阅读说明：本技术 一种通用型快速提取生物样本总核酸的试剂盒及方法 (General kit and method for rapidly extracting total nucleic acid of biological sample ) 是由 唐勇军 刘小龙 黄婷 罗俭康 于 2021-08-18 设计创作，主要内容包括：本发明公开一种通用型快速提取生物样本总核酸的试剂盒及方法,通用型快速提取生物样本总核酸试剂盒,包括：Buffer ATL、Proteinase K、MBs磁珠、Buffer WSI、Buffer WSII、Buffer WSIII、Buffer ES、Buffer MDS。与现有核酸提取技术相比,本发明技术方案的有益效果为：无需对血液制品进行预处理,裂解结合一步完成,大大节省了全血基因组核酸提取的时间；使用Buffer WSI、Buffer WSII、Buffer WSIII三种洗涤液可以洗去样本中绝大部分的蛋白、酚类、多糖等抑制杂质,最大限度的减少杂质的污染对下游实验的干扰；本发明试剂盒及方法可通用于多种来源样本的核酸提取,适用范围广。(The invention discloses a general kit and a method for rapidly extracting total nucleic acid of a biological sample, wherein the general kit for rapidly extracting the total nucleic acid of the biological sample comprises the following components: buffer ATL, protease K, MBs magnetic beads, Buffer WSI, Buffer WSII, Buffer WSIII, Buffer ES and Buffer MDS. Compared with the prior nucleic acid extraction technology, the technical scheme of the invention has the beneficial effects that: the blood product is not required to be pretreated, and the cracking is completed in one step, so that the time for extracting the whole blood genome nucleic acid is greatly saved; the three washing solutions of Buffer WSI, Buffer WSII and Buffer WSIII can be used for washing away most of protein, phenols, polysaccharides and other inhibitory impurities in the sample, so that the interference of the pollution of the impurities on downstream experiments is reduced to the maximum extent; the kit and the method can be universally used for extracting nucleic acid from various source samples, and have wide application range.)

1. A general kit for rapidly extracting total nucleic acid from a biological sample is characterized by comprising: buffer ATL, protease K, MBs magnetic beads, Buffer WSI, Buffer WSII, Buffer WSIII and Buffer ES;

the Buffer ATL comprises: the pH value of Buffer ATL is 5.0-8.0; wherein the concentration of guanidine hydrochloride is 1-6mol/L, the concentration of sodium lauroyl sarcosine is 1-15 wt%, the concentration of isopropanol is 40-60% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L;

the protease K comprises: protease K, KCl and glycerol in Tris-HCl buffer solution, wherein the pH value of the protease K is 5.0-8.0; wherein the concentration of the protease k is 20-50mg/mL, the concentration of the KCl is 50-150mmol/L, the concentration of the glycerol is 30-50% v/v, and the concentration of the Tris-HCl buffer solution is 10-50 mmol/L;

the MBs magnetic bead comprises: hydroxyl magnetic beads or carboxyl magnetic beads, NaCl and absolute ethyl alcohol, wherein the pH value of the MBs magnetic beads is 5.0-8.0; wherein the concentration of the hydroxyl magnetic beads or the carboxyl magnetic beads is 1-5mg/mL, and the particle size is 200-1500 nm; the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L;

the Buffer WSI comprises: Tris-HCl buffer solution of NaCl, isopropanol and PEG; the pHs of Buffer WSI are 5.0-8.0; wherein, the concentration of NaCl is 500-1000mmol/L, the concentration of isopropanol is 75-85% v/v, the concentration of PEG is 35-45 wt%, wherein the molecular weight range of PEG is 4000-10000, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L;

the Buffer WSII is Tris-HCl Buffer solution containing isopropanol, and the pH value of the Buffer WSII is 5.0-8.0; wherein the concentration of the isopropanol is 75-85% v/v, and the concentration of the Tris-HCl buffer solution is 50-100 mmol/L;

the Buffer WSIII is Tris-HCl Buffer solution containing KCl, and the pH value of the Buffer WSIII is 6.8-9.0; wherein, the concentration of KCl is 0.85-2 wt%, and the concentration of Tris-HCl buffer solution is 40-120 mmol/L;

the Buffer ES is trihydroxymethyl aminomethane hydrochloride containing ethylene diamine tetraacetic acid disodium, wherein the concentration of the ethylene diamine tetraacetic acid disodium is 1.0 +/-0.2 mmol/L, the concentration of the trihydroxymethyl aminomethane hydrochloride is 10 +/-1 mmol/L, and the pH value of the Buffer ES is 6.0-8.0.

2. The universal kit for rapidly extracting total nucleic acid from a biological sample according to claim 1, further comprising: the Buffer MDS is Tris-HCl Buffer solution containing NaCl and absolute ethyl alcohol, and the pH value of the Buffer MDS is 5.0-8.0; wherein the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 6-16 mmol/L; the Buffer MDS is a diluent of MBs magnetic beads; the Buffer MDS is applied to automation operation.

3. A universal method for rapidly extracting total nucleic acid from a biological sample, which is based on the universal kit for rapidly extracting total nucleic acid from a biological sample of claim 1, and comprises the following steps: the method comprises the following steps:

s1: taking a container, adding protease K, a sample, MBs magnetic beads and Buffer ATL, uniformly mixing, heating at 50-60 ℃ for 4-20 minutes, and reversely mixing once every 1-2 minutes; wherein, Buffer ATL: protease K: MBs magnetic beads: the volume ratio of the sample is: 50-80: 2-4: 4-8: 8-30 parts of;

s2: after incubation is finished, cooling the container to room temperature, transferring the container to a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, and performing magnetic adsorption on the magnetic frame in a standing mode until the solution is clarified.

S3: taking down the container, adding Buffer WSI, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSI: the volume ratio of Buffer ATL is 6-8: 5-8;

s4: taking down the container, adding Buffer WSII, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSII: volume of Buffer WSI 1: 1;

s5: taking down the container, adding Buffer WSIII, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSIII: volume of Buffer WSI 1: 1;

s6: placing the container on a magnetic frame, opening the cover at room temperature, and drying until the surface of the hydroxyl magnetic beads or the carboxyl magnetic beads is free of floodlight, which indicates that the container is fully dried;

s7: adding Buffer ES, mixing, and heating at 60-70 deg.C for 2-15 min; wherein, Buffer ES: volume ratio of Buffer WSI 1: 6-10;

s8: and after the incubation is finished, slightly oscillating the container to mix the hydroxyl magnetic beads or the carboxyl magnetic beads uniformly, placing the container on a magnetic frame, carrying out magnetic attraction until the solution is clarified, carefully transferring the supernatant to a storage tube, and storing the supernatant at a temperature below-20 ℃ for later use.

4. The method for universal rapid extraction of total nucleic acid in biological samples according to claim 3, wherein the mixing is performed by vortex oscillation for 10-100 seconds; the magnetic enrichment time is 5-60 seconds, and the magnetic adsorption time is 10-100 seconds.

5. A universal rapid extraction method of total nucleic acid in a biological sample, which is characterized in that the universal rapid extraction kit of total nucleic acid in a biological sample according to claim 2 is applied to an automatic device operation, and the universal rapid extraction kit of total nucleic acid in a biological sample comprises the following steps:

SS 1: preparing a sterile 96-depth well plate, wherein the 96-depth well plate is 8 rows by 12 columns; wherein 500-800. mu.L Buffer ATL and 20-40. mu.L protease K are added into the hole of the 1 st column and/or the 7 th column; adding 40-80 mu L of MBs magnetic beads and 800 mu L of Buffer MDS 600-; adding 800 mu L of Buffer WSI 600-; adding 800 mu L of Buffer WSII 600-; adding 800 mu L of Buffer WSIII 600-; adding Buffer ES80-100 μ L into the hole of the 6 th row and/or the 12 th row;

SS 2: adding 50-200 mu L of sample into the hole containing Buffer ATL and protease K;

SS 3: the 96 deep-hole plate is sent into an instrument, the sequence of the rows of the plate from inside to outside is A-H, and the following procedures are carried out:

SS 31: lysis, mixing the solution in the 1 st and/or 7 th row well, heating at 50-60 deg.C for 4-20 min;

SS 32: moving magnetism, carrying out magnetic attraction for 30-90 seconds, adsorbing hydroxyl magnetic beads or carboxyl magnetic beads in the holes of the 2 nd row and/or the 8 th row, adding the mixture into the holes of the 1 st row and/or the 7 th row, and fully mixing the mixture;

SS 33: combining, fully mixing the solution in the holes of the 1 st column and/or the 7 th column, heating for 4-20 minutes at 50-60 ℃, then magnetically attracting for 60-120 seconds, and adsorbing hydroxyl magnetic beads or carboxyl magnetic beads;

SS 34: first cleaning, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS33 to the holes in the 3 rd column and/or the 9 th column, fully mixing, then magnetically attracting for 60-120 seconds, and adsorbing the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 35: second cleaning, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS34 to the holes in the 4 th column and/or the 10 th column, fully mixing, and then magnetically attracting for 60-120 seconds to adsorb the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 36: washing for the third time, transferring the hydroxyl magnetic beads or the carboxyl magnetic beads adsorbed in the step SS35 to the holes in the 5 th column and/or the 11 th column, fully mixing, and then magnetically attracting for 20-80 seconds to adsorb the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 37: eluting, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS36 to the holes in the 6 th column and/or the 12 th column, fully mixing, heating at 60-70 ℃ for 3-20 minutes, then magnetically adsorbing for 20-80 seconds, and adsorbing the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 38: removing magnetism, and transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS37 to the holes in the 2 nd column and/or the 8 th column;

SS 4: the whole liquid in the well of column 6 and/or column 12 is transferred to a storage tube and stored below-20 ℃ for later use.

6. The method as claimed in claim 5, wherein if the sample is insufficient, the sample is supplemented with physiological saline or PBS.

7. The method as claimed in claim 5, wherein the maximum liquid loading of a single well of the 96-well reaction plate is less than 1.0 mL.

Technical Field

The invention relates to the technical field of biology, in particular to a universal kit and a method for rapidly extracting total nucleic acid of a biological sample.

Background

Nucleic acid detection is increasingly being used in the fields of clinical disease diagnosis, blood transfusion safety, forensic science identification, environmental microorganism detection, food safety detection, and the like. At present, two techniques, sequencing and fluorescence quantitative PCR, have become the main tools for detecting gene polymorphism, and digital PCR is also being used. However, neither sequencing nor qPCR, extraction and purification of nucleic acids is an essential technical step;

among the traditional and commercial methods for nucleic acid extraction, centrifugal column extraction and phenol-chloroform extraction are the two most classical and most commonly used methods, and are commonly used in pathogen detection and research facilities. Although the two methods extract high quality nucleic acid, the operation time is long, the efficiency is low and the automation difficulty is large. Most importantly, toxic reagents such as phenol, chloroform and the like are used in experiments, which cause great harm to operators and ecological environment. The nano magnetic bead separation and purification technology does not need centrifugation, does not need to contact toxic reagents, is simple and convenient to operate, is easy to realize automation, and realizes rapid and high-quality sample nucleic acid extraction. At present, the application of the magnetic bead method for separating nucleic acid from samples such as blood, animal tissues, food, pathogenic microorganisms and the like is gradually enriched, but the magnetic bead method generally has single function and is difficult to deal with the use scene of complex samples. In order to solve the problem of complicated nucleic acid analysis, it is necessary to develop a kit and a method for extracting nucleic acids from various samples.

Accordingly, the prior art is deficient and needs improvement.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a general kit and a method for rapidly extracting total nucleic acid of a biological sample, which are convenient for extracting nucleic acid from different samples.

The technical scheme of the invention is as follows: provides a general kit for rapidly extracting total nucleic acid of a biological sample, which comprises: buffer ATL, protease K, MBs magnetic beads, Buffer WSI, Buffer WSII, Buffer WSIII, Buffer ES and Buffer MDS.

The Buffer ATL comprises: the pH value of Buffer ATL is 5.0-8.0; wherein the concentration of guanidine hydrochloride is 1-6mol/L, the concentration of sodium lauroyl sarcosine is 1-15 wt%, the concentration of isopropanol is 40-60% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The protease K comprises: protease K, KCl and glycerol in Tris-HCl buffer solution, wherein the pH value of the protease K is 5.0-8.0; wherein the concentration of the protease k is 20-50mg/mL, the concentration of the KCl is 50-150mmol/L, the concentration of the glycerol is 30-50% v/v, and the concentration of the Tris-HCl buffer solution is 10-50 mmol/L.

The MBs magnetic bead comprises: hydroxyl magnetic beads or carboxyl magnetic beads, NaCl and absolute ethyl alcohol, wherein the pH value of the MBs magnetic beads is 5.0-8.0; wherein the concentration of the hydroxyl magnetic beads or the carboxyl magnetic beads is 1-5mg/mL, and the particle size is 200-1500 nm; the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The Buffer WSI comprises: Tris-HCl buffer solution of NaCl, isopropanol and PEG; the pHs of Buffer WSI are 5.0-8.0; wherein, the concentration of NaCl is 500-1000mmol/L, the concentration of isopropanol is 75-85% v/v, the concentration of PEG is 35-45 wt%, wherein the molecular weight range of PEG is 4000-10000, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The Buffer WSII is Tris-HCl Buffer solution containing isopropanol, and the pH value of the Buffer WSII is 5.0-8.0; wherein the concentration of the isopropanol is 75-85% v/v, and the concentration of the Tris-HCl buffer solution is 50-100 mmol/L.

The Buffer WSIII is Tris-HCl Buffer solution containing KCl, and the pH value of the Buffer WSIII is 6.8-9.0; wherein, the concentration of KCl is 0.85-2 wt%, and the concentration of Tris-HCl buffer solution is 40-120 mmol/L.

The Buffer ES is trihydroxymethyl aminomethane hydrochloride containing ethylene diamine tetraacetic acid disodium, wherein the concentration of the ethylene diamine tetraacetic acid disodium is 1.0 +/-0.2 mmol/L, the concentration of the trihydroxymethyl aminomethane hydrochloride is 10 +/-1 mmol/L, and the pH value of the Buffer ES is 6.0-8.0.

The Buffer MDS is a Tris-HCl Buffer solution containing NaCl and absolute ethyl alcohol, and the pH value of the Buffer MDS is 5.0-8.0; wherein the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 6-16 mmol/L; the Buffer MDS is a diluent of MBs magnetic beads; the Buffer MDS is applied to automation operation.

The invention provides two specific general methods for quickly extracting total nucleic acid of a biological sample, wherein the first method is a manual operation method, and the kit for quickly extracting the total nucleic acid of the biological sample based on the general method comprises the following steps:

s1: taking a container, adding protease K, a sample, MBs magnetic beads and Buffer ATL, uniformly mixing, heating at 50-60 ℃ for 4-20 minutes, and reversely mixing once every 1-2 minutes; wherein, Buffer ATL: protease K: MBs magnetic beads: the volume ratio of the sample is: 50-80: 2-4: 4-8: 8-30 parts of; preferably, Buffer ATL is 500-800 μ L, protease K is 20-40 μ L, MBs magnetic beads are 40-80 μ L, and samples are 80-300 μ L;

s2: after incubation is finished, cooling the container to room temperature, transferring the container to a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, and performing magnetic adsorption on the magnetic frame in a standing mode until the solution is clarified.

S3: taking down the container, adding Buffer WSI, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSI: the volume ratio of Buffer ATL is 6-8: 5-8; preferably, the Buffer WSI is 600-

S4: taking down the container, adding Buffer WSII, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSII: volume of Buffer WSI 1: 1; preferably, the Buffer WSII is 600-

S5: taking down the container, adding Buffer WSIII, mixing uniformly, placing the container on a magnetic frame, reversing the magnetic frame up and down after magnetic enrichment, washing hydroxyl magnetic beads or carboxyl magnetic beads possibly remaining on a tube cover into the solution in the tube, standing the magnetic frame, performing magnetic adsorption until the solution is clarified, and sucking and discarding the supernatant; wherein, Buffer WSIII: volume of Buffer WSI 1: 1; preferably, the Buffer WSIII is 600-

S6: placing the container on a magnetic frame, opening the cover at room temperature, and drying until the surface of the hydroxyl magnetic beads or the carboxyl magnetic beads is free of floodlight, which indicates that the container is fully dried;

s7: adding Buffer ES, mixing, and heating at 60-70 deg.C for 2-15 min; wherein, Buffer ES: volume ratio of Buffer WSI 1: 6-10; preferably, Buffer ES is 80-100 μ L;

s8: and after the incubation is finished, slightly oscillating the container to mix the hydroxyl magnetic beads or the carboxyl magnetic beads uniformly, placing the container on a magnetic frame, carrying out magnetic attraction until the solution is clarified, carefully transferring the supernatant to a storage tube, and storing the supernatant at a temperature below-20 ℃ for later use.

Further, uniformly mixing by vortex oscillation for 10-100 seconds; the magnetic enrichment time is 5-60 seconds, and the magnetic adsorption time is 10-100 seconds.

Preferably, the container is a centrifuge tube.

The invention provides two specific general methods for rapidly extracting total nucleic acid of a biological sample, wherein the second method is applied to automatic equipment operation and comprises the following steps:

SS 1: preparing a sterile 96-depth well plate, wherein the 96-depth well plate is 8 rows by 12 columns; wherein 500-800. mu.L Buffer ATL and 20-40. mu.L protease K are added into the hole of the 1 st column and/or the 7 th column; adding 40-80 mu L of MBs magnetic beads and 800 mu L of Buffer MDS 600-; adding 800 mu L of Buffer WSI 600-; adding 800 mu L of Buffer WSII 600-; adding 800 mu L of Buffer WSIII 600-; adding 80-100 mu L of Buffer ES into the hole of the 6 th row and/or the 12 th row;

SS 2: adding 50-200 mu L of sample into the hole containing Buffer ATL and protease K;

SS 3: the 96 deep-hole plate is sent into an instrument, the sequence of the rows of the plate from inside to outside is A-H, and the following procedures are carried out:

SS 31: lysis, mixing the solution in the 1 st and/or 7 th row well, heating at 50-60 deg.C for 4-20 min;

SS 32: moving magnetism, carrying out magnetic attraction for 30-90 seconds, adsorbing hydroxyl magnetic beads or carboxyl magnetic beads in the holes of the 2 nd row and/or the 8 th row, adding the mixture into the holes of the 1 st row and/or the 7 th row, and fully mixing the mixture;

SS 33: combining, fully mixing the solution in the holes of the 1 st column and/or the 7 th column, heating for 4-20 minutes at 50-60 ℃, then magnetically attracting for 60-120 seconds, and adsorbing hydroxyl magnetic beads or carboxyl magnetic beads;

SS 34: first cleaning, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS33 to the holes in the 3 rd column and/or the 9 th column, fully mixing, then magnetically attracting for 60-120 seconds, and adsorbing the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 35: second cleaning, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS34 to the holes in the 4 th column and/or the 10 th column, fully mixing, and then magnetically attracting for 60-120 seconds to adsorb the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 36: washing for the third time, transferring the hydroxyl magnetic beads or the carboxyl magnetic beads adsorbed in the step SS35 to the holes in the 5 th column and/or the 11 th column, fully mixing, and then magnetically attracting for 20-80 seconds to adsorb the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 37: eluting, transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS36 to the holes in the 6 th column and/or the 12 th column, fully mixing, heating at 60-70 ℃ for 3-20 minutes, then magnetically adsorbing for 20-80 seconds, and adsorbing the hydroxyl magnetic beads or the carboxyl magnetic beads;

SS 38: removing magnetism, and transferring the hydroxyl magnetic beads or carboxyl magnetic beads adsorbed in the step SS37 to the holes in the 2 nd column and/or the 8 th column;

SS 4: the whole liquid in the well of column 6 and/or column 12 is transferred to a storage tube and stored below-20 ℃ for later use.

If the sample is insufficient (less than 200. mu.L), the sample is supplemented with physiological saline or PBS.

The maximum liquid loading of the single hole of the 96-hole reaction plate needs to be less than 1.0 mL.

By adopting the scheme, the invention provides the universal kit and the method for rapidly extracting the total nucleic acid of the biological sample, the whole blood, the blood plasma/the blood serum to be extracted do not need to be pretreated, the protease K, the MBs magnetic beads and the Buffer ATL are directly mixed with the blood product, the cracking can be completed in one step, and the time is greatly shortened. In addition, the kit can be applied to the extraction of nucleic acid of biological products of various species, including various organisms such as mammals, birds, bacteria and the like; the extraction process can be manually completed, and can also be automatically completed by a full-automatic nucleic acid extractor; the nucleic acid extracted by using the kit can be directly applied to scientific researches such as PCR amplification, qPCR genotyping, gene sequencing and the like or analysis of forensic identification or clinical diagnosis. Compared with the prior general nucleic acid extraction technology, the technical scheme of the invention has the beneficial effects that:

1. the blood product is not required to be pretreated, and the cracking is completed in one step, so that the time for extracting the whole blood genome nucleic acid is greatly saved;

2. the three washing solutions of Buffer WSI, Buffer WSII and Buffer WSIII can be used for washing away most of protein, phenols, polysaccharides and other inhibitory impurities in the sample, so that the interference of the pollution of the impurities on downstream experiments is reduced to the maximum extent;

3. the kit and the method can be universally used for extracting nucleic acid from samples with various sources, and have wide application range;

4. the kit is applied to a nucleic acid extractor, can finish the extraction of whole blood nucleic acid in one step, can simultaneously extract 1-16 or even higher fluxes of whole blood sample nucleic acid, and realizes the automation of nucleic acid extraction.

Drawings

FIG. 1 shows the results of electrophoresis of nucleic acids of 16 human blood samples extracted by the method of example 1;

FIG. 2 shows the results of electrophoresis of nucleic acids from three different blood samples extracted by the method of example 2;

FIG. 3 shows the results of electrophoresis of nucleic acids extracted from samples of bacterial cultures by the method of example 2;

FIG. 4 shows the results of electrophoresis of nucleic acids from plasma and serum samples extracted by the method of example 2;

FIG. 5 shows the results of PCR electrophoresis of nucleic acids from 6 human whole blood samples extracted by the method of example 2.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments.

The invention provides a general kit for rapidly extracting total nucleic acid of a biological sample, which comprises: buffer ATL, protease K, MBs magnetic beads, Buffer WSI, Buffer WSII, Buffer WSIII, Buffer ES and Buffer MDS.

The Buffer ATL comprises: the pH value of Buffer ATL is 5.0-8.0; wherein the concentration of guanidine hydrochloride is 1-6mol/L, the concentration of sodium lauroyl sarcosine is 1-15 wt%, the concentration of isopropanol is 40-60% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The protease K comprises: protease K, KCl and glycerol in Tris-HCl buffer solution, wherein the pH value of the protease K is 5.0-8.0; wherein the concentration of the protease k is 20-50mg/mL, the concentration of the KCl is 50-150mmol/L, the concentration of the glycerol is 30-50% v/v, and the concentration of the Tris-HCl buffer solution is 10-50 mmol/L.

The MBs magnetic bead comprises: hydroxyl magnetic beads or carboxyl magnetic beads, NaCl and absolute ethyl alcohol, wherein the pH value of the MBs magnetic beads is 5.0-8.0; wherein the concentration of the hydroxyl magnetic beads or the carboxyl magnetic beads is 1-5mg/mL, and the particle size is 200-1500 nm; the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The Buffer WSI comprises: Tris-HCl buffer solution of NaCl, isopropanol and PEG; the pHs of Buffer WSI are 5.0-8.0; wherein, the concentration of NaCl is 500-1000mmol/L, the concentration of isopropanol is 75-85% v/v, the concentration of PEG is 35-45 wt%, wherein the molecular weight range of PEG is 4000-10000, and the concentration of Tris-HCl buffer solution is 10-50 mmol/L.

The Buffer WSII is Tris-HCl Buffer solution containing isopropanol, and the pH value of the Buffer WSII is 5.0-8.0; wherein the concentration of the isopropanol is 75-85% v/v, and the concentration of the Tris-HCl buffer solution is 50-100 mmol/L.

The Buffer WSIII is Tris-HCl Buffer solution containing KCl, and the pH value of the Buffer WSIII is 6.8-9.0; wherein, the concentration of KCl is 0.85-2 wt%, and the concentration of Tris-HCl buffer solution is 40-120 mmol/L.

The Buffer ES is trihydroxymethyl aminomethane hydrochloride containing ethylene diamine tetraacetic acid disodium, wherein the concentration of the ethylene diamine tetraacetic acid disodium is 1.0 +/-0.2 mmol/L, the concentration of the trihydroxymethyl aminomethane hydrochloride is 10 +/-1 mmol/L, and the pH value of the Buffer ES is 6.0-8.0.

The Buffer MDS is a Tris-HCl Buffer solution containing NaCl and absolute ethyl alcohol, and the pH value of the Buffer MDS is 5.0-8.0; wherein the concentration of NaCl is 100-500mmol/L, the concentration of absolute ethyl alcohol is 15-25% v/v, and the concentration of Tris-HCl buffer solution is 6-16 mmol/L; the Buffer MDS is a diluent of MBs magnetic beads; the Buffer MDS is applied to automation operation.

Example 1

The invention provides a universal method for quickly extracting total nucleic acid of a biological sample, which is a manual operation method and is based on the universal kit for quickly extracting the total nucleic acid of the biological sample, and the kit comprises the following steps.

S1: a1.5 mL centrifuge tube was added with 20. mu.L of protease K, 200. mu.L of the sample, 40. mu.L of magnetic beads MBs and 500. mu.L of Buffer ATL, vortexed for 30 seconds, and then vortexed and mixed by inversion at 2 min intervals in a 56 ℃ water or metal bath for 10 min.

S2: after incubation, the centrifuge tube is transferred to a magnetic frame after cooling to room temperature, the magnetic frame is inverted up and down after magnetic enrichment is carried out for 10 seconds, magnetic beads possibly remaining on a tube cover are washed into the solution in the tube, and the magnetic frame is kept still for magnetic adsorption for 50 seconds until the solution is clarified.

S3: taking down the centrifugal tube, adding 600 mu L of Buffer WSI, carrying out vortex oscillation for 30 seconds to mix uniformly, placing the centrifugal tube on a magnetic frame, carrying out magnetic enrichment for 10 seconds, turning the magnetic frame upside down, washing magnetic beads possibly remaining on a tube cover into the solution in the centrifugal tube, standing the magnetic frame, carrying out magnetic adsorption for 50 seconds until the solution is clarified, and sucking and discarding the supernatant.

S4: taking down the centrifugal tube, adding 600 mu L of Buffer WSII, carrying out vortex oscillation for 30 seconds to mix uniformly, placing the centrifugal tube on a magnetic frame, carrying out magnetic enrichment for 10 seconds, turning the magnetic frame upside down, washing magnetic beads possibly remaining on a tube cover into the solution in the centrifugal tube, standing the magnetic frame, carrying out magnetic adsorption for 50 seconds until the solution is clarified, and removing the supernatant by suction.

S5: taking down the centrifugal tube, adding 600 mu L of Buffer WSIII, carrying out vortex oscillation for 30 seconds to mix uniformly, placing the centrifugal tube on a magnetic frame, carrying out magnetic enrichment for 10 seconds, turning the magnetic frame upside down, washing magnetic beads possibly remaining on a tube cover into the solution in the centrifugal tube, standing the magnetic frame, carrying out magnetic adsorption for 50 seconds until the solution is clarified, and removing the supernatant by suction.

S6: and (4) placing the centrifuge tube on a magnetic frame, uncapping and drying at room temperature for 5-10 minutes until the surfaces of the magnetic beads are free from floodlight, and indicating that the centrifuge tube is fully dried.

S7: add 100. mu.L Buffer ES, vortex and shake for 3-5 seconds or blow and suck, mix well, and water bath or metal bath at 65 ℃ for 5 minutes.

S8: after the incubation is finished, the centrifugal tube is slightly shaken to mix the magnetic beads evenly, the centrifugal tube is placed on a magnetic frame to be magnetically absorbed for 1 minute until the solution is clear, and the supernatant is carefully transferred to a preservation tube and is preserved at the temperature of minus 20 ℃ for standby.

Referring to fig. 1, fig. 1 shows the result of electrophoresis of nucleic acid of 16 human blood samples extracted according to example 1 by using the universal rapid extraction biological sample total nucleic acid kit of the present invention, and as can be seen from fig. 1, the obtained nucleic acid sample has good integrity, the nucleic acid fragments are mainly concentrated above 15kbp (>1700bp), no obvious degradation occurs, and the sample quality requirements of NGS sequencing and most technologies are met properly.

Example 2

The invention provides a universal method for quickly extracting total nucleic acid of a biological sample, which is a machine operation method and is based on the universal kit for quickly extracting the total nucleic acid of the biological sample, and the kit comprises the following steps. SS 1: a96-well plate was prepared, and the respective components were counted in the 96-well plate according to Table 1.

TABLE 1

Column(s) of	Components	Column(s) of	Components
				1/7	Buffer ATL 500μL+Proteinase K 20μL	4/10	Buffer WSII 600μL
2/8	MBs magnetic bead 40 uL + Buffer MDS 600 uL	5/11	Buffer WSIII 600μL
				3/9	Buffer WSI 600μL	6/12	Buffer ES 100μL

SS 2: adding 200 mu L of sample into the hole containing Buffer ATL and protease K;

SS 3: the 96 deep-well plate is sent into the instrument, the sequence of the plate rows from inside to outside is A-H, and the method is carried out according to the program steps shown in the table 2:

TABLE 2

SS 4: the whole liquid in the well of column 6 and/or column 12 is transferred to a storage tube and stored below-20 ℃ for later use.

If the sample is insufficient (less than 200. mu.L), the sample is supplemented with physiological saline or PBS.

The maximum liquid loading of the single hole of the 96-hole reaction plate needs to be less than 1.0 mL.

Table 3 shows that the general kit for rapidly extracting total nucleic acid from biological samples can be used for extracting nucleic acid samples from three different blood samples in a nucleic acid extractor according to the method of example 2. FIG. 2 shows the results of electrophoresis of the nucleic acid samples obtained in Table 3, and it can be seen from FIG. 2 that excellent uniformity was achieved in a variety of different blood nucleic acid extractions, the results were stable, and the integrity was good without degradation of nucleic acid fragments.

TABLE 3

Referring to fig. 3, fig. 3 is a diagram showing the result of electrophoresis of nucleic acid of a bacterial culture sample extracted by a nucleic acid extractor according to the method of example 2 by using the universal type kit for rapidly extracting total nucleic acid of a biological sample of the present invention, and as can be seen from fig. 3, compared with the single separation and purification of DNA and RNA, the present invention can simultaneously obtain DNA (>15kb) and RNA (<7kb) in a bacterial sample, and is simple and rapid without separate separation of DNA and RNA.

Referring to fig. 4, fig. 4 is a diagram illustrating an electrophoresis result of extracting nucleic acids from plasma and serum by using the universal kit for rapidly extracting total nucleic acids from biological samples according to the method of embodiment 2, wherein serial numbers 1, 2, and 3 are plasma samples, and serial numbers 4, 5, and 6 are serum samples; as can be seen from FIG. 4, the scheme can recover nucleic acid with low content and wide fragment distribution range in blood plasma and blood serum, can adsorb and recover nucleic acid with different fragment sizes, and has strong universality.

Referring to fig. 5, fig. 5 is a diagram showing the electrophoresis result of PCR performed on the nucleic acid of a whole blood sample of 6 individuals (two samples for each person) extracted by a nucleic acid extractor according to the method of example 2, wherein serial No. 1 is a blank sample, serial nos. 2 and 3 are two samples for 1 individual, serial nos. 4 and 5 are two samples for the same person, serial nos. 6 and 7 are two samples for the same person, serial nos. 8 and 9 are two samples for the same person, serial nos. 10 and 11 are two samples for the same person, and serial nos. 12 and 13 are two samples for the same person; as can be seen from FIG. 5, the PCR product has clear and uniform bands, strong specificity, no residue of inhibitory components and good purity.

As can be seen from the results of FIGS. 1 to 5, the nucleic acid samples of different biological products extracted by the general kit and the method for rapidly extracting total nucleic acid of biological samples have less degradation, better integrity and good quality. Excellent homogeneity, no need of separate DNA and RNA separation, strong specificity and no residue of inhibitory components. Can be applied to the extraction of nucleic acid of biological products of a plurality of species, fully meets the general requirements, is very favorable for popularization and application, and has good market prospect.

In summary, the invention provides a universal kit and a method for rapidly extracting total nucleic acid from a biological sample, wherein pretreatment is not required for whole blood, blood plasma/blood serum to be extracted, protease K, MBs magnetic beads and Buffer ATL are directly mixed with a blood product, and the lysis can be completed in one step, so that the time is greatly shortened. In addition, the kit can be applied to the extraction of nucleic acid of biological products of various species, including various organisms such as mammals, birds, bacteria and the like; the extraction process can be manually completed, and can also be automatically completed by a full-automatic nucleic acid extractor; the nucleic acid extracted by using the kit can be directly applied to scientific researches such as PCR amplification, qPCR genotyping, gene sequencing and the like or analysis of forensic identification or clinical diagnosis. Compared with the prior general nucleic acid extraction technology, the technical scheme of the invention has the beneficial effects that:

1. the blood product is not required to be pretreated, and the cracking is completed in one step, so that the time for extracting the whole blood genome nucleic acid is greatly saved;

2. the three washing solutions of Buffer WSI, Buffer WSII and Buffer WSIII can be used for washing away most of protein, phenols, polysaccharides and other inhibitory impurities in the sample, so that the interference of the pollution of the impurities on downstream experiments is reduced to the maximum extent;

3. the kit and the method can be universally used for extracting nucleic acid from samples with various sources, and have wide application range;

4. the kit is applied to a nucleic acid extractor, can finish the extraction of whole blood nucleic acid in one step, can simultaneously extract 1-16 or even higher fluxes of whole blood sample nucleic acid, and realizes the automation of nucleic acid extraction.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.