CN108728518A - Real-time fluorescence quantitative PCR probe assay and its reaction solution and kit - Google Patents
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Abstract
The invention discloses a kind of real-time fluorescence quantitative PCR probe assay and its reaction solution and kits.The reaction solution includes the trehalose of PCR buffer solutions, Taq thermal startings enzyme, dNTP, the glycine betaine of final concentration of 0.04~0.05M, final concentration no more than 0.05M and the tetramethyl ammonium chloride of final concentration of 30~40mM, it is also further added with magnesium chloride in the reaction solution, until final concentration of 3.0~5.0mM of magnesium chloride.By glycine betaine and trehalose that certain concentration is added in reaction solution; the normal activity of Taq thermal starting enzymes can be protected at high temperature; the tetramethyl ammonium chloride of certain concentration is added; the specificity of PCR reactions can be improved; and further improve the concentration of magnesium chloride in reaction solution; while ensureing to have no effect on specific amplification, the yield and efficiency of amplification are significantly improved, can effectively shorten and expand the required time.
Description
Technical field
The present invention relates to biochemistry detection technical field, more particularly, to a kind of real-time fluorescence quantitative PCR probe assay and
Its reaction solution and kit.
Background technology
Real-time fluorescence quantitative PCR (Real-time fluorescent quantitave PCR, qPCR) is by the U.S.
Applied Biosystems companies took the lead in releasing in 1996, be widely used in Gene expression differential display, core at present
Basic scientific researches and the clinics such as sour copy number detection, single nucleotide polymorphism analysis, oncogene detection, pre-natal diagnosis
The fields such as diagnosis, the detection of pathogenic microorganism, disease research and medicament research and development.The technology can be divided into non-specific fluorescence dye method
With specificity fluorescent probe assay, wherein detected and quantitative aspect in specific gene, using it is most be that Taqman and MGB is visited
Needle detection method also has the reaction solution qPCR probe mix of corresponding quantitative fluorescent PCR probe assay in the market.Using real
When quantitative fluorescent PCR application in, sample is often more precious and content is low, and the length in reaction time is to stability and detection spirit
On the contrary sensitivity influences very big, and the reaction time is short to be advantageously ensured that stability and improve the sensitivity of detection, then can influence detection and tie
Fruit.However, most of the reaction solution qPCR probe mix of real-time fluorescence quantitative PCR probe assay currently on the market react
Time, the reaction time was long in 2h or so；QPCR probe mix of some commercializations shorten the reaction time, but stability meeting
It substantially reduces, it is difficult to while reaching higher amplification efficiency and detection sensitivity.
Invention content
Based on this, it is necessary to provide one kind be conducive to shorten the reaction time, ensure stability and can improve detection it is sensitive
The real-time fluorescence quantitative PCR probe assay and its reaction solution and kit of degree.
A kind of reaction solution of real-time fluorescence quantitative PCR probe assay, including PCR buffer solutions, Taq thermal startings enzyme, dNTP,
The trehalose of glycine betaine, final concentration no more than 0.05M of final concentration of 0.04~0.05M and the tetramethyl of final concentration of 30~40mM
Ammonium chloride is also further added with magnesium chloride in the reaction solution, until final concentration of 3.0~5.0mM of magnesium chloride.
It is 8.0~9.0 that the PCR buffer solutions, which include a concentration of 20~55mM, pH, in one of the embodiments,
The magnesium chloride of Tris-HCl, the potassium chloride of a concentration of 25~75mM and a concentration of 2.0~2.5mM.
The Taq thermal startings enzyme is TAKARA Ex Taq HS thermal starting enzymes in one of the embodiments,.
The final concentration of the thermal starting enzyme is 0.025~0.05U/ μ l in one of the embodiments,.
The final concentration of the dNTP is 0.2~0.25mM in one of the embodiments,.
The final concentration of 0.05M of glycine betaine described in the reaction solution in one of the embodiments,；The trehalose
Final concentration of 0.05M；The final concentration of 35mM of the tetramethyl ammonium chloride；The final concentration of 3.0mM of the magnesium chloride；It is described
PCR buffer solutions include the Tris-HCl that a concentration of 50mM, pH are 8.8, the potassium chloride of a concentration of 50mM and a concentration of 2.0mM
Magnesium chloride；The final concentration of the Taq thermal startings enzyme is 0.05U/ μ l；The final concentration of the dNTP is 0.2mM.
A kind of kit of real-time fluorescence quantitative PCR probe assay, including it is real-time glimmering described in any of the above-described embodiment
The reaction solution of Fluorescent Quantitative PCR probe assay.
The kit further includes wild type Kras primer and probes in one of the embodiments,；In the primer
Upstream primer sequence is selected from least one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4,
Downstream primer sequence is selected from least one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8；
The sequence of the probe is selected from least one of SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11.
The upstream primer sequence in the primer is SEQ ID No.4, downstream primer sequence in one of the embodiments,
It is SEQ ID No.6；The sequence of the probe is SEQ ID No.10.
A kind of real-time fluorescence quantitative PCR probe assay, includes the following steps：
Step 1：Template DNA, primer and probe are added into reaction solution, reaction system is made, the reaction solution includes
PCR buffer solutions, Taq thermal startings enzyme, dNTP, the glycine betaine of final concentration of 0.04~0.05M, final concentration are no more than the sea of 0.05M
The tetramethyl ammonium chloride of algae sugar and final concentration of 35mM is also further added with magnesium chloride in the reaction solution, until magnesium chloride
Final concentration of 3.0~5.0mM；
Step 2：Real-time fluorescence quantitative PCR amplification, fluorescence intensity are carried out to the reaction system.
It is 8.0~9.0 that the PCR buffer solutions, which include a concentration of 20~55mM, pH, in one of the embodiments,
The magnesium chloride of Tris-HCl, the potassium chloride of a concentration of 25~75mM and a concentration of 2.0~2.5mM；
The Taq thermal startings enzyme is TAKARA Ex Taq HS thermal starting enzymes, and the final concentration of the thermal starting enzyme is 0.025
~0.05U/ μ l；
The final concentration of the dNTP is 0.2~0.25mM
The PCR buffer solutions include the Tris-HCl, concentration that a concentration of 50mM, pH are 8.8 in one of the embodiments,
For the potassium chloride of 50mM and the magnesium chloride of a concentration of 3.0mM；
The final concentration of the thermal starting enzyme is 0.05U/ μ l；
The final concentration of the dNTP is 0.2mM.
In one of the embodiments, real-time fluorescence quantitative PCR amplification be using DNA be denaturalized-extend two-step method into
Row PCR amplification.
Reaction solution, kit and the real-time fluorescence quantitative PCR probe in detecting of above-mentioned real-time fluorescence quantitative PCR probe assay
Method can protect Taq hot at high temperature by the glycine betaine (Bet) and trehalose (Tre) of the addition certain concentration in reaction solution
The normal activity for starting enzyme, is added the tetramethyl ammonium chloride (TMAC) of certain concentration, can improve the specificity of PCR reactions, and
And the concentration of magnesium chloride in reaction solution is further improved, while ensureing to have no effect on specific amplification, significantly improve
The yield and efficiency of amplification can effectively shorten and expand the required time.
Use the reaction solution of above-mentioned real-time fluorescence quantitative PCR probe assay, kit and real-time fluorescence quantitative PCR probe
Detection method only needs 1 hour or so to obtain a result, and amplification efficiency high (up to 95~110%), the good (standard of stability
Deviation is between 0.02~1.12%), and it is able to detect that the target gene of 0.03 copy, a series of this index all reaches
The standard of quantitative experiment.
Description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of gDNA；
Fig. 2 is (corresponding respectively to 100ng, 10ng, 1ng with Genstar Mix using the reaction solution of one embodiment of the invention
A, b, gDNA c) compare the amplification curve of wild type Kras genes in figure；
Fig. 3 is using the reaction solution of one embodiment of the invention with Genstar Mix to 100ng, 10ng, 1ng (corresponding diagram respectively
Middle a, b, gDNA c) compare the amplification curve of GAPDH genes；
Fig. 4 is Kras primed probe amplification efficiency curves；
Fig. 5 is GAPDH primed probe amplification efficiency curves.
Specific implementation mode
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing
Data represent presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this
Literary described embodiment.Make understanding to the disclosure more on the contrary, purpose of providing these embodiments is
Thorough and comprehensive.
Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention
The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the Listed Items of pass.
Embodiment 1
Main agents explanation involved by 1.
Glycine betaine (Bet) and trehalose (Tre) analysis are pure, are purchased from sigma companies；Tetramethyl ammonium chloride and magnesium chloride analysis
It is pure, it is purchased from Shanghai biotechnology Services Co., Ltd；Micro-example genome DNA extracting reagent kit (centrifugal column type) is purchased from
Tiangeng biochemical technology Co., Ltd；TAKARA Ex Taq HS are purchased from upper Hypon bioengineering Co., Ltd；Genstar qPCR
Probe Mix are purchased from Beijing bio tech ltd Kang Run.
TAKARA Ex Taq HS thermal starting enzymes are used in the reaction solution of real-time fluorescence quantitative PCR probe assay, enzyme
Dosage be 0.025~0.05U/ μ L, dNTP concentration be 0.2~0.25mM, and using containing 20~55mM pH8.0~
The PCR buffer solutions of 9.0Tris-HCl, 2.0~2.5mM magnesium chlorides and 250~750mM potassium chloride；The present embodiment on this basis,
By control variate method, a certain amount of PCR reinforcing agents are added：Concentration is not higher than 0.05M Bet, concentration is not higher than 0.05M Tre,
Concentration is not higher than 3.0mM MgCl2And concentration is not higher than 40mM TMAC.
On the basis of the above, a kind of PCR reagents recombinations are finally determined, have also determined that final qPCR probe assays
Reaction solution ingredient it is as follows：The TAKARA Ex Taq HS thermal startings enzyme of 0.05U/ μ L, the dNTP of 0.2mM, 50mM pH8.0
The potassium chloride of Tris-HCl, 50mM, the magnesium chloride (for the final concentration in reaction solution) of 3.0mM, the Bet of 0.05M, 0.05M's
The TMAC of Tre, 35mM.
The primer and probe of wild type Kras (wild type Kras)：
Sense primer is any one in following 4：
5'-TGAATCGAAACTTGTGGTAG-3'(SEQ ID No.1)、
5'-ATCGAAACTTGTGGTAGTTG-3'(SEQ ID No.2)、
5'-TGACTGAATATAAACTTGTGGTAG-3'(SEQ ID No.3)、
5'-TTATTATAAGGCCTGCTGAAA-3'(SEQ ID No.4)；
Downstream primer is any one in following 4：
5'-TCCTGCACCAGTAATATGC-3'(SEQ ID No.5)、
5'-CTATTGTTGGATCATATTCGTC-3'(SEQ ID No.6)、
5'-CTGAATTAGCTGTATCGTCAAG-3'(SEQ ID No.7)、
5'-CCTTGTGTGTGACATGTTCTA-3'(SEQ ID No.8)；
Probe sequence is any one in following 3：
FAM-GAGCTGGTGGCGTAGG-MGB(SEQ ID No.9)、
FAM-CTGGTGGCGTAGGCAAG-MGB(SEQ ID No.10)、
FAM-GAGCTGGTGGCGTAGGCAAG-MGB(SEQ ID No.11)。
The primer and probe sequence of GAPDH：
Sense primer：5'-GCTCCCTCTTTCTTTGCAGCAAT-3'(SEQ ID No.12)；
Downstream primer：5'-TACCATGAGTCCTTCCACGATAC-3'(SEQ ID No.13)；
Probe sequence：VIC-TCCTGCACCACCAACTGCTTAGCACC-BHQ1(SEQ ID No.14).
2. key step
2.1 extract the genomic DNA of people with micro-example genome DNA extracting reagent kit, use agarose gel electrophoresis
Identify gDNA, the results are shown in Figure 1, it will be seen from figure 1 that extraction gDNA successes.
By Nanodrop measurement nucleic acid concentrations and purity, (between 1.8~2.0, A260/280 is more than A260/230
2.0) after normal, which can be just used as to the template DNA of follow-up realtime fluorescent quantitative PCR experiment.
The confirmatory experiment of 2.2 qPCR probe assays
2.2.1 the primer and probe of wild type Kras carries out qPCR experiments.
QPCR reaction solutions and Genstar qPCR probe mix obtained by the present embodiment is used to prepare the reactant of 20 μ L respectively
System.The upstream and downstream primer of wild type Kras and probe (SEQ ID No.4, SEQ ID No.6 and SEQ in reaction system
ID No.10) dosage be 200nmol, the additive amount of template is 5 μ L.Template is will to extract gained gDNA templates by 10 times of ladders
Degree is diluted to 100ng/5 μ L, 10ng/5 μ L, 1ng/5 μ L, 0.1ng/5 μ L, 0.01ng/5 μ L, and each concentration gradient is with this reality
The reaction solution and Genster qPCRprobe mix for applying example are compared, and 3 parallel laboratory tests are arranged in each concentration gradient.
2 footworks of qPCR experimental arrangements：95 DEG C, 1min；(95 DEG C of 5s, 58 DEG C, 30s) 45 cycles.QPCR instruments count
Entire quantitative fluorescent PCR reaction process only needs 61min can be completed.
2.2.2 the primer and probe of GAPDH carries out the primer and probe of qPCR experiment and above-mentioned wild type Kras
Progress qPCR experimental setups are essentially identical, and what difference was is the primer and probe of GAPDH.
3. experimental result
Table 1 is Cq values of the gDNA to wild type Kras gene magnifications of 100ng, 10ng, 1ng, by the present embodiment
The reaction solution and Genstar qPCR probe mix of qPCR probe assays are compared, the results showed that, same concentrations template
In the case of, small about 1.5 cycles of Cq values of the qPCR reaction solution ratio Genstar qPCR probe mix of the present embodiment.
Table 1
Table 2 is the Cq values that the gDNA of 100ng, 10ng, 1ng expand housekeeping gene GAPDH, by the present embodiment the present embodiment
The reaction solution and Genstar qPCR probe mix of gained qPCR probe assays are compared, the results showed that, same concentrations
In the case of template, the Cq values of qPCR reaction solution ratio Genstar qPCR probe mix obtained by the present embodiment are 3.7~5.5 small
Cycle.
Table 2
By the Cq values comparison of Tables 1 and 2 and the amplification curve comparison of Fig. 2 and Fig. 3, the present embodiment is all illustrated
QPCR reaction solutions are either in the amplification of colorectal cancer related gene Kras, or in the amplification of housekeeping gene GAPDH genes, all
It is substantially better than Genstar qPCR probe mix, when same template concentration, the Cq ratios of the qPCR reaction solutions of the present embodiment
The Cq of Genstar qPCR probe mix wants forward, and the more forward qPCR reaction solutions for illustrating the present embodiment of Cq values are to low concentration
Detection when template amplification has the advantage of bigger.In figure 2 and figure 3, the qPCR reaction solution end point fluorescence values and song of the present embodiment
Linear index phase slope ratio Genstar qPCR probe mix's will be high, and exponent phase slope has reacted amplification efficiency, bent
The linear index phase, slope was bigger, and amplification efficiency is higher, illustrated the qPCR reaction solution amplification efficiency highers of the present embodiment.
Using the logarithm of template concentrations as abscissa, corresponding Cq values are that ordinate does figure, and slope k and the amplification of straight line are imitated
The relationship of rate E：E=10-1/k- 1, see that Fig. 4 wild type Kras primed probe amplification efficiency curves and Fig. 5 GAPDH primers are visited
Needle amplification efficiency curve, the amplification efficiency to Kras are 107%, R2=0.998, the amplification efficiency to GAPDH is 95%, R2=
0.988.Linearly dependent coefficient (R2) >=0.98, amplification efficiency between 89.57%~110.17% (- 3.1 >=slope >=-
3.6)), all meet the international specification to fluorescent quantitative PCR experiment, also indicate that the qPCR reaction solutions of the present embodiment draw what is had
When object and probe are expanded, there is very high amplification efficiency.Moreover, the detection that the qPCR reaction solutions of the present embodiment can be stablized
To the gDNA of 0.01ng, and Genstar is just difficult to stable detection for 1ng templates below and arrives, it can be seen that the present embodiment
QPCR reaction solutions can detect the sample size of lower concentration.
3 Duplicate Samples, the standard deviation of calculating illustrate the present embodiment all between 0.02-1.12% in table 3 and table 4
QPCR reaction solutions there is good stability and repeatability, (European Union's regulation is fixed well below European Union's specified value for standard deviation
In amount experiment 25%) relative standard deviation RSD is less than or equal to.
The corresponding Cq values of qPCR reaction solutions amplification wild type Kras of the present embodiment when 3 different templates concentration of table
The corresponding Cq values of qPCR reaction solutions amplification GAPDH of the present embodiment when 4 different templates concentration of table
| GDNA concentration (ng) | Cq1 | Cq2 | Cq3 | Average Cq values | Standard deviation |
| 100 | 25.28 | 25.28 | 25.27 | 25.27667 | 0.02% |
| 10 | 28.56 | 28.54 | 28.65 | 28.58333 | 0.20% |
| 1 | 31.86 | 31.67 | 31.73 | 31.75333 | 0.31% |
| 0.1 | 34.92 | 35.63 | 35.33 | 35.29333 | 1.01% |
The present embodiment passes through to the dense of magnesium ion, glycine betaine (Bet), trehalose (Tre) and tetramethyl ammonium chloride (TMAC)
Degree is in optimized selection, it is determined that the reaction solution of preferable qPCR probe assays, process is the study found that magnesium ion concentration pair
The influence of PCR amplification efficiency is very big, and excessive concentration can reduce the specificity of PCR amplification, and concentration is too low, influence PCR amplification yield very
To making PCR amplification fail, therefore, on the basis of magnesium ion concentration of the present embodiment in traditional PCR buffer solutions, further add
Add certain density magnesium ion, while ensureing and not influencing specific amplification, is remarkably improved the amplification yield of PCR；
It, can effective protection Taq enzyme be just at high temperature simultaneously by adding certain density glycine betaine and trehalose in reaction solution
Often activity；And a certain concentration tetramethyl ammonium chloride is added, the specificity of PCR reactions can be significantly improved, ensure the inspection of probe
Survey sensitivity.
For the reaction solution of the qPCR probe assays of the present embodiment when qPCR is detected, the entire PCR reaction time only needs 1h
Left and right can obtain a result, and amplification efficiency is high (95-110%), stability it is good (standard deviation 0.02-1.12% it
Between), it is able to detect that the wild type Kras of 0.03 copy, a series of this index have reached the standard of quantitative experiment.And
And by housekeeping gene GAPDH and colorectal cancer related gene Kras, the feasibility of the qPCR probe mix is demonstrated.It should
The reaction solution of qPCR probe assays can steadily detect the template of lower concentration, and amplification efficiency is high, these are all real
When fluorescent quantitative PCR technique extensive use lay a good foundation.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
SEQUENCE LISTING
<110>Shunde designing and research sciences（Shunde innovative design research institute）
<120>Real-time fluorescence quantitative PCR probe assay and its reaction solution and kit
<160> 14
<170> PatentIn version 3.3
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tgaatcgaaa cttgtggtag 20
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atcgaaactt gtggtagttg 20
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tgactgaata taaacttgtg gtag 24
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Claims (10)
1. a kind of reaction solution of real-time fluorescence quantitative PCR probe assay, which is characterized in that opened including PCR buffer solutions, Taq heat
Dynamic enzyme, dNTP, the glycine betaine of final concentration of 0.04~0.05M, final concentration no more than the trehalose of 0.05M and final concentration of 30~
The tetramethyl ammonium chloride of 40mM is also further added with magnesium chloride in the reaction solution, until final concentration of the 3.0 of magnesium chloride~
5.0mM。
2. the reaction solution of real-time fluorescence quantitative PCR probe assay as described in claim 1, which is characterized in that the PCR is slow
Fliud flushing includes the Tris-HCl that a concentration of 20~55mM, pH are 8.0~9.0, the potassium chloride and concentration of a concentration of 25~75mM
For the magnesium chloride of 2.0~2.5mM.
3. the reaction solution of real-time fluorescence quantitative PCR probe assay as described in claim 1, which is characterized in that the Taq heat
It is TAKARA Ex Taq HS thermal starting enzymes to start enzyme.
4. the reaction solution of real-time fluorescence quantitative PCR probe assay as claimed in claim 3, which is characterized in that the heat opens
The final concentration of dynamic enzyme is 0.025~0.05U/ μ l.
5. the reaction solution of real-time fluorescence quantitative PCR probe assay as described in claim 1, which is characterized in that the dNTP
Final concentration be 0.2~0.25mM.
6. a kind of kit of real-time fluorescence quantitative PCR probe assay, which is characterized in that including appointing in such as Claims 1 to 5
The reaction solution of real-time fluorescence quantitative PCR probe assay described in one.
7. the kit of real-time fluorescence quantitative PCR probe assay as claimed in claim 6, which is characterized in that further include open country
Raw type Kras primer and probes；Upstream primer sequence in the primer is selected from SEQ ID No.1, SEQ ID No.2, SEQ ID
At least one of No.3 and SEQ ID No.4, downstream primer sequence are selected from SEQ ID No.5, SEQ ID No.6, SEQ ID
At least one of No.7 and SEQ ID No.8；The sequence of the probe is selected from SEQ ID No.9, SEQ ID No.10 and SEQ
At least one of ID No.11.
8. a kind of real-time fluorescence quantitative PCR probe assay, which is characterized in that include the following steps：
Step 1：Template DNA, primer and probe are added into reaction solution, reaction system is made, the reaction solution includes PCR slow
Fliud flushing, Taq thermal startings enzyme, dNTP, the glycine betaine of final concentration of 0.04~0.05M, final concentration no more than 0.05M trehalose and
The tetramethyl ammonium chloride of final concentration of 35mM is also further added with magnesium chloride in the reaction solution, until the final concentration of magnesium chloride
For 3.0~5.0mM；
Step 2：Real-time fluorescence quantitative PCR amplification, fluorescence intensity are carried out to the reaction system.
9. real-time fluorescence quantitative PCR probe assay as claimed in claim 8, which is characterized in that the PCR buffer solutions include
The potassium chloride for the Tris-HCl, a concentration of 25~75mM that a concentration of 20~55mM, pH are 8.0~9.0 and a concentration of 2.0~
The magnesium chloride of 2.5mM；
The Taq thermal startings enzyme is TAKARA Ex Taq HS thermal starting enzymes, the final concentration of the thermal starting enzyme is 0.025~
0.05U/μl；
The final concentration of the dNTP is 0.2~0.25mM.
10. real-time fluorescence quantitative PCR probe assay as claimed in claim 9, which is characterized in that the real time fluorescent quantitative
PCR amplification is to be denaturalized-extend two-step method using DNA to carry out PCR amplification.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804652A (en) * | 2019-12-03 | 2020-02-18 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for rapid detection of real-time quantitative PCR of DNA |
| CN111218502A (en) * | 2020-04-23 | 2020-06-02 | 圣湘生物科技股份有限公司 | Composition for improving qPCR detection performance, reaction solution, application and method |
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| CN105483289A (en) * | 2015-12-29 | 2016-04-13 | 深圳澳东检验检测科技有限公司 | Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system |
| CN105755172A (en) * | 2016-04-05 | 2016-07-13 | 深圳澳东检验检测科技有限公司 | Primer and probe for direct fluorescence RT-PCR detection on typing of foot-and-mouth disease viruses and kit |
| CN106498060A (en) * | 2016-11-03 | 2017-03-15 | 江苏然科生物技术有限公司 | A kind of fluorescence quantitative PCR reaction solution and method |
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| CN110804652A (en) * | 2019-12-03 | 2020-02-18 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for rapid detection of real-time quantitative PCR of DNA |
| CN110804652B (en) * | 2019-12-03 | 2023-07-25 | 郑州安图生物工程股份有限公司 | Additive, kit and reaction method for real-time quantitative PCR (polymerase chain reaction) for rapidly detecting DNA (deoxyribonucleic acid) |
| CN111218502A (en) * | 2020-04-23 | 2020-06-02 | 圣湘生物科技股份有限公司 | Composition for improving qPCR detection performance, reaction solution, application and method |
| CN111218502B (en) * | 2020-04-23 | 2020-07-21 | 圣湘生物科技股份有限公司 | Composition for improving qPCR detection performance, reaction solution, application and method |
| WO2021212771A1 (en) * | 2020-04-23 | 2021-10-28 | 圣湘生物科技股份有限公司 | Composition for improving qpcr test performance, reaction liquid, use, and method |
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