CN112301097B - Sample lysis and PCR reaction composition - Google Patents
Sample lysis and PCR reaction composition Download PDFInfo
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- CN112301097B CN112301097B CN201910954105.3A CN201910954105A CN112301097B CN 112301097 B CN112301097 B CN 112301097B CN 201910954105 A CN201910954105 A CN 201910954105A CN 112301097 B CN112301097 B CN 112301097B
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Abstract
The invention provides a sample lysis and PCR reaction composition, which comprises: a first component comprising EDTA, EGTA, sodium dodecyl sulfate, saponin, proteinase K, polyethylene glycol 3350, tris-HCl, water; and/or a second component comprising mannitol, sucrose, potassium chloride, magnesium chloride, bovine serum albumin, dNTPs, polyoxyethylene lauryl ether, HEPES, DNA polymerase, reverse transcriptase and RNase inhibitor, water. The first component of the composition can be effectively used for sample lysis and nucleic acid extraction, and the second component can be effectively used for PCR amplification reaction. The first component and the second component in the composition are combined for use, so that the lysis of a sample, the extraction of nucleic acid and the preparation of a fluorescent quantitative PCR reagent can be completed without professionals, the technical requirements on operators are reduced, and meanwhile, the PCR amplification effect is excellent.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a sample lysis and PCR reaction composition, and more particularly to a composition and freeze-dried powder.
Background
The existing fluorescent quantitative PCR reaction is generally divided into 4 steps: firstly, cracking a sample and extracting nucleic acid, then preparing a fluorescent quantitative PCR reaction reagent, then adding the sample into a PCR reaction system, and finally performing on-machine detection.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventors found that, on the one hand, the reagents phenol, chloroform, guanidine hydrochloride, ethanol, guanidine isothiocyanate and the like used during the sample extraction are not only harmful to the human body, but also serious and even carcinogenic; some reagents are inflammable, and have higher requirements on laboratory environment and safety risk control; in addition, these agents are also prone to contamination of the atmosphere and water sources. On the other hand, the preparation and dispensing of the fluorescent quantitative PCR reagent are required to be carried out in a standard reagent preparation room to prevent sample pollution, and the preparation and dispensing operations are complicated and can be carried out by specially trained personnel. Based on the above problems, the inventors studied and developed a composition in which a first component can be effectively used for lysis of a sample and extraction of nucleic acid, and a second component can be effectively used for a PCR amplification reaction, the first component has significantly reduced toxicity and contamination, significantly improved safety and stability, is advantageous for transportation and storage, and has reduced requirements for a sample, compared to existing reagents; compared with the prior art, the second component has the advantages of obviously improved stability, favorable transportation and storage and reduced requirements on experimental environment; the combined use of the first component and the second component can lead the cracking of the sample, the extraction of nucleic acid and the preparation of the fluorescent quantitative PCR reagent to be completed without professional personnel, thereby reducing the technical requirements on operators and having excellent PCR amplification effect.
To this end, in a first aspect of the invention, the invention proposes a composition for a PCR reaction. According to an embodiment of the invention, the composition comprises: a first component comprising a metal ion chelating agent, sodium Dodecyl Sulfate (SDS), saponin (saponin), proteinase K (protease K), polyethylene glycol 3350 (PEG 3350), tris-HCl, water; and/or a second component comprising mannitol, sucrose, chloride salts, bovine Serum Albumin (BSA), dNTPs, polyoxyethylene dodecyl ether (Brij 35), HEPES, DNA polymerase, reverse transcriptase and RNase inhibitor, water. In some embodiments, the metal ion chelating agent is EDTA and EGTA. In some embodiments, the chloride salts are potassium chloride and magnesium chloride. According to the composition disclosed by the embodiment of the invention, the first component is combined with the heating function of other instruments to be effectively used for sample cracking and nucleic acid extraction, and the second component is effectively used for PCR amplification reaction; compared with the prior art, the second component is stable and effective, is beneficial to transportation and storage, and reduces the requirements on transportation and storage temperature; the combined use of the first component and the second component can complete the sample lysis, the nucleic acid extraction and the preparation of the fluorescent quantitative PCR reagent without professional personnel, thereby reducing the technical requirements on operators and having excellent PCR amplification effect.
According to an embodiment of the present invention, the above composition may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the concentration of EDTA is 0.1 to 10mmol/L, such as 0.5, 0.7, 1.0, 2.0, 3.0 or 4.0mmol/L, the concentration of EGTA is 0.1 to 15mmol/L, such as 0.5, 0.7, 3, 5, 7, 10 or 13mmol/L, the concentration of proteinase K is 5 to 150U/mL, such as 7, 10, 15, 30, 45, 60, 75, 90 or 120U/mL, the concentration of sodium dodecyl sulfate is 0.1 to 3.0%, such as 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5%, the concentration of saponin is 0.1 to 3.0%, such as 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5%, the concentration of polyethylene glycol is 0.1 to 5%, such as 0.5, 2.0, 4.5%, or 0% based on the total volume of the first component. The concentration of the sodium dodecyl sulfate, the saponin and the polyethylene glycol 3350 is mass volume concentration, which means the mass of the sodium dodecyl sulfate, the saponin or the polyethylene glycol 3350 in each 100mL of solution, and the unit is g. For example, the concentration of sodium lauryl sulfate is 0.1 to 3.0%, which means that the mass of sodium lauryl sulfate per 100mL of the first component is 0.1 to 3.0g. The inventor finds that when the concentration of each component in the first component is within the range, the first component can be further effectively used for sample lysis and nucleic acid extraction, and meanwhile, the first component is lower in toxicity and pollution, higher in safety, lower in requirement on the sample, and further, better in subsequent PCR amplification effect.
According to the embodiment of the invention, based on the total volume of the first component, the concentration of EDTA is 0.5-5 mmol/L, the concentration of EGTA is 0.5-10 mmol/L, the concentration of proteinase K is 10-100U/mL, the concentration of sodium dodecyl sulfate is 0.5-2.5%, the concentration of saponin is 0.5-2.5%, and the concentration of polyethylene glycol 3350 is 0.5-4.5%. The inventor finds that when the concentration of each component in the first component is within the range, the first component can be further effectively used for sample lysis and nucleic acid extraction, and meanwhile, the first component is lower in toxicity and pollution, higher in safety, lower in requirement on a sample, and further better in subsequent PCR amplification effect.
According to an embodiment of the invention, the Tris-HCl is provided in a form dissolved in water. It should be noted that Tris-HCl is a buffer substance commonly used in the art, and can be configured by itself or purchased directly.
According to an embodiment of the invention, the Tris-HCl solution in water has a pH of 7.5 to 8.2, such as 7.6, 7.7, 7.8, 7.9, 8.0 or 8.1. It should be noted that the pH does not refer to the pH of the first component, but is the pH of a solution of Tris-HCl in water. According to the embodiment of the invention, when the pH value of the solution formed by dissolving Tris-HCl in water is 7.5-8.2, the buffer effect of Tris-HCl on the first component is better, the sample cracking and nucleic acid extraction are more facilitated, the stability is better, and further, the subsequent PCR amplification effect 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 between 1 and 25mmol/L, such as 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24mmol/L, based on the total volume of the first component. According to the embodiment of the invention, when the concentration of Tris-HCl is 1-25 mmol/L, the buffer effect of Tris-HCl on the first component is better, the sample cracking and nucleic acid extraction are more facilitated, the stability is better, and the subsequent PCR amplification effect is better. In some embodiments, the concentration of Tris-HCl is between 5 and 20mmol/L.
According to an embodiment of the invention, the potassium chloride is present in a concentration of 10 to 150mmol/L, such as 15, 20, 30, 40, 60, 80, 100 or 120mmol/L, the magnesium chloride is present in a concentration of 0.5 to 10.0mmol/L, such as 0.7, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0 or 10.0mmol/L, the dNTP is present in a concentration of 150 to 250. Mu. Mol/L, such as 180, 200 or 230. Mu. Mol/L, the DNA polymerase is present in a concentration of 10 to 250U/mL, such as 13, 15, 18, 20, 30, 50, 80, 100, 120, 150, 180, 200 or 230U/mL, the reverse transcriptase is present in a concentration of 5 to 100U/mL, such as 7, 10, 20, 30, 40, 50, 70 or 90U/mL, the RNase inhibitor is at a concentration of 100 to 1000U/mL, such as 150, 200, 300, 500, 700 or 900U/mL, the mannitol is at a concentration of 0.1 to 10%, such as 0.2, 0.4, 0.5, 1.0, 3.0, 5.0, 7.0 or 9.0%, the sucrose is at a concentration of 0.1 to 10%, such as 0.2, 0.4, 0.5, 1.0, 3.0, 5.0, 7.0 or 9.0%, the bovine serum albumin is at a concentration of 0.1 to 5mg/mL, such as 0.2, 0.4, 0.6, 0.8, 1.0, 2.09, 3.0 or 4.0mg/mL, the polyoxyethylene lauryl ether is at a concentration of 0.01 to 0.10%, such as 0.02, 0.03, 0.04, 0.05, 0.08 or 0.06%. The concentration of the sucrose and the polyoxyethylene lauryl ether is a mass volume concentration, and means the mass of the sucrose or the polyoxyethylene lauryl ether per 100mL of the solution, and the unit is g. For example, the concentration of sucrose is 0.1 to 10%, which means that the mass of sucrose per 100mL of the second component is 0.1 to 10g. The concentration of the mannitol is 0.1-10%, which means that the volume of the mannitol in each 100mL of the second component is 0.1-10 mL. The inventors found that when the concentrations of the components in the second component are within this range, the second component can be further effectively used in a PCR amplification reaction, and the PCR amplification effect is better and the stability is higher.
According to the embodiment of the invention, based on the total volume of the second component, the concentration of potassium chloride is 20-100 mmol/L, the concentration of magnesium chloride is 1.0-5.0 mmol/L, the concentration of dNTP is 200 [ mu ] mol/L, the concentration of DNA polymerase is 20-200U/mL, the concentration of reverse transcriptase is 10-50U/mL, the concentration of RNase inhibitor is 200-1000U/mL, the concentration of mannitol is 0.5-8%, the concentration of sucrose is 0.5-8%, the concentration of bovine serum albumin is 0.1-1mg/mL, and the concentration of polyoxyethylene lauryl ether is 0.05%. The inventors found that when the concentrations of the components in the second component are within this range, the second component can be further effectively used in a PCR amplification reaction, and the PCR amplification effect is better and the stability is higher.
According to an embodiment of the present invention, the HEPES is provided in a water-soluble form. It should be noted that HEPES is a buffer substance commonly used in the art, and can be configured by itself or purchased directly.
According to an embodiment of the invention, the pH of the solution of HEPES in water is 8.0 to 8.5, such as 8.1, 8.2, 8.25, 8.3 or 8.4. It should be noted that the pH does not refer to the pH of the second component, but the pH of a solution of HEPES in water. According to the embodiment of the invention, when the pH value of the solution formed by dissolving HEPES in water is 8.0-8.5, the HEPES has a better buffering effect on the second component, is more beneficial to PCR amplification reaction, has a better PCR amplification effect and is higher in stability. In some embodiments, the solution of HEPES in water has a pH of 8.25.
According to an embodiment of the invention, the concentration of the HEPES is 5 to 55mmol/L, such as 10, 15, 20, 25, 30, 35, 40, 45 or 50mmol/L, based on the total volume of the second component. According to the embodiment of the invention, when the concentration of the HEPES is 5-55 mmol/L, the HEPES is more beneficial to PCR amplification reaction, the PCR amplification effect is better, and the stability is higher. In some embodiments, the concentration of HEPES is 10 to 50mmol/L.
According to an embodiment of the present invention, the saponin includes at least one selected from the group consisting of tea saponin, ginsenoside, paridis saponin, soyasaponin.
According to an embodiment of the present invention, the DNA polymerase includes at least one selected from Taq enzyme, tth DNA polymerase.
According to an embodiment of the present invention, the reverse transcriptase comprises at least one selected from the group consisting of M-MLV reverse transcriptase, AMV reverse transcriptase.
According to an embodiment of the present invention, the rnase inhibitor comprises at least one selected from the group consisting of diethyl pyrophosphate, guanidine isothiocyanate, vanadyl riboside complex, RNasin, urea, diatomaceous earth.
In a second aspect of the invention, a composition is provided. According to an embodiment of the invention, the composition comprises: a first component comprising EDTA at a concentration of 0.5 to 5mmol/L, EGTA at a concentration of 0.5 to 10mmol/L, proteinase K at a concentration of 10 to 100U/mL, sodium dodecyl sulfate at a concentration of 0.5 to 2.5%, saponin at a concentration of 0.5 to 2.5%, polyethylene glycol 3350 at a concentration of 0.5 to 4.5%, tris-HCl at a concentration of 5 to 20mmol/L, and water, based on the total volume of the first component; and/or a second component comprising potassium chloride at a concentration of 20 to 100mmol/L, magnesium chloride at a concentration of 1.0 to 5.0mmol/L, dNTP at a concentration of 200. Mu. Mol/L, DNA polymerase at a concentration of 20 to 200U/mL, reverse transcriptase at a concentration of 10 to 50U/mL, RNase inhibitor at a concentration of 200 to 1000U/mL, mannitol at a concentration of 0.5 to 8%, sucrose at a concentration of 0.5 to 8%, bovine serum albumin at a concentration of 0.1 to 1mg/mL, polyoxyethylene dodecyl ether at a concentration of 0.05%, HEPES at a concentration of 10 to 50mmol/L, and water, based on the total volume of the second component. The inventor finds that the combined use of the first component and the second component can complete the sample lysis, the nucleic acid extraction and the fluorescent quantitative PCR reagent preparation without professional personnel, reduce the technical requirements on operators and simultaneously have excellent PCR amplification effect.
In a third aspect of the invention, a lyophilized powder is provided. According to an embodiment of the invention, the lyophilized powder is prepared from the composition of any of the above. The inventor finds that when the first component and/or the second component exist in the form of freeze-dried powder, the stability is greatly improved, the first component and/or the second component can be stored and transported at normal temperature, the environmental requirements on storage and transportation are greatly reduced, the freeze-dried powder can be redissolved after being mixed with a proper buffer solution, and the original functions are maintained.
In a fourth aspect of the present invention, the present invention provides a PCR reaction system. Referring to fig. 8, 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 diluent accommodating unit 200, in which a diluent is disposed within the diluent accommodating unit 200, and the diluent accommodating unit 200 is provided with a diluent 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 diluent 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 outlet/inlet 411, the second liquid outlet/inlet 411 is connected with the first liquid outlet/inlet 110 through a first pipeline 910, the second liquid outlet/inlet 411 is connected with the diluent outlet 210 through a second pipeline 920, and the second liquid outlet/inlet 411 is connected with the lysed sample mixed liquid inlet 310 through a third pipeline 930; wherein: the cracking raw material freeze-dried powder comprises a metal ion chelating agent, sodium dodecyl sulfate, saponin, proteinase K, polyethylene glycol 3350, tris-HCl and water; the reverse transcriptase and PCR raw material freeze-dried powder comprises mannitol, sucrose, chloride salt, bovine serum albumin, dNTP, polyoxyethylene dodecyl ether, HEPES, DNA polymerase, reverse transcriptase and RNA enzyme inhibitor and water. In some embodiments, the metal ion chelating agent is EDTA and EGTA. In some embodiments, the chloride salts are potassium chloride and magnesium chloride. It should be noted that the description of the composition in the two aspects is 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 diluent accommodating unit 200, the PCR reaction unit 300, and the piston unit 400 to each other through a microfluidic circuit; meanwhile, each unit is an independently arranged unit, so that different reactants can be stored in each unit before use, and the reactants can be stored for a long time under the condition of nonuse. 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. 8, first, the piston 420 is pulled outward to a position such that the diluent in the diluent containing unit 200 flows toward the injection chamber 410; then, the piston 420 is moved back and forth, so that part of the diluent in the injection chamber 410 enters the sample containing unit 100 and is uniformly mixed with the lysis freeze-dried powder and the sample (i.e. the lysis raw material freeze-dried powder) 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 sample mixed liquid cracked in the injection chamber 410 returns to the diluent containing unit 200 and is uniformly mixed with the diluent remaining in the diluent containing unit 200, thereby diluting the sample mixed liquid cracked and reducing the concentration of impurities therein; thereafter, the piston 420 is pulled outward again to a certain position, so that the diluted sample mixture in the diluent storage 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 diluent outlet through the fourth pipeline, so that the pressure system communication between the PCR reaction liquid outlet and the diluent outlet is formed, and 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 diluent-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 diluent 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 test results according to example 1 of the present invention;
FIG. 2 is a graph showing the test results according to example 2 of the present invention;
FIG. 3 is a graph showing the test results of comparative example 1 according to the present invention;
FIG. 4 is a graph showing the test results of comparative example 2 according to the present invention;
FIG. 5 is a graph showing the test results of comparative example 3 according to the present invention;
FIG. 6 is a graph showing the test results of comparative example 4 according to the present invention;
FIG. 7 is a graph showing the test results of comparative example 5 according to the present invention;
FIG. 8 is a schematic diagram of a PCR system according to an embodiment of the present invention.
Reference numerals are as follows:
100: sample containing unit
110: first liquid outlet/inlet
200: diluent containing unit
210: diluent 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: the second pipeline
930: third pipeline
940: fourth pipeline
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
It is to be noted that, unless otherwise specified, the meaning of each component and each concentration in the present invention is understood in accordance with the conventional explanation in the art, such as EDTA and EGTA. In addition, amplification Plot represents Amplification curve; cycle represents the number of cycles.
It should be noted that, except that the sample lysis and nucleic acid extraction composition or lyophilized powder thereof of the present invention is the development result of the inventor, other related reagents used in the following steps can be purchased or obtained by referring to the prior art, unless otherwise specified. Those skilled in the art can purchase related reagents according to actual needs, or consult the existing technology to obtain related reagents.
1. Preparation of freeze-dried powder for sample cracking and nucleic acid extraction
1. Weighing or aspirating a quantity of a target component to formulate a predetermined volume of a mixed solution in which: EDTA of 0.5mM-5mM, EGTA of 0.5mM-10mM, sodium dodecyl sulfate of 0.5% -2.5%, saponin of 0.5% -2.5%, proteinase K of 10-100U/mL, polyethylene glycol 3350 of 0.5-4.5%, tris-HCl of 5mM-20mM, prepared to a predetermined volume with water, and the pH of the absorbed Tris-HCl solution is 7.5-8.2.
2. Sucking 50 μ L of the above mixed solution, lyophilizing by conventional lyophilization method (such as lyophilizing on dry ice, and drying and sublimating at-30 deg.C in a lyophilizer), and preparing lyophilized powder for sample lysis and nucleic acid extraction.
2. Preparation of fluorescent quantitative PCR reagent freeze-dried powder
1. Weighing or pipetting a quantity of a target component to formulate a predetermined volume of a mixed solution having: 0.5 to 8% of mannitol, 0.5 to 8% of sucrose, 20 to 100mM of potassium chloride, 1.0 to 5mM of magnesium chloride, 0.1 to 1mg/mL of bovine serum albumin, 200. Mu.M of dNTP, 0.05% of Brij 35, 10 to 50mM of HEPES, 20 to 200U/mL of DNA polymerase, 10 to 50U/mL of M-MLV reverse transcriptase, 200 to 1000U/mL of RNase inhibitor, prepared with water to a predetermined volume, and the pH of the extracted HEPES solution is 8.0 to 8.5.
2. And filtering the mixed solution, and freeze-drying according to a conventional freeze-drying method to prepare a certain amount of fluorescent quantitative PCR reagent freeze-dried powder.
3. Property measurement
1) Toxicity
The components of the freeze-dried powder for sample lysis and nucleic acid extraction do not contain toxic substances such as phenol, chloroform, guanidine hydrochloride or guanidine isothiocyanate and the like, and the toxicity is low.
2) Stability of
The fluorescent quantitative PCR reagent freeze-dried powder is preserved at room temperature, and the stability is good.
3) Validation test for sample lysis and nucleic acid extraction and fluorescent quantitative PCR
Sample lysis:
flu A influenza A virus is added to the lysate in a certain ratio. And carrying out warm bath at 95 ℃ for a certain time to carry out sample cracking.
Fluorescent quantitative PCR verification test:
and adding the lysis sample into a redissolved fluorescent quantitative PCR reaction system according to a certain proportion, and adding a corresponding Flu A primer and a probe. And (4) performing fluorescent quantitative PCR reaction on the sample. The reaction procedure is as follows: 50 ℃ for 5min; 2min at 95 ℃;95 ℃ 15s,60 ℃ 1min,40cycles. Wherein the sequences of the primers and the probes are shown as follows:
FluA-Forward：CAGAGACTTGAAGATGTTTTTGC(SEQ ID NO：1)
FluA-Reverse：CTACGCTGCAGTCCTCGCTC(SEQ ID NO：2)
FluA-Prob：CY3-CAAGACCAATCCTGTCACCTCTGA-BHQ2(SEQ ID NO：3)
the present invention is described in further detail below by way of specific examples.
Example 1
1. Sample cracking and preparation of freeze-dried powder for nucleic acid extraction
Weighing or aspirating a quantity of a target component to formulate a predetermined volume of a mixed solution in which: EDTA of 2mM, EGTA of 2mM, sodium dodecyl sulfate of 1%, saponin of 1.0%, proteinase K of 20U/mL, polyethylene glycol 3350 of 1%, tris-HCl of 10mM, water to a predetermined volume, and the pH of the absorbed Tris-HCl solution of 7.6.
Sucking 50 μ L of the above mixed solution, adding into eight-connected tube, centrifuging to tube bottom, and freezing at-80 deg.C overnight. Taking out, putting into a freeze dryer for freeze-drying overnight, covering after freeze-drying, and storing at room temperature. The temperature of the lyophilizer was ensured to be below-45 deg.C, the vacuum pressure was <450Torr, and the sample in the tube was placed on dry ice for at least 30 minutes.
2. Preparation of fluorescent quantitative PCR reagent freeze-dried powder
Weighing or aspirating a quantity of a target component to formulate a predetermined volume of a mixed solution in which: mannitol 4%, sucrose 1.5%, potassium chloride 80mM, magnesium chloride 3.5mM, bovine serum albumin 0.5mg/mL, dNTP 200. Mu.M, brij 35 0.05%, HEPES 2mM, DNA polymerase 40U/mL, M-MLV reverse transcriptase 20U/mL, RNase inhibitor 500U/mL, prepared in a predetermined volume with water, and the pH of the extracted HEPES solution was 8.25.
3. Validation test for sample lysis and nucleic acid extraction and fluorescent quantitative PCR
Sample lysis and nucleic acid extraction as well as reagent preparation and lyophilization for fluorescent quantitative PCR were performed as described above. Storing for 3 months at room temperature, adding water for redissolving, then adding Flu A virus for sample lysis, further adding the lysed sample into a fluorescent quantitative PCR reaction system of the Flu A primer and the probe after redissolving according to the proportion, and performing a fluorescent quantitative PCR verification test on the sample, wherein the test result is shown in figure 1.
And (4) conclusion: as can be seen from figure 1, the lyophilized powder reagent can still be normally amplified after being redissolved after being stored for 3 months at room temperature. The reagent formula of the first component and the second component is re-dissolved after being freeze-dried, and still maintains the sample cracking and PCR detection activity.
Example 2
1. Preparation of freeze-dried powder for sample cracking and nucleic acid extraction
Weighing or pipetting a quantity of a target component to formulate a predetermined volume of a mixed solution having: EDTA 4mM, EGTA 4mM, sodium dodecyl sulfate 2%, saponin 2%, proteinase K80U/mL, polyethylene glycol 3350 4%, tris-HCl 18mM, water to predetermined volume, suction Tris-HCl solution pH 7.6.
Sucking 50 μ L of the above mixed solution, adding into eight-connected tube, centrifuging to tube bottom, and freezing at-80 deg.C overnight. Taking out, putting into a freeze dryer for freeze-drying overnight, covering after freeze-drying, and storing at room temperature. The temperature of the lyophilizer was ensured to be below-45 deg.C, the vacuum pressure was <450Torr, and the sample in the tube was placed on dry ice for at least 30 minutes.
2. Preparation of fluorescent quantitative PCR reagent freeze-dried powder
Weighing or pipetting a quantity of a target component to formulate a predetermined volume of a mixed solution having: mannitol 6%, sucrose 3%, potassium chloride 40mM, magnesium chloride 5mM, bovine serum albumin 1mg/mL, dNTP 200. Mu.M, brij 35 0.05%, HEPES 40mM, DNA polymerase 100U/mL, M-MLV reverse transcriptase 40U/mL, RNase inhibitor 800U/mL, prepared with water to a predetermined volume, and the pH of the extracted HEPES solution 8.25.
3. Validation test for sample lysis and nucleic acid extraction and fluorescent quantitative PCR
Sample lysis and nucleic acid extraction as well as reagent preparation and lyophilization for fluorescent quantitative PCR were performed as described above. Storing for 3 months at room temperature, adding water for redissolving, then adding Flu A virus for sample lysis, further adding the lysed sample into a fluorescent quantitative PCR reaction system of the Flu A primer and the probe after redissolving according to the proportion, and performing a fluorescent quantitative PCR verification test on the sample, wherein the test result is shown in figure 2.
And (4) conclusion: as can be seen from FIG. 2, the lyophilized powder reagent can still be normally amplified after being reconstituted after being stored at room temperature for 3 months. The first component and the second component reagent formula are re-dissolved after freeze-drying, and the sample lysis and PCR detection activity are still maintained.
Comparative example 1
The raw material ratio is different from that of the example 1 only: the proteinase K in the first component is 2U/mL, and the proportion of other components is unchanged. The test method was the same as in example 1.
The amplification results are shown in FIG. 3.
And (4) conclusion: as can be seen from FIG. 3, when the concentration of proteinase K is too low, the effect of the first component on the cleavage of the sample is affected, and the PCR amplification result is further affected. It is important to specify the stoichiometric concentration of proteinase K in the first component.
Comparative example 2
The raw material ratio is different from that of the example 1 only: the concentrations of EDTA and EGTA in the first component are both 0mM, and the proportion of the other components is unchanged. The test method was the same as in example 1.
The amplification results are shown in FIG. 4.
And (4) conclusion: as can be seen from FIG. 4, when the EDTA and EGTA components are removed, the effect of the first component on sample lysis is affected, and the PCR amplification result is affected. It is important to demonstrate the role of EDTA, EGTA in the first component.
Comparative example 3
The raw material ratio is different from that of the example 1 only: the concentration of saponin in the first component is 10%, and the proportion of other components is unchanged. The test method was the same as in example 1.
The amplification results are shown in FIG. 5.
And (4) conclusion: as can be seen from FIG. 5, when the concentration of saponin is too high, the effect of the first component on cracking the sample is affected, and the PCR amplification result is affected. It is important to specify the stoichiometric concentration of saponin in the first component.
Comparative example 4
The raw material ratio is different from that of the embodiment 1 only in that: the concentration of magnesium chloride in the second component is 0.2mM, and the proportion of other components is unchanged. The test method was the same as in example 1.
The amplification results are shown in FIG. 6.
And (4) conclusion: as can be seen from FIG. 6, when the concentration of magnesium chloride is too low, the results of the second PCR reaction set are affected. It is important to specify the stoichiometric concentration of magnesium chloride in the second component.
Comparative example 5
The raw material ratio is different from that of the embodiment 1 only in that: the concentration of HEPES in the second component is 2mM, and the proportion of other components is unchanged. The test method was the same as in example 1.
The amplification results are shown in FIG. 7.
And (4) conclusion: as can be seen from FIG. 7, when the concentration of HEPES is too low, the results of the second PCR reaction are affected. It is important to note the stoichiometric concentration of HEPES in the second component.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means 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 will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, 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 (3)
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;
a diluent containing unit in which a diluent is disposed and which has a diluent 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 diluent 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 diluent outlet through a fourth pipeline;
the use method of the PCR reaction system comprises the following steps:
pulling the piston to enable the diluent of the diluent containing unit to enter the injection chamber;
moving the piston back and forth to enable part of diluent in the injection chamber to enter the sample containing unit so as to enable the diluent to be mixed with the sample and the lysis raw material freeze-dried powder and perform lysis;
pulling the piston again to make the sample mixed liquid cracked in the sample containing unit enter the injection chamber;
moving the piston back and forth again to make the lysed sample mixed liquid in the injection chamber enter the diluent containing unit, and the lysed sample mixed liquid is mixed with the rest diluent in the diluent containing unit;
continuously pulling the piston to enable the diluted sample mixed liquid in the diluent containing unit to enter the injection chamber;
continuously moving the piston back and forth to enable the diluted sample mixed solution in the injection chamber to enter the PCR reaction unit, mixing the diluted sample mixed solution with the reverse transcriptase and the freeze-dried powder of the PCR raw material, and carrying out PCR amplification reaction; wherein:
the cracking raw material freeze-dried powder comprises a metal ion chelating agent, sodium dodecyl sulfate, saponin, proteinase K, polyethylene glycol 3350, tris-HCl and water;
the reverse transcriptase and PCR raw material freeze-dried powder comprises mannitol, sucrose, chloride, bovine serum albumin, dNTP, polyoxyethylene dodecyl ether, HEPES, DNA polymerase, reverse transcriptase and RNase inhibitor and water.
2. The PCR reaction system of claim 1, wherein the metal ion chelating agent is EDTA and EGTA.
3. The PCR reaction system of claim 1, wherein the chloride salt is potassium chloride and magnesium chloride.
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| 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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