CN112176038A - Buffer composition - Google Patents

Buffer composition Download PDF

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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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buffer
concentration
tris
hcl
pcr reaction
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韩巧玲
徐强
崔相民
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Shenyi Biotech Hangzhou Co Ltd
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Shenyi Biotech Hangzhou Co Ltd
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Priority to EP20834221.2A priority Critical patent/EP3995563A4/en
Priority to PCT/CN2020/097142 priority patent/WO2021000750A1/en
Publication of CN112176038A publication Critical patent/CN112176038A/en
Priority to US17/551,153 priority patent/US20220106626A1/en
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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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    • 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
    • C12Q1/6806—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

Buffer composition
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:
line 1 represents the amplification result of the subsequent PCR reaction system directly added to the solution (lysis stock solution) after the sample is subjected to lysis treatment;
line 2 shows the amplification result of the same PCR reaction system added after the lysis stock solution is diluted by the dilution buffer;
line 3 represents the above lysis batch via H2Diluting the mixture by equal times, and adding the same PCR reaction system for amplification;
line 4 represents negative quality control, i.e., amplification results obtained by adding the same PCR reaction system using dilution buffer as a template.
And (4) conclusion:
line 1 is the result of amplification of the subsequent PCR reaction system directly added to the solution after the sample is subjected to the lysis treatment, and shows that an amplification curve cannot be amplified. Line 2 is the lysis stock solution diluted by the dilution buffer and added into the same PCR reaction system, and shows that a positive amplification curve can be obtained. Line 3 is the above lysis stock solution through H2The dilution of O is equal to the multiple, the sample is amplified, but the Ct value is obviously higher than that of the dilution sample of the dilution buffer (the buffer solution of the invention), which shows that the buffer effect of the dilution buffer is better than that of H2And O. Line 4 is a dilution buffer as a template added into the same PCR reaction system as a negative quality control, and the negative quality control has no amplification curve, which shows that the dilution buffer can effectively buffer the lysate and the PCR reaction system.
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:
line 1 represents the amplification result of the subsequent PCR reaction system directly added to the solution (lysis stock solution) after the sample is subjected to lysis treatment;
line 2 shows the amplification result of the same PCR reaction system added after the lysis stock solution is diluted by the dilution buffer;
line 3 represents the above lysis batch via H2Diluting the mixture by equal times, and adding the same PCR reaction system for amplification;
line 4 shows negative quality control, and dilution buffer is used as a template to add the amplification result of the same PCR reaction system.
And (4) conclusion:
line 1 is the result of amplification of the subsequent PCR reaction system directly added to the solution after the sample is subjected to the lysis treatment, and shows that an amplification curve cannot be amplified. Line 2 is the lysis stock solution diluted by the dilution buffer and added into the same PCR reaction system, and shows that a positive amplification curve can be obtained. Line 3 is the above lysis stock solution through H2The dilution of O is equal to the multiple, the sample is amplified, but the Ct value is obviously higher than that of the dilution sample of the dilution buffer (the buffer solution of the invention), which shows that the buffer effect of the dilution buffer is better than that of H2And O. Line 4 is a dilution buffer as a template added into the same PCR reaction system as a negative quality control, and the negative quality control has no amplification curve, which shows that the dilution buffer can effectively buffer the lysate and the PCR reaction system.
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:
line 1 represents the amplification result of the subsequent PCR reaction system directly added to the solution (lysis stock solution) after the sample is subjected to lysis treatment;
line 2 shows the amplification result of the same PCR reaction system added after the lysis stock solution is diluted by the dilution buffer;
line 3 represents the above lysis batch via H2Diluting the mixture by equal times, and adding the same PCR reaction system for amplification;
line 4 shows negative quality control, and dilution buffer is used as a template to add the amplification result of the same PCR reaction system.
And (4) conclusion:
line 1 is the result of amplification of the subsequent PCR reaction system directly added to the solution after the sample is subjected to the lysis treatment, and shows that an amplification curve cannot be amplified. Line 2 is the lysis stock solution diluted by the dilution buffer and added into the same PCR reaction system, and shows that a positive amplification curve can be obtained. Line 3 is the above lysis stock solution through H2The dilution of O is equal to the multiple, the sample is amplified, but the Ct value is obviously higher than that of the dilution sample of the dilution buffer (the buffer solution of the invention), which shows that the buffer effect of the dilution buffer is better than that of H2And O. Line 4 is a dilution buffer as a template added into the same PCR reaction system as a negative quality control, and the negative quality control has no amplification curve, which shows that the dilution buffer can effectively buffer the lysate and the PCR reaction system.
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:
line 1 represents the amplification result of the subsequent PCR reaction system directly added to the solution (lysis stock solution) after the sample is subjected to lysis treatment;
line 2 shows the amplification result of the same PCR reaction system added after the lysis stock solution is diluted by the dilution buffer;
line 3 represents the above lysis batch via H2Diluting the mixture by equal times, and adding the same PCR reaction system for amplification;
line 4 shows negative quality control, and dilution buffer is used as a template to add the amplification result of the same PCR reaction system.
And (4) conclusion:
line 1 is the result of amplification of the subsequent PCR reaction system directly added to the solution after the sample is subjected to the lysis treatment, and shows that an amplification curve cannot be amplified. Line 2 is the lysis stock solution diluted by the dilution buffer and added into the same PCR reaction system, and shows that a positive amplification curve can be obtained. Line 3 is the above lysis stock solution through H2The dilution of O is equal to the multiple, the sample is amplified, but the Ct value is obviously higher than that of the dilution sample of the dilution buffer (the buffer solution of the invention), which shows that the buffer effect of the dilution buffer is better than that of H2And O. Line 4 is a dilution buffer as a template added into the same PCR reaction system as a negative quality control, and the negative quality control has no amplification curve, which shows that the dilution buffer can effectively buffer the lysate and the PCR reaction system.
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.
CN201910953826.2A 2019-07-01 2019-10-09 Buffer composition Pending CN112176038A (en)

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Application Number Priority Date Filing Date Title
EP20834221.2A EP3995563A4 (en) 2019-07-01 2020-06-19 Novel method for performing pcr reaction using comprehensive pcr reaction system
PCT/CN2020/097142 WO2021000750A1 (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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