阅读说明：本技术 一种用于核酸提取的裂解结合液及提取方法 (Cracking binding solution for nucleic acid extraction and extraction method ) 是由 不公告发明人 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种用于核酸提取的裂解结合液及提取方法,裂解结合液包括以下物质成分：5～500mM的Tr i s-HCl缓冲液、100～400mM的乙二胺四乙酸、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、0.1％～2％的烯丙基聚氧乙烯醚、0.08％～4％的月桂酸单甘油酯、余量为纯水。(The invention discloses a cracking binding solution for nucleic acid extraction and an extraction method, wherein the cracking binding solution comprises the following components: 5-500 mM Tris s-HCl buffer solution, 100-400 mM ethylene diamine tetraacetic acid, 50-200 mM guanidine isothiocyanate, 0.4-3% of tris (hydroxymethyl) aminomethane, 0.02-2% of nonylphenol polyoxyethylene ether, 0.1-2% of allyl polyoxyethylene ether, 0.08-4% of lauric monoglyceride 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, 100-400 mM ethylene diamine tetraacetic acid, 50-200 mM guanidine isothiocyanate, 0.4-3% of Tris (hydroxymethyl) aminomethane, 0.02-2% of nonylphenol polyoxyethylene ether, 0.1-2% of allyl polyoxyethylene ether, 0.08-4% of lauric monoglyceride and the balance of pure water.

2. The lysis binding solution for nucleic acid extraction according to claim 1, wherein the lysis binding 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 between 6.2 and 6.8.

4. The lysis binding solution for nucleic acid extraction according to claim 2, wherein the magnetic beads are superparamagnetic nanospheres with a diameter of 300-600 nm.

5. The method for extracting a lysis binding solution for nucleic acid extraction according to claim 1, wherein the method comprises 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 6000-12000 rpm/min and the centrifugation time is controlled to be 6-20 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 Mesel sonM and the like separated DNA by a density gradient centrifugation method 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 acid have been improved, various reagents such as sodium dodecyl sulfate, 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.

The existing nucleic acid extraction methods mainly comprise a pyrolysis method, a phenol chloroform extraction method, a centrifugal column method and a magnetic bead method; the heating cracking method is that cracking solution is added into a sample, and cells are cracked by the heating method to release nucleic acid, the method is simple and convenient to operate, but the extracted nucleic acid has low purity and low flux, is not beneficial to subsequent detection and is easy to cause pollution; phenol chloroform extraction has high toxicity to operators due to the use of a large amount of toxic solvents, and is difficult to realize automatic operation; the centrifugal column method has low nucleic acid extraction efficiency and high cost, needs a high-speed centrifuge, is difficult to realize automation, and cannot be widely applied to large-scale clinical detection. The magnetic bead method nucleic acid extraction technology developed in recent years adopts superparamagnetic nanoparticles, and utilizes the principle of adsorbing nucleic acid under high salt and low pH and then separating nucleic acid under low salt and high pH to separate and purify sample nucleic acid, and can realize high-throughput automatic standardized operation because the nucleic acid can be separated and purified only under the action of a magnetic field; however, the commercial reagents based on the magnetic bead method in the current market are too complex to operate, require incubation with proteinase K and the like at a certain temperature, and require multiple washing steps, resulting in low automation degree, poor repeatability of nucleic acid extraction and low extraction efficiency. The magnetic bead method for extracting nucleic acid mainly comprises the following steps: (1) cracking; (2) combining; (3) washing; (4) and separating the four major parts. Specifically, under the action of a lysis solution, DNA/RNA is released and then is subjected to specific binding with surface-modified superparamagnetic nano magnetic beads to form a nucleic acid-magnetic bead compound, then washing liquid is added to wash away impurities such as nonspecifically adsorbed proteins, finally, the magnetic beads are separated and enriched under the condition of a magnetic field, and finally, the nucleic acid is eluted from the magnetic beads by using eluent to obtain the DNA/RNA to be extracted.

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, 100-400 mM ethylene diamine tetraacetic acid, 50-200 mM guanidine isothiocyanate, 0.4-3% of Tris (hydroxymethyl) aminomethane, 0.02-2% of nonylphenol polyoxyethylene ether, 0.1-2% of allyl polyoxyethylene ether, 0.08-4% of lauric monoglyceride 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 6.2-6.8.

Further, the magnetic beads are superparamagnetic nano microspheres with the diameter of 300-600 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, 100mM ethylene diamine tetraacetic acid, 50mM guanidinium isothiocyanate, 0.4% trihydroxymethyl aminomethane, 0.02% nonylphenol polyoxyethylene ether, 0.1% allyl polyoxyethylene ether, 0.08% lauric acid monoglyceride and the balance of pure water.

The lysis binding solution further comprises 4% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.2, and the magnetic beads are superparamagnetic nano microspheres with the diameter of 300 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, 400mM ethylene diamine tetraacetic acid, 200mM guanidinium isothiocyanate, 3% trihydroxymethyl aminomethane, 2% nonylphenol polyoxyethylene ether, 2% allyl polyoxyethylene ether, 4% lauric acid monoglyceride 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 6.8, 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, 200mM ethylene diamine tetraacetic acid, 100mM guanidinium isothiocyanate, 1.2% trihydroxymethyl aminomethane, 0.8% nonylphenol polyoxyethylene ether, 0.7% allyl polyoxyethylene ether, 0.9% lauric acid monoglyceride and the balance of pure water.

The lysis binding solution further comprises 5% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.4, 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 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, 300mM ethylene diamine tetraacetic acid, 150mM guanidinium isothiocyanate, 2% trihydroxymethyl aminomethane, 1.5% nonylphenol polyoxyethylene ether, 1.8% allyl polyoxyethylene ether, 3.5% lauric acid monoglyceride and the balance of pure water.

The lysis binding solution further comprises 7% of magnetic beads, the pH value of the lysis binding solution is controlled to be 6.6, 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 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.

Stool samples are selected, 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.