CN104313152A - Fluorogenic quantitative PCR (Polymerase Chain Reaction) liquid as well as kit and fluorogenic quantitative PCR method - Google Patents
Fluorogenic quantitative PCR (Polymerase Chain Reaction) liquid as well as kit and fluorogenic quantitative PCR method Download PDFInfo
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- CN104313152A CN104313152A CN201410578009.0A CN201410578009A CN104313152A CN 104313152 A CN104313152 A CN 104313152A CN 201410578009 A CN201410578009 A CN 201410578009A CN 104313152 A CN104313152 A CN 104313152A
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention relates to the technical field of biologics, and in particular relates to a fluorogenic quantitative PCR (Polymerase Chain Reaction) liquid as well as a kit and a fluorogenic quantitative PCR method. The fluorogenic quantitative PCR liquid is prepared from the following raw materials: dimethylsulfoxide, bovine serum albumin, DNTP (Diethyl-nitrophenyl Thiophosphate), 10*SYB fluorochrome, 10*PCR buffer, glycerite of which the volume percentage of solute is 30%, and Ex-Taq enzyme. By adopting the method, the fluorogenic quantitative PCR liquid for PCR experiment is optimized, particularly, due to addition of the glycerite of certain ratio, the activity of the Ex-Taq enzyme is ensured, and due to addition of the composition of BSA and DMSO, the stability and activity of Tag enzyme are ensured, formation of a primer dimer is reduced, the time for preparing the fluorogenic quantitative PCR liquid is shortened, and the prepared PCR liquid can be preserved at minus 20 DEG C for a long time and is convenient to use.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of fluorescence quantitative PCR reaction solution and test kit and fluorescence quantifying PCR method.
Background technology
Quantitative fluorescent PCR (realtime fluores-cence quantitative PCR, RTFQ PCR) be the new quantitative test technology of one released by Applied Biosystems company of the U.S. for 1996, it is by fluorescence dye or fluorescently-labeled specific probe, carry out mark to PCR primer to follow the tracks of, real time and on line monitoring reaction process, can analyze product in conjunction with corresponding software, calculate the starting point concentration of testing sample template.
SYBR Green I is the most frequently used DNA binding dye of quantitative fluorescent PCR, with double-stranded DNA non-specific binding.Under unbound state, SYBR Green I sends faint fluorescence, but once be combined with double-stranded DNA, its fluorescence increases by 1000 times.So the double-stranded DNA amount of whole fluorescent signal that reaction sends and outlet is proportional, and can increase with the increase of amplified production.
The advantage of stranded DNA binding dye: experimental design is simple, only need 2 primers, do not need designing probe, namely can the multiple gene of quick test without the need to designing multiple probe, and can melting point curve analysis be carried out, the specificity of inspection amplified reaction, low initial cost, versatility is good, therefore domestic and international use in scientific research commonplace.
Conventional fluorescent quantitative PCR system configurations, often the reagent such as DNTP, Buffer, Ex-Taq enzyme are preserved separately, in time will carrying out pcr amplification, sample is added to respectively in same PCR pipe, in operation except often forget add indivedual reagent shortcoming except, taking repeatedly can pollute reagent, affects follow-up use.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of reduce fluorescence quantitative PCR reaction solution setup time fluorescence quantitative PCR reaction solution and test kit and fluorescence quantifying PCR method.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: provide a kind of fluorescence quantitative PCR reaction solution, comprise following raw material to be prepared from: methyl-sulphoxide, bovine serum albumin, DNTP, 10 × SYBR fluorescence dye, 10 × PCR buffer, solute percent by volume is glycerine solution and the Ex-Taq enzyme of 30%.
Another technical scheme of the present invention is for providing a kind of PCR kit for fluorescence quantitative, and it comprises above-mentioned fluorescence quantitative PCR reaction solution.
Another technical scheme of the present invention, for providing a kind of fluorescence quantifying PCR method, comprises the step using above-mentioned fluorescence quantitative PCR reaction solution.
Beneficial effect of the present invention is: the fluorescence quantitative PCR reaction solution that present invention optimizes PCR experiment, particularly add a certain proportion of glycerine solution, to ensure the activity of Ex-Taq enzyme, the combination adding BSA and DMSO ensure that the stability of Tag enzyme and active and reduce the formation of primer dimer, reduce fluorescence quantitative PCR reaction solution setup time, gained PCR reaction solution can at-20 DEG C, shelf time is 2 years, existing collocation method is as methyl-sulphoxide by the medicine required for PCR reaction solution, bovine serum albumin, DNTP, Ex-Taq enzyme etc. is kept in its optimum temperuture separately, mixed etc. when will configure quantitative fluorescent PCR system, all now with the current during each configuration scheme, relative trouble.The present invention by research and development are a kind of can the test kit not affecting again its amplification efficiency of disposable configuration fluorescence quantitative PCR reaction solution.Be convenient to commercial production.Realize once configuring, take at any time, significantly reduce the setup time of quantitative fluorescent PCR.
Accompanying drawing explanation
Fig. 1 is AK109848 gene amplification graphic representation;
Fig. 2 is that diagram is dissolved in AK109848 gene amplification;
Fig. 3 is AK070626 gene by fluorescence quantitative canonical plotting.
Embodiment
By describing technology contents of the present invention in detail, realized object and effect, accompanying drawing is coordinated to be explained below in conjunction with embodiment.
The design of most critical of the present invention is: the present invention adds glycerine in fluorescence quantitative PCR reaction solution, BSA and DMSO ensures Ex-Taq enzyme and other components stability and activity, make it can be kept at for a long time in-20 ° of C, the amplification efficiency improving PCR can be taken at any time.
Embodiment 1
A kind of fluorescence quantitative PCR reaction solution, comprise following raw material to be prepared from: 5-10 μ l methyl-sulphoxide, 5-10 μ l quality percent by volume is 0.1% bovine serum albumen solution, 5-20 μ l 10mM DNTP solution, 10-30 μ l 10 × SYBR fluorescence dye, 10-30 μ l 10 × PCR buffer, 10-50 μ l solute percent by volume is 30% glycerine solution and 3-6 μ l Ex-Taq enzyme.
A kind of fluorescence quantitative PCR reaction solution, comprise that following raw material is mixed to be formed: 5-10 μ l methyl-sulphoxide, 5-10 μ l quality percent by volume is 0.1% bovine serum albumen solution, 5-20 μ l 10mM DNTP solution, 10-30 μ l 10 × SYBR fluorescence dye, 10-30 μ l 10 × PCR buffer, 10-50 μ l solute percent by volume is 30% glycerine solution and 3-6 μ l Ex-Taq enzyme.
A kind of fluorescence quantitative PCR reaction solution, comprise following raw material to mix: 5-10 μ l methyl-sulphoxide, 5-10 μ l quality percent by volume is 0.1% bovine serum albumen solution, 5-20 μ l 10mM DNTP solution, 10-30 μ l 10 × SYBR fluorescence dye, 10-30 μ l 10 × PCR buffer, 10-50 μ l solute percent by volume is 30% glycerine solution and 3-6 μ l Ex-Taq enzyme.
Embodiment 2
A kind of fluorescence quantitative PCR reaction solution, mixed by following raw material: 7.5 μ l methyl-sulphoxides, 7.5 μ l quality percent by volumes are 0.1% bovine serum albumen solution, 10 μ l 10mM DNTP solution, 20 μ l 10 × SYBR fluorescence dyes, 20 μ l 10 × PCR buffer, 30 μ l solute percent by volumes are 30% glycerine solution and 5 μ l Ex-Taq enzymes.By rear centrifugal for the solution mixing configured, deposit in-20 DEG C of refrigerators.
Further, in above-mentioned fluorescence quantitative PCR reaction solution, the PH that described 10 × PCR buffer comprises 50-200mM is the Tris-Hcl damping fluid of 8.3,200-800mM Kcl and 10-20mM Mgcl
2.
The use of embodiment 3 fluorescence quantitative PCR reaction solution of the present invention
The making of quantitative fluorescent PCR typical curve: with paddy rice AK070626 gene for template construct typical curve, carries out diluted sample by with following table 1 pair of AK070626 gene.
Table 1
| Extension rate | Collocation method | |
| Standard substance 1 | Original solubility 1 | 1 μ l AK070626 gene PCR amplified production |
| Standard substance 2 | 10 | 1 μ l standard substance 1+9 μ l distilled water |
| Standard substance 3 | 100 | 1 μ l standard substance 2+9 μ l distilled water |
| Standard substance 4 | 1000 | 1 μ l standard substance 3+9 μ l distilled water |
| Standard substance 5 | 10000 | 1 μ l standard substance 4+9 μ l distilled water |
| Standard substance 6 | 100000 | 1 μ l standard substance 5+9 μ l distilled water |
From refrigerator, take out embodiment 2 fluorescence quantitative PCR reaction solution, configuration qRT-PCR system, should avoid strong illumination in layoutprocedure.The PCR system configuration of 20 μ l is as follows: 2 × fluorescence quantitative PCR reaction solution 10 μ l, primer R 1 μ l, primers F 1 μ l, masterplate 1 μ l, aqua sterilisa 7 μ l.Fluorescence quantitative PCR detection can be carried out after configuring
Pcr amplification program is as follows:
Solubility curve routine analyzer is as follows
95℃ 15s
60℃ 15s
95 DEG C are progressively warmed up to from 60 DEG C in 10min
95℃ 15s
END
Analysis is carried out and production standard curve to experimental result, the Y-axis of typical curve is the CT value of each standard substance, the X-axis of typical curve is the Log2 (extension rate) that a standard substance is corresponding, wherein Log2 (1)=0, Log2 (10)=3.32, Log2 (100)=6.65, Log2 (1000)=9.97, Log2 (10000)=13.29, Log2 (100000)=16.61.
Fig. 1 is AK109848 gene amplification graphic representation, Fig. 2 is that diagram is dissolved in AK109848 gene amplification, and Fig. 3 is AK070626 gene by fluorescence quantitative canonical plotting, and table 2 is the CT value of different templates, the PCR primer can finding out amplification from solubility curve figure is single band, without nonspecific products.Can find out that from canonical plotting this test kit has good expanding effect.
Table 2
| Template | CT value |
| Standard substance 1 | 23.69 |
| Standard substance 2 | 19.88 |
| Standard substance 3 | 15.75 |
| Standard substance 4 | 12.84 |
| Standard substance 5 | 8.81 |
| Standard substance 6 | 4.63 |
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalents utilizing specification sheets of the present invention and accompanying drawing content to do, or be directly or indirectly used in relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (6)
1. a fluorescence quantitative PCR reaction solution, is characterized in that, comprises following raw material and is prepared from: methyl-sulphoxide, bovine serum albumin, DNTP, 10 × SYBR fluorescence dye, 10 × PCR buffer, and solute percent by volume is glycerine solution and the Ex-Taq enzyme of 30%.
2. fluorescence quantitative PCR reaction solution according to claim 1, it is characterized in that, comprise following raw material to be prepared from: 5-10 μ l methyl-sulphoxide, 5-10 μ l quality percent by volume is the bovine serum albumen solution of 0.1%, 5-20 μ l10mM DNTP solution, 10-30 μ l10 × SYBR fluorescence dye, 10-30 μ l10 × PCR buffer, 10-50 μ l solute percent by volume is 30% glycerine solution and 3-6 μ l Ex-Taq enzyme.
3. fluorescence quantitative PCR reaction solution according to claim 1, it is characterized in that, comprise following raw material to be prepared from: 7.5 μ l methyl-sulphoxides, 7.5 μ l quality percent by volumes are 0.1% bovine serum albumen solution, 10 μ l10mM DNTP solution, 20 μ l10 × SYBR fluorescence dyes, 20 μ l10 × PCR buffer, 30 μ l solute percent by volumes are 30% glycerine solution and 5 μ l Ex-Taq enzymes.
4. fluorescence quantitative PCR reaction solution according to claim 1, is characterized in that, the PH that described 10 × PCR buffer comprises 50-200mM is the Tris-Hcl damping fluid of 8.3,200-800mM Kcl and 10-20mM Mgcl
2.
5. a PCR kit for fluorescence quantitative, is characterized in that, it comprises the fluorescence quantitative PCR reaction solution described in any one of claim 1-4.
6. a fluorescence quantifying PCR method, is characterized in that, comprises the step of the fluorescence quantitative PCR reaction solution used described in any one of claim 1-4.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107794297A (en) * | 2017-11-09 | 2018-03-13 | 先正达参股股份有限公司 | The copy number based on real-time fluorescence quantitative PCR is improved using dimethyl sulfoxide (DMSO) to analyze |
| CN109337965A (en) * | 2018-09-04 | 2019-02-15 | 江苏中济万泰生物医药有限公司 | A kind of fluorescent dye and electrophoresis PCR double-purpose buffer |
| CN109423526A (en) * | 2017-08-29 | 2019-03-05 | 湖北省疾病预防控制中心 | Mosquito matchmaker's infectious disease pathogens premix fluorescence PCR detection reagent and kit |
| CN109536587A (en) * | 2018-12-31 | 2019-03-29 | 吴江近岸蛋白质科技有限公司 | A kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109423526A (en) * | 2017-08-29 | 2019-03-05 | 湖北省疾病预防控制中心 | Mosquito matchmaker's infectious disease pathogens premix fluorescence PCR detection reagent and kit |
| CN107794297A (en) * | 2017-11-09 | 2018-03-13 | 先正达参股股份有限公司 | The copy number based on real-time fluorescence quantitative PCR is improved using dimethyl sulfoxide (DMSO) to analyze |
| WO2019090971A1 (en) * | 2017-11-09 | 2019-05-16 | 先正达参股股份有限公司 | Improvement in copy number analysis based on real-time fluorescence quantitative pcr by using dimethyl sulfoxide |
| CN109337965A (en) * | 2018-09-04 | 2019-02-15 | 江苏中济万泰生物医药有限公司 | A kind of fluorescent dye and electrophoresis PCR double-purpose buffer |
| CN109337965B (en) * | 2018-09-04 | 2022-01-25 | 江苏中济万泰生物医药有限公司 | Fluorescent dye and electrophoresis PCR (polymerase chain reaction) dual-purpose buffer solution |
| CN109536587A (en) * | 2018-12-31 | 2019-03-29 | 吴江近岸蛋白质科技有限公司 | A kind of efficient multiple fluorescence quantitative PCR kit and preparation method thereof |
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Application publication date: 20150128 |