CN117587131A - Detection method for dynamically monitoring ctDNA and application thereof - Google Patents
Detection method for dynamically monitoring ctDNA and application thereof Download PDFInfo
- Publication number
- CN117587131A CN117587131A CN202410027965.3A CN202410027965A CN117587131A CN 117587131 A CN117587131 A CN 117587131A CN 202410027965 A CN202410027965 A CN 202410027965A CN 117587131 A CN117587131 A CN 117587131A
- Authority
- CN
- China
- Prior art keywords
- amplification reaction
- dna
- ctdna
- mbda
- library
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 238000012544 monitoring process Methods 0.000 title claims abstract description 13
- 230000003321 amplification Effects 0.000 claims abstract description 51
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 51
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 238000000746 purification Methods 0.000 claims abstract description 21
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 17
- 239000003708 ampul Substances 0.000 claims abstract description 6
- 239000011324 bead Substances 0.000 claims abstract description 6
- 238000005070 sampling Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 39
- 230000008569 process Effects 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 6
- 150000002632 lipids Chemical class 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 230000004543 DNA replication Effects 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 3
- 102000006382 Ribonucleases Human genes 0.000 claims description 3
- 108010083644 Ribonucleases Proteins 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 230000006037 cell lysis Effects 0.000 claims description 3
- 239000008004 cell lysis buffer Substances 0.000 claims description 3
- 210000000170 cell membrane Anatomy 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- AINBZKYUNWUTRE-UHFFFAOYSA-N ethanol;propan-2-ol Chemical compound CCO.CC(C)O AINBZKYUNWUTRE-UHFFFAOYSA-N 0.000 claims description 3
- 238000001727 in vivo Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000010076 replication Effects 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 239000002096 quantum dot Substances 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 claims 4
- 108020004414 DNA Proteins 0.000 description 32
- 238000007481 next generation sequencing Methods 0.000 description 26
- 239000000523 sample Substances 0.000 description 16
- 238000012163 sequencing technique Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 238000000926 separation method Methods 0.000 description 9
- 239000013076 target substance Substances 0.000 description 9
- 239000003480 eluent Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 108091093088 Amplicon Proteins 0.000 description 6
- 239000003463 adsorbent Substances 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 108700028369 Alleles Proteins 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 239000000107 tumor biomarker Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- C12Q1/6876—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
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the field of ctDNA detection, and provides a detection method for dynamically monitoring ctDNA and application thereof, comprising the following steps: DNA sample, amplification reaction, adding an amplification reaction adapter and an NGS library, performing mBDA after sampling DNA, performing a purification column, forming an amplification reaction, performing a secondary purification column after adding the adapter for PCR, adding the adapter for the amplification reaction, adding an index for PCR, selecting the size of ampoule beads, and finally entering the NGS library; the invention solves the problems of high difficulty, high cost and the like of the MRD detection of solid tumors by using an amplification reaction and a purification column mBDA.
Description
Technical Field
The invention relates to the field of ctDNA detection, in particular to a detection method for ctDNA and application thereof.
Background
The circulating tumor gene, namely ctDNA, refers to tumor cell somatic DNA which is released into the circulatory system after shedding or apoptosis, is a characteristic tumor biomarker, tumor trails in blood can be detected through ctDNA detection, the sum of free DNA in blood plasma is called cfDNA, the cfDNA exists in various body fluids of a human body and changes in concentration along with tissue injury, cancer, inflammatory reaction and the like, and the cfDNA released into the circulatory system by tumor patient cells comprises ctDNA.
Most of the existing detection uses imaging detection and MRD detection, namely human imaging is carried out through various imaging devices, so that basis is provided for disease diagnosis, including X-ray detection, CT detection, MRI, ultrasonic detection and the like, and ctDNA detection can prompt tumor recurrence earlier than imaging detection, wherein the median advance time is 88 days, but the difficulty of solid tumor MRD detection is high, and the reasons are tumor heterogeneity, low early ctDNA content and lower ctDNA relative to tissue abundance, and a detection method with extremely high sensitivity is more needed.
In summary, the present invention thus provides a detection method for ctDNA and application thereof to solve the above problems.
Disclosure of Invention
In order to solve the technical problems, the invention provides a detection method for dynamically monitoring ctDNA and application thereof, so as to solve the problems of high difficulty in detecting the MRD of the solid tumor in the prior art.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
detection methods for dynamic monitoring of ctDNA and applications thereof, comprising:
s1, DNA sampling, (1) cell lysis and cell lysis buffer solution are firstly used for cell membrane lysis, and then lipid is decomposed by a detergent and a surfactant;
(2) Digesting the protein by adding protease, digesting the RNA by adding RNase, and then separating the cell debris, digested protein, lipid and RNA by adding concentrated salt and then centrifuging;
(3) Finally, precipitating DNA with ice-cold ethanol or isopropanol ethanol, wherein the ionic strength of sodium acetate can be used for improving precipitation, and the precipitated DNA is linear in a final solution;
s2, an amplification reaction, namely an amplification reaction of a specific template catalyzed by enzyme under the guidance of a primer, simulates an in-vivo DNA replication process, and the problem that ctDNA is difficult to detect in a small amount of DNA is solved, and the amplification reaction needs to be revealed through other processes from the replication and amplification process;
s3, adding an amplification reaction adapter, wherein the two amplification reaction devices are connected with the NGS library device by adding the adapter and are matched with the two amplification reaction devices in current, so that the amplification reaction device and the NGS library device can cooperatively operate; the additional PCR amplification reaction equipment is connected with equipment in the NGS library workstation, so that data generated by the amplification reaction can be rapidly input into the NGS library, and rapid transmission of the data is realized.
S4, inputting the DNA subjected to the amplification reaction into an NGS library, wherein the DNA is generally divided into four steps, namely cutting DNA or RNA fragments, repairing and connecting ends, amplifying and purifying a library and controlling the quality of the library, and then monitoring ctDNA and tumor treatment processes.
Preferably, the DNA is sampled and subjected to an mBDA, and the mBDA is subjected to a purification column and then to an amplification reaction. Wherein mBDA is a multiplex inhibition probe displacement amplification technique that detects low frequency genetic variation of VAF as low as 0.019% with only 250X sequencing depth. Unlike other mutant allele enrichment techniques, mBDA can be well extended to multiple Panel (Panel of 80-fold amplicon can achieve a median fold enrichment of 300 fold). The VAF of the low-frequency variation allele in the sample is amplified by the library enriched by the mBDA technology and far exceeds the NGS system error, so that the low-frequency variation can be detected only by relatively low sequencing depth without additional error correction, and the detection specificity is greatly improved; the enrichment efficiency of mBDA is extremely stable between different samples, different libraries, and initial sample variation of 2-fold difference can be accurately quantified with 95% accuracy.
Preferably, an adapter is added to the PCR after the amplification reaction, and the purification column is performed once again after the addition of the PCR adapter is completed.
Preferably, the amplification reaction is adapted to be added and then the PCR is performed with the primers added, the size of the ampoule beads is selected after the PCR is performed with the primers added, and then the detection data is input into the NGS library.
Preferably, after the data are input into the NGS library, the qubit and the biological analyzer are analyzed, and then the data in other libraries are combined to finally form the NGS. The mBDA-NGS technology greatly reduces the sequencing data volume (100 times can be reduced), and can enable various medium-low flux sequencers to realize the detection of low-frequency gene variation with lower sequencing cost.
Preferably, the method should be in an mBDA detection ctDNA kit.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention detects the mixed cell line by using the mBDA-NGS method, the initial sample size is 300ng genome DNA, and the lowest LoD can reach 0.019%; in terms of variant sequence proportion (VRF), compared with 0.2% of the NGS sequencing method of a standard multiplex amplicon library, the mBDA method can reach 30%, and the enrichment efficiency is more than 150 times (median 300 times); on the premise that the mid-target rate is basically the same (mBDA 81.4% vs. 89.6% of standard amplicon method), the mBDA method greatly reduces the amount of sequencing data required for detection, thereby reducing the sequencing cost; compared with a probe capture method, the mBDA can shorten the library construction time from 24 hours to 6 hours, and solves the problems of high difficulty, high cost and the like of solid tumor MRD detection;
drawings
FIG. 1 is a flow chart of a ctDNA detection method of the present invention;
FIG. 2 is a graph showing experimental flow and low frequency mutation detection performance of the mbDA-NGS method of the invention;
FIG. 3 is a graph showing the variation trend of the equivalent level of ctDNA of the present invention;
FIG. 4 is a graph of VM value level change trend for the present invention;
FIG. 5 shows the absolute number of ctDNA molecules of the test samples of the present invention;
FIG. 6 is a risk assessment of a past sample of a subject of the present invention;
FIG. 7 is a graph showing the tendency of ctDNA change relative to initial detection according to the present invention.
Detailed Description
Embodiments of the present invention are described in further detail below with reference to the accompanying drawings and examples. The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
The invention provides a detection method for dynamically monitoring ctDNA and application thereof, comprising the following steps:
s1, DNA sampling:
s1, DNA sampling, (1) cell lysis and cell lysis buffer solution are firstly used for cell membrane lysis, and then lipid is decomposed by a detergent and a surfactant;
(2) Digesting the protein by adding protease, digesting the RNA by adding RNase, and then separating the cell debris, digested protein, lipid and RNA by adding concentrated salt and then centrifuging;
(3) Finally, precipitating DNA with ice-cold ethanol or isopropanol ethanol, wherein the ionic strength of sodium acetate can be used for improving precipitation, and the precipitated DNA is linear in a final solution;
s2, an amplification reaction, namely an amplification reaction of a specific template catalyzed by enzyme under the guidance of a primer, simulates an in-vivo DNA replication process, and the problem that ctDNA is difficult to detect in a small amount of DNA is solved, and the amplification reaction needs to be revealed through other processes from the replication and amplification process;
s3, adding an amplification reaction adapter, wherein the two amplification reaction devices are connected with the NGS library device by adding the adapter and are matched with the two amplification reaction devices in current, so that the amplification reaction device and the NGS library device can cooperatively operate;
s4, inputting the DNA subjected to the amplification reaction into an NGS library, wherein the steps of DNA or RNA fragment shearing, end repair and connection, library amplification and purification and library quality control are generally divided into four steps, and then ctDNA and tumor treatment processes are monitored; preferably, the DNA is sampled and subjected to an mBDA, and the mBDA is subjected to a purification column and then to an amplification reaction.
As an embodiment of the present invention: and (3) performing the mBDA after the DNA is sampled, performing a purification column after the mBDA project is performed, and performing an amplification reaction, wherein the low-frequency gene variation of the VAF as low as 0.019% can be detected by the mBDA technology only by using a sequencing depth of 250X. Unlike other mutant allele enrichment techniques, mBDA can be well extended to multiple Panel (Panel of 80-fold amplicon can achieve a median fold enrichment of 300 fold). The VAF of the low-frequency variation allele in the sample is amplified by the library enriched by the mBDA technology and far exceeds the NGS system error, so that the low-frequency variation can be detected only by relatively low sequencing depth without additional error correction, and the detection specificity is greatly improved;
as an embodiment of the present invention: the method comprises the steps of adding an adapter to PCR after amplification reaction, and carrying out a purification column again after the addition of the PCR adapter, wherein the purification column is a common separation and purification technology, and the working principle is that a target substance in a mixture is selectively adsorbed by using an adsorbent filled in the column, so that the separation and purification of the target substance are realized;
as an embodiment of the present invention: the amplification reaction is adaptively added and then carries out PCR adding primers, the size of ampoule beads is selected after the PCR adding primers, then detection data is input into an NGS library, the PCR adding primers can be selectively paired with two ends of a DNA sequence to be amplified, so that the PCR amplification reaction can be started, and the establishment of the NGS library is mainly used for converting a DNA or RNA sample into a library which can be read by a sequencer;
as an embodiment of the present invention: the method generally comprises four steps of cutting DNA or RNA fragments, end repairing and connecting, amplifying and purifying the library and controlling the quality of the library, cutting the DNA or RNA fragments into short fragments, carrying out end repairing and connecting on the DNA or RNA, adding a proper connector to realize the end repairing and connecting, amplifying and purifying the library after the connecting, and finally controlling the quality of the library, wherein the aim of the quality control of the library is to detect the quality and purity of the library so as to ensure that the library can be correctly read by a sequencer.
The specific working principle is as follows:
as shown in fig. 1-2, DNA needs to be sampled first, DNA precipitated after a series of operations is finally in a linear form in a solution, and then an mBDA procedure is performed on the DNA, so that the enrichment efficiency of mBDA is extremely stable between different samples and different libraries, and the variation of an initial sample with a 2-fold difference can be accurately quantified with 95% accuracy. The mBDA-NGS technology greatly reduces the sequencing data volume (100 times can be reduced), and can enable various medium-low flux sequencers to realize the detection of low-frequency gene variation with lower sequencing cost;
as shown in fig. 2, the experimental flow of the mBDA-NGS method and the low-frequency mutation detection performance verification experiment show that, when the mBDA-NGS method is used for detecting the mixed cell line, the initial sample size is 300ng of genome DNA, the LoD can be as low as 0.019%, in terms of variant sequence proportion (variant-read fraction, VRF), compared with the standard multiplex amplicon library NGS sequencing method, the mBDA method can reach 30%, the enrichment efficiency is more than 150 times (median 300 times), and on the premise that the targeting rate is basically the same (mBDA 81.4% vs. standard amplicon method 89.6%), the sequencing data size required by detection is greatly reduced by the mBDA method, so that the sequencing cost is reduced, compared with the probe capturing method, the mBDA can shorten the library construction time from 24 hours to 6 hours, and the problems of high cost, high difficulty and the like of solid tumor MRD detection are solved by the method;
after the end of mBDA, the first purification column is carried out, the purification column is loaded with a sample, the sample enters the purification column through a sample inlet at the top of the column, through an adsorbent filled in the column, a target substance can be selectively adsorbed on the surface of the adsorbent, a non-target substance can be discharged through an outlet at the bottom of the column, and in the process, the flow rate and the concentration of the sample can influence the adsorption effect, so that the sample needs to be adjusted according to practical conditions, such as: in some related experiments, the higher the column effect is for long and thin or the higher the column effect is for short and thick, wherein the two column effects can reach ideal values, but the intermediate value is not taken, so that the process needs to be adjusted according to actual conditions, the elution is performed, after the sample is loaded, some non-target substances remain on the surface of the adsorbent in the column, the non-target substances need to be removed through elution, and the choice of the eluent needs to be adjusted according to actual conditions, such as: the eluent with larger polarity can cause the poor separation effect in the column chromatography, and the eluent with larger polarity can cause substances in the column chromatography to flow faster along the flow direction of the column chromatography, thereby the poor separation effect of the substances. And (3) expanding: column chromatography is a separation technique that allows the mixture to be separated into individual components, thereby achieving higher purity. The separation effect of the column chromatography depends on the polarity of the eluent, the greater the polarity is, the better the separation effect is, on the contrary, the smaller the polarity is, the worse the separation effect is, so the selection of the eluent and the optimization of the elution condition are critical to the separation effect of the purification column, the recovery is carried out finally, the target substance is desorbed from the surface of the adsorbent after elution and enters the eluent, at this time, the eluent needs to be discharged through the outlet of the column bottom, the target substance needs to be recovered through the outlet of the column top, and the recovered target substance can be further analyzed or applied;
the method comprises the steps of carrying out amplification reaction after the first purification column is finished, wherein the amplification reaction is mainly used for DNA mapping, DNA sequencing and the like, adding an adapter for PCR after the amplification reaction, carrying out a second purification column after the amplification reaction, adding the adapter for the amplification reaction, adding an index for PCR, then selecting ampoule beads with proper size, putting the ampoule beads into an NGS library, carrying out DNA or RNA fragment shearing, end repair and connection, library amplification and purification and library quality control, and finally forming the whole process of NGS by utilizing a biological quantum analyzer, thereby completing detection of patients.
The invention provides the following experimental data for further explaining the application of the mBDA-NGS technology in the actual scene:
ctDNA (circulating tumor DNA ) is a free DNA secreted after tumor cell necrosis and apoptosis. By detecting and analyzing ctDNA in peripheral blood of a patient, the influence of tumor tissue heterogeneity can be overcome, and the tumor load state can be reflected more accurately.
Claims (6)
1. A detection method for dynamically monitoring ctDNA, comprising:
s1, DNA sampling:
(1) Firstly, cell lysis and cell lysis buffer solution are used for cell membrane lysis, and then lipid is decomposed by a detergent and a surfactant;
(2) Digesting the protein by adding protease, digesting the RNA by adding RNase, and then separating the cell debris, digested protein, lipid and RNA by adding concentrated salt and then centrifuging;
(3) Finally, precipitating DNA with ice-cold ethanol or isopropanol ethanol, wherein the ionic strength of sodium acetate can be used for improving precipitation, and the precipitated DNA is linear in a final solution;
s2, an amplification reaction, namely an amplification reaction of a specific template catalyzed by enzyme under the guidance of a primer, simulates an in-vivo DNA replication process, and the problem that ctDNA is difficult to detect in a small amount of DNA is solved, and the amplification reaction needs to be revealed through other processes from the replication and amplification process;
s3, adding an amplification reaction adapter, wherein the two amplification reaction devices are connected with the NGS library device by adding the adapter and are matched with the two amplification reaction devices in current, so that the amplification reaction device and the NGS library device can cooperatively operate;
s4, inputting the DNA subjected to the amplification reaction into an NGS library, wherein the DNA is generally divided into four steps, namely cutting DNA or RNA fragments, repairing and connecting ends, amplifying and purifying a library and controlling the quality of the library, and then monitoring ctDNA and tumor treatment processes.
2. The detection method for dynamically monitoring ctDNA according to claim 1, wherein: the DNA in S1 is sampled and subjected to mBDA, and the mBDA project is subjected to purification column and then amplification reaction.
3. The detection method for dynamically monitoring ctDNA according to claim 1, wherein: and (2) adding an adapter to the PCR after the amplification reaction in the step (S2), and carrying out a purification column again after the addition of the PCR adapter is finished.
4. The detection method for dynamically monitoring ctDNA according to claim 1, wherein: and (3) carrying out PCR (polymerase chain reaction) primer addition after the amplification reaction in the step (S3) is adaptively added, selecting the size of ampoule beads after the primer addition, and inputting detection data into an NGS library.
5. The detection method for dynamically monitoring ctDNA according to claim 1, wherein: and S4, inputting the data into an NGS library, analyzing the data by a qubit and biological analyzer, and finally combining the data in other libraries to form the NGS.
6. Use of the method of any one of claims 1-5 in an mBDA detection ctDNA kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410027965.3A CN117587131A (en) | 2024-01-09 | 2024-01-09 | Detection method for dynamically monitoring ctDNA and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410027965.3A CN117587131A (en) | 2024-01-09 | 2024-01-09 | Detection method for dynamically monitoring ctDNA and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117587131A true CN117587131A (en) | 2024-02-23 |
Family
ID=89911826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410027965.3A Pending CN117587131A (en) | 2024-01-09 | 2024-01-09 | Detection method for dynamically monitoring ctDNA and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117587131A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN111683958A (en) * | 2018-02-20 | 2020-09-18 | 威廉马歇莱思大学 | Systems and methods for allele enrichment using multiple suppression probe displacement amplification |
| WO2022050654A1 (en) * | 2020-09-01 | 2022-03-10 | 주식회사 아이엠비디엑스 | Method for increasing ratio of intrinsic fragment used in ngs analysis for detecting low-frequency mutation of cfdna |
-
2024
- 2024-01-09 CN CN202410027965.3A patent/CN117587131A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN111683958A (en) * | 2018-02-20 | 2020-09-18 | 威廉马歇莱思大学 | Systems and methods for allele enrichment using multiple suppression probe displacement amplification |
| WO2022050654A1 (en) * | 2020-09-01 | 2022-03-10 | 주식회사 아이엠비디엑스 | Method for increasing ratio of intrinsic fragment used in ngs analysis for detecting low-frequency mutation of cfdna |
Non-Patent Citations (1)
| Title |
|---|
| SONG，P.等: "Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing", BIORXIV, 28 April 2020 (2020-04-28), pages 1 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109628277B (en) | System and method for separating and detecting tumor marker miRNA in exosome | |
| CN111172287B (en) | Application of exosome lncRNA RN7SL5P as internal reference gene in gastric cancer lncRNA detection | |
| JP7204004B2 (en) | Reagent kit for batch detection of multiple gene mutations in lung cancer | |
| CN110760580A (en) | Early diagnosis equipment for liver cancer | |
| CN105838777A (en) | Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology | |
| CN109234394A (en) | A kind of diagnosing cancer of liver marker and its screening technique | |
| WO2018228028A1 (en) | Gene marker for use in detecting liver cancer and use thereof | |
| CN106048021B (en) | Primer for detecting P53 gene mutation in microcomponent combines and its application | |
| Yoon et al. | Plasma circulating tumor DNA in patients with primary central nervous system lymphoma | |
| CN117587131A (en) | Detection method for dynamically monitoring ctDNA and application thereof | |
| CN112280867A (en) | Early warning method for liver cancer, detection kit for early warning and detection method | |
| CN110938685A (en) | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof | |
| CN114214416B (en) | Biomarkers associated with pre-cervical lesion occurrence and uses thereof | |
| WO2015179672A1 (en) | Methods of isolation of cell free complexes and circulating cell-free nucleic acid | |
| CN112501287B (en) | DNA methylation marker of psoriatic arthritis, diagnostic reagent and application thereof | |
| WO2009049147A2 (en) | Method of tumor screening by detection of tumour marker in low molecular weight nucleic acid from urine | |
| CN108315323A (en) | The detection of PIK3CA gene mutation sites in urine ctDNA | |
| CN109694912B (en) | Application of methylation sites, nucleic acid composition for detecting methylation, kit and detection method thereof | |
| CN111968702A (en) | Early malignant tumor screening system based on circulating tumor DNA | |
| WO2012043183A1 (en) | Method for synthesizing target nucleic acid in feces | |
| CN116875721B (en) | Application of cfDNA of cryptococcus in diagnosis of cryptococcus infection | |
| CN116397024B (en) | Multi-gene combined detection kit for screening lung cancer patients | |
| CN113981059B (en) | Primer composition for detecting thalassemia mutant gene and reagent thereof | |
| Yeh | Pre-analytical assessment of circulating cell-free DNA prepared by an isolation-free enrichment technology | |
| CN113584155B (en) | Kit for evaluating testicular microscopic semen extraction effect of idiopathic non-obstructive azoospermia patient |
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 |