CN111057705B - Kit for extracting free nucleic acid and use method - Google Patents
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Abstract
本发明提供一种提取游离核酸的试剂盒及使用方法，涉及核酸提取技术领域，包括蛋白酶K、裂解缓冲液Buffer CL、吸附结合液Buffer CB、异丙醇、第一次清洗液Buffer GW1、第二次清洗液Buffer GW2和洗脱缓冲液Buffer EBL；取体液样本离心后取上清置干净的离心管中，向收集的上清液中加入蛋白酶K和裂解缓冲液Buffer CL，向裂解混合物中加入混有异丙醇的吸附结合液Buffer CB，将混合溶液转入硅胶吸附柱进行负压抽滤；依次使用第一次清洗液Buffer GW1和第二次清洗液Buffer GW2和无水乙醇对硅胶吸附柱进行洗涤，离心处理后在室温条件下晾干，得到去除杂质的吸附有cfDNA的硅胶吸附柱；Buffer EBL对硅胶吸附柱进行洗脱，离心收集洗脱液，得到cfDNA。本发明兼容性好，提取纯化的cfDNA产量高、质量好，成本低。
The present invention provides a kit for extracting free nucleic acid and a method for using it, and relates to the technical field of nucleic acid extraction, including proteinase K, lysis buffer Buffer CL, adsorption binding solution Buffer CB, isopropanol, first cleaning solution Buffer GW1, The secondary washing solution Buffer GW2 and the elution buffer Buffer EBL; after centrifuging the body fluid samples, take the supernatant and place it in a clean centrifuge tube, add proteinase K and lysis buffer Buffer CL to the collected supernatant, and add to the lysis mixture. Add the adsorption binding solution Buffer CB mixed with isopropanol, transfer the mixed solution to a silica gel adsorption column for negative pressure suction filtration; use the first cleaning solution Buffer GW1 and the second cleaning solution Buffer GW2 and absolute ethanol in turn to filter the silica gel. The adsorption column was washed, centrifuged and air-dried at room temperature to obtain a silica gel adsorption column with cfDNA adsorbed to remove impurities; the silica gel adsorption column was eluted with Buffer EBL, and the eluate was collected by centrifugation to obtain cfDNA. The invention has good compatibility, and the extracted and purified cfDNA has high yield, good quality and low cost.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a kit for extracting free nucleic acid and a using method thereof.
Background
Eukaryotic DNA exists primarily as chromosomes in the nucleus, but a small portion of DNA exists extracellularly as single-stranded or double-stranded DNA, and is a fragmented extracellular DNA in a cell-free state called episomal DNA (cfdna). cfDNA is mainly derived from necrotic or apoptotic cells, exosomes secreted by cells, and the like, and almost all cells, including healthy cells, fetal cells, tumor cells, transplanted cells, and the like can release cfDNA fragments into body fluids such as blood, urine, interstitial fluid, and the like. Most cfDNA is double-stranded DNA in the form of nucleoprotein, and fragments are small, typically between tens to hundreds of bp in length. cfDNA is widely studied and becomes a new molecular marker for noninvasive diagnosis and research of diseases. cfDNA is proved to be an important molecular marker in tumor and prenatal diagnosis research, and the application of the cfDNA in noninvasive prenatal detection and liquid biopsy of tumors is two clinical fields of fire and heat. In addition, the application value of cfDNA in non-tumor diseases such as organ transplantation, blood diseases, cardiovascular diseases, autoimmune diseases and the like is increasingly clear. With the continuous development of liquid biopsy technology, cfDNA detection is expected to become an important method for replacing tissue biopsy.
The first step in detection using cfDNA is to extract and purify cfDNA from various body fluids. The accuracy and reliability of subsequent detection results are directly influenced by the yield and quality of extracted products, and a high-purity and high-concentration nucleic acid sample is one of important conditions for obtaining good experimental results in the molecular biology experimental process. However, the content of cfDNA in body fluid is very low, the content of cfDNA in serum is usually lower than 10ng/mL, the cfDNA fragment is small, the length is generally between 40 and 220bp, and the cfDNA fragment is unstable and easy to degrade, and the cfDNA fragment and the method all put higher requirements on extraction and purification of free nucleic acid. In addition, the components in different body fluid samples are different, for example, urine, blood of infected patients, cerebrospinal fluid and other body fluids often contain a large amount of bacteria, so that cfDNA is easier to degrade, the effusion of some pathological tissues contains substances such as hyaluronic acid, proteoglycan, glycosaminoglycan and the like, the viscosity is higher, and the components in various body fluid storage buffer solutions are different. Moreover, the free nucleic acids in different body fluids vary from content to size distribution, all of which increase the difficulty of free nucleic acid extraction.
Currently, the most common methods for extracting cfDNA are a magnetic bead adsorption method and a silica gel membrane adsorption column method. The magnetic bead method has high extraction efficiency on the free small-fragment DNA of the blood plasma (or blood serum), the extraction process does not need centrifugation or filtration, and the extraction can be manually operated or completed in an automatic working platform mode. However, the extraction by the magnetic bead method is only suitable for extracting a small amount of free nucleic acid with low concentration, and the adsorption amount of the magnetic beads per unit area is limited, so the magnetic bead method has a very limited concentration multiple for large-volume samples. Although the extraction efficiency of the silica gel membrane adsorption column method for small fragment DNA is lower than that of the magnetic bead method, the silica gel membrane adsorption column method is simple, convenient and quick, the extracted cfDNA or RNA has high purity and good repeatability, and can effectively remove various PCR inhibitors in body fluid, and is particularly suitable for extracting and enriching large amount of free nucleic acid with high concentration from large amount of samples such as hydrothorax, ascites and urine. At present, the most main product of the silica gel membrane adsorption column method is the QIAamp Circulating Nucleic Acid Kit of Qiagen company, but the imported Kit is very expensive, and the cost problem becomes a restriction factor when a plurality of laboratories process body fluid free Nucleic Acid.
In recent years, various domestic manufacturers have developed corresponding extraction methods and kits aiming at different body fluid sample types including blood, serum, cerebrospinal fluid, urine, hydrothorax and ascites and the like, but the methods are suitable for all body fluid sample types, have good compatibility, and are relatively lack of rapid and low-cost extraction methods at present. Therefore, improving the traditional centrifugal column method and improving the compatibility and extraction efficiency of the free nucleic acid extraction reagent become urgent matters for cfDNA research and clinical popularization and application.
Therefore, it is urgently needed to provide a kit for extracting free nucleic acid and a using method thereof, wherein the kit is good in compatibility, high in yield of the extracted and purified cfDNA, good in quality and low in cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for extracting free nucleic acid and a using method thereof.
The invention provides the following technical scheme:
a kit for extracting free nucleic acid comprises protease K, lysis Buffer CL, adsorption binding solution Buffer CB, isopropanol, first cleaning solution Buffer GW1, second cleaning solution Buffer GW2 and elution Buffer EBL;
the lysis Buffer CL includes 50-500mM Tris-HCl, 1-10M guanidinium isothiocyanate solution, 0.1-30% (w/v) SDS Buffer, 0.1-5M sodium chloride, 2-20mM EDTA Buffer, 1% dimethyl sulfoxide and water, the lysis Buffer CL has a pH value of 6.0-8.5, the adsorption binding solution Buffer CB includes 0.1-10% (w/v) sodium citrate Buffer, 0.1-10M guanidinium isothiocyanate solution, 0.5-1.5M sodium chloride, 0.5-5% Triton X-100, 15-25mM HEPS-KOH, 3M thiourea and 10-30% PEG (v/v), the adsorption binding solution CB has a pH value of 6-9, 10-30% (w/v) isopropanol is added thereto before the adsorption binding solution Buffer CB is used, the first cleaning solution Buffer GW1 comprises 100-500mM Tris-HCl, 0.1-10M guanidine hydrochloride, 0.5-1.5M sodium chloride, 10-60mM EDTA Buffer solution and 20-70% absolute ethyl alcohol, the pH value of the first cleaning solution Buffer GW1 is 6.0-8.0, and the second cleaning solution Buffer GW2 comprises: 10-500mM Tris-HCl and 50-80% ethanol, wherein the pH value of the second washing solution, namely Buffer GW2, is 6.0-8.0, and the elution Buffer EBL comprises: 1-10mM Tris-HCl and water, and the pH value of the elution Buffer EBL is 6.0-8.0.
Preferably, the lysis Buffer CL comprises the following components in percentage by weight: 100mM Tris-HCl, 6M guanidinium isothiocyanate solution, 10% (w/v) SDS Buffer, 0.3M sodium chloride, 20mM EDTA Buffer, 1% dimethyl sulfoxide and water, wherein the pH value of the lysis Buffer CL is 7.0.
Preferably, the lysis Buffer CB comprises the following components in percentage by weight: 3.5% (w/v) sodium citrate Buffer, 5M guanidinium isothiocyanate solution, 1M sodium chloride, 1% Triton X-100 and 20mM HEPS-KOH, 3M thiourea, 20% PEG (v/v), the pH of the lysis Buffer CB being 7.0, 20% (w/v) isopropanol being added before the lysis Buffer CB is used.
Preferably, the content of the components in the first cleaning solution Buffer GW1 is as follows: 100mM Tris-HCl, 6M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA Buffer solution and 60% absolute ethyl alcohol, wherein the pH value of the first washing solution Buffer GW1 is 8.0.
Preferably, the Buffer GW2 of the second cleaning solution comprises the following components: 100mM Tris-HCl and 80% ethanol, and the pH value of the second washing solution, namely Buffer GW2, is 8.0.
Preferably, the elution Buffer EBL comprises the following components: 5mM Tris-HCl and water, the elution Buffer EBL having a pH of 8.0.
The use method of the kit for extracting the free nucleic acid comprises the following steps:
s1, taking 1-10ml of body fluid sample, centrifuging at 3000-5000Xg for 5-10min, taking the supernatant, putting the supernatant into a clean centrifugal tube, centrifuging at 10000-16000rpm for 5-10min, and collecting the supernatant;
s2, adding protease K and lysis Buffer CL into the collected supernatant, mixing uniformly, and performing incubation treatment to obtain a lysis mixture;
s3, adding an adsorption binding solution Buffer CB mixed with isopropanol into the cracking mixture, uniformly mixing, carrying out ice bath for 3-5min, and transferring the mixed solution into a silica gel adsorption column for negative pressure suction filtration;
s4, after the suction filtration is finished, washing the silica gel adsorption column by using a first washing liquid, namely Buffer GW1, a second washing liquid, namely Buffer GW2 and absolute ethyl alcohol in sequence, and drying the silica gel adsorption column at room temperature after centrifugal treatment to obtain the impurity-removed silica gel adsorption column with cfDNA adsorbed;
s5, adding 20-150 mu L of eluent Buffer EBL to elute the silica gel adsorption column, and centrifugally collecting eluent to obtain cfDNA.
Preferably, in step S2, the volume ratio of the urine or pleural effusion sample to proteinase K is 8:1, the volume ratio of the plasma or serum sample to proteinase K is 10:1, and the enzyme activity concentration of proteinase K is 20 mg/mL.
Drawings
The invention is described in detail below with reference to the accompanying drawings
Fig. 1 shows the results of fragment size detection of plasma cfDNA samples, urine cfDNA, and pleural effusion cfDNA in the test group and the control group.
The invention has the beneficial effects that: the unique lysis and combination buffer system is adopted, and the method can be compatible with various body fluid sample types. The lysis buffer solution can be compatible with various body fluid sample types, including fresh or frozen serum, plasma, amniotic fluid, hydrothorax and ascites, urine, tissue exudate and the like, and under the synergistic action of proteinase K, the free nucleic acid and the nucleic acid binding protein in various body fluid samples can be rapidly and thoroughly separated; then, the separated free nucleic acid is efficiently combined with the silica gel membrane under the high-salt condition provided by the adsorption and combination buffer solution, and impurities such as denatured protein, lipid and the like are prevented from blocking the silica gel membrane; finally, two-step cleaning operation is adopted, so that protein, pigment, lipid and other inhibitory impurity pollution are removed to the maximum extent, and the extracted and purified cfDNA has high yield and good quality; the kit for extracting the free nucleic acid from a large number of body fluid samples in the silica gel membrane adsorption column method provided by the invention adopts a negative pressure method, is provided with the extension tube, can be matched with a negative pressure device to improve the sample treatment capacity to 20ml, can treat dozens of samples at the same time, is simple to operate, can complete one-time batch extraction within 1-1.5 hours, and is low in cost; the kit for extracting free nucleic acid from a large number of body fluid samples in the silica gel membrane adsorption column method provided by the invention combines a target fragment with a high-efficiency adsorption column, effectively improves the recovery rate of nucleic acid, can flexibly change the elution volume between 20 and 150 mu L, can concentrate low-concentration nucleic acid, meets the requirement on the total mass of the free nucleic acid in the multi-field research and application of molecular biology, and has wide application prospect and market popularization value.
Detailed Description
Example 1
The kit is used for extracting and testing the free nucleic acid from 10 plasma samples (sources: clinical laboratory samples of Taizhou Jian medical inspection laboratory Co., Ltd.), and comprises the following steps:
s1, taking a 10ml plasma sample, centrifuging for 10min at 3000xg, taking the supernatant, placing the supernatant into a clean centrifugal tube, centrifuging for 5min at a high speed of 12000rpm, collecting the supernatant, and mainly removing cells and impurities in the plasma sample through centrifugation to prevent the subsequent introduction of cell genome DNA pollution or microorganism DNA pollution such as bacteria, fungi and the like in the sample;
s2, adding 300 mu L of protease K (20mg/mL) into 3mL of collected supernatant, adding 2400 mu L of lysis Buffer CL after vortexing for 5s, performing incubation treatment at 60 ℃ for 30min after vortexing for 30s to obtain a lysis mixture, and separating free nucleic acid and nucleic acid binding protein in a plasma sample to obtain the lysis mixture;
s3, adding 5400 mu L of adsorption binding solution Buffer CB mixed with isopropanol into the cracking mixture, swirling for 20s, then carrying out ice bath for 5min, transferring the mixed solution into a silica gel adsorption column for negative pressure suction filtration, adding the mixed solution into an extension tube, starting and adjusting the negative pressure to-900 to-800 mbar, slowly sucking away the solution in the tube, and taking away the extension tube to enable the separated free nucleic acid to be efficiently adsorbed onto the silica gel membrane column;
s4, after the suction filtration is finished, sequentially adding 1mL of first cleaning solution, namely Buffer GW1, 1mL of second cleaning solution, namely Buffer GW2 and 1mL of absolute ethyl alcohol into the adsorption column, closing a negative pressure switch after the solution is completely absorbed, taking down the adsorption column when the pressure is restored to 0mbar, placing the adsorption column into a new collecting pipe, centrifuging the collection pipe for 2min at 12000rpm, pouring off waste liquid in the collecting pipe, and placing the adsorption column at room temperature for completely airing to obtain the impurity-removed silica gel adsorption column with cfDNA adsorbed;
s5, placing the adsorption column in a new collecting pipe of 1.5ml, adding 40 mu L of eluent Buffer EBL to elute the adsorption column, placing for 3min at room temperature, centrifuging for 1min at 12000rpm, and collecting eluent to obtain cfDNA.
The plasma samples from example 1 were extracted with free Nucleic Acid using the QIAamp Circulating Nucleic Acid Kit, a QIAGEN commercial Kit, as a control, and the extraction method was performed according to the instructions in the QIAamp Circulating Nucleic Acid Kit.
Example 2
The kit is used for extracting and testing free nucleic acid from 5 urine samples and 5 pleural effusion samples (source: Taizhou Jian is clinical laboratory sample of medical examination experiment Limited), and comprises the following steps:
s1, taking a 10ml urine/chest and abdomen water sample, centrifuging at 3000xg for 10min, taking the supernatant, putting the supernatant into a clean centrifugal tube, centrifuging at 12000rpm for 5min, and collecting the supernatant;
s2, adding 375 mu L of protease K (20mg/mL) into 3mL of collected supernatant, adding 3mL of lysis Buffer CL after swirling for 5s, performing incubation treatment at 60 ℃ for 30min after swirling for 30s to obtain a lysis mixture, and separating free nucleic acid and nucleic acid binding protein in the urine/pleural and abdominal water sample to obtain the lysis mixture;
s3, adding 7.2mL of adsorption binding solution Buffer CB mixed with isopropanol into the cracking mixture, swirling for 20s, then carrying out ice bath for 5min, transferring the mixed solution into a silica gel adsorption column for negative pressure suction filtration, adding the mixed solution into an extension tube, starting and adjusting the negative pressure to-900 to-800 mbar, slowly sucking away the solution in the tube, and taking away the extension tube to enable the separated free nucleic acid to be efficiently adsorbed onto the silica gel membrane column;
s4, after the suction filtration is finished, sequentially adding 1mL of first cleaning solution, namely Buffer GW1, 1mL of second cleaning solution, namely Buffer GW2 and 1mL of absolute ethyl alcohol into the adsorption column, closing a negative pressure switch after the solution is completely absorbed, taking down the adsorption column when the pressure is restored to 0mbar, placing the adsorption column into a new collecting pipe, centrifuging the collection pipe for 2min at 12000rpm, pouring off waste liquid in the collecting pipe, and placing the adsorption column at room temperature for completely airing to obtain the impurity-removed silica gel adsorption column with cfDNA adsorbed;
s5, placing the adsorption column in a new collecting pipe with the volume of 1.5mL, adding 40 mu L of eluent Buffer EBL to elute the adsorption column, placing the adsorption column at room temperature for 3min, centrifuging the adsorption column for 1min at 12000rpm, and collecting the eluent to obtain cfDNA.
The urine/thoracic ascites specimen in example 2 was subjected to free Nucleic Acid extraction using a QIAGEN commercial Kit QIAamp Circulating Nucleic Acid Kit, and used as a control, and the extraction method was performed with reference to the manual in the QIAamp Circulating Nucleic Acid Kit.
Quality detection and result of extracted cfDNA
The cfDNA extracted in example 1, example 2 was measured for concentration using Qubit, DNA purity using Nanodrop, and 10 μ L of cfDNA fragment size was measured using agilent 2100 bioanalyzer, the results are shown in fig. 1 and table 1.
Fig. 1 shows the results of fragment size detection of 2 plasma cfDNA samples, 2 urine cfDNA samples, and 2 pleural effusion cfDNA samples, wherein the three upper panels, from left to right, are the results of fragment size detection of plasma, urine, pleural effusion cfDNA samples of the test group; the three upper panels from left to right are the results of the detection of the sizes of the plasma, urine, and pleural effusion cfDNA fragments of the control group. The results show that the cfDNA fragment size distribution of the test group in example 1 and example 2 was substantially identical to the control group with no significant difference.
TABLE 1 comparison of the effect of two methods for extracting DNA from microorganisms
Table 1 shows the test results of the concentration and total amount of extracted cfDNA of the test group and the control group, wherein the total amount of extracted cfDNA of 10 plasma samples of the test group is 1623.44ng, the total amount of extracted cfDNA of 5 urine samples is 415.2ng, the total amount of extracted cfDNA of 5 pleural effusion samples is 3860.4ng, the total amount of extracted cfDNA of 10 plasma samples of the control group is 1474.6 ng, the total amount of extracted cfDNA of 5 urine samples is 384ng, the total amount of extracted cfDNA of 5 pleural effusion samples is 3382.4ng, and the total amount of extracted cfDNA of the test group is more than that of the control group; the mean value of cfDNA extracted by the test group was 1.8995, the mean value of cfDNA extracted by the control group was 1.8835, and the purity of both groups was substantially equivalent.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A kit for extracting free nucleic acid is characterized by comprising protease K, lysis Buffer CL, adsorption binding solution Buffer CB, isopropanol, first cleaning solution Buffer GW1, second cleaning solution Buffer GW2 and elution Buffer EBL; the lysis Buffer CL comprises the following components in content: 100mM Tris-HCl, 6M guanidinium isothiocyanate solution, 10% (w/v) SDS Buffer, 0.3M sodium chloride, 20mM EDTA Buffer, 1% dimethyl sulfoxide and water, wherein the pH value of the lysis Buffer CL is 7.0; the adsorption binding solution Buffer CB comprises the following components in percentage by weight: 3.5% (w/v) sodium citrate Buffer, 5M guanidinium isothiocyanate solution, 1M sodium chloride, 1% Triton X-100 and 20mM HEPS-KOH, 3M thiourea, 20% PEG (v/v), pH of the adsorption conjugate Buffer CB being 7.0, 20% (w/v) isopropanol being added before the adsorption conjugate Buffer CB is used; the content of the components in the first cleaning solution Buffer GW1 is as follows: 100mM Tris-HCl, 6M guanidine hydrochloride, 1M sodium chloride, 10mM EDTA Buffer solution and 60% absolute ethyl alcohol, wherein the pH value of the first cleaning solution Buffer GW1 is 8.0; the Buffer GW2 of the second cleaning liquid comprises the following components in percentage by weight: 100mM Tris-HCl and 80% ethanol, wherein the pH value of the Buffer GW2 of the second cleaning solution is 8.0; the elution Buffer EBL comprises the following components in percentage by weight: 5mM Tris-HCl and water, the elution Buffer EBL having a pH of 8.0.
2. The method for using the kit for extracting free nucleic acid according to claim 1, comprising the following steps:
s1, taking 1-10ml of body fluid sample, centrifuging at 3000-5000Xg for 5-10min, taking the supernatant, putting the supernatant into a clean centrifugal tube, centrifuging at 10000-16000rpm for 5-10min, and collecting the supernatant;
s2, adding protease K and lysis Buffer CL into the collected supernatant, mixing uniformly, and performing incubation treatment to obtain a lysis mixture;
s3, adding an adsorption binding solution Buffer CB mixed with isopropanol into the cracking mixture, uniformly mixing, carrying out ice bath for 3-5min, and transferring the mixed solution into a silica gel adsorption column for negative pressure suction filtration;
s4, after the suction filtration is finished, washing the silica gel adsorption column by using a first washing liquid, namely Buffer GW1, a second washing liquid, namely Buffer GW2 and absolute ethyl alcohol in sequence, and drying the silica gel adsorption column at room temperature after centrifugal treatment to obtain the impurity-removed silica gel adsorption column with cfDNA adsorbed; s5, adding 20-150 mu L of eluent Buffer EBL to elute the silica gel adsorption column, and centrifugally collecting eluent to obtain cfDNA.
3. The method for using the kit for extracting free nucleic acid according to claim 2, wherein the enzyme activity concentration of proteinase K in step S2 is 20 mg/mL.
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| CN112680513A (en) * | 2020-11-04 | 2021-04-20 | 深圳思勤医疗科技有限公司 | Means for determining the presence of cancer based on peripheral blood |
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