACTTCAACCA
CAAAGTTGGGAG TTGATGTCAGTTCTGGCTCCTGTTCTTCAGCCTTCTGCCCCTGTGAGGGCTCCTCCTCACCAT
CAAATGTTGGAGGG ATGCTGTTGTTGATGTTGAAAGTGACCGTGATCTTTTCTCCGGCAACTTTGCGCAATAATT
TAGCCTCCGTGCCGTT CACCTCCAGCTCCCAATCTCCAGACATCTTGGGAAGGGACTTGTGTTTCTGGATCTTCT
TTTCTTCCTTAATTTCAT CAGTCAAGAATTCAACGAAGGCCTTGTCTCCTTCCGTGTGCAGCAT-3’。
On the one hand of the invention wherein provides a kind of efficient multiple fluorescence quantitative PCR kit, primer pair is primers F
With primer R, primers F sequence such as SEQ ID No.2, primer R sequence such as SEQ ID No.3, probe sequence such as SEQ ID No.4.
Primers F: 5 '-CCAGTCCAGGGAATCTGTGT-3 '
Primer R:5 '-GAAGATGAGGCCGAGAGTGA-3 '
Probe: 5 '-CCCTCCACTCAGAGTCACCAGTGGCC-3 '
On the one hand of the invention wherein provides a kind of preparation method of efficient multiple fluorescence quantitative PCR kit, mix
Close 200~700mM PH 8.3Tris-HCl, 0.3~1.5mM dNTPs, 25~75mM KCl, 75~200mM (NH4)
The ingredients such as 2SO4,12~20mM MgCl2 solution and 8%~25% glycerol prepare multiple fluorescence quantitative PCR reaction solution, the present invention
Efficient multiple fluorescence quantitative PCR kit include multiple fluorescence quantitative PCR reaction solution.
On the one hand of the invention wherein provides a kind of preparation method of efficient multiple fluorescence quantitative PCR kit, system
Preparation Method is mixing 50~150 μ l 2M Tris-HCl PH, 8.3 solution, 5~20 μ l 25mM dNTPs solution, 5~15 μ l
2M KCl solution, 15~40 μ l 2M (NH4)2SO4Solution, 10~20 μ l 0.5M MgCl2Solution, 10~30 μ l mass fractions are
5% BSA solution, the glycerite that 70~200 μ l mass fractions are 50% and 3~10 μ l 1M DTT solution compositions are multiple glimmering
Fluorescent Quantitative PCR reaction solution, then to prepare remaining ingredient spare.
The present invention also provides a kind of efficient multiple fluorescence quantitative PCR methods, using more described in any one of the above
Weight fluorescence quantitative PCR reaction solution or multiple fluorescence quantitative PCR kit carry out multiple fluorescence quantitative PCR detection.By to reagent
Sensitivity and the detection of amplification capability, the multiple fluorescence quantitative PCR reagent that discovery invention provides have more higher than conventional reagents
Sensitivity and amplification capability.
Detailed description of the invention
It Fig. 1, is experimental group amplification curve in one embodiment of the invention；
Fig. 2, a group fluorescent quantitation standard curve is tested in one embodiment for the present invention；
It Fig. 3, is present invention control group amplification curve in one embodiment；
It Fig. 4, is present invention control group fluorescent quantitation standard curve in one embodiment；
It Fig. 5, is present invention experimental group multiple fluorescence PCR amplification curve in one embodiment；
It Fig. 6, is present invention control group multiple fluorescence PCR amplification curve in one embodiment.
Specific embodiment
Following embodiments is some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.The present invention will be further explained below with reference to the accompanying drawings.
Embodiment 1: the configuration of efficient 5 × multiple fluorescence quantitative PCR reaction solution
A kind of efficient 5 × multiple fluorescence quantitative PCR reaction solution, mainly by basis buffer and protein protection liquid group
It include: 50~150 μ l 2M Tris-HCl PH, 8.3 solution at, specific ingredient, 5~20 μ l 25mM dNTPs solution, 5~
15 μ l 2M KCl solution, 15~40 μ l 2M (NH4)2SO4Solution, 10~20 μ l 0.5M MgCl2Solution, 10~30 μ l mass
The BSA solution that score is 5%, the glycerite and 3~10 μ l 1M DTT solution that 70~200 μ l mass fractions are 50%.
Embodiment 2: the sensitivity technique of efficient 5 × multiple fluorescence quantitative reaction solution
1. the dilution mode of template
2.PCR amplification program
3. primer and probe sequence
F:CCAGTCCAGGGAATCTGTGT
R:GAAGATGAGGCCGAGAGTGA
Probe: CCCTCCACTCAGAGTCACCAGTGGCC
4. reaction system
| PCR reaction system | 20μl |
| 5 × multiple fluorescence quantitative reaction solution | 4μl |
| Primers F/R (10 μM) | 0.8μl/0.8μl |
| Probe (10 μM) | 0.4μl |
| Thermal starting archaeal dna polymerase (5U/ μ l) | 0.25μl |
| Template | 1μl |
| DEPC water | 12.75μl |
5. establishing standard curve
Standard curve is analyzed experimental result and is made, the Y-axis of standard curve is the Ct value of each standard items, standard
The X-axis of curve is the corresponding Log10 of each standard items (copy number), is corresponding in turn to as Log10 (standard items 1)=7.51, Log10
(standard items 2)=6.51, Log10 (standard items 3)=5.51, Log10 (standard items 4)=4.51, Log10 (standard items 5)=
3.51, Log10 (standard items 6)=2.51.
6. experimental group and the detection of control group sensitivity results
This experiment contrast product are luxuriant and rich with fragrance roc product (MD026).Fig. 1 is experimental group amplification curve, and Fig. 2 is experimental group fluorescent quantitation
Standard curve, Fig. 3 are control group amplification curve, and Fig. 4 is control group fluorescent quantitation standard curve, and table one is experimental group in different moulds
Ct value under plate, table two are Ct value of the control group under different templates.It can be seen that this kit than control tool from amplification curve diagram
There is higher sensitivity, can be seen that this kit has good amplification capability from canonical plotting.
Table one
Table two
| Template | Ct value |
| Standard items 1 | 15.56 |
| Standard items 2 | 19.02 |
| Standard items 3 | 22.35 |
| Standard items 4 | 25.62 |
| Standard items 5 | 29.11 |
| Standard items 6 | 32.15 |
| It is negative | 32.85 |
Embodiment 3: the use of efficient 5 × multiple fluorescence quantitative reaction solution
1. reaction system
2. reaction condition
3. multiplex amplification experimental result
Fig. 5 is experimental group amplification curve, and Fig. 6 is control group amplification curve, and table three is the Ct value of experimental group, and table four is control
The Ct value of group.It can be seen that this kit has higher sensitivity than control from amplification curve diagram.
Table three
| Channel | Ct value |
| FAM | 19.18 |
| HEX | 20.67 |
| ROX | 16.70 |
| CY5 | 13.98 |
Table four
The above embodiments are some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.
Sequence table
<110>Wujiang Alongshore Protein Technology Co., Ltd.
<120>a kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof
<130> 2018
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 654
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>engineer
<400> 1
tcagtggtgg tggtggtggt gctcgagctg gttcttgaca aagcttttga ggtcctcaag 60
aaaggtgata tattcctggt gctccagggc tgtgctgagc tccaccaact catccgcaaa 120
agtgttatcc acccctcggt ccgcaaggaa atccattagg tggtcataca aggcccagtc 180
cagggaatct gtgttgagtg tatagtttgt atccctccac tcagagtcac cagtggcctg 240
aaagctaact tccttgatag agaaaatatc actctcggcc tcatcttcgt gtccaatctc 300
atcctcagga tagtgacagt ccagtacaag ggtcttcttg ccatcagtct ttgtaacttc 360
aaccacaaag ttgggagttg atgtcagttc tggctcctgt tcttcagcct tctgcccctg 420
tgagggctcc tcctcaccat caaatgttgg agggatgctg ttgttgatgt tgaaagtgac 480
cgtgatcttt tctccggcaa ctttgcgcaa taatttagcc tccgtgccgt tcacctccag 540
ctcccaatct ccagacatct tgggaaggga cttgtgtttc tggatcttct tttcttcctt 600
aatttcatca gtcaagaatt caacgaaggc cttgtctcct tccgtgtgca gcat 654
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>engineer
<400> 2
ccagtccagg gaatctgtgt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>engineer
<400> 3
gaagatgagg ccgagagtga 20
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223>engineer
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ccctccactc agagtcacca gtggcc 26
Claims (10)
1. a kind of efficient multiple fluorescence quantitative PCR reaction solution, which is characterized in that the efficient multiple fluorescence quantitative PCR is anti-
Answering liquid includes basis buffer and protein protection liquid.
2. a kind of efficient multiple fluorescence quantitative PCR reaction solution according to claim 1, which is characterized in that the albumen
It includes glycerol, BSA and DTT that liquid is protected in quality guarantee.
3. a kind of efficient multiple fluorescence quantitative PCR reaction solution according to claim 1, which is characterized in that the basis
The ingredient of buffer includes Tris-HCl, dNTPs, KCl, (NH4)2SO4、MgCl2。
4. a kind of efficient multiple fluorescence quantitative PCR reaction solution according to claim 1, which is characterized in that described multiple
Fluorescence quantitative PCR reaction solution is by Tris-HCl solution, dNTPs solution, KCl solution, (NH4)2SO4Solution, MgCl2Solution, BSA
Solution, glycerite and DTT solution composition.
5. a kind of efficient multiple fluorescence quantitative PCR reaction solution according to claim 4, which is characterized in that described multiple
Fluorescence quantitative PCR reaction solution include 200~700mM PH 8.3Tris-HCl, 0.3~1.5mM dNTPs, 25~75mM KCl,
75~200mM (NH4)2SO4, 12~20mM MgCl2Solution and 8%~25% glycerol.
6. a kind of efficient multiple fluorescence quantitative PCR kit, which is characterized in that the multiple fluorescence quantitative PCR kit packet
Containing any one multiple fluorescence quantitative PCR reaction solution described in claim 1 to 5.
7. a kind of efficient multiple fluorescence quantitative PCR kit according to claim 6, which is characterized in that described multiple
PCR kit for fluorescence quantitative also includes primer pair, probe, thermal starting archaeal dna polymerase.
8. a kind of efficient multiple fluorescence quantitative PCR kit according to claim 7, which is characterized in that the primer
To for primers F and primer R, the primers F sequence such as SEQ ID No.2, the primer R sequence such as SEQ ID No.3, the spy
Needle sequence such as SEQ ID No.4.
9. a kind of preparation method of efficient multiple fluorescence quantitative PCR kit, which is characterized in that mixing includes 200~700mM
PH 8.3Tris-HCl, 0.3~1.5mM dNTPs, 25~75mM KCl, 75~200mM (NH4)2SO4, 12~20mM MgCl2
The ingredient of solution and 8%~25% glycerol prepares multiple fluorescence quantitative PCR reaction solution, the efficient multiple fluorescence quantitative PCR
Kit includes the multiple fluorescence quantitative PCR reaction solution.
10. a kind of efficient multiple fluorescence quantitative PCR method, which is characterized in that using any one in the claim 1 to 8
Multiple fluorescence quantitative PCR reaction solution or multiple fluorescence quantitative PCR kit described in kind carry out multiple fluorescence quantitative PCR detection.
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| WO2023170152A1 (en) * | 2022-03-08 | 2023-09-14 | Rarity Bioscience Ab | Method for detecting nucleic acid amplification products using blocking agents |
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