CN112176038A - Buffer composition - Google Patents
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- CN112176038A CN112176038A CN201910953826.2A CN201910953826A CN112176038A CN 112176038 A CN112176038 A CN 112176038A CN 201910953826 A CN201910953826 A CN 201910953826A CN 112176038 A CN112176038 A CN 112176038A
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- 239000000872 buffer Substances 0.000 title claims abstract description 49
- 239000000203 mixture Substances 0.000 title claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 91
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 59
- 239000007853 buffer solution Substances 0.000 claims abstract description 49
- 230000009089 cytolysis Effects 0.000 claims abstract description 40
- 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 abstract description 36
- 239000004094 surface-active agent Substances 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 150000003841 chloride salts Chemical class 0.000 claims abstract description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 28
- 235000011187 glycerol Nutrition 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 17
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 14
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 14
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 14
- 238000002347 injection Methods 0.000 claims description 11
- 239000007924 injection Substances 0.000 claims description 11
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 9
- 229920001223 polyethylene glycol Polymers 0.000 claims description 9
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 claims description 8
- 229920005862 polyol Polymers 0.000 claims description 6
- 150000003077 polyols Chemical class 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 102100034343 Integrase Human genes 0.000 claims description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 3
- 235000019329 dioctyl sodium sulphosuccinate Nutrition 0.000 claims description 3
- YHAIUSTWZPMYGG-UHFFFAOYSA-L disodium;2,2-dioctyl-3-sulfobutanedioate Chemical compound [Na+].[Na+].CCCCCCCCC(C([O-])=O)(C(C([O-])=O)S(O)(=O)=O)CCCCCCCC YHAIUSTWZPMYGG-UHFFFAOYSA-L 0.000 claims description 3
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 claims description 3
- OABYVIYXWMZFFJ-ZUHYDKSRSA-M sodium glycocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 OABYVIYXWMZFFJ-ZUHYDKSRSA-M 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- 150000005846 sugar alcohols Polymers 0.000 claims 2
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 claims 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 abstract description 8
- 238000012408 PCR amplification Methods 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 7
- 108020004707 nucleic acids Proteins 0.000 abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 abstract description 7
- 102000039446 nucleic acids Human genes 0.000 abstract description 7
- 230000010354 integration Effects 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 61
- 230000003321 amplification Effects 0.000 description 45
- 238000003199 nucleic acid amplification method Methods 0.000 description 45
- 239000013024 dilution buffer Substances 0.000 description 31
- 238000012360 testing method Methods 0.000 description 17
- 239000011550 stock solution Substances 0.000 description 16
- 238000003908 quality control method Methods 0.000 description 12
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000006166 lysate Substances 0.000 description 7
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 7
- 238000007865 diluting Methods 0.000 description 6
- 108020004463 18S ribosomal RNA Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000012139 lysis buffer Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012928 buffer substance Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The present invention provides a buffer composition comprising: glycerol, chloride salt, Tris-HCl, surfactant and water. The buffer solution composition can play a buffering role in sample lysis and PCR amplification reaction simultaneously, thereby providing technical support for integration of nucleic acid extraction and fluorescence PCR reaction of a sample.
Description
Technical Field
The present invention relates to the field of molecular biology, in particular, the present invention relates to buffer compositions, more particularly, the present invention relates to buffers.
Background
The existing fluorescent quantitative PCR reaction is generally divided into 4 steps: firstly, carrying out sample cracking and nucleic acid extraction, then preparing a fluorescent quantitative PCR reaction reagent, then adding the sample into a PCR reaction system, and finally carrying out on-machine detection. A sample lysis buffer is required during lysis of the sample and extraction of nucleic acids, and a PCR buffer is required during a PCR reaction.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventor finds that the existing sample lysis buffer and PCR buffer are different greatly in formula and components, the sample lysis buffer is specially used for sample lysis and sample nucleic acid extraction, the PCR buffer is specially used for PCR amplification reaction, and the two buffers cannot be used commonly. Based on the above problems, the inventors studied and developed a buffer composition through a great deal of experimental research, which can simultaneously play a buffering role in sample lysis and PCR amplification reactions, thereby providing technical support for the integration of nucleic acid extraction and fluorescence PCR reaction of a sample.
To this end, in a first aspect of the invention, the invention proposes a buffer. According to an embodiment of the invention, the buffer comprises: polyol, chloride salt, Tris-HCl, surfactant and water. In some embodiments, the polyol is glycerol. The buffer solution has a certain buffering effect on the sample lysate and a PCR reaction system, and the lysate after sample lysis can be directly added into the PCR reaction system to complete the PCR reaction without purification through the buffer dilution of the buffer solution, so that technical support is provided for the integration of nucleic acid extraction and PCR detection of the sample.
According to an embodiment of the present invention, the buffer solution may further include at least one of the following additional features:
according to an embodiment of the present invention, the surfactant includes at least one selected from the group consisting of Tween20 (Tween20), Tween 80, polyethylene glycol octylphenyl ether, Sodium Dodecyl Sulfate (SDS), sodium dodecylbenzene sulfonate, sodium dioctyl sulfosuccinate, sodium glycocholate.
According to an embodiment of the invention, the chloride salt comprises at least one selected from potassium chloride, sodium chloride, magnesium chloride.
According to an embodiment of the invention, the chloride salt is magnesium chloride and sodium chloride, the magnesium chloride has a concentration of 0.5 to 15mmol/L, such as 1.0, 1.5, 2.0, 3.0, 5.0, 6.0, 8.0, 10.0 or 12.0mmol/L, the sodium chloride has a concentration of 1 to 150mmol/L, such as 2, 4, 5, 10, 20, 40, 50, 60, 80, 100 or 120mmol/L, the surfactant has a concentration of 0.1 to 7%, such as 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 or 7.0%, the glycerol has a concentration of 2 to 20%, such as 3, 5, 7, 8, 9, 12, 15 or 18%, based on the total volume of the buffer. When the surfactant is a solid such as sodium lauryl sulfate, the concentration of the surfactant is a mass volume concentration, which means the mass of the surfactant per 100mL of the solution, and is expressed in g, for example, the concentration of the sodium lauryl sulfate is 0.1 to 7%, which means the mass of the sodium lauryl sulfate per 100mL of the buffer is 0.1 to 7 g. When the surfactant is liquid such as polyethylene glycol octyl phenyl ether or tween20, the concentration of the surfactant is volume concentration, which means the volume of the surfactant in each 100mL of solution, and the unit is mL, for example, the concentration of the polyethylene glycol octyl phenyl ether or tween20 is 0.1-7%, which means the volume of the polyethylene glycol octyl phenyl ether or tween20 in each 100mL of buffer solution is 0.1-7 mL. The concentration of glycerol is 2-20% by volume, and the volume of glycerol in each 100mL of buffer solution is 2-20 mL. The inventors have found that when the concentrations of the respective components in the buffer are within this range, the buffer can more effectively perform a buffering function in both the sample lysis and the PCR amplification reaction.
According to the embodiment of the invention, the chloride salt is magnesium chloride and sodium chloride, the concentration of the magnesium chloride is 1.5-10 mmol/L, the concentration of the sodium chloride is 5-100 mmol/L, the concentration of the surfactant is 0.1-5%, and the concentration of the glycerol is 5-10% based on the total volume of the buffer solution. The inventors have found that when the weight parts of the respective components in the buffer solution are within this range, the buffer solution can more effectively perform a buffering function in both the sample lysis and the PCR amplification reaction.
According to an embodiment of the present invention, the Tris-HCl is provided in a form dissolved in water, meaning that the buffer of the present invention is added in a form of Tris-HCl aqueous solution. It should be noted that aqueous Tris-HCl is a buffer substance commonly used in the art, and may be self-prepared, for example, by slowly adjusting to a predetermined pH with Tris base and hydrochloric acid, or may be purchased directly.
According to an embodiment of the invention, the Tris-HCl in water solution (Tris-HCl in water) has a pH of 7.5 to 8.0, such as 7.6, 7.7, 7.8, 7.9 or 8.0. It should be noted that the pH does not mean the pH of the buffer, but the pH of a solution in which Tris-HCl is dissolved in water. According to the embodiment of the invention, when the pH value of the solution formed by dissolving Tris-HCl in water is 7.5-8.0, the buffering effect of Tris-HCl on the buffer solution is better. In some embodiments, the solution of Tris-HCl in water has a pH of 7.6.
According to an embodiment of the invention, the concentration of Tris-HCl is 1-100 mmol/L, such as 3, 5, 7, 10, 20, 30, 40, 50, 70, 90 or 100mmol/L, based on the total volume of the buffer. The concentration of Tris-HCl is understood in the light of routine knowledge of those skilled in the art. According to the embodiment of the invention, when the concentration of Tris-HCl is 1-100 mmol/L, the buffer solution has a better buffering effect of Tris-HCl. In some embodiments, the concentration of Tris-HCl is 5 to 50 mmol/L.
In a second aspect of the invention, a buffer is provided. According to an embodiment of the invention, the buffer comprises, based on the total volume of the buffer: the detergent composition comprises magnesium chloride with the concentration of 1.5-10 mmol/L, sodium chloride with the concentration of 5-100 mmol/L, a surfactant with the concentration of 0.1-5%, glycerol with the concentration of 5-10%, Tris-HCl with the concentration of 5-50 mmol/L and water, wherein the surfactant is Tween20, sodium dodecyl sulfate or polyethylene glycol octyl phenyl ether. The buffer solution provided by the embodiment of the invention can more effectively play a buffering role in sample lysis and PCR amplification reaction simultaneously.
In a third aspect of the invention, the invention provides a PCR reaction system. Referring to fig. 7, the system includes, according to an embodiment of the present invention: a sample-containing unit 100, in which a lysis material lyophilized powder is disposed in the sample-containing unit 100, and the sample-containing unit 100 is provided with a first liquid outlet/inlet 110; a buffer solution containing unit 200, wherein a buffer solution is arranged in the buffer solution containing unit 200, and the buffer solution containing unit 200 is provided with a buffer solution outlet 210; the PCR reaction unit 300 is internally provided with reverse transcriptase and PCR raw material freeze-dried powder, the PCR reaction unit 300 is provided with a cracked sample mixed liquid inlet 310 and a PCR reaction liquid outlet 320, and the PCR reaction liquid outlet 320 is connected with the buffer liquid outlet 210 through a fourth pipeline 940; and a piston unit 400, wherein the piston unit 400 comprises an injection chamber 410 and a piston 420, the injection chamber 410 is provided with a second liquid inlet/outlet 411, the second liquid inlet/outlet 411 is connected with the first liquid inlet/outlet 110 through a first pipeline 910, the second liquid inlet/outlet 411 is connected with the buffer liquid outlet 210 through a second pipeline 920, and the second liquid inlet/outlet 411 is connected with the lysed sample mixed liquid inlet 310 through a third pipeline 930; wherein the buffer comprises: polyol, chloride salt, Tris-HCl, surfactant and water. In some embodiments, the polyol is glycerol. It should be noted that the above two descriptions regarding the buffer solution are also applicable to the PCR reaction system.
The PCR reaction system according to the embodiment of the present invention connects the sample accommodating unit 100, the buffer accommodating unit 200, the PCR reaction unit 300, and the piston unit 400 to each other through the microfluidic circuit; meanwhile, each unit is an independently arranged unit so as to store different reactants before use, and the long-term storage of the reactants under the condition of nonuse is facilitated. For example, the independent arrangement of the sample-accommodating unit 100 facilitates the separate addition of the sample, simplifies the sample addition operation, and also facilitates the long-term preservation of the sample. Referring to fig. 7, first, the piston 420 is pulled outward to a position such that the buffer solution in the buffer solution containing unit 200 flows to the injection chamber 410; then, the piston 420 is moved back and forth, so that part of the buffer solution in the injection chamber 410 enters the sample containing unit 100 and is uniformly mixed with the lysis freeze-dried powder and the sample in the sample containing unit 100; heating the sample accommodating unit 100 to a set temperature, so that the sample in the sample accommodating unit 100 is fully cracked at the set temperature; after the lysis is completed, the piston 420 is pulled outward to a certain position again, so that the lysed sample mixture in the sample-containing unit 100 flows to the injection chamber 410; then, the piston 420 is moved back and forth, so that the lysed sample mixed liquid in the injection chamber 410 returns to the buffer solution containing unit 200, and is uniformly mixed with the residual buffer solution in the buffer solution containing unit 200, thereby diluting the lysed sample mixed liquid and reducing the concentration of impurities therein; thereafter, the piston 420 is pulled outward to a certain position again, so that the diluted sample mixture in the buffer solution containing unit 200 flows to the injection chamber 410; then, the piston 420 is moved back and forth, so that the diluted sample mixed solution in the injection chamber 410 enters the PCR reaction unit 300 and is uniformly mixed with the reverse transcriptase in the PCR reaction unit 300 and the freeze-dried powder of the PCR raw material; and finally, performing PCR temperature heating control on the PCR reaction unit 300 so as to finally complete the PCR amplification reaction. According to the PCR reaction system provided by the embodiment of the invention, the PCR reaction liquid outlet is connected with the buffer liquid outlet through the fourth pipeline, so that the pressure system communication between the PCR reaction liquid outlet and the buffer liquid outlet is formed, and therefore, excessive reaction liquid in the PCR reaction unit can smoothly flow out to the fourth pipeline through the reaction liquid outlet. Further, in the PCR reaction system according to the embodiment of the present invention, a valve or other switch may be flexibly designed at a suitable position of the microfluidic circuit so as to control a communication state of the piston unit 400 with the sample-accommodating unit 100, the buffer-accommodating unit 200, or the PCR reaction unit 300. In addition, the movement of the piston and the control of valves or other switches can be flexibly designed into other mechanical devices for automation. Therefore, according to the PCR reaction system provided by the embodiment of the invention, the piston unit is respectively connected with the sample containing unit, the buffer solution containing unit and the PCR reaction unit, so that a full-automatic process from sample nucleic acid extraction to reagent mixing and finally to PCR reaction is realized, the problem that a professional person needs to operate in a professional experiment environment in the traditional PCR experiment process is solved, the PCR reaction can be completed without the professional person, errors caused by manual operation are reduced, the working efficiency of PCR reaction is greatly improved, and the cost of human resources is greatly saved.
Drawings
FIG. 1 is a graph showing the results of property testing according to example 1 of the present invention, in which amplification plot represents amplification curve and cycle represents cycle number;
FIG. 2 is a graph showing the results of property testing according to example 2 of the present invention, in which amplification plot represents amplification curve and cycle represents cycle number;
FIG. 3 is a graph showing the results of property testing according to example 3 of the present invention, in which amplification plot represents amplification curve and cycle represents cycle number;
FIG. 4 is a graph showing the results of property testing according to example 4 of the present invention, in which amplification plot represents amplification curve and cycle represents cycle number;
FIG. 5 is a graph showing the results of the property test of comparative example 1 according to the present invention, wherein amplification plot represents amplification curve and cycle represents cycle number;
FIG. 6 is a graph showing the results of the property test of comparative example 2 according to the present invention, wherein amplification plot indicates amplification curve and cycle indicates cycle number;
FIG. 7 is a schematic diagram of a PCR system according to an embodiment of the present invention.
Reference numerals:
100: sample containing unit
110: first liquid outlet/inlet
200: buffer solution containing unit
210: buffer outlet
300: PCR reaction unit
310: sample mixed liquid inlet after cracking
320: PCR reaction liquid outlet
400: piston unit
410: injection chamber
411: second liquid inlet/outlet
420: piston
910: first pipeline
920: second pipeline
930: third pipeline
940: fourth pipeline
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and not to be construed as limiting the invention.
It should be noted that, unless otherwise specified, the meaning of each component in the present invention is understood in accordance with the conventional explanation in the art, such as Tris-HCl and Tween 20.
Firstly, preparation of buffer solution
Weighing or aspirating a quantity of a target component to dispense a predetermined volume of buffer solution in which: 0.5-15 mmol/L of magnesium chloride, 1-150 mmol/L of sodium chloride, 0.1-7% of surfactant and 2-20% of glycerol, wherein the components are prepared to a preset volume by water, the concentration of the absorbed Tris-HCl solution is 1-100 mM, and the pH value is 7.5-8.0. The surfactant is Tween20, Tween 80, polyethylene glycol octyl phenyl ether, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium dioctyl sulfosuccinate or sodium glycocholate.
Second, property testing
Diluting 10. mu.L of the treated lysate with 90. mu.L of the dilution buffer, adding 5. mu.L of the diluted solution to the PCR reaction system, performing amplification reaction, and reading the Ct value.
Example 1
1. Formula composition of buffer solution
5% glycerol, 2mM magnesium chloride, 5mM sodium chloride, 5mM Tris-HCl, 0.5% Tween20, Tris-HCl added at pH 7.6.
PCR reaction system formula composition
Taq enzyme 1U/reaction, dNTP 4 mM/reaction, 18s rRNA Assay (Thermo Fisher)1 uL/reaction, template 5 uL/reaction.
2. Results of Property testing
Property measurements were performed as described above and Ct values were read.
The test results are shown in fig. 1. Wherein:
And (4) conclusion:
Example 2
1. Formula composition of buffer solution
8% glycerol, 6mM magnesium chloride, 50mM sodium chloride, 40mM Tris-HCl, 3% Tween20, Tris-HCl added at pH 8.0.
PCR reaction system formula composition
Taq enzyme 1U/reaction, dNTP 4 mM/reaction, 18s rRNA Assay (Thermo Fisher)1 uL/reaction, template 5 uL/reaction.
2. Results of Property testing
Property measurements were performed as described above and Ct values were read.
The test results are shown in fig. 2. Wherein:
And (4) conclusion:
Example 3
1. Formula composition of buffer solution
Glycerol 8%, magnesium chloride 6mM, sodium chloride 50mM, Tris-HCl 40mM, sodium lauryl sulfate 1%, Tris-HCl added at pH 8.0.
PCR reaction system formula composition
Taq enzyme 1U/reaction, dNTP 4 mM/reaction, 18s rRNA Assay (Thermo Fisher)1 uL/reaction, template 5 uL/reaction.
2. Results of Property testing
Property measurements were performed as described above and Ct values were read.
The test results are shown in fig. 3. Wherein:
And (4) conclusion:
Example 4
1. Formula composition of buffer solution
5% of glycerol, 2mM of magnesium chloride, 5mM of sodium chloride, 5mM of Tris-HCl, 0.1% of polyethylene glycol octylphenyl ether, the pH of the added Tris-HCl being 8.0.
PCR reaction system formula composition
Taq enzyme 1U/reaction, dNTP 4 mM/reaction, 18s rRNA Assay (Thermo Fisher)1 uL/reaction, template 5 uL/reaction.
2. Results of Property testing
Property measurements were performed as described above and Ct values were read.
The test results are shown in fig. 4. Wherein:
And (4) conclusion:
Comparative example 1
Only the raw material ratio is different from that of the example 1: the concentration of Tween20 was 8%, and the rest was the same as in example 1.
The test results are shown in fig. 5.
Analysis and conclusion:
after the formula is changed, the sample diluted by the dilution Buffer can not amplify a positive result (line 2), and the sample diluted by water can still amplify a positive result (line 1), which indicates that the sample is effective, but the Buffer solution in the formula proportion can not amplify a result. The proportion of Tween20 was shown to have a significant effect on the technical effect of the buffer compositions of the present application.
Comparative example 2
Only the raw material ratio is different from that of the example 1: the concentration of glycerin was 30%, and the rest was the same as in example 1.
The test results are shown in fig. 6.
Analysis and conclusion:
after the formula is changed, the sample diluted by the dilution Buffer can not amplify a positive result (line 2), and the sample diluted by water can still amplify a positive result (line 1), which indicates that the sample is effective, but the Buffer solution in the formula proportion can not amplify a result. Indicating that the ratio of glycerol has a significant effect on the technical effect of the buffer composition of the present application.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A PCR reaction system, comprising:
a sample containing unit in which a lysis raw material freeze-dried powder is disposed, and which has a first liquid outlet/inlet;
the buffer solution accommodating unit is internally provided with a buffer solution and is provided with a buffer solution outlet;
the PCR reaction unit is internally provided with reverse transcriptase and PCR raw material freeze-dried powder and is provided with a PCR reaction liquid outlet and a cracked sample mixed liquid inlet; and
a piston unit including an injection chamber and a piston, the injection chamber having a second liquid outlet/inlet;
the second liquid outlet/inlet is connected with the first liquid outlet/inlet through a first pipeline,
the second liquid outlet/inlet is connected with the buffer liquid outlet through a second pipeline,
the second liquid outlet/inlet is connected with the cracked sample mixed liquid inlet through a third pipeline,
the PCR reaction liquid outlet is connected with the buffer liquid outlet through a fourth pipeline;
wherein the buffer comprises: polyhydric alcohol, chloride salt, Tris-HCl, surfactant and water;
optionally, the polyol is glycerol.
2. A buffer, comprising: polyhydric alcohol, chloride salt, Tris-HCl, surfactant and water;
optionally, the polyol is glycerol.
3. The buffer of claim 2, wherein the surfactant comprises at least one selected from the group consisting of tween20, tween 80, octyl polyethylene glycol phenyl ether, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium dioctyl sulfosuccinate, and sodium glycocholate.
4. The buffer of claim 2, wherein the chloride salt comprises at least one selected from the group consisting of potassium chloride, sodium chloride, and magnesium chloride.
5. The buffer solution of claim 2, wherein the chloride salt is magnesium chloride and sodium chloride, and the concentration of the magnesium chloride is 0.5 to 15mmol/L, the concentration of the sodium chloride is 1 to 150mmol/L, the concentration of the surfactant is 0.1 to 7%, and the concentration of the glycerol is 2 to 20%, based on the total volume of the buffer solution.
6. The buffer solution of claim 2, wherein the chloride salt is magnesium chloride and sodium chloride, and the concentration of the magnesium chloride is 1.5 to 10mmol/L, the concentration of the sodium chloride is 5 to 100mmol/L, the concentration of the surfactant is 0.1 to 5%, and the concentration of the glycerol is 5 to 10%, based on the total volume of the buffer solution.
7. The buffer of claim 2, wherein the Tris-HCl is provided in a form dissolved in water.
8. The buffer solution of claim 7, wherein the solution of Tris-HCl in water has a pH of 7.5 to 8.0; preferably, the pH of the solution of Tris-HCl in water is 7.6.
9. The buffer solution according to claim 2, wherein the concentration of Tris-HCl is 1 to 100mmol/L based on the total volume of the buffer solution; preferably, the concentration of the Tris-HCl is 5-50 mmol/L.
10. A buffer, comprising, based on the total volume of the buffer: the detergent composition comprises magnesium chloride with the concentration of 1.5-10 mmol/L, sodium chloride with the concentration of 5-100 mmol/L, a surfactant with the concentration of 0.1-5%, glycerol with the concentration of 5-10%, Tris-HCl with the concentration of 5-50 mmol/L and water, wherein the surfactant is Tween20, sodium dodecyl sulfate or polyethylene glycol octyl phenyl ether.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2020/097142 WO2021000750A1 (en) | 2019-07-01 | 2020-06-19 | Novel method for performing pcr reaction using comprehensive pcr reaction system |
| EP20834221.2A EP3995563A4 (en) | 2019-07-01 | 2020-06-19 | Novel method for performing pcr reaction using comprehensive pcr reaction system |
| US17/551,153 US20220106626A1 (en) | 2019-07-01 | 2021-12-14 | Novel method for performing pcr reaction using comprehensive pcr reaction system |
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