CN108179191A - A kind of kit for detecting mankind's ctDNA gene mutations - Google Patents
A kind of kit for detecting mankind's ctDNA gene mutations Download PDFInfo
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- CN108179191A CN108179191A CN201810112801.5A CN201810112801A CN108179191A CN 108179191 A CN108179191 A CN 108179191A CN 201810112801 A CN201810112801 A CN 201810112801A CN 108179191 A CN108179191 A CN 108179191A
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- 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
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- 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
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- 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
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of kits for detecting mankind's ctDNA gene mutations.The kit includes multiplexed PCR amplification primer and extension primer, and the multiplexed PCR amplification primer is by SEQ ID NO:1~SEQ ID NO:10 compositions, the extension primer is by SEQ ID NO:11~SEQ ID NO:15 compositions.The detecting step of the kit includes sample to be tested carrying out PCR amplification, gained pcr amplification product carries out SAP processing, Single base extension is carried out to amplified production using PCR, pass through enrichment with magnetic bead extension products after extension, extension products are detected using Matrix-assisted laser desorption ionization technology, distinguish flight time length and analyze data.The kit of the present invention can detect multiple mutational sites simultaneously, have the characteristics that high-throughput, high-precision, low cost.
Description
Technical field
The present invention relates to detection in Gene Mutation technical fields, and in particular, to a kind of detection mankind's ctDNA gene mutations
Kit.
Background technology
Dissociative DNA (Cell Free nucleic acids, cfDNA) refers to that there are blood plasma or serum, cerebrospinal fluid etc.
The nucleic acid fragment of extracellular free state, about 160~180bp is product of the cell DNA under physiology or pathological conditions.Research
Show that cfDNA present in tumour patient peripheral blood is significantly higher than healthy population, and these cfDNA carry tumor patient
The variation information of gene（Mutation including oncogene etc.）, referred to as Circulating tumor DNA (circulating tumor DNA,
ctDNA）.CtDNA is mainly derived from the tumour cell of necrosis, circulating tumor cell, the tumour cell of apoptosis and from tumour
The outer secretory body of cell.It can be clinical judgment patient's prognosis by the detection of ctDNA, determine that therapeutic regimen provides reference.
Clinical discovery EGFR genetic mutation is with having correlation the effect of NSCLC targeted therapies, and in asian ancestry crowd,
EGFR genetic mutation accounts for 35% of non-small cell lung cancer or so.The patient that the overwhelming majority carries EGFR genetic mutation is significant in efficacy.Greatly
Quantity research data shows that EGFR genetic mutation is concentrated mainly on tyrosine kinase area (tyrosine kinase coding
Domain) on exons 1 8~21, the mutation of the L861Q on 21 exons of kinase domain is encoded and are resulted in EGFR
In amino acid replacement in 861 bit codons from leucine to glutamine.Account for about in the lung cancer that this mutation is mutated in EGFR
2%.To the detection of EGFR genetic mutation for instructing the medication of NSCLC patients clinicals that there is important reference value.
When research finds that KRAS genes are abnormal, lead to cell continued propagation, and prevent cell self-destruction, it is impossible to produce
Raw normal RAS albumen makes Cellular Signaling Transduction Mediated disorderly, uncontrolled cellular proliferation and canceration.KRAS- mutation betide a variety of cancers
Disease, such as lung cancer, colon cancer etc..90% mutation is happened at 12,13 bit codons, and KRAS-G12D mutation cause in KRAS 12
Amino acid replacement of the bit codon from glycine to aspartic acid, KRAS-G12S mutation cause in 12 bit codons of KRAS from sweet
To the amino acid replacement of serine, KRAS-G13D is mutated to be caused in 13 bit codons of KRAS from glycine to aspartic acid propylhomoserin
Amino acid replacement.These mutation can lead to the anomalous variation of the growth signals of p21-ras albumen.The gene is TKIs treatments
Target molecule, the patient for carrying KRAS gene mutation are insensitive to TKIs treatments.A large amount of clinical researches show targeted drug to not
Patient's effective percentage that KRAS gene mutation occurs can reach 60%, and to occurred the patient of KRAS gene mutation then entirely without
Effect.KRAS genetic tests be current doctor understand patient oncogene situation most directly, most efficient method.By detecting KRAS bases
Because either with or without mutation, anti-EGFR can be filtered out（EGF-R ELISA）Targeted drug treats effective PATIENTS WITH LARGE BOWEL,
The individualized treatment of tumour patient is realized, so as to extend patient survival.For the patient of no mutation, then it is unnecessary to reduce
Medical expense and toxic side effect.
Research shows that in a variety of human malignancies, such as lung cancer, malignant mela noma, colorectal cancer exist not
BRAF gene mutation in proportion.About 80~90% BRAF gene mutation is happened on 1799 nucleotide of exon15, and T dashes forward
Become A, its glutamic acid encoded is caused to be replaced (V600E) by valine.It is now recognized that V600E mutation can simulate T599 and
The Phosphorylation events in two sites of S602, so as to activate BRAF abnormal proteins.The generation of BRAF mutation status and kinds of tumors,
Development and clinical effectiveness are related.Clinical studies show, there may be primary resistance to EGFR targeted drugs by BRAF gene mutation patient
Medicine, therefore NCCN suggests, KRAS gene wild type patients are coped with when using targeted drug and further detect BRAF gene state.
BRAF gene mutation detects the specific aim that can improve clinical treatment, instructs the reasonable employment of targeted drug, avoids invalid or treatment
Patient's state of an illness caused by improper is delayed, and reduces Operative risk.
At present to the detection means of ctDNA including ddPCR, NGS and qPCR etc., but these types of technology is each have their own scarce
Point, as ddPCR is once only capable of one SNP site of detection, it is impossible to realize high throughput, NGS is faced with the problem of cost is excessively high.Matrix
Assisted laser desorption ionisation（matrix assisted laser desorption/ionization, MALDI）It is a kind of arteries and veins
Rush formula Soft ionization techniques.Sample through ionization, which is transferred to from ion source in mass analyzer, to be analyzed, and obtains molecular weight.Due to
The ion that MALDI ion sources generate often uses the flight time（time of flight, TOF）Mass analyzer is analyzed, so
MALDI often connects together use, referred to as Matrix-assisted laser desorption ionization with TOF（MALDI-TOF-MS）.
It has not yet to see and mass spectrometry method is applied in the detection of ctDNA gene mutations in the prior art.
Invention content
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, one group of detection mankind's ctDNA gene is provided and is dashed forward
The primer sets of change.
It is a further object to provide a kind of kits for detecting mankind's ctDNA gene mutations, which can
Multiple mutational sites are detected simultaneously in primary experiment, workload is significantly reduced, improves detection flux, and reduce detection
Expense has the characteristics that high-throughput, high-precision, low cost.
To achieve these goals, the present invention is achieved by following scheme：
The primer sets of one group of detection mankind's ctDNA gene mutation, are made of, multiplex PCR multiplexed PCR amplification primer and extension primer
The sequence of amplimer such as SEQ ID NO:Shown in 1~10, the sequence such as SEQ ID NO of extension primer:Shown in 11~15.
A kind of kit for detecting mankind's ctDNA gene mutations includes detection mankind's ctDNA gene mutations as described above
Primer sets, primer sets are made of multiplexed PCR amplification primer and extension primer, the sequence such as SEQ ID of multiplexed PCR amplification primer
NO:Shown in 1~10, the sequence such as SEQ ID NO of extension primer:Shown in 11~15.
The multiplex PCR that the present invention is previously mentioned refers to amplification reaction system of the mixing Multiple components in reacting hole, comes
Derived from the genomic DNA to sample extraction as template.PCR amplification primer and extension according to kit of the present invention are drawn
Object can simultaneously expand in primary experiment and extend multiple SNP sites, realize high-throughput.The kit is directed to 5 specificity
SNPs sites, used PCR amplification primer and Single base extension primer are respectively：
For the COSM6213 in EGFR gene（L861Q）Site, used PCR amplification primer are SEQ ID NO:1 and SEQ
ID NO:2, used Single base extension primer is SEQ ID NO:11；
For the COSM517 on KRAS genes（G12S）Site, used PCR amplification primer are SEQ ID NO:3 and SEQ
ID NO:4, used Single base extension primer is SEQ ID NO:12；
For the COSM521 on KRAS genes（G12D）Site, used PCR amplification primer are SEQ ID NO:5 and SEQ
ID NO:6, used Single base extension primer is SEQ ID NO:13；
For the COSM532 on KRAS genes（G13D）Site, used PCR amplification primer are SEQ ID NO:7 and SEQ
ID NO:8, used Single base extension primer is SEQ ID NO:14；
For the COSM476 in BRAF gene（V600E）Site, used PCR amplification primer are SEQ ID NO:9 and SEQ
ID NO:10, used Single base extension primer is SEQ ID NO:15.
The kit further includes high pressure sterilization purified water, multiplex PCR buffer solution, magnesium chloride buffer solution, dNTP mixtures,
Multiplex PCR enzyme, phosphatase buffer, phosphoric acid digestion enzyme extend buffer solution, catalytic reaction enzyme, and extension terminates mixed liquor, washing
Liquid competes liquid, Streptavidin MagneSphere.
The detection architecture of the kit is by pcr amplification reaction system, SAP reaction systems, extension system and enrichment
Reaction system forms.
Preferably, the pcr amplification reaction system into being grouped into：4.3 μ L of high pressure sterilization purified water, multiplex PCR delay
2 μ L of fliud flushing, 0.8 μ L, dNTP mixture of magnesium chloride buffer solution 0.1 μ L, 0.8 μ L of multiplex PCR enzyme, the mixing of multiplexed PCR amplification primer
Object 2 μ L, 10 μ L of DNA sample to be measured, totally 20 μ L；
The reaction condition of pcr amplification reaction system is as follows：
Preferably, the SAP reaction systems into being grouped into：20 μ L of pcr amplification reaction product, high pressure sterilization purified water 3.06
μ L, 0.34 μ L of phosphatase buffer, 0.6 μ L of phosphoric acid digestion enzyme；The reaction condition of SAP reaction systems is 37 DEG C of reactions 40
Minutes, 85 DEG C of reaction minutes, 4 DEG C of preservations.
Preferably, the extension system into being grouped into：24 μ L of SAP reaction products, high pressure sterilization purified water
1.42 μ L, extend 0.4 μ L of buffer solution, 0.08 μ L of catalytic reaction enzyme, and extension terminates mixed liquor 0.1 μ L, 2 μ of extension primer mixture
L；The reaction condition of extension system is as follows：
Preferably, the enrichment reaction system into being grouped into：28 μ L of extension product, 16 μ L of high pressure sterilization purified water,
33.5 μ L of cleaning solution compete 41 μ L of liquid, 4.25 μ L of Streptavidin MagneSphere；It is described enrichment reaction system reaction condition be：90
DEG C reaction 5minutes.
Preferably, detection method includes the following steps for the kit：
S1. sample to be tested is subjected to PCR amplification；
S2. gained pcr amplification product carries out SAP processing；
S3. Single base extension is carried out to amplified production using PCR；
S4. pass through enrichment with magnetic bead extension products after extending；
S5. extension products are detected using Matrix-assisted laser desorption ionization technology, when distinguishing flight
Between length and analyze data.
The present invention analyzes the gene mutation of ctDNA according to the length of extension products flight time in vacuum tubule, leads to
It crosses enrichment with magnetic bead and improves accuracy of detection.Extension products are carried out using Matrix-assisted laser desorption ionization technology
Detection, flight time of the detection extension products in vacuum tubule, the mutation analysis for ctDNA provides foundation.
Compared with prior art, the invention has the advantages that：
The kit of the present invention can be used for the mutation of external qualitative detection cancer patient ctDNA, including EGFR-L861Q,
The V600E mutation in 3 kinds of mutation and BRAF gene on the 2nd exon of KRAS genes.By applying time-of-flight mass spectrometry (TOFMS)
To clinical judgment patient's prognosis, there is good directive significance to the personalized medicine of lung cancer.
The kit of the present invention can detect multiple mutational sites simultaneously in primary experiment, significantly reduce workload,
Detection flux is improved, and reduces testing cost, there is high-throughput, high-precision, low cost.
Description of the drawings
Fig. 1 is the mass spectrum peak figure in application examples 1；Wherein, result figures of the Figure 1A for a wild type KRAS-G12D, Figure 1B
For the result figure of the one 5% horizontal KRAS-G12D of mutation, result figures of Fig. 1 C for the one 1% horizontal KRAS-G12D of mutation, Fig. 1 D
For the result figure of the one 0.1% horizontal KRAS-G12D of mutation, sample is cfDNA standard items.
Fig. 2 is the mass spectrum peak figure in application examples 2；Wherein, Fig. 2A is the result figure of the one 1% horizontal BRAF-V600E of mutation,
Fig. 2 B are the result figure of the one 1% horizontal KRAS-G13D of mutation, and Fig. 2 C are the result figure of the one 1% horizontal EGFR-L861Q of mutation,
Sample is plasmid.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
A kind of kit for detecting mankind's ctDNA gene mutations, constituent are：One group of detection mankind's ctDNA gene mutation
Primer sets, high pressure sterilization purified water, multiplex PCR buffer solution, magnesium chloride buffer solution, dNTP mixtures, multiplex PCR enzyme, phosphatase
Buffer solution, phosphoric acid digestion enzyme extend buffer solution, catalytic reaction enzyme, and extension terminates mixed liquor（T）, cleaning solution, competition liquid, strepto-
Avidin magnetic bead.The primer sets of mankind's ctDNA gene mutations are wherein detected, are made of multiplexed PCR amplification primer and extension primer,
The sequence of multiplexed PCR amplification primer such as SEQ ID NO:Shown in 1~10, the sequence such as SEQ ID NO of extension primer:11~15 institutes
Show.
The detection architecture of the kit is by pcr amplification reaction system, SAP reaction systems, extension system and enrichment
Reaction system forms；
The pcr amplification reaction system into being grouped into：4.3 μ L of high pressure sterilization purified water, 2 μ L of multiplex PCR buffer solution, chlorination
0.8 μ L, dNTP mixture of magnesium buffer solution 0.1 μ L, 0.8 μ L of multiplex PCR enzyme, multiplexed PCR amplification primer mixture 2 μ L, DNA to be measured
10 μ L of sample, totally 20 μ L；
The reaction condition of pcr amplification reaction system is as follows：
The SAP reaction systems into being grouped into：20 μ L of pcr amplification reaction product, 3.06 μ L of high pressure sterilization purified water, phosphoric acid
0.34 μ L of enzyme buffer liquid, 0.6 μ L of phosphoric acid digestion enzyme；
The reaction condition of SAP reaction systems is 37 DEG C of 40 minutes of reaction, and 85 DEG C of reaction minutes, 4 DEG C preserve.
The extension system into being grouped into：24 μ L of SAP reaction products, 1.42 μ L of high pressure sterilization purified water, prolong
0.4 μ L of buffer solution, 0.08 μ L of catalytic reaction enzyme are stretched, extension terminates 0.1 μ L of mixed liquor, 2 μ L of extension primer mixture；
The reaction condition of extension system is as follows：
The enrichment reaction system into being grouped into：28 μ L of extension product, 16 μ L of high pressure sterilization purified water, cleaning solution
33.5 μ L compete 41 μ L of liquid, 4.25 μ L of Streptavidin MagneSphere；Enrichment reaction system reaction condition be：90 DEG C of reactions
5minutes。
Detection method includes the following steps for the kit：
S1. sample to be tested is subjected to PCR amplification；
S2. gained pcr amplification product carries out SAP processing；
S3. Single base extension is carried out to amplified production using PCR；
S4. pass through enrichment with magnetic bead extension products after extending；
S5. extension products are detected using Matrix-assisted laser desorption ionization technology, when distinguishing flight
Between length and analyze data.
Application examples 1
The DNA sample of detection wild type KRAS-G12D, the 5% horizontal KRAS-G12D of mutation are removed using kit described in embodiment 1
DNA sample, the DNA sample of the 1% horizontal KRAS-G12D of mutation, the DNA sample of the 0.1% horizontal KRAS-G12D of mutation.
First, sample collection, transport and preservation：
1. sample collection：CfDNA standard items.
2. transport and preservation：Storage period is 12 months；Sample transport uses curling stone is on the rocks or bubble chamber is on the rocks to seal progress
Transport.
2nd, detecting step and interpretation of result：
The concentration of 1.cfDNA standard items is about 40ng/uL, and the cfDNA standard items of wild type, 5%, 1%, 0.1% mutation level are taken
1uL is diluted in 39uL high pressure sterilization purified waters, then takes 10uL i.e. in total about 10ng cfDNA for once testing.
2.PCR is expanded：With embodiment 1.
3.SAP processing：With embodiment 1.
4. Single base extension：With embodiment 1.
5. enrichment with magnetic bead：With embodiment 1.
Mass spectrograph and data analysis on 6：With embodiment 1.Whether SNP site is analyzed according to the signal value that mass spectrograph detects
There is mutation, as a result collection of illustrative plates is as shown in Figure 1A, 1B, 1C, 1D.
Application examples 2
It goes to detect EGFR-L861Q, BRAF-V600E, KRAS-G13D SNP sites exception using kit described in embodiment 1
DNA sample.
First, sample configuration：Match with EGFR-L861Q, BRAF-V600E, KRAS-G13D mutant plasmid and without mutant plasmid
It is set to more SNP sites mutation mixed liquor of 1% mutation level.
2nd, the reaction step of kit and reaction system are detected as described in embodiment 1.
Whether the signal value analysis SNP site the 3rd, detected according to mass spectrograph has mutation, as a result collection of illustrative plates such as Fig. 2A, Fig. 2 B
Shown in Fig. 2 C.
Sequence table
<110>Guangzhou Da Rui Biotechnology Ltd.
<120>A kind of kit for detecting mankind's ctDNA gene mutations
<160> 15
<170> SIPOSequenceListing 1.0
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<211> 30
<212> DNA
<213>People (hunman)
<400> 1
acgttggatg tttgcctcct tctgcatggt 30
<210> 2
<211> 30
<212> DNA
<213>People (hunman)
<400> 2
acgttggatg aaacaccgca gcatgtcaag 30
<210> 3
<211> 30
<212> DNA
<213>People (hunman)
<400> 3
acgttggatg ataaggcctg ctgaaaatga 30
<210> 4
<211> 30
<212> DNA
<213>People (hunman)
<400> 4
acgttggatg ctgaattagc tgtatcgtca 30
<210> 5
<211> 30
<212> DNA
<213>People (hunman)
<400> 5
acgttggatg ataaggcctg ctgaaaatga 30
<210> 6
<211> 30
<212> DNA
<213>People (hunman)
<400> 6
acgttggatg tctgaattag ctgtatcgtc 30
<210> 7
<211> 30
<212> DNA
<213>People (hunman)
<400> 7
acgttggatg ataaggcctg ctgaaaatga 30
<210> 8
<211> 30
<212> DNA
<213>People (hunman)
<400> 8
acgttggatg gattctgaat tagctgtatc 30
<210> 9
<211> 30
<212> DNA
<213>People (hunman)
<400> 9
acgttggatg ttcttcatga agacctcaca 30
<210> 10
<211> 30
<212> DNA
<213>People (hunman)
<400> 10
acgttggatg tccacaaaat ggatccagac 30
<210> 11
<211> 16
<212> DNA
<213>People (hunman)
<400> 11
ctcttccgca cccagc 16
<210> 12
<211> 16
<212> DNA
<213>People (hunman)
<400> 12
ctcttgccta cgccac 16
<210> 13
<211> 16
<212> DNA
<213>People (hunman)
<400> 13
actcttgcct acgcca 16
<210> 14
<211> 16
<212> DNA
<213>People (hunman)
<400> 14
ggcactcttg cctacg 16
<210> 15
<211> 16
<212> DNA
<213>People (hunman)
<400> 15
ctcttgccta cgccac 16
Claims (8)
1. the primer sets of one group of detection mankind's ctDNA gene mutation, which is characterized in that drawn by multiplexed PCR amplification primer and extension
Object forms, the sequence such as SEQ ID NO of multiplexed PCR amplification primer:Shown in 1~10, the sequence such as SEQ ID NO of extension primer:
Shown in 11~15.
2. a kind of kit for detecting mankind's ctDNA gene mutations, which is characterized in that the kit includes claim 1 institute
The primer sets of detection mankind's ctDNA gene mutations stated, primer sets are made of multiplexed PCR amplification primer and extension primer, multiple
The sequence of PCR amplification primer such as SEQ ID NO:Shown in 1~10, the sequence such as SEQ ID NO of extension primer:Shown in 11~15.
3. kit according to claim 2, which is characterized in that the kit further includes high pressure sterilization purified water, more
Weight PCR buffer solutions, magnesium chloride buffer solution, dNTP mixtures, multiplex PCR enzyme, phosphatase buffer, phosphoric acid digestion enzyme, extension are slow
Fliud flushing, catalytic reaction enzyme, extension terminate mixed liquor, and cleaning solution competes liquid, Streptavidin MagneSphere.
4. kit according to claim 2, which is characterized in that the detection architecture of the kit is by pcr amplification reaction
System, SAP reaction systems, extension system and enrichment reaction system composition.
5. kit according to claim 4, which is characterized in that the pcr amplification reaction system into being grouped into：It is high
Pressure sterilizing 4.3 μ L of purified water, 2 μ L of multiplex PCR buffer solution, 0.8 μ L, dNTP mixture of magnesium chloride buffer solution, 0.1 μ L, multiplex PCR
0.8 μ L of enzyme, multiplexed PCR amplification primer mixture 2 μ L, 10 μ L of DNA sample to be measured, totally 20 μ L；
The reaction condition of pcr amplification reaction system is as follows：95℃ 2minutes；95 DEG C of 30seconds, 56 DEG C
30seconds, 72 DEG C of 1minute, 45 cycles；72℃ 5minutes；4 DEG C of preservations.
6. kit according to claim 4, which is characterized in that the SAP reaction systems into being grouped into：PCR expands
Increase 20 μ L of reaction product, 3.06 μ L of high pressure sterilization purified water, 0.34 μ L of phosphatase buffer, 0.6 μ L of phosphoric acid digestion enzyme；
The reaction condition of SAP reaction systems is 37 DEG C of 40 minutes of reaction, and 85 DEG C of reaction minutes, 4 DEG C preserve.
7. kit according to claim 4, which is characterized in that the extension system into being grouped into：SAP is anti-
24 μ L of product, 1.42 μ L of high pressure sterilization purified water are answered, extends 0.4 μ L of buffer solution, 0.08 μ L of catalytic reaction enzyme, extension terminates mixing
0.1 μ L of liquid, 2 μ L of extension primer mixture；
The reaction condition of extension system is as follows：95℃ 30seconds；95 DEG C of 5seconds, 52 DEG C of 5seconds, 80
DEG C 5seconds, 72 DEG C of 3minutes, 5 cycles, 40 cycles；4 DEG C of preservations.
8. kit according to claim 4, which is characterized in that the enrichment reaction system into being grouped into：Extension
28 μ L of reaction product, 16 μ L of high pressure sterilization purified water, 33.5 μ L of cleaning solution compete 41 μ L of liquid, 4.25 μ L of Streptavidin MagneSphere；
It is described enrichment reaction system reaction condition be：90 DEG C of reaction 5minutes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110144399A (en) * | 2019-04-09 | 2019-08-20 | 中源协和(天津)医学检验所有限公司 | Detect primer sets, kit and the application method of lung cancer related gene mutation in mankind's Circulating tumor DNA |
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