CN111500677A - Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA - Google Patents
Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA Download PDFInfo
- Publication number
- CN111500677A CN111500677A CN202010419473.0A CN202010419473A CN111500677A CN 111500677 A CN111500677 A CN 111500677A CN 202010419473 A CN202010419473 A CN 202010419473A CN 111500677 A CN111500677 A CN 111500677A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- acid precipitation
- dna
- extracting
- free dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 38
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 38
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 38
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000003916 acid precipitation Methods 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 19
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 9
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 9
- 239000001632 sodium acetate Substances 0.000 claims abstract description 9
- 229920002527 Glycogen Polymers 0.000 claims abstract description 7
- 229940096919 glycogen Drugs 0.000 claims abstract description 7
- -1 Acryl Chemical group 0.000 claims abstract 3
- 230000005298 paramagnetic effect Effects 0.000 claims description 4
- 230000008021 deposition Effects 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 108010067770 Endopeptidase K Proteins 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 21
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 19
- 101100091155 Homo sapiens RNF43 gene Proteins 0.000 description 11
- 230000035772 mutation Effects 0.000 description 9
- 239000011324 bead Substances 0.000 description 7
- 230000005291 magnetic effect Effects 0.000 description 6
- 206010064571 Gene mutation Diseases 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 201000007270 liver cancer Diseases 0.000 description 5
- 208000014018 liver neoplasm Diseases 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 108700039887 Essential Genes Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011528 liquid biopsy Methods 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001331845 Equus asinus x caballus Species 0.000 description 1
- 101000692702 Homo sapiens E3 ubiquitin-protein ligase RNF43 Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000007886 magnetic bead extraction Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid precipitation aid and an improved method for extracting circulating tumor DNA, and belongs to the technical field of biology. The invention discloses a nucleic acid precipitation aid, which is prepared by combining at least one of Carrier RNA, Acryl Carrier, polyA and glycogen with sodium acetate. The improved method for extracting the circulating tumor DNA disclosed by the invention has the advantage that the detection yield of free DNA in a sample is improved by adding the settling agent into a plasma sample. The invention improves the extraction yield of free DNA in a sample, improves the detection rate of tumor mutant genes and is beneficial to the clinical auxiliary effect on tumor diagnosis.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid precipitation promoter and an improved method for extracting circulating tumor DNA.
Background
Circulating tumor gene (ctDNA), an extracellular DNA in a cell-free state, is derived from somatic mutation DNA fragments of dead tumor cells, and exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid as free DNA. The tumor marker has wide application prospect, high sensitivity and high specificity, plays an important role in the aspects of tumor diagnosis, treatment, prognosis detection and the like, and can be applied to liquid biopsy of tumors for early screening and diagnosis of the tumors. The content of ctDNA is very low, so that the efficient extraction of ctDNA becomes one of the keys of the tumor liquid biopsy technology, and the detection rate of ctDNA mutation can be improved by improving the extraction rate of ctDNA, so that the accuracy of early tumor screening is improved.
The method for separating and purifying nucleic acid generally comprises the following main steps of cell lysis, enzyme treatment, separation of nucleic acid from other biomacromolecules, purification of nucleic acid and the like. The separation and purification of nucleic acid is a key step, the high-charge phosphate skeleton of nucleic acid makes it more hydrophilic than other biomacromolecule substances of protein, polysaccharide and fat, etc., and according to the difference of their physicochemical properties, the nucleic acid can be separated and purified by using the methods of selective precipitation, chromatography and density gradient centrifugation, etc. The nucleic acid extraction effect obtained by different methods is different.
The separation and purification methods of free DNA (cfDNA) mainly comprise a phenol-chloroform method, a silica gel membrane adsorption column method and a magnetic bead method. The traditional phenol-chloroform method has low extraction efficiency, poor repeatability, large dosage of toxic solvent, complex operation and easy pollution. The silica gel membrane adsorption column method realizes the adsorption of nucleic acid by combining specific buffer solution, accurate pH and salt concentration. The method has the advantages of high speed, high nucleic acid purity and good reproducibility, and has the main defects of low extraction efficiency of small fragment nucleic acid compared with a magnetic bead method, common chaotropic salt condition and complex and long subsequent salt washing. A serious problem of the column extraction method is that the shearing force caused by high-speed centrifugation is easy to degrade DNA, and the integrity of the DNA is influenced. The major products for extracting free nucleic acid by a silica gel membrane adsorption column method in the current market are kits of Qiagen companies, the price is expensive, and the recovery rate is only about 40%. The magnetic bead method nucleic acid purification technology adopts nanometer magnetic bead microbeads, the surfaces of which are marked with a functional group which can perform adsorption reaction with nucleic acid. The magnetic bead extraction technology can be used for batch processing of various samples, is one of the best choices of automation and high flux, is simple and convenient to operate, and consumes short time, but most of commercially available extraction kits have low extraction rate of free DNA, are not suitable for extracting the free DNA with extremely low content such as tumor circulating DNA, and cannot be well applied to the tumor liquid biopsy technology.
Therefore, it is an urgent need to solve the problems of the art to provide a nucleic acid precipitation promoter and an improved method for extracting circulating tumor DNA.
Disclosure of Invention
In view of the above, the invention provides a nucleic acid precipitation aid and an improved method for extracting circulating tumor DNA, which improves the existing method for extracting free DNA by a magnetic bead method, improves the extraction yield of the free DNA by adding the nucleic acid precipitation aid into a sample, has good effect on extracting the circulating tumor DNA, and can be applied to the liquid biopsy of the tumor.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nucleic acid deposition aid for extracting circulating tumor DNA is prepared from at least one of 0.5-1.5 μ g/μ l carrier RNA, l-3 μ g/μ l acrylic carrier, 1-5 μ g/μ l polyA, and 2-6 μ g/μ l glycogen and 0.1-0.6mol/μ l sodium acetate.
Further, a nucleic acid precipitation aid, which is composed of 1.0. mu.g/. mu.l of CarrierRNA, 3. mu.g/. mu.l of glycogen and 0.2 mol/. mu.l of sodium acetate.
Further, a nucleic acid precipitation aid, which is composed of 1.5. mu.g/. mu.l of acrylic Carrier, 4.0. mu.g/. mu.l of PolyA and 0.3 mol/. mu.l of sodium acetate.
Further, an improved method for extracting circulating tumor DNA comprises the following specific steps:
(1) separating the plasma sample;
(2) adding the nucleic acid precipitation aid into the plasma sample separated in the step (1), adding 200 mu L ProteinaseK, and extracting free DNA according to a free DNA kit by a paramagnetic particle method;
(3) and (3) carrying out PCR detection on the free DNA extracted in the step (2).
According to the technical scheme, compared with the prior art, the invention discloses the nucleic acid precipitation aid and the improved extraction method of the circulating tumor DNA, and aims at the situation that most of the existing extraction kits are not high in free DNA extraction rate and are not suitable for extracting free DNA with extremely low content such as tumor circulating DNA. The invention improves the extraction yield of free DNA in a sample, improves the detection rate of tumor mutant genes and is beneficial to the clinical auxiliary effect on tumor diagnosis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the result of fluorescent quantitative PCR verification according to the present invention;
wherein, A: no nucleic acid precipitating agent is added; b: adding a nucleic acid precipitation aid;
FIG. 2 is a graph showing the detection rate of the mutation positivity of RNF43 gene according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The free DNA kit by the magnetic bead method is purchased from Jiangsu Mule Biotech GmbH; glycogen is purchased from Hefei Qufeng Biotech limited; polyA was purchased from jejie biotechnology limited, shanghai.
Example 1 extraction of cfDNA from plasma
(1) Respectively taking 2ml of blood plasma samples obtained after blood separation of 10 normal people, respectively dividing the blood plasma samples into two parts, wherein each part is 1ml, and adding the two parts into a 2m L centrifugal tube;
(2) to one of the plasma samples of each example, nucleic acid precipitation promoter was added (CarrierRNA1.0. mu.g/. mu.l, glycogen 3. mu.g/. mu.l, sodium acetate 0.2 mol/. mu.l) and 200. mu. L ProteinaseK was added, and two plasma samples of each plasma sample were simultaneously extracted for free DNA according to the paramagnetic free DNA kit.
(3) The extracted free DNA was verified by fluorescent quantitative PCR using housekeeping gene GAPDH, and the detection result was judged according to the Ct value shown by the fluorescent PCR amplifier (the smaller the Ct value is, the higher the amount of the initial template is), and the detailed Ct value is shown in FIG. 1.
As can be seen from the results of fig. 1: under the same extraction conditions, the Ct value of the amplified plasma sample without the addition of the settling agent is larger, which indicates that the DNA fragment of housekeeping gene GAPDH in the sample is less and the extraction rate of free DNA is lower; the Ct value of the qPCR amplification of the same plasma sample added with the settling agent is smaller, which shows that the number of DNA fragments of housekeeping genes in the sample is obviously increased, and the extraction yield of free DNA is higher. The qPCR verification of housekeeping genes can judge that the extraction yield of free DNA in blood plasma can be obviously improved by adding the nucleic acid precipitation aid.
Example 2 detection of mutation in RNF43 Gene
The protease coded by the RNF43 gene can participate in the metabolism detoxification of carcinogens such as nitrosamine and benzpyrene, and when the gene is mutated, the detoxification capability of the coded protease is reduced, so that the incidence rate of liver cancer is increased. Meanwhile, researches show that 462 th site mutation of the RNF43 gene can obviously increase the risk of smoking people suffering from liver cancer. The invention verifies the influence of the plasma sample added with the settling agent on the mutation detection of the RNF43 gene by detecting the mutation of the RNF43 gene, and the specific steps are as follows:
(1) collecting 20 cases of liver cancer patients with anticoagulation blood 2-3m L with cfDNA blood collection tubes, centrifuging at 4 deg.C for 10min within 4 hr, collecting supernatant to 2m L centrifuge tube, and storing at-20 deg.C;
(2) 2ml of plasma samples obtained after blood separation of 20 liver cancer patients are respectively taken and divided into two parts, wherein each part is 1ml, and the two parts are added into a 2m L centrifugal tube;
(3) to one plasma sample of each example, nucleic acid precipitation promoter was added (AcrylCarrier 1.5. mu.g/. mu.l, PolyA4.0. mu.g/. mu.l, sodium acetate 0.3 mol/. mu.l) and 200. mu. L ProteinaseK was added, and two plasma samples of each plasma sample were simultaneously subjected to extraction of free DNA according to the paramagnetic beads method free DNA kit.
(4) And (3) carrying out digital PCR (polymerase chain reaction) on the extracted free DNA, detecting the mutation condition of the RNF43 gene, and calculating the positive detection rate of the RNF43 gene mutation, wherein the detailed detection result is shown in figure 2.
As can be seen from fig. 2: under the same extraction conditions, the detection rate of the RNF43 gene mutation in the plasma sample without the addition of the precipitation aid is 60%, while the detection rate of the RNF43 gene mutation in the same plasma sample with the addition of the nucleic acid precipitation aid is 80%. Through the verification of the detection rate of the RNF43 gene mutation, the addition of the nucleic acid precipitation aid can be judged to obviously improve the extraction efficiency of plasma ctDNA, so that the detection rate of the gene mutation in tumor free DNA can be obviously improved.
Besides the mutation detection of the liver cancer mutant gene RNF43, the kit can also be used for the mutation detection of other tumor genes.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. A nucleic acid deposition aid for extracting circulating tumor DNA is characterized by being prepared by combining 0.5-1.5 mu g/mu l of Carrier rRNA, l-3 mu g/mu l of Acryl Carrier, 1-5 mu g/mu l of PolyA and 2-6 mu g/mu l of glycogen with 0.1-0.6 mol/mu l of sodium acetate.
2. The nucleic acid precipitation aid of claim 1, wherein the nucleic acid precipitation aid is prepared from 1.0 μ g/μ l of carrierna, 3 μ g/μ l of glycogen and 0.2mol/μ l of sodium acetate.
3. The nucleic acid precipitation aid of claim 1, wherein the nucleic acid precipitation aid comprises 1.5 μ g/μ l Acryl Carrier, 4.0 μ g/μ l PolyA, and 0.3mol/μ l sodium acetate.
4. An improved method for extracting circulating tumor DNA is characterized by comprising the following specific steps:
(1) separating the plasma sample;
(2) adding the nucleic acid precipitation aid of any one of claims 1-3 into the plasma sample separated in the step (1), adding 200 mu L protease K, and extracting free DNA according to a free DNA kit by a paramagnetic particle method;
(3) and (3) carrying out PCR detection on the free DNA extracted in the step (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010419473.0A CN111500677A (en) | 2020-05-18 | 2020-05-18 | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010419473.0A CN111500677A (en) | 2020-05-18 | 2020-05-18 | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111500677A true CN111500677A (en) | 2020-08-07 |
Family
ID=71868374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010419473.0A Pending CN111500677A (en) | 2020-05-18 | 2020-05-18 | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111500677A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673783A (en) * | 2015-01-19 | 2015-06-03 | 宝瑞源生物技术(北京)有限公司 | Kit for extracting DNA/RNA of virus through magnetic bead method and using method |
| CN107312771A (en) * | 2017-01-11 | 2017-11-03 | 华东医药（杭州）基因科技有限公司 | A kind of method of efficient extracting serum and plasma DNA |
| CN109371018A (en) * | 2018-12-30 | 2019-02-22 | 北京优迅医疗器械有限公司 | For dissociate Circulating tumor DNA extraction reinforcing agent, extract peripheral blood in dissociate Circulating tumor DNA kit and method |
| CN109762878A (en) * | 2017-11-29 | 2019-05-17 | 铭时基因科技(上海)有限公司 | A kind of the ddPCR detection method and application in the site lung cancer EGFR L858R |
| CN109762874A (en) * | 2018-12-30 | 2019-05-17 | 北京优迅医疗器械有限公司 | Nucleic acid settling agent, pregnant woman blood plasma dissociative DNA extracts kit and method |
-
2020
- 2020-05-18 CN CN202010419473.0A patent/CN111500677A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673783A (en) * | 2015-01-19 | 2015-06-03 | 宝瑞源生物技术(北京)有限公司 | Kit for extracting DNA/RNA of virus through magnetic bead method and using method |
| CN107312771A (en) * | 2017-01-11 | 2017-11-03 | 华东医药（杭州）基因科技有限公司 | A kind of method of efficient extracting serum and plasma DNA |
| CN109762878A (en) * | 2017-11-29 | 2019-05-17 | 铭时基因科技(上海)有限公司 | A kind of the ddPCR detection method and application in the site lung cancer EGFR L858R |
| CN109371018A (en) * | 2018-12-30 | 2019-02-22 | 北京优迅医疗器械有限公司 | For dissociate Circulating tumor DNA extraction reinforcing agent, extract peripheral blood in dissociate Circulating tumor DNA kit and method |
| CN109762874A (en) * | 2018-12-30 | 2019-05-17 | 北京优迅医疗器械有限公司 | Nucleic acid settling agent, pregnant woman blood plasma dissociative DNA extracts kit and method |
Non-Patent Citations (1)
| Title |
|---|
| 张孝欢等: "《循环肿瘤DNA检测及相关研究进展》", 《实用医院临床杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| He et al. | Based on magnetic beads to develop the kit for extraction of high-quality cell-free DNA from blood of breast cancer patients | |
| CN110129314B (en) | Amino acid buffer solution and plasma free DNA extraction kit | |
| CN108624586A (en) | A kind of nucleic acid extraction kit and its application process | |
| US20220372471A1 (en) | Apparatuses systems and methods for enrichment and separation of nucleic acids by size | |
| CN107779451A (en) | A kind of mankind's dissociative DNA extracting method and its kit | |
| EP1882738A1 (en) | Methods for recovering microorganism and nucleic acid using fine particle and kit to be used for the methods | |
| CN108796075B (en) | Application of reagent for detecting circRNF13 and LOC284454 and kit | |
| EP3452613B1 (en) | Profiling microvesicle nucleic acids and uses thereof as signatures in diagnosis of renal transplant rejection | |
| CN109321563A (en) | The preparation of excretion body and the method for constructing excretion body tiny RNA library | |
| US10323241B2 (en) | Method for recovering short-chain nucleic acids | |
| Tsubota et al. | Association of enzymes with replicating and nonreplicating simian virus 40 chromosomes | |
| CN112322614A (en) | Kit for extracting blood genome DNA by paramagnetic particle method and use method thereof | |
| CN111500677A (en) | Nucleic acid precipitation promoter and improved method for extracting circulating tumor DNA | |
| CN104450683B (en) | A kind of method that grape genomic DNA is extracted from grape wine | |
| CN115896316A (en) | Tuberculosis detection method | |
| US20210207125A1 (en) | Method of isolating nucleic acids for long sequencing reads | |
| Chaisomchit et al. | A simple method for extraction and purification of genomic DNA from dried blood spots on filter paper | |
| CN102807979A (en) | Method for extracting and pretreating microorganism deoxyribonucleic acid (DNA) on biological active carbon | |
| CN108866042B (en) | Extraction method of trace RNA | |
| CN114085894A (en) | Method for converting nucleic acid methylated cytosine | |
| CN113832141A (en) | Method and kit for specifically purifying human genome DNA of excrement | |
| CN112063615A (en) | Genome DNA extracting solution, genome DNA extracting method and application thereof | |
| CN117487922B (en) | Methylation detection kit for assisting in identifying benign and malignant lung nodules | |
| CN115125238B (en) | Method for separating and purifying tumor extracellular vesicle DNA | |
| CN114200127B (en) | Hepatitis B virus enrichment fluorescent PCR detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200807 |