CN108315324A - The detection in BRAF gene mutation site in urine ctDNA - Google Patents
The detection in BRAF gene mutation site in urine ctDNA Download PDFInfo
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Abstract
The present invention relates to the detections in BRAF gene mutation site in urine ctDNA, specially from detaching the method for ctDNA in urine specimen, the method for sequencing library, sequencing library are built based on urine specimen, carry out method for nucleic acid sequencing, the equipment for detaching ctDNA from urine specimen and the system for determining individual drugs sensibility based on urine specimen.The method of separation ctDNA includes from urine specimen：EDTA is added into urine specimen, to obtain the first mixed liquor；Mixed liquor is subjected to centrifugal treating, collects supernatant；Supernatant is subjected to concentration, to obtain concentrate；Concentrate and anion exchange resin are subjected to mixed processing, to obtain the second mixed liquor；Second mixed liquor is subjected to chromatographic isolation processing, to obtain eluent；And eluent is subjected to purification process, to obtain ctDNA.The present invention can from a large amount of urine specimens quickly, extract ctDNA in large quantities, and nucleic acid sequencing can be carried out to it, individual drugs sensibility can also be determined, to improve the application prospect of urine ctDNA.
Description
Technical field
The present invention relates to biological fields.In particular it relates in urine ctDNA BRAF gene mutation site inspection
It surveys.
Background technology
Circulating tumor DNA (circulating tumor DNA, ctDNA) is to be discharged into the circulatory system such as by tumour cell
Genome small fragment in blood derives from all tumor locus of body, has that content is extremely low, inter-individual difference is big, partly declines
Phase is short, homogeneity, carries the features such as tumour-specific science of heredity/epigenetics variation.These are from Oncogenome
DNA fragmentation can carry certain feature, including mutation, lack, and be inserted into, and reset, and copy number exception and methylate.
However, still having for the acquisition pattern of ctDNA and its application at present to be developed.
Invention content
The present invention is directed to solve at least one the technical problems existing in the prior art at least to a certain extent.For this purpose,
The present invention propose it is a kind of from urine specimen detach ctDNA method, based on urine specimen build sequencing library method, survey
Preface library is carried out method for nucleic acid sequencing, the equipment for detaching ctDNA from urine specimen based on urine specimen and determined a
The system of body drug susceptibility.The present invention can from a large amount of urine specimens quickly, extract ctDNA in large quantities, and can be right
It carries out nucleic acid sequencing, individual drugs sensibility can also be determined, to improve the application prospect of urine ctDNA.
For this purpose, in one aspect of the invention, the present invention proposes a kind of method detaching ctDNA from urine specimen.
According to an embodiment of the invention, the method includes：EDTA is added into the urine specimen, to obtain the first mixed liquor；
First mixed liquor is subjected to centrifugal treating, collects supernatant；The supernatant is subjected to concentration, to be concentrated
Liquid；The concentrate and anion exchange resin are subjected to mixed processing, to obtain the second mixed liquor；Described second is mixed
Liquid carries out chromatographic isolation processing, to obtain eluent；And the eluent is subjected to purification process, it is described to obtain
ctDNA.Inventor has found, using ion-exchange chromatography method can quickly, in large quantities in isolated urine specimen
ctDNA。
According to an embodiment of the invention, the above-mentioned method that ctDNA is detached from urine specimen can also have following additional
Technical characteristic：
According to an embodiment of the invention, the method includes：It is added into 110 milliliters of urine specimens final concentration of
The EDTA of 10mmoL/L, to obtain the first mixed liquor；By first mixed liquor in 18~23 DEG C of temperature, turn of 1800g
The lower centrifugation of speed 10 minutes, collects supernatant；By the supernatant concentration to 4 milliliters, to obtain concentrate；By the concentrate
Mixed processing is carried out with 750 microlitres of Q Sepharose anion exchanger resins, to obtain the second mixed liquor；Described second is mixed
Liquid carries out chromatographic column elution processing, to obtain eluent；And the eluent is subjected to purification process, it is described to obtain
ctDNA。
Inventor has found, centrifugal treating condition, concentration volume and its can influence to carry with the amount ratio of anion exchange resin
Take effect.In turn, inventor has found by many experiments, and mixed liquor is centrifuged under 18~23 DEG C of temperature, the rotating speed of 1800g
10 minutes, it can effectively take out cell fragment, impurity.Then, due in urine specimen ctDNA contents it is relatively low, in turn
By will the supernatant concentration containing ctDNA to 4 milliliters, ctDNA contents are higher in obtained concentrate, convenient for follow-up right
The utilization of ctDNA.Further, inventor has found by many experiments, and ion is carried out using Q Sepharose anion exchanger resins
Displacement chromatography can effectively adsorb ctDNA, to improve the recovery rate of ctDNA.In addition, based on 4 milliliters of concentrate, it is cloudy
The dosage of ion exchange resin is 750 microlitres, under conditions of the two amount ratio, ctDNA can be fully adsorbed, if anion
The dosage of exchanger resin is very few, have a small amount of ctDNA it is not to be adsorbed, collect, if the dosage of anion exchange resin is excessive, improve
Separation costs.In addition, the addition of EDTA can inhibit the activity of nuclease, prevent it from ctDNA degrading.However, other are centrifuged
Treatment conditions, concentration volume and its ineffective with the amount ratio of resin.It is according to the ... of the embodiment of the present invention from urine sample as a result,
In this method of separation ctDNA further quickly, the ctDNA in isolated urine specimen in large quantities.
In another aspect of this invention, the present invention proposes a kind of method building sequencing library based on urine specimen.Root
According to the embodiment of the present invention, the method includes：Based on urine specimen, the side of ctDNA is detached from urine specimen according to front
Method detaches ctDNA；The ctDNA is expanded, to obtain amplified production；And it is added in the end of the amplified production
Joint sequence, to obtain connection product, the connection product constitutes the sequencing library.Inventor find, using front from
The method that ctDNA is detached in urine specimen detaches ctDNA, using ctDNA as template, sequencing library is built, it is possible thereby to realize
Analysis to ctDNA.
According to an embodiment of the invention, institute is carried out using the primer and probe in the mutational sites ctDNA described in specific recognition
Amplification is stated, the primer and probe is respectively provided with such as SEQ ID NO：Nucleotide sequence shown in 1~4；Or the primer and
Probe is respectively provided with such as SEQ ID NO：Nucleotide sequence shown in 5~8.
BRAF gene is located at human chromosome 7q34, is about 190kb, is that Ras-Raf-MEK-ERK signal transduction pathways are important
Transduced element, mainly played a role by there is the serine threonine protein kinase in silk-fibroin kinase pathway, the enzyme will
The receptor and RAS albumen of cell surface are connected by MEK and ERK with the transcription factor in core, are started a variety of factors and are participated in adjusting
Control intracellular various biological event, such as cell growth, differentiation and apoptosis.BRAF is in the high, medium and low differentiation of colorectal cancer
The positive rate difference of expression is apparent, and tumor differentiation degree is lower, and BRAF positive expression rates are higher, can be colorectal cancer diagnosis,
Treatment provides new foundation.
Inventor obtains above-mentioned primer sequence by many experiments, and 2 pairs of primers and probe are directed to BRAF gene (tool respectively
Body sequence is referring to https://www.ncbi.nlm.nih.gov/nuccore/NM_004333.4) in 2 mutational sites
Target area design, it can be specifically binding to mutational site, to be expanded to it, convenient for subsequent detection, analysis
It needs.
It should be noted that any pair of primer and probe or multipair primer and probe can be selected according to actual needs
It is expanded, such as the research in single mutation site, can only expand the target area in required mutational site；For multimutation
The research in site can select multipair primer and probe, and the amplification of target area is realized in one or more amplification system.
The nucleotide sequence of 1 primer of table and probe
According to an embodiment of the invention, the amplification is carried out using digital pcr, based on 50 microlitres of PCR reaction systems, institute
Stating PCR reaction systems includes：1 × archaeal dna polymerase of 25 μ L；1 × oil droplet stabilizer of 2 μ L；12.5 μM of the forward direction of 2 μ L is drawn
Object；12.5 μM of the reverse primer of 2 μ L；5 μM of the positive probe of 2 μ L；5 μM of the reversed probe of 2 μ L；1~15 μ L's is described
Urine ctDNA, the urine ctDNA are diluted with the carrier RNA of 5ng/ μ L in advance；And remaining distilled water.
A kind of nucleic acid molecules absolute quantitation technology of digital pcr.Compared to qPCR, digital pcr can directly count DNA molecular
Number, be the absolute quantitation to initial sample.Inventor utilizes RainDrop using ctDNA as template, by PCR reaction systems
Individual molecule is dispersed in Water-In-Oil drop by Source (RainDance Technologies) system, the oil droplet warp prepared
First step PCR amplification captures target area, destroys oil droplet and discharges amplicon, then is suitable for by second step PCR additions
The joint sequence of illumina microarray datasets, paramagnetic particle method purify DNA.Inventor obtains preferable PCR by many experiments and reacts
System, thereby, it is possible to quickly and accurately expand capture target area.Wherein, since ctDNA contents are less, it is easily hung at reaction
On tube wall, nucleic acid concentration that carry RNA can be in increase system so that be largely in the wall built-up liquid of same volume
Carry RNA are conducive to the enrichment reaction of ctDNA, and carryRNA will not react next stage and have an impact.
It will be appreciated to those of skill in the art that above for described by the method for detaching ctDNA from urine specimen
Feature and advantage, be equally applicable to this based on urine specimen build sequencing library method, details are not described herein.
In still another aspect of the invention, the present invention proposes a kind of sequencing library.According to an embodiment of the invention, the survey
Preface library is that the method for building sequencing library based on urine specimen by front obtains.
It will be appreciated to those of skill in the art that being retouched above for the method for building sequencing library based on urine specimen
The feature and advantage stated are equally applicable to the sequencing library, and details are not described herein.
In still another aspect of the invention, the present invention proposes a kind of based on urine specimen progress method for nucleic acid sequencing.It is described
Method includes：Based on urine specimen, sequencing library is built according to the method that front builds sequencing library based on urine specimen；And
Nucleic acid sequencing is carried out to the sequencing library.It is according to the ... of the embodiment of the present invention as a result, that nucleic acid sequencing side is carried out based on urine specimen
Method can accurately and rapidly realize nucleic acid sequencing.
It will be appreciated to those of skill in the art that being retouched above for the method for building sequencing library based on urine specimen
The feature and advantage stated are equally applicable to this and are based on urine specimen progress method for nucleic acid sequencing, and details are not described herein.
In still another aspect of the invention, the present invention proposes a kind of equipment for detaching ctDNA from urine specimen.Root
According to the embodiment of the present invention, the equipment includes：Adding device, suitable for EDTA is added into the urine specimen, to obtain
First mixed liquor；Centrifugal unit, the centrifugal unit are connected with the adding device, suitable for by first mixed liquor carry out from
Heart processing, collects supernatant；Upgrading unit, the upgrading unit are connected with the centrifugal unit, be suitable for by the supernatant into
Row concentration, to obtain concentrate；Mixed cell, the mixed cell are connected with the upgrading unit, and being suitable for will be described
Concentrate carries out mixed processing with anion exchange resin, to obtain the second mixed liquor；Chromatographic isolation unit, the chromatography point
It is connected with the mixed cell from unit, is suitable for second mixed liquor carrying out chromatographic isolation processing, to obtain eluent；
And purification unit, the purification unit are connected with the chromatographic isolation unit, are suitable for the eluent carrying out purification process,
To obtain the ctDNA.The equipment according to the ... of the embodiment of the present invention for detaching ctDNA from urine specimen can as a result,
Quickly, the ctDNA in isolated urine specimen in large quantities.
According to an embodiment of the invention, the equipment includes：Adding device is suitable for into 110 milliliters of urine specimens
The EDTA of final concentration of 10mmoL/L is added, to obtain the first mixed liquor；Centrifugal unit, the centrifugal unit and the addition
Unit is connected, and is suitable under 18~23 DEG C of temperature, the rotating speed of 1800g centrifuging first mixed liquor 10 minutes, in collection
Clear liquid；Upgrading unit, the upgrading unit are connected with the centrifugal unit, are suitable for the supernatant concentration to 4 milliliters, so as to
Obtain concentrate；Mixed cell, the mixed cell are connected with the upgrading unit, are suitable for the concentrate and 750 microlitres of Q
Sepharose anion exchanger resin carries out mixed processing, to obtain the second mixed liquor；Chromatographic isolation unit, the chromatographic isolation
Unit is connected with the mixed cell, is suitable for second mixed liquor carrying out chromatographic column elution processing, to obtain eluent；
And purification unit, the purification unit are connected with the chromatographic isolation unit, are suitable for the eluent carrying out purification process,
To obtain the ctDNA.The equipment according to the ... of the embodiment of the present invention for detaching ctDNA from urine specimen can as a result,
Further quickly, the ctDNA in isolated urine specimen in large quantities.
In still another aspect of the invention, the present invention proposes a kind of system of determining individual drugs sensibility.According to this hair
Bright embodiment, the system comprises：Nucleic acid separation equipment, the nucleic acid separation equipment are to be described previously for from urine sample
The equipment that ctDNA is detached in this；Library construction equipment, the library construction equipment are connected with the nucleic acid separation equipment, are used for
Sequencing library is built based on the ctDNA obtained in the nucleic acid separation equipment；Sequencing equipment, the sequencing equipment and the text
Library builds equipment and is connected, and the sequencing equipment is for being sequenced the sequencing library, to obtain sequencing result；And point
Desorption device, the analytical equipment are connected with the sequencing equipment, and the analytical equipment is based on described in sequencing result determination
Whether scheduled mutational site is carried in ctDNA.
Inventor has found, can accurately and rapidly determine scheduled mutation position whether is carried in ctDNA using the system
Point, the application prospect with larger scientific research or clinical diagnosis, treatment.Such as it can be using the mutational site as targeting medication
Site, by determining in ctDNA whether carry scheduled mutational site, in order to direction of medication usage.Specifically, if being taken in ctDNA
It is the instruction that individual drugs have the mutational site sensibility, which can be regarded as target with scheduled mutational site
To medication site；It is that individual drugs do not have sensibility to the mutational site if not carrying scheduled mutational site in ctDNA
Instruction.It can quickly and accurately be determined using the system of determining individual drugs sensibility according to the ... of the embodiment of the present invention as a result,
Whether scheduled mutational site is carried in ctDNA, so that it is determined that individual drugs sensibility.
In addition, why library construction equipment selects to build library with the ctDNA in urine specimen, mainly have following former
Cause：
Prior art generally use venous blood collection, is detected for extracting sample of nucleic acid.But venous blood collection can be brought
Clinic and transport on many troubles, such as due to vein tube wall is fragile, in narrow or blood sampling caused by being difficult to position
Inconvenience, and at present blood sampling need specific section office and clinical staff cooperation under could carry out, do not accomplish truly just
It is prompt noninvasive.In addition, also have the limitation of itself as clinical diagnosis auxiliary characteristics using ctDNA detections, its usual height fragmentation,
Proportion and its pettiness in the free nucleic acid of entire blood circulation system, it is therefore desirable to largely acquire blood sample, ability
The fully variation situation of reflection Tumour DNA.However advanced tumor patient health status is poor, a large amount of blood samplings can still make patient body
At certain wound, sample is very few to cause DNA content deficiency that can not be used for Molecular Detection, and for the tumour of late period transfer
Patient, the characteristics of due to Tumor Heterogeneity, the very few good overall view that can cause analysis result that can not react patient of DNA quantity.
In turn, inventor has found that, using urine specimen as the sources ctDNA, the collection of urine specimen is more convenient, and collects
Amount is big, is convenient for subsequent detection.
According to an embodiment of the invention, the individual drugs for treat melanoma, colorectal cancer, thyroid cancer or
Lung cancer.Inventor's discovery, predefines in ctDNA whether carry scheduled mutational site, can if carrying scheduled mutational site
To apply to it for treating melanoma, colorectal cancer, thyroid cancer or the drug of lung cancer, which can act on this
Therapeutic purposes are realized in mutational site.
It will be appreciated to those of skill in the art that above for the equipment institute for detaching ctDNA from urine specimen
The feature and advantage of description, the system for being equally applicable to the determination individual drugs sensibility, details are not described herein.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obviously, or practice through the invention is recognized.
Specific implementation mode
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it is carried out according to technology or condition described in document in the art or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
Embodiment 1
In this embodiment, determine in urine specimen whether carry scheduled mutational site in ctDNA in following manner：
1, the separation of urine ctDNA
To patients with lung cancer medication (Gefitinib), to urine specimen continuous sampling, before medication and medication after every 21 days into
Row sampling, acquires patient's empty stomach urina sanguinis.110ml urines are collected with 120ml collection cups, EDTA, which is added, keeps its final concentration of
10mmol/L, for inhibiting the activity of nuclease.Mixed liquor is centrifuged at a temperature of the rotating speed of 1800g and 18 DEG C~23 DEG C
10min collects supernatant, and supernatant is concentrated into 4mL through super filter tube, by the Q Sepharose anion of sample and 750 μ l after concentration
Exchanger resin mixes, and is placed on gyroscope and is incubated at room temperature, and the precipitation of collection is utilized Micro Bio-SpinTM(BioRad) chromatography
It is eluted after column cleaning, the DNA after elution is further purified with QiaQuick columns (Qiagen).
2, sequencing library is built
It is crushed instrument with Covaris and urine ctDNA is broken into the segment that length is 200bp sizes at random, respectively as mould
Plate prepares first step PCR system, and PCR reaction systems are utilized RainDrop Source (RainDance Technologies)
Individual molecule is dispersed in Water-In-Oil drop by system, and the oil droplet prepared captures target area through first step PCR amplification, is destroyed
Oil droplet discharges amplicon, then the joint sequence by second step PCR additions suitable for illumina microarray datasets, paramagnetic particle method purifying
DNA detects clip size to complete DNA library 2100 detectors of Agilent, accurately fixed using library quantification kit
DNA concentration is measured, the library of quality inspection qualification carries out subsequent experimental.
Wherein, PCR reaction systems are as follows：
1 × archaeal dna polymerase of 25 μ L；
1 × oil droplet stabilizer of 2 μ L；
12.5 μM of the forward primer of 2 μ L；
12.5 μM of the reverse primer of 2 μ L；
5 μM of the positive probe of 2 μ L；
5 μM of the reversed probe of 2 μ L；
The urine ctDNA of 10 μ L, urine ctDNA are diluted with the carrier RNA of 5ng/ μ L in advance；And it is surplus
Remaining distilled water.
Specific primer and probe sequence are as shown in the table.
3, high-flux sequence
For the extremely low Circulating tumor DNA segment of content in dissociative DNA, target be sequenced depth be 20000 ×, according to capture
Section size calculates library input quantity.We are concentrated mainly between 160-200bp target acquistion region segments length, selection
The HiSeq X Ten sequenators that PE100 sequencing strategies lead to illumina companies carry out high-flux sequence, and lower machine data Q30 exists
The subsequently raw letter analysis of 85% or more progress.
4, bioinformatic analysis
The lower machine data of acquisition are split by index, obtain primitive sequencer sequence (Raw Reads).To Raw
The results such as data volume, base contents, the base quality of Reads are counted, and are filtered underproof data, are obtained Clean
Reads.In the case where having reference sequences or reference gene group (hg19), Clean Data are compared in the way of being segmented and comparing
Onto reference gene group.Revised comparison result will be used as input file to carry out abrupt climatic change, annotation and statistics.In body cell
In the detection process of SNV, Indel, normal sample (normal) and tumor sample (tumor) are analyzed in pairs, with detection
Go out in body cell and relevant mutation occurs with tumour；In mutation annotates result, SIFT scores and PolyPhen2 are chosen
HDIV scores carry out conservative prediction and pathogenic analysis to obtained nonsynonymous mutation.
Wherein, ctDNA copy numbers are more than 6, are the instructions that patient has mutational site, and ctDNA copy numbers are less than or equal to 6,
It is the instruction that patient does not have mutational site.
Embodiment 2
In this embodiment, verification is using urine ctDNA as the accuracy of detection object.
Specifically, three control groups are respectively set：
Control group 1：To patients with lung cancer medication (Gefitinib), tissue biopsy sample is collected within every 21 days before medication and after medication
This, carries out paraffin embedding, by estimating Tumor Cell Content after haematoxylin eosin stains, including tumor tissues by the sample of collection
Sample can be used for subsequent analysis, use commercial kit QIAamp DNA FFPE Tissue Kit (Qiagen) extract cancer
Tissue DNA.
Control group 2：It to patients with lung cancer medication (Gefitinib), before medication and is sampled within every 21 days, is acquired after medication
Empty stomach vein peripheral blood on the day of patient.Blood sample is acquired with the vacuum blood collection tube of streck companies EDTA anti-freezings, after blood sampling
Separated plasma (1800g, 10min, 18 DEG C -23 DEG C) in 30min, by less than -70 DEG C preservations of the blood plasma of separation.The haemocyte of separation
Genomic DNA is extracted with commercial kit AllPrep DNA/RNA Mini Kit (Qiagen).
Control group 3：By 2 obtained blood plasma of control group using QIAamp free nucleic acids extracts kit (Qiagen) according to
Operating instruction extracts the ctDNA in 1.5-4.0ml blood plasma and is used for subsequent analysis.
Respectively using 1~3 obtained cancerous tissue DNA of control group, haemocyte DNA and blood plasma ctDNA as template, successively
Carry out the operation of step 2~4.
The result shows that urine ctDNA is consistent with the abrupt information of cancerous tissue DNA, haemocyte DNA and blood plasma ctDNA, say
Bright urine ctDNA can be accurately determined with the presence or absence of mutational site, be of great significance to direction of medication usage.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
SEQUENCE LISTING
<110>Suo Zhen（Beijing）Medical science and technology Co., Ltd
<120>The detection in BRAF gene mutation site in urine ctDNA
<130> PIDC3171001
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer
<400> 1
tccacaaaat ggatccagac 20
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer
<400> 2
tcataatgct tgctctgata gg 22
<210> 3
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe
<400> 3
cgctcttccg atctctgtcc acaaaatgga tccagac 37
<210> 4
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe
<400> 4
tgctcttccg atctgactca taatgcttgc tctgatagg 39
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer
<400> 5
catgccactt tcccttgtag 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Primer
<400> 6
tgtttggctt gacttgactt 20
<210> 7
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe
<400> 7
cgctcttccg atctctgcat gccactttcc cttgtag 37
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223>Probe
<400> 8
tgctcttccg atctgactgt ttggcttgac ttgactt 37
Claims (10)
1. a kind of method detaching ctDNA from urine specimen, which is characterized in that including：
EDTA is added into the urine specimen, to obtain the first mixed liquor；
First mixed liquor is subjected to centrifugal treating, collects supernatant；
The supernatant is subjected to concentration, to obtain concentrate；
The concentrate and anion exchange resin are subjected to mixed processing, to obtain the second mixed liquor；
Second mixed liquor is subjected to chromatographic isolation processing, to obtain eluent；And
The eluent is subjected to purification process, to obtain the ctDNA.
2. according to the method described in claim 1, it is characterised in that it includes：
The EDTA of final concentration of 10mmoL/L is added into 110 milliliters of urine specimens, to obtain the first mixed liquor；
First mixed liquor is centrifuged 10 minutes under 18~23 DEG C of temperature, the rotating speed of 1800g, collects supernatant；
By the supernatant concentration to 4 milliliters, to obtain concentrate；
The concentrate and 750 microlitres of Q Sepharose anion exchanger resins are subjected to mixed processing, to obtain the second mixing
Liquid；
Second mixed liquor is subjected to chromatographic column elution processing, to obtain eluent；And
The eluent is subjected to purification process, to obtain the ctDNA.
3. a kind of method that sequencing library is built based on urine specimen, including：
Based on urine specimen, ctDNA is detached according to method as claimed in claim 1 or 2；
The ctDNA is expanded, to obtain amplified production；And
Joint sequence is added in the end of the amplified production, to obtain connection product, the connection product constitutes the survey
Preface library.
4. according to the method described in claim 3, it is characterized in that, drawing using the mutational sites ctDNA described in specific recognition
Object and probe carry out the amplification,
The primer and probe is respectively provided with such as SEQ ID NO：Nucleotide sequence shown in 1~4；Or
The primer and probe is respectively provided with such as SEQ ID NO：Nucleotide sequence shown in 5~8,
Optionally, the amplification is carried out using digital pcr,
Based on 50 microlitres of PCR reaction systems, the PCR reaction systems include：
1 × archaeal dna polymerase of 25 μ L；
1 × oil droplet stabilizer of 2 μ L；
12.5 μM of the forward primer of 2 μ L；
12.5 μM of the reverse primer of 2 μ L；
5 μM of the positive probe of 2 μ L；
5 μM of the reversed probe of 2 μ L；
The urine ctDNA of 1~15 μ L, the urine ctDNA are diluted with the carrier RNA of 5ng/ μ L in advance；With
And
Remaining distilled water.
5. a kind of sequencing library, which is characterized in that the sequencing library is obtained by the method described in claim 3 or 4.
6. one kind carrying out method for nucleic acid sequencing based on urine specimen, which is characterized in that including：
Based on urine specimen, sequencing library is built according to the method described in claim 3 or 4；And
Nucleic acid sequencing is carried out to the sequencing library.
7. a kind of equipment for detaching ctDNA from urine specimen, which is characterized in that including：
Adding device, suitable for EDTA is added into the urine specimen, to obtain the first mixed liquor；
Centrifugal unit, the centrifugal unit are connected with the adding device, are suitable for first mixed liquor carrying out centrifugal treating,
Collect supernatant；
Upgrading unit, the upgrading unit are connected with the centrifugal unit, are suitable for the supernatant carrying out concentration, so as to
Obtain concentrate；
Mixed cell, the mixed cell are connected with the upgrading unit, are suitable for the concentrate and anion exchange resin
Mixed processing is carried out, to obtain the second mixed liquor；
Chromatographic isolation unit, the chromatographic isolation unit are connected with the mixed cell, are suitable for carrying out second mixed liquor
Chromatographic isolation processing, to obtain eluent；And
Purification unit, the purification unit are connected with the chromatographic isolation unit, are suitable for the eluent carrying out purification process,
To obtain the ctDNA.
8. equipment according to claim 7, which is characterized in that including：
Adding device, suitable for the EDTA of final concentration of 10mmoL/L is added into 110 milliliters of urine specimens, to obtain
One mixed liquor；
Centrifugal unit, the centrifugal unit are connected with the adding device, are suitable for first mixed liquor at 18~23 DEG C
Temperature, 1800g rotating speed under centrifuge 10 minutes, collect supernatant；
Upgrading unit, the upgrading unit are connected with the centrifugal unit, are suitable for the supernatant concentration to 4 milliliters, so as to
Obtain concentrate；
Mixed cell, the mixed cell are connected with the upgrading unit, are suitable for the concentrate and 750 microlitres of Q agaroses
Anion exchange resin carries out mixed processing, to obtain the second mixed liquor；
Chromatographic isolation unit, the chromatographic isolation unit are connected with the mixed cell, are suitable for carrying out second mixed liquor
Chromatographic column elution is handled, to obtain eluent；And
Purification unit, the purification unit are connected with the chromatographic isolation unit, are suitable for the eluent carrying out purification process,
To obtain the ctDNA.
9. a kind of system of determining individual drugs sensibility, which is characterized in that including：
Nucleic acid separation equipment, the nucleic acid separation equipment are described in claim 7 or 8 for being detached from urine specimen
The equipment of ctDNA；
Library construction equipment, the library construction equipment are connected with the nucleic acid separation equipment, for being detached based on the nucleic acid
CtDNA obtained in equipment builds sequencing library；
Sequencing equipment, the sequencing equipment are connected with the library construction equipment, and the sequencing equipment is used for sequencing text
Library is sequenced, to obtain sequencing result；And
Analytical equipment, the analytical equipment are connected with the sequencing equipment, and the analytical equipment is determined based on the sequencing result
Whether scheduled mutational site is carried in the ctDNA.
10. system according to claim 9, which is characterized in that the individual drugs are for treating melanoma, Colon and rectum
Cancer, thyroid cancer or lung cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710211468.9A CN108315324A (en) | 2017-03-31 | 2017-03-31 | The detection in BRAF gene mutation site in urine ctDNA |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710211468.9A CN108315324A (en) | 2017-03-31 | 2017-03-31 | The detection in BRAF gene mutation site in urine ctDNA |
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| CN201710211468.9A Pending CN108315324A (en) | 2017-03-31 | 2017-03-31 | The detection in BRAF gene mutation site in urine ctDNA |
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Cited By (3)
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| CN108315323A (en) * | 2017-03-31 | 2018-07-24 | 索真（北京）医学科技有限公司 | The detection of PIK3CA gene mutation sites in urine ctDNA |
| CN108315322A (en) * | 2017-03-31 | 2018-07-24 | 索真（北京）医学科技有限公司 | The detection in EGFR genetic mutation site in urine ctDNA |
| WO2021209549A1 (en) * | 2020-04-17 | 2021-10-21 | F. Hoffmann-La Roche Ag | Devices and methods for urine sample analysis |
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| CN108315323A (en) * | 2017-03-31 | 2018-07-24 | 索真（北京）医学科技有限公司 | The detection of PIK3CA gene mutation sites in urine ctDNA |
| CN108315322A (en) * | 2017-03-31 | 2018-07-24 | 索真（北京）医学科技有限公司 | The detection in EGFR genetic mutation site in urine ctDNA |
| WO2021209549A1 (en) * | 2020-04-17 | 2021-10-21 | F. Hoffmann-La Roche Ag | Devices and methods for urine sample analysis |
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Application publication date: 20180724 |