阅读说明：本技术 一种核酸提取的裂解结合液及其提取方法 (Lysis binding solution for nucleic acid extraction and extraction method thereof ) 是由 不公告发明人 于 2021-10-13 设计创作，主要内容包括：本发明公开了一种核酸提取的裂解结合液及其提取方法,裂解结合液包括以下物质成分：5～500mM的Tris-HCl缓冲液、20～1000mM的多巴胺、100～400mM的三羟甲基氨基甲烷、50～200mM的异硫氰酸胍、0.4％～3％的三羟甲基氨基甲烷、0.02％～2％的壬基酚聚氧乙烯醚、0.03％～1.5％的聚氧乙烯壬基酚磷酸酯、0.1％～2％的烯丙基聚氧乙烯醚、0.05％～1.8％的聚丙二醇二缩水甘油醚、0.08％～4％的月桂酸单甘油酯、0.06％～3.5％的单乙酸甘油酯、余量为纯水。(The invention discloses a lysis binding solution for nucleic acid extraction and an extraction method thereof, wherein the lysis binding solution comprises the following components: 5-500 mM Tris-HCl buffer solution, 20-1000 mM dopamine, 100-400 mM trihydroxymethyl aminomethane, 50-200 mM guanidinium isothiocyanate, 0.4-3% trihydroxymethyl aminomethane, 0.02-2% nonylphenol polyoxyethylene ether, 0.03-1.5% polyoxyethylene nonylphenol phosphate, 0.1-2% allyl polyoxyethylene ether, 0.05-1.8% polypropylene glycol diglycidyl ether, 0.08-4% lauric acid monoglyceride, 0.06-3.5% monoacetin glyceride and the balance of pure water.)

1. A cracking binding solution for nucleic acid extraction is characterized by comprising the following components: 5-500 mM Tris-HCl buffer solution, 20-1000 mM dopamine, 100-400 mM trihydroxymethyl aminomethane, 50-200 mM guanidinium isothiocyanate, 0.4-3% trihydroxymethyl aminomethane, 0.02-2% nonylphenol polyoxyethylene ether, 0.03-1.5% polyoxyethylene nonylphenol phosphate, 0.1-2% allyl polyoxyethylene ether, 0.05-1.8% polypropylene glycol diglycidyl ether, 0.08-4% lauric acid monoglyceride, 0.06-3.5% monoacetin glyceride and the balance of pure water.

2. The lysis solution for nucleic acid extraction according to claim 1, wherein the lysis solution further comprises 4-8% magnetic beads.

3. The lysis-binding solution for nucleic acid extraction according to claim 1, wherein the pH of the lysis-binding solution is controlled to be 6.5-7.2.

4. The lysis binding solution for nucleic acid extraction according to claim 2, wherein the magnetic beads are superparamagnetic nanospheres with a diameter of 400-600 nm.

5. The method for extracting a cleavage binding solution for nucleic acid extraction according to claim 1, comprising the steps of:

s1: adding the cracking binding solution and the magnetic beads into a centrifuge tube, uniformly mixing by shaking, then adding a sample to be tested, uniformly shaking by vortex, heating to 40-80 ℃, and centrifuging;

s2: standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding enzyme, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant;

s3: adding a washing solution into the centrifuge tube, and repeating the step S2 once;

s4: and (4) drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use.

6. The method for extracting the lysis binding solution for nucleic acid extraction according to claim 1, wherein the centrifugation rate is controlled to be 6000 to 12000rpm/min and the centrifugation time is controlled to be 6 to 20 min.

Technical Field

The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a cracking binding solution for nucleic acid extraction and an extraction method.

Background

Nucleic acid extraction is a very critical step in the field of nucleic acid molecule detection. Since MeselsonM and the like separated DNA by density gradient centrifugation for the first time in 1957, various nucleic acid extraction methods have been reported, many researchers have conducted continuous research on the nucleic acid extraction methods, various materials and reagents for nucleic acids have been improved, various reagents such as sodium dodecylsulfate, phenol, urea and guanidine salt have been applied to nucleic acid extraction experiments, and various commercial kits for nucleic acid extraction have come into force. The solid phase methods usually need to adopt steps of rapid centrifugation, vacuum filtration and the like for several times to realize separation, and have the disadvantages of large sample demand, more sample consumption, inconvenience for high-throughput and automatic operation, and serious limitation on the application in the field of clinical gene diagnosis. With the development of molecular biology technology, in order to meet the experimental requirements of high-throughput sample processing and high-quality nucleic acid acquisition, the nucleic acid extraction and separation technology tends to be developed more simply, conveniently, quickly, with high quality, high purity and high throughput, and the quality of nucleic acid directly determines the success or failure of subsequent experiments. In recent years, molecular diagnostic techniques have been rapidly developed, and with the maturity of techniques such as gene sequencing, the cost is further reduced, and the application in clinic is more and more common. The types of samples for molecular diagnosis and detection are dozens of samples, and the detection items suitable for the processed products also cover various molecular biology technical platforms such as fluorescent quantitative PCR, gene sequencing, gene chips, biological mass spectrometry and the like. However, regardless of the type of test item, the accurate test result is undoubtedly dependent on the specimen of high quality and the specimen pretreatment process. Therefore, the automation trend of clinical molecular diagnosis will gradually become dominant in the domestic clinical laboratory, and the automation is firstly realized on the nucleic acid extraction which is popular in the current manual operation and has the greatest influence on the result. By adopting the magnetic bead extraction method, the extraction rate and the extraction purity of nucleic acid are mainly influenced by extracting solutions, such as lysis solution, binding solution, washing solution, eluent and the like, and if the amount and the purity of the extracted sample are not too high, the nucleic acid cannot be effectively detected, even the detection result is influenced, and even the subsequent experimental requirements cannot be completely met.

As an important substance carrying genetic information, nucleic acids are generally present in complex actual samples such as blood, cells, feces, leaves, and the like. The nucleic acids in these samples are often not directly available for molecular biology experiments and assays, and further purification is required to remove interfering substances for some downstream experiments and analyses. Methods for isolating and extracting nucleic acids have been reported in the literature (e.g. Chapter 2(DNA) and Chapter4(RNA) of F.Ausubelety., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York, 1993). These methods generally require resuspending the sample in a solution and disrupting the cells by chemical or enzymatic means to release the nucleic acids. This process is called lysis. The released nucleic acids reversibly bind to the nucleic acid-adsorbing material in solution. These materials include glass particles, glass fibers, magnetic beads, diatomaceous earth, silica gel, and the like, or variations or combinations thereof. High concentrations of chaotropic salts will facilitate binding of nucleic acids to the above-mentioned materials. In the next step, these nucleic acid-binding materials are collected by centrifugation or by applying an external force such as a magnetic field. These nucleic acid-binding materials are then washed with a particular solution, such as 80% ethanol, to remove impurities. Finally, the nucleic acid binding material is treated with an eluent to elute the nucleic acids, typically by heating to 60 degrees and eluting with redistilled water. Based on this principle, researchers have reported a number of methods for extracting nucleic acids.

Disclosure of Invention

The invention aims to provide a lysis binding solution for nucleic acid extraction, which comprises the following components: 5-500 mM Tris-HCl buffer solution, 20-1000 mM dopamine, 100-400 mM trihydroxymethyl aminomethane, 50-200 mM guanidinium isothiocyanate, 0.4-3% trihydroxymethyl aminomethane, 0.02-2% nonylphenol polyoxyethylene ether, 0.03-1.5% polyoxyethylene nonylphenol phosphate, 0.1-2% allyl polyoxyethylene ether, 0.05-1.8% polypropylene glycol diglycidyl ether, 0.08-4% lauric acid monoglyceride, 0.06-3.5% monoacetin glyceride and the balance of pure water.

Further, the lysis binding solution further comprises 4% -8% of magnetic beads.

Further, the pH value of the cracking binding solution is controlled to be 6.5-7.0.

Further, the magnetic beads are superparamagnetic nano microspheres with the diameter of 400-600 nm.

The extraction method comprises the following steps:

s1: and adding the cracking binding solution and the magnetic beads into a centrifugal tube, uniformly mixing by shaking, then adding a sample to be tested, uniformly shaking by vortex, heating to 40-80 ℃, and centrifuging.

S2: and (4) standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding enzyme, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant.

S3: the washing solution was added to the centrifuge tube, and the above step S2 was repeated once.

S4: and (4) drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use.

Further, the enzyme is a ribonuclease.

Further, the centrifugation speed is controlled to be 6000-12000 rpm/min, and the centrifugation time is controlled to be 6-20 min.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the nucleic acid extraction and cracking combined solution can be used for cracking and adsorbing the sample at the same time, so that the operation steps are reduced, and the purity and the concentration of the extracted nucleic acid are high; and each component in the cracking combination liquid has better synergistic effect, and the concentration and quality of the extracted DNA can be greatly improved by synergistic use.

Detailed Description

The following embodiments of the present invention are described in detail, and the embodiments are implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Example 1

A method for extracting a cracking binding solution for nucleic acid extraction comprises the following components: 5mM Tris-HCl buffer solution, 20mM dopamine, 100mM trihydroxymethyl aminomethane, 50mM guanidinium isothiocyanate, 0.4% trihydroxymethyl aminomethane, 0.02% nonylphenol polyoxyethylene ether, 0.03% polyoxyethylene nonylphenol phosphate, 0.1% allyl polyoxyethylene ether, 0.05% polypropylene glycol diglycidyl ether, 0.08% lauric acid monoglyceride, 0.06% monoacetin glyceride, and the balance being pure water.

The lysis binding solution further comprises 4% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.5, and the magnetic beads are superparamagnetic nano microspheres with the diameter of 400 nm.

The extraction method comprises the following steps:

s1: and adding the cracking binding solution and the magnetic beads into a centrifuge tube, uniformly mixing by shaking, adding a sample to be tested, uniformly shaking by vortex, heating to 40 ℃, and centrifuging.

S2: and (4) standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding ribonuclease, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant.

S3: the washing solution was added to the centrifuge tube, and the above step S2 was repeated once.

S4: air-drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use; wherein, the centrifugation speed is controlled at 6000rpm/min and the centrifugation time is controlled at 6min in the centrifugation process.

Example 2

A method for extracting a cracking binding solution for nucleic acid extraction comprises the following components: 500mM Tris-HCl buffer solution, 1000mM dopamine, 400mM Tris, 200mM guanidine isothiocyanate, 3% Tris, 2% nonylphenol polyoxyethylene ether, 1.5% polyoxyethylene nonylphenol phosphate, 2% allyl polyoxyethylene ether, 1.8% polypropylene glycol diglycidyl ether, 4% lauric acid monoglyceride, 3.5% monoacetin glyceride and the balance of pure water.

The lysis binding solution further comprises 8% of magnetic beads, the pH value of the lysis binding solution is controlled to be 7.0, and the magnetic beads are superparamagnetic nano microspheres with the diameter of 600 nm.

The extraction method comprises the following steps:

s1: and adding the cracking binding solution and the magnetic beads into a centrifuge tube, uniformly mixing by shaking, adding a sample to be tested, uniformly shaking by vortex, heating to 80 ℃, and centrifuging.

S2: and (4) standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding ribonuclease, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant.

S3: the washing solution was added to the centrifuge tube, and the above step S2 was repeated once.

S4: air-drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use; wherein, the centrifugation speed is controlled at 12000rpm/min and the centrifugation time is controlled at 20min in the centrifugation process.

Example 3

A method for extracting a cracking binding solution for nucleic acid extraction comprises the following components: 200mM Tris-HCl buffer solution, 500mM dopamine, 200mM Tris-hydroxymethyl-aminomethane, 100mM guanidine isothiocyanate, 0.9% Tris-hydroxymethyl-aminomethane, 0.6% nonylphenol polyoxyethylene ether, 0.85% polyoxyethylene nonylphenol phosphate, 0.8% allyl polyoxyethylene ether, 0.8% polypropylene glycol diglycidyl ether, 2.2% lauric acid monoglyceride, 1.2% monoacetin glyceride, and the balance being pure water.

The lysis binding solution further comprises 5% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.7, and the magnetic beads are superparamagnetic nano microspheres with the diameter of 500 nm.

The extraction method comprises the following steps:

s1: and adding the cracking binding solution and the magnetic beads into a centrifuge tube, uniformly mixing by shaking, adding a sample to be tested, uniformly shaking by vortex, heating to 60 ℃, and centrifuging.

S2: and (4) standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding ribonuclease, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant.

S3: the washing solution was added to the centrifuge tube, and the above step S2 was repeated once.

S4: air-drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use; wherein the centrifugation speed is controlled at 8000rpm/min and the centrifugation time is controlled at 10min in the centrifugation process.

Example 4

A method for extracting a cracking binding solution for nucleic acid extraction comprises the following components: 400mM Tris-HCl buffer solution, 800mM dopamine, 300mM Tris-hydroxymethyl-aminomethane, 150mM guanidine isothiocyanate, 2.6% Tris-hydroxymethyl-aminomethane, 1.8% nonylphenol polyoxyethylene ether, 1.3% polyoxyethylene nonylphenol phosphate, 1.8% allyl polyoxyethylene ether, 1.5% polypropylene glycol diglycidyl ether, 3.5% lauric acid monoglyceride, 3.2% monoacetin glyceride, and the balance being pure water.

The lysis binding solution further comprises 7% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.8, and the magnetic beads are superparamagnetic nano microspheres with the diameter of 550 nm.

The extraction method comprises the following steps:

s1: and adding the cracking binding solution and the magnetic beads into a centrifuge tube, uniformly mixing by shaking, adding a sample to be tested, uniformly shaking by vortex, heating to 70 ℃, and centrifuging.

S2: and (4) standing the liquid centrifuged in the step S1, adding the supernatant into another centrifuge tube, adding ribonuclease, uniformly standing, adding magnetic beads, placing on a magnetic frame for adsorption, and removing the supernatant.

S3: the washing solution was added to the centrifuge tube, and the above step S2 was repeated once.

S4: air-drying the centrifuge tube, adding the eluent, shaking uniformly, centrifuging again, and taking the supernatant for later use; wherein, the centrifugation speed is controlled at 10000rpm/min and the centrifugation time is controlled at 15min in the centrifugation process.

Examples of the experiments

The same fecal samples are selected, the samples in examples 1-4 are used for detecting the concentration and quality of DNA extracted from the same batch of samples, and the results are shown in the following table 1:

table 1. test results:

as can be seen from Table 1, the lysis binding solutions of examples 1 to 4 of the present invention have excellent DNA mass concentration.