CN113403300A - Method for enriching circulating free nucleic acid in body fluid by using MOF material and application thereof - Google Patents
Method for enriching circulating free nucleic acid in body fluid by using MOF material and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of separation and enrichment of circulating free nucleic acid in body fluid, in particular to a method for enriching circulating free nucleic acid in body fluid by using MOF materials, which comprises the following steps: (1) treating the body fluid sample such that circulating free nucleic acids bound to proteins in the body fluid sample are released from the protein-nucleic acid complex; (2) adding an MOF material into the solution treated in the step (1), and adsorbing circulating free nucleic acid in the solution; (3) separating the MOF material having the circulating free nucleic acids adsorbed in step (2); (4) disrupting the MOF material having adsorbed circulating free nucleic acids of step (3) with EDTA to release the adsorbed nucleic acids; (5) and (4) purifying the nucleic acid released in the step (4) to obtain circulating free DNA and circulating free RNA. The method has the advantages of high enrichment efficiency, low cost, wide application range and high compatibility.
Description
Technical Field
The invention relates to the technical field of separation and enrichment of circulating free nucleic acid in body fluid, in particular to a method for enriching circulating free nucleic acid in body fluid by using an MOF material and application thereof.
Background
The morbidity and mortality of human cancers worldwide is largely due to poor therapeutic intervention after late diagnosis of the disease. At this stage, few clinically proven biomarkers are widely available for diagnosing and treating patients. Recent analysis of circulating free DNA (cfDNA) in blood has shown that detection analysis of cfDNA may provide new opportunities for early diagnosis. In healthy individuals, the concentration of cfDNA in plasma is often between 1 and 10 ng/mL. Increased cfDNA levels were first reported in the serum of cancer patients in 1977; cfDNA concentrations can also be elevated in other physiological conditions or clinical situations, such as acute trauma, cerebral infarction, exercise, transplantation, and infection. In 1997, the identification of fetal DNA sequences in maternal plasma by Lo et al opened up a variety of applications of cfDNA in prenatal medicine, including sex determination, identification of monogenic diseases and non-invasive prenatal examinations (NIPT, including down syndrome). In 2007, Lo et al demonstrated NIPT for the first time and rapidly entered into widespread clinical use. The content of cfDNA in blood is very small but is an important diagnostic marker for the diagnosis of diseases and prenatal diagnosis. However, studies on circulating free rna (cfRNA) in blood have just begun, and whether cfRNA has become a biomarker to determine disease and physiological status is yet to be further determined. The content of cctDNA and ccRNA in 1ml serum or plasma is approximately as follows: about 5ng/mL cfDNA in blood of healthy people; up to 50ng/mL in the blood of cancer patients. The cfRNA in the blood of healthy people is approximately 5-10ng/mL, with a higher proportion of cancer patients [ data sources Qiagen QIAamp ccfDNA/RNA kit instructions ]. Therefore, the efficient enrichment of cfDNA in blood plays a decisive role in whether cfDNA applications can be popularized and advanced more; the efficient enrichment of the cfRNA in blood has profound significance for determining the function and application research of the cfRNA.
Metal-organic Framework Materials (MOFs), also known as Metal-organic Framework materials, Metal-organic complexes or coordination polymers, are crystalline porous materials formed by connecting inorganic Metal ions or Metal clusters and organic ligands, and have the common characteristics of organic materials and inorganic materials. The huge specific surface area and abundant structural diversity of MOFs make them have important application prospects in the aspect of guest molecule loading, especially the loading and release of biomolecules.
Disclosure of Invention
One of the purposes of the invention is to provide a method for enriching circulating free nucleic acid in body fluid by using MOF material, which has the advantages of high enrichment efficiency, low cost, wide application range and high compatibility.
It is a further object of the invention to provide the use of a method for enriching circulating free nucleic acids in a body fluid using MOF material.
The scheme adopted by the invention for realizing one of the purposes is as follows: a method of enriching circulating free nucleic acids in a body fluid using MOF material comprising the steps of:
(1) treating the body fluid sample such that circulating free nucleic acids bound to proteins in the body fluid sample are released from the protein-nucleic acid complex;
(2) adding an MOF material into the solution treated in the step (1), and adsorbing circulating free nucleic acid in the solution;
(3) separating the MOF material having the circulating free nucleic acids adsorbed in step (2);
(4) disrupting the MOF material having adsorbed circulating free nucleic acids of step (3) with EDTA to release the adsorbed nucleic acids;
(5) and (4) purifying the nucleic acid released in the step (4) to obtain circulating free DNA and circulating free RNA.
Preferably, in the step (1), guanidine thiocyanate and polyethylene glycol octylphenyl ether are added to the body fluid to denature the protein, and ZnCl is added2The protein is allowed to precipitate out, the protein is separated from the supernatant, and circulating free nucleic acid is present in the supernatant. More preferably, the body fluid contains guanidine thiocyanate, octyl phenyl ether of polyethylene glycol and ZnCl2The final concentrations of (B) were 0.2-1M, 20-200mM and 100-400mM, respectively.
Preferably, in the step (1), the MOF material is at least one of Co-IRMOF-74-III, Co-IRMOF-74-IV, Co-IRMOF-74-V, Ni-IRMOF-74-III, Ni-IRMOF-74-IV and Ni-IRMOF-74-V.
Preferably, in the step (1), the body fluid sample is any one of whole blood, plasma, serum, urine, saliva, pleural effusion, cerebrospinal fluid, lymph fluid and ascites.
Preferably, in the step (2), the amount of the MOF material added is 200-500 μ g/mL.
Preferably, in the step (2), monovalent and divalent metal cations are added into the adsorption reaction system; more preferably, the extraction efficiency is improved by adding monovalent and divalent metal cations, such as K, to the adsorption reaction system+，Na+，Mg2+And the like.
Preferably, in the step (4), EDTA aqueous solution is added into the separated MOF material until the final concentration of EDTA is 1-10mM, and the mixture is repeatedly blown or shaken until white MOF white organic ligand insoluble appears to release the nucleic acid from the MOF material.
Preferably, in the step (5), the released nucleic acid is purified by using a glacial ethanol precipitation method or a nucleic acid purification kit; the concrete operation of the glacial ethanol precipitation method is as follows: adding an absolute ethanol solution into the nucleic acid solution obtained in the step (4), placing at the temperature of (-20) - (-70) DEG C to precipitate a nucleic acid solid from the solution, and performing low-temperature centrifugal separation to obtain a precipitated nucleic acid solid; the specific operation of the nucleic acid purification kit is as follows: and (3) adding ethanol into the nucleic acid solution obtained in the step (4), adsorbing the nucleic acid solution onto a glass fiber material in a purification column, and eluting the nucleic acid from the purification column by using water to achieve the purpose of purifying the nucleic acid.
Preferably, the method further comprises the step (6): and (3) treating the circulating free nucleic acid obtained in the step (5) with RNA hydrolase or DNA hydrolase respectively to obtain single cfDNA or cfRNA.
The enriched circulating free nucleic acid of the invention comprises circulating free DNA (cfDNA) and circulating free RNA (cfRNA). If a single cfDNA is desired, the enriched circulating free nucleic acids need only be treated with RNA hydrolase, which hydrolyzes the RNA leaving only cfDNA. If a single cfRNA is desired, the enriched circulating free nucleic acids need only be treated with DNA hydrolase to hydrolyze the DNA leaving only cfDNA.
The second scheme adopted by the invention for achieving the purpose is as follows: the method for enriching the circulating free nucleic acid in the body fluid by using the MOF material is applied to the enrichment detection of the tumor cfDNA.
The invention has the following advantages and beneficial effects:
the invention discloses a method for enriching circulating free nucleic acids (cell free nucleic acids) in body fluid, wherein the nucleic acids obtained by enrichment and purification can be subjected to downstream analysis and detection, and the method comprises the steps of real-time fluorescence quantitative PCR and second-generation sequencing technology quantitative and qualitative analysis of circulating free DNA.
The method creatively uses the MOF material for enriching circulating free nucleic acid in body fluid, and has the advantages that: (1) the enrichment efficiency of the circulating free nucleic acid in body fluid is very high, because the MOF material has high porosity and huge specific surface area, and has high-efficiency adsorption effect on nucleic acid molecules. (2) The cost of enriching the circulating free nucleic acid is low, the method only needs to simply mix the material and the body fluid sample, and a loaded device preparation process is not needed. (3) The tolerance range of the enriched circulating free nucleic acid of the invention to the solution volume is very wide, and the enriched circulating free nucleic acid can be enriched from a small amount of 200uL body fluid and can also be extracted from a few milliliters of body fluid, which is mainly different from the enrichment principle of the method of the invention and a commercial kit. The commercial kit mainly utilizes the reduction of the solubility of nucleic acid in ethanol solvent, precipitates and adsorbs the nucleic acid in the glass fiber column material, and then uses water to elute the nucleic acid adsorbed in the column material, wherein the elution efficiency of water is low, and if the loss of small amount of nucleic acid adsorbed in the glass fiber is very large. The three points make the method for enriching the free nucleic acid by circulating the body fluid have high advantages in price, the variation range of the use amount of the body fluid and adsorption efficiency compared with other commercialized enrichment methods. (4) Compared with cfDNA, the content of cfRAN is lower in the obtained circulating free nucleic acid comprising cfDNA and cfRNA, and only cfDNA can be obtained by a plurality of enrichment methods. (5) The method of the invention can not only separate and enrich the circulating free nucleic acid in various types of blood (including whole blood, plasma and serum), but also be applicable to the enrichment of circulating free nucleic acid in other various types of body fluid, including but not limited to urine, saliva, pleural effusion, cerebrospinal fluid, lymph fluid, ascites and the like.
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FIG. 1 is a schematic flow diagram of the process of the present invention;
FIG. 2A is a diagram showing the quantitative detection of the content of cfDNA enriched by the MOF method and the kit method by the Qubit fluorometer;
FIG. 3 content of cfDNA enriched by real-time fluorescent quantitative PCR versus MOF method and kit method;
FIG. 4 shows the difference of the components of cfDNA obtained by comparing the MOF method and the kit method.
Detailed Description
The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.
This example compares the enrichment method of the present invention with cfDNA enriched with a commercial kit (QIAamp ccfDNA/RNA kit) in order to clarify the efficiency and superiority of the enrichment method as set forth in the present invention.
In the embodiment of the invention, three materials of Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V are used as enrichment materials, blood plasma is used as a research sample, and in order to accurately compare the difference between cfDNAs obtained by an MOF enrichment method and a kit enrichment method, blood plasma samples of several people are mixed and used for subsequent experiments.
Example 1: method for enriching cfDNA in blood plasma by using MOF material and kit
In this example, three materials, namely Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V, were used to adsorb cfDNA in 500. mu.L of mixed plasma, and FIG. 1 is a schematic flow chart of the method in this example.
1. Treatment of plasma samples
(1) A500. mu.L sample of plasma was taken in a 1mL EP tube, added with a Guanidine thiocyanate solution (final concentration: 500mM) and polyethylene glycol octyl phenyl ether (Triton X-100) (final concentration: 50mM), vortexed for 5 seconds, and allowed to stand at room temperature for 3min after being mixed well.
(2) Adding ZnCl2The solution (final concentration 300mM) was immediately vortexed for 1min, whereupon a milky insoluble material appeared, and placed on ice for 3 min.
(3) Centrifugation at 12000g for 3min showed a milky white precipitate, and the supernatant was a clear, pale yellow liquid, which was transferred to a clean EP tube for use, and 500. mu.L of plasma gave approximately 500. mu.L of supernatant.
Adsorbing cfDNA in plasma with MOF material:
the reaction system for the adsorption process was configured as follows:
| species of | Volume of | |
| Supernatant obtained in step 1 | 500μL | |
| MOF(20mg/mL) | 20μL | |
| 10× | 60μL | |
| 10×MgCl2(200mM) | 60μL | |
| Volume | 640μL |
The above reaction was carried out on a rotator at room temperature for 2 h.
3. After the reaction was completed, 12000g of the above reaction was centrifuged for 15min, the supernatant was removed, and the precipitate (precipitated as a pink solid) was collected.
4. Disruption of MOF material releases cfDNA: mu.L of enzyme-free water was added to the pellet, 10. mu.L of EDTA (150mM) solution was added, and the mixture was mixed with a tip for 3min (shaking was also possible for 3 min). The pink solid was seen to disappear and a white flocculent insoluble material appeared.
5. Purification step 4 yielded cfDNA: there are two purification modes, i.e., a method of ethanol precipitation and a method of DNA purification kit:
(1) the glacial ethanol method comprises the following steps: adding 5 mu L of sodium acetate (3mol/L, pH 5.2) into the mixed solution obtained in the step 4, and fully and uniformly mixing to ensure that the final concentration is 0.3 mol/L;
(2) adding 150 μ L of pre-cooled glacial ethanol, mixing, fully and uniformly mixing, and placing at-80 ℃ for 30 minutes to 2 hours;
(3) centrifugation at 12000g for 10 min, careful removal of the supernatant, aspiration of all droplets from the tube walls;
(4) adding 75% of glacial ethanol with the capacity of 1/2 centrifuge tubes, blowing for three times, centrifuging for 2 minutes at 12000g, carefully removing supernatant, and sucking all droplets on the tube walls;
(5) the uncapped EP tube was placed on a laboratory table at room temperature for 5 minutes to evaporate the remaining liquid to dryness;
(6) add 20. mu.L of enzyme-free water to dissolve the cfDNA solids at the bottom of the EP tube and place in a freezer at-20 ℃ for use. DNA purification kit method (DNA purification kit from ZYMO RESEARCH, DNA Clean)&ConcentratorTM-5)：
(1) mu.L of Oligo Binding Buffer was added to the above 50. mu.L of reaction solution, and mixed well.
(2) Add 400. mu.L of 100% absolute ethanol, mix well and transfer to a zymo column (the column is placed in a collection tube).
(3) The column was centrifuged at 12000g for 1min, the effluent discarded, and the collection tube continued to be fitted over the column.
(4) 750 μ L of DNA washing buffer was added to the column, and after centrifugation at 12000g for 1min the waste was discarded, the collection tube was returned to the column and centrifuged again at 12000g for 2 min.
(5) The column was transferred to a clean enzyme-free EP tube, 20. mu.L of enzyme-free water was added to the center of the column, and after standing at room temperature for 3min, it was centrifuged at 12000g for 2 min. The resulting purified cfDNA was placed in a-20 degree refrigerator for subsequent experiments.
Comparative example: kit method for extracting cfDNA in blood plasma
Using a circulating free nucleic acid enrichment kit (QIAamp ccfDNA/RNA kit) from Qiagen, the plasma sample was 500. mu.L of the mixed plasma sample described above, and the detailed procedure was as follows according to the instructions provided in the kit, and the obtained cfDNA was stored at-20 ℃ for use.
Example 2: comparative analysis of content and composition of large cfDNA enriched by MOF material enrichment methods and kit methods
Qubit quantification of cfDNA:
the instrument comprises the following steps: qubit fluorometer (Invitrogen Qubit 4) reagents: the kit for detecting the Qubit 1x dsDNA HS.
According to the detection instruction of the Qubit detection kit and the use instruction of the instrument, the contents of the cfDNA separated and enriched by the Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V and the cfDNA enriched by the kit are respectively detected. The detection result is shown in figure 2, the same amount of plasma (500 μ L of cfDNA obtained by enriching three materials of Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V, the cfDNA content obtained by Co-IRMOF-74-V is the highest, 24.8ngde cfDNA is obtained, the same amount of plasma, the cfDNA content obtained by using the kit is 29.8ng, the cfDNA amount extracted by using the method of Co-IRMOF-74-V is close to the cfDNA content obtained by using the method of the kit, and the enrichment method of the invention and the method of the commercial kit achieve the same effect.
quantitative comparison of qPCR: and detecting the content of the cfDNA enriched by the Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V separation and enrichment cfDNA and the cfDNA enriched by the kit by using a fluorescent quantitative PCR method. An ALU gene is selected as a detection target gene, and qPCR primers are designed as follows: 5'-CCTGAGGTCAGGAGTTCGAG-3', and 5'-CCCGAGTAGCTGGGATTACA-3'. The qPCR test results are shown in FIG. 3, and the ct values (Cycle Threshold) of the Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V samples are sequentially increased, which shows that the amount of cfDNA obtained by enrichment is sequentially increased. The ct values of the cfDNA obtained by the Co-IRMOF-74-V and the kit are relatively close to each other and are consistent with the results obtained by the Qubit quantitative method.
The second generation sequencing technology analyzes the component difference of cfDNA obtained by enrichment among different methods: the enriched cfDNA separated from Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V and the enriched cfDNA of the Kit are respectively subjected to construction of a sequencing Library, the Kit (NEBNext Ultra DNA Library Prep Kit for Illumina) is used, and the detailed steps refer to the method of the Kit specification. The data obtained by sequencing is subjected to main component analysis, the result is shown in fig. 4, the main component difference of the cfDNA obtained by enriching the Co-IRMOF-74-III, the Co-IRMOF-74-IV and the Co-IRMOF-74-V and the kit is very small, namely the component difference of the cfDNA in the four samples is very small, which shows that the cfDNA obtained by enriching the method is almost the same in component.
While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.
Claims (10)
1. A method of enriching circulating free nucleic acids in a body fluid using MOF material comprising the steps of:
(1) treating the body fluid sample such that circulating free nucleic acids bound to proteins in the body fluid sample are released from the protein-nucleic acid complex;
(2) adding an MOF material into the solution treated in the step (1), and adsorbing circulating free nucleic acid in the solution;
(3) separating the MOF material having the circulating free nucleic acids adsorbed in step (2);
(4) disrupting the MOF material having adsorbed circulating free nucleic acids of step (3) with EDTA to release the adsorbed nucleic acids;
(5) and (4) purifying the nucleic acid released in the step (4) to obtain circulating free DNA and circulating free RNA.
2. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (1), guanidine thiocyanate and polyethylene glycol octyl phenyl ether are added into body fluid to denature protein, and ZnCl is added2The protein is allowed to precipitate out, the protein is separated from the supernatant, and circulating free nucleic acid is present in the supernatant.
3. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (1), the MOF material is at least one of Co-IRMOF-74-III, Co-IRMOF-74-IV, Co-IRMOF-74-V, Ni-IRMOF-74-III, Ni-IRMOF-74-IV and Ni-IRMOF-74-V.
4. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (1), the body fluid sample is any one of whole blood, plasma, serum, urine, saliva, pleural effusion, cerebrospinal fluid, lymph fluid and ascites.
5. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (2), the addition amount of the MOF material is 200-500 mu g/mL.
6. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (2), univalent and divalent metal cations are added into an adsorption reaction system.
7. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (4), EDTA aqueous solution is added into the separated MOF material until the final concentration of EDTA is 1-10mM, and the mixture is repeatedly blown or shaken until white MOF white organic ligand insoluble substances appear, so that nucleic acid is released from the MOF material.
8. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: in the step (5), the released nucleic acid is purified by adopting an ice ethanol precipitation method or a nucleic acid purification kit; the concrete operation of the glacial ethanol precipitation method is as follows: adding an absolute ethanol solution into the nucleic acid solution obtained in the step (4), placing at the temperature of (-20) - (-70) DEG C to precipitate a nucleic acid solid from the solution, and performing low-temperature centrifugal separation to obtain a precipitated nucleic acid solid; the specific operation of the nucleic acid purification kit is as follows: and (3) adding ethanol into the nucleic acid solution obtained in the step (4), adsorbing the nucleic acid solution onto a glass fiber material in a purification column, and eluting the nucleic acid from the purification column by using water to achieve the purpose of purifying the nucleic acid.
9. The method of enriching circulating free nucleic acids in a body fluid using MOF material of claim 1, wherein: further comprising the step (6): and (3) treating the circulating free nucleic acid obtained in the step (5) with RNA hydrolase or DNA hydrolase respectively to obtain single cfDNA or cfRNA.
10. Use of a method of enriching circulating free nucleic acids in a body fluid using a MOF material of any one of claims 1 to 9 in a tumor cfDNA enrichment assay.
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| CN109364899A (en) * | 2018-12-21 | 2019-02-22 | 上海纳米技术及应用国家工程研究中心有限公司 | The preparation method and products thereof of magnetic ZIF-8 Nano composite granules |
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