CN114133991A - Nucleic acid cleaning combined reagent and use method thereof - Google Patents
Nucleic acid cleaning combined reagent and use method thereof Download PDFInfo
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- CN114133991A CN114133991A CN202110998279.7A CN202110998279A CN114133991A CN 114133991 A CN114133991 A CN 114133991A CN 202110998279 A CN202110998279 A CN 202110998279A CN 114133991 A CN114133991 A CN 114133991A
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 73
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 73
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 73
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 28
- 238000004140 cleaning Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000008367 deionised water Substances 0.000 claims abstract description 21
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000012459 cleaning agent Substances 0.000 claims abstract description 18
- 239000011521 glass Substances 0.000 claims abstract description 18
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 10
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 10
- 229920006317 cationic polymer Polymers 0.000 claims abstract description 10
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- 239000004094 surface-active agent Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 14
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 235000006708 antioxidants Nutrition 0.000 claims description 9
- 239000003381 stabilizer Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 229960005070 ascorbic acid Drugs 0.000 claims description 6
- 235000010323 ascorbic acid Nutrition 0.000 claims description 6
- 239000011668 ascorbic acid Substances 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 6
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 claims description 6
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000004952 Polyamide Substances 0.000 claims description 5
- 235000006408 oxalic acid Nutrition 0.000 claims description 5
- 229920002647 polyamide Polymers 0.000 claims description 5
- 229920000768 polyamine Polymers 0.000 claims description 5
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013543 active substance Substances 0.000 claims description 3
- 238000000889 atomisation Methods 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 108010024636 Glutathione Proteins 0.000 claims description 2
- 229960003180 glutathione Drugs 0.000 claims description 2
- 235000003969 glutathione Nutrition 0.000 claims description 2
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 2
- 235000019136 lipoic acid Nutrition 0.000 claims description 2
- 229960002663 thioctic acid Drugs 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 59
- 108020004414 DNA Proteins 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 239000003344 environmental pollutant Substances 0.000 description 4
- 231100000719 pollutant Toxicity 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- -1 azide compounds Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
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- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/667—Neutral esters, e.g. sorbitan esters
-
- B08B1/143—
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B08—CLEANING
- B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
- B08B11/00—Cleaning flexible or delicate articles by methods or apparatus specially adapted thereto
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B08—CLEANING
- B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
- B08B13/00—Accessories or details of general applicability for machines or apparatus for cleaning
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/20—Organic compounds containing oxygen
- C11D3/2003—Alcohols; Phenols
- C11D3/2006—Monohydric alcohols
- C11D3/201—Monohydric alcohols linear
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/37—Polymers
- C11D3/3703—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C11D3/3719—Polyamides or polyimides
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/39—Organic or inorganic per-compounds
- C11D3/3942—Inorganic per-compounds
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/39—Organic or inorganic per-compounds
- C11D3/3947—Liquid compositions
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
- C11D7/22—Organic compounds
- C11D7/26—Organic compounds containing oxygen
- C11D7/265—Carboxylic acids or salts thereof
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
- C11D7/22—Organic compounds
- C11D7/26—Organic compounds containing oxygen
- C11D7/267—Heterocyclic compounds
Abstract
The invention relates to the field of molecular biology, in particular to a nucleic acid cleaning combined reagent and a using method thereof, and the technical scheme is as follows: the nucleic acid cleaning combined reagent is formed by combining two cleaning agents, namely solution A and solution B, wherein the solution A comprises an oxidizing agent, a nonpolar solvent and a surfactant, and the solution B comprises a water-soluble cationic polymer, an antioxidant and deionized water. The nucleic acid cleaning combined reagent and the use method have the advantages of chemical stability, heat resistance, convenience in use, quickness in taking effect and high removal rate, and can be used for well removing the nucleic acid pollution remained on the surface of organic glass.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nucleic acid cleaning combined reagent and a using method thereof.
Background
In molecular biology laboratories, since PCR can increase target nucleic acids several million-fold in a short time, a large amount of PCR product is liable to cause nucleic acid contamination if it enters the laboratory air. In addition, even minute amounts of PCR products can cause false positive results, leading to incorrect judgment of experimental results, and nucleic acid contaminants are cumulative and persistent and difficult to remove. How to effectively remove nucleic acid pollutants in a laboratory is an urgent problem to be solved in the field of molecular diagnosis.
Aiming at the problems, most of the traditional nucleic acid cleaning agents modify and degrade DNA or modify DNA bases by adding DNA enzyme or chemical methods, ensure the integrity of the DNA to prevent the principles of DNA polymerase sequence reading and the like to inactivate nucleic acid molecules, and the nucleic acid cleaning agents can effectively remove nuclease and nucleic acid pollution in laboratories and reduce the times of repeated experiments.
The scheme solves the problem of nucleic acid pollution in molecular biology laboratories, but genetic information encoded in the inactivated DNA strand of the traditional nucleic acid cleaning agent is only covered but not destroyed, and the DNA cannot be completely degraded. In PCR amplification, the information of these molecules is again available and can also be amplified by enzymatic reactions, causing secondary contamination. In addition, the traditional nucleic acid cleaning agent often contains chemical components with potential corrosive risks, including azide compounds, mineral acid such as phosphoric acid and hydrochloric acid, peroxide with strong irritation, alkaline substances such as sodium hydroxide and the like, and enzyme reagents are corrosive, so that the surface of equipment is corroded after long-term use, and precision instruments are slowly worn.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nucleic acid cleaning combined reagent and a use method thereof, which have the advantages of chemical stability, heat resistance, convenience in use, quickness in taking effect and high removal rate, and can better remove the nucleic acid pollution remained on the surface of organic glass.
The technical purpose of the invention is realized by the following technical scheme: a nucleic acid cleaning combined reagent and a use method thereof are formed by combining two cleaning agents of solution A and solution B, wherein the solution A comprises an oxidant, a surfactant, a nonpolar solvent and deionized water, and the solution B comprises a water-soluble cationic polymer, an antioxidant, a stabilizer and deionized water.
By adopting the technical scheme, the places where pollution is accumulated, such as the bench surface of a super-clean workbench, the experimental wall surface and the shell of a liquid-transferring gun, are respectively sprayed and wiped with the nucleic acid cleaning agent A and the nucleic acid cleaning agent B, so that the pollution of residual nucleic acid aerosol is taken away, and the condition that false positive occurs when a PCR result is interfered is effectively improved.
Preferably, the component and mass percentage of the solution A are 4 to 15 percent of oxidant, 0.8 to 10 percent of surfactant, 5 to 20 percent of nonpolar solvent and the balance of deionized water. Wherein the oxidant is hydrogen peroxide or peracetic acid, the active agents are tween 20, tween 100 and triton X-100, and the nonpolar solvent is ethanol or acetic acid.
By adopting the technical scheme, the hydrogen peroxide conversion rate and the hydroxyl radical content in the reagent are high, and the reagent has chemical stability and heat resistance.
Preferably, the component and the mass percentage of the solution B are 1 to 10 percent of water-soluble cationic polymer, 5 to 12 percent of antioxidant, 0.5 to 3 percent of stabilizer and the balance of deionized water. Wherein the water-soluble cationic polymer is polyamide polyamine epichlorohydrin (PAE) or polydiallyldimethylammonium chloride (PDADMAC), the antioxidant is ascorbic acid, glutathione or lipoic acid, and the stabilizer is sodium bicarbonate or oxalic acid.
Through adopting above-mentioned technical scheme, under the combined action of active ingredient in the solution, change the electric charge distribution of the nucleic acid of adsorbing on instrument and laboratory bench surface, promote the nucleic acid pollutant to break away from by the adsorption surface, then combine the shearing force that the in-process produced of wiping and further make the nucleic acid pollutant dissociate from the adsorption surface to reach the effect of getting rid of surface nucleic acid pollution.
Preferably, the method for using the nucleic acid washing combined reagent comprises the following steps:
(1) respectively preparing a solution A and a solution B nucleic acid cleaning agent and carrying out atomization treatment;
(2) wiping the surface of the organic glass polluted by the nucleic acid by using paper towels wetted by the solution A and the solution B in sequence.
By adopting the technical scheme, the nucleic acid pollution in a laboratory can be conveniently and quickly removed.
Preferably, the content of the oxidizing agent, the surfactant and the nonpolar solvent in the solution a is 4%, 10% and 5%, respectively.
Preferably, the content of the oxidizing agent, the surfactant and the nonpolar solvent in the solution a is 15%, 0.8% and 20%, respectively.
Preferably, the oxidant in the solution A is hydrogen peroxide, the active agent is Tween 20, and the nonpolar solvent is ethanol.
Preferably, the content of the water-soluble cationic polymer in the solution B is 1%, the content of the antioxidant is 12%, and the content of the stabilizer is 0.5%.
Preferably, the content of the water-soluble cationic polymer in the solution B is 10%, the content of the antioxidant is 5%, and the content of the stabilizer is 3%.
Preferably, the water-soluble cationic polymer in the solution B is polyamide polyamine epichlorohydrin (PAE), the antioxidant is ascorbic acid, and the stabilizer is oxalic acid.
Preferably, the application method of the nucleic acid cleaning combined reagent is that the solution A cleaning agent is sprayed and wiped twice repeatedly, so that the nucleic acid pollution on the surface of the organic glass can be better removed.
In order to prove the clearing effect of the nucleic acid cleaning combined reagent, the efficiency of cleaning different cleaning reagents on the surface of organic glass is detected by adopting a real-time fluorescence quantitative PCR method and is compared, 6 mu L of nucleic acid samples are respectively absorbed by a pipette and dropped on the surface of the organic glass, and the organic glass is placed on an ultraclean workbench to be blown and dried; after drying, wiping the surface of the organic glass with paper towels wetted by tap water, ethanol, the solution A, the solution B and the solution C respectively; and after drying for 2-5min, wiping the residual surface of the nucleic acid by using a paper towel wetted by deionized water, placing the wiped paper towel in the deionized water, uniformly mixing by using a clean forceps, after all sample tubes are operated, centrifuging for a short time on a centrifugal machine, collecting supernatant, transferring the supernatant into a new centrifugal tube, and performing real-time fluorescence quantitative detection, wherein experimental results show that the removal efficiency of the solution A and the solution B is higher.
The invention further verifies the combined use cleaning efficiency of the liquid A and the liquid B, and compared with two experimental groups of the liquid A, the liquid A and the liquid B, and the liquid B, the experimental result shows that the combined use of the liquid A and the liquid B can effectively improve the nucleic acid clearance rate.
In conclusion, the invention has the following beneficial effects:
firstly, the nucleic acid can be conveniently, quickly and quickly removed after being sprayed;
secondly, the coating has chemical stability and heat resistance and can be stored for a long time;
thirdly, no damage is caused to instrument and equipment: has no corrosiveness, and can be directly sprayed on the surface of an instrument.
Drawings
FIG. 1 shows the Δ Ct values of five nucleic acid washing reagents for fluorescence quantitative PCR detection in the present invention;
FIG. 2 is a qPCR amplification curve of the nucleic acid detergent of the present invention for cleaning the genome on the surface of the organic glass;
FIG. 3 shows the nucleic acid clearance rate of the combination of the solutions A and B in the real-time fluorescent quantitative PCR assay of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and examples.
Example 1
A nucleic acid cleaning combined reagent and a using method thereof are formed by combining a liquid A and a liquid B, wherein the liquid A is composed of 15% of hydrogen peroxide, 0.8% of Tween 20, 20% of ethanol and the balance of deionized water. The B liquid consists of 10 percent of polyamide polyamine epichlorohydrin (PAE), 5 percent of ascorbic acid, 3 percent of oxalic acid and the balance of deionized water.
The experimental groups were: tap water, ethanol, solution C (0.4% sodium hypochlorite and deionized water), solution A and solution B.
The formula for the calculation of nucleic acid clearance is as follows:
Delta-Ct: sample Ct-Positive control Ct
Origin quality: negative control Ct-positive control Ct;
the five cleaning agents are compared on the efficiency of removing the high-concentration PCR product, and the specific experimental steps are as follows: dripping 6 mu L of nucleic acid samples respectively absorbed by a pipette on the surface of organic glass, and placing the organic glass on an ultraclean workbench for air blowing and drying; after drying, wiping the surface of the organic glass with paper towels wetted by tap water, ethanol, the solution A, the solution B and the solution C respectively; and after drying for 2-5min, wiping the residual surface of the nucleic acid by using a paper towel wetted by deionized water, putting the wiped paper towel into the deionized water, uniformly mixing by using a clean forceps, after all sample tubes are operated, centrifuging for a short time on a centrifugal machine, collecting supernatant, transferring the supernatant into a new centrifugal tube, and performing real-time fluorescence quantitative detection.
As shown in figure 1, the cleaning efficiency of different cleaning reagents on the surface of organic glass is detected by a real-time fluorescent quantitative PCR method and compared, and the experimental result shows that the cleaning efficiency of the solution A and the solution B is higher.
Example 2
A nucleic acid cleaning combined reagent and a using method thereof are formed by combining a liquid A and a liquid B, wherein the liquid A is composed of 4% of hydrogen peroxide, 0.8% of Tween 100, 20% of ethanol and the balance of deionized water. The B liquid consists of 1 percent of polyamide polyamine epichlorohydrin (PAE), 12 percent of ascorbic acid, 3 percent of oxalic acid and the balance of deionized water.
The experimental groups were: tap water, ethanol, solution A, solution B, a combination of solution A and solution B, a negative control (buffer without nucleic acid), and a positive control (high concentration nucleic acid).
As shown in FIG. 2, the five nucleic acid detergents are used for cleaning the surface genome of organic glass and extracting, and then the fluorescence quantitative determination is carried out to obtain a qPCR amplification curve. Experimental results show that a positive control, namely high-concentration nucleic acid, is smeared on the surface of the organic glass, a large amount of nucleic acid pollution is detected by fluorescent quantitative PCR, part of nucleic acid pollution can be eliminated by independently spraying and wiping the solution A and the solution B, the nucleic acid pollution can be obviously eliminated by the combined use of the solution A and the solution B, and the elimination efficiency is higher than that of ethanol and deionized water.
Example 3:
a nucleic acid cleaning combined reagent and a using method thereof are formed by combining two cleaning agents of solution A and solution B, wherein the solution A is composed of 15% of hydrogen peroxide, 0.8% of triton X-100, 20% of acetic acid and the balance of deionized water. The solution B consists of 10% polydiallyldimethylammonium chloride (PDADMAC), 12% ascorbic acid, 3% sodium bicarbonate and the balance deionized water.
The experimental groups were: liquid A, liquid A & liquid A (spraying and wiping are repeated twice), liquid B & liquid B (spraying and wiping are repeated twice), liquid A & liquid B combined use, negative control (buffer solution without nucleic acid), and positive control (high-concentration nucleic acid).
As shown in FIG. 2, the five nucleic acid cleaning agents are used for cleaning the surface genome of the organic glass, extracting, then performing fluorescence quantitative determination, and calculating the Ct value by adopting a clearance formula of nucleic acid clearance. The experimental result shows that compared with the solution A, the cleaning efficiency of the cleaning agent of the solution A and the solution A is improved by 20%, and the repeated cleaning is probably one of the influence factors for improving the cleaning rate. In addition, the cleaning agent used by combining the solution A and the solution B has higher efficiency for removing nucleic acid pollutants, and compared with two experimental groups of the solution A, the solution A and the solution B, the combined use of the solution A and the solution B can effectively improve the nucleic acid removal rate.
Example 4:
a method for using the nucleic acid washing combined reagent comprises the following steps:
(1) respectively preparing a solution A and a solution B nucleic acid cleaning agent and carrying out atomization treatment;
(2) wiping the surface of the organic glass polluted by the nucleic acid by using paper towels wetted by the solution A and the solution B in sequence.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (6)
1. A nucleic acid cleaning combined reagent and a using method thereof are characterized in that: the nucleic acid cleaning combined reagent is formed by combining two cleaning agents, namely a solution A and a solution B, wherein the solution A comprises an oxidizing agent, a surfactant, a nonpolar solvent and deionized water, and the solution B comprises a water-soluble cationic polymer, an antioxidant, a stabilizer and deionized water.
2. The nucleic acid washing combined reagent according to claim 1, wherein: the component and mass percentage of the solution A are 4-15% of oxidant, 0.8-10% of surfactant, 5-20% of non-polar solvent and the balance of deionized water.
3. The nucleic acid washing combined reagent according to claim 2, wherein: the oxidant in the solution A is hydrogen peroxide or peracetic acid, the active agents are tween 20, tween 100 and triton X-100, and the nonpolar solvent is ethanol or acetic acid.
4. The nucleic acid washing combined reagent according to claim 1, wherein: the liquid B comprises, by mass, 1-10% of a water-soluble cationic polymer, 5-12% of an antioxidant, 0.5-3% of a stabilizer and the balance of deionized water.
5. The nucleic acid washing combined reagent according to claim 4, wherein: the water-soluble cationic polymer in the solution B is polyamide polyamine epichlorohydrin (PAE) or polydiallyldimethylammonium chloride (PDADMAC), the antioxidant is ascorbic acid, glutathione or lipoic acid, and the stabilizer is sodium bicarbonate or oxalic acid.
6. The method for using the nucleic acid washing combined reagent according to claim 1, characterized by comprising the following steps:
(1) respectively preparing a solution A and a solution B nucleic acid cleaning agent and carrying out atomization treatment;
(2) wiping the surface of the organic glass polluted by the nucleic acid by using paper towels wetted by the solution A and the solution B in sequence.
Priority Applications (1)
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| CN115820343A (en) * | 2022-11-17 | 2023-03-21 | 浙江泰林医学工程有限公司 | Nucleic acid scavenger, preparation method and application thereof |
| CN115873670A (en) * | 2022-11-29 | 2023-03-31 | 苏州近岸蛋白质科技股份有限公司 | Biological enzyme scavenger for eliminating nucleic acid pollution and preparation method thereof |
| CN115873663A (en) * | 2022-12-11 | 2023-03-31 | 熠创鼎惠(上海)生物科技有限公司 | Aqueous solution for removing object surface nucleic acid pollution and wet tissue thereof |
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