CN109385469A - A kind of high sensitivity double-strand Circulating tumor DNA detection method and kit - Google Patents
A kind of high sensitivity double-strand Circulating tumor DNA detection method and kit Download PDFInfo
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Abstract
The present invention relates to biotechnology and DNA detection technique field more particularly to a kind of highly sensitive double-strand Circulating tumor DNA detection method and kits.The method of the present invention includes carrying out the reparation of the end DNA and tailing reaction to plasma DNA, being attached with the adapter containing UMI and react, AMPure XP magnetic beads for purifying, qPCR reaction, carry out using qPCR result library high-fidelity PCR amplification, to library progress oncogene targeted capture.It can get higher library yield using the method for the present invention, the defect for overcoming ctDNA quality difference to cause Library Quality unstable fundamentally improves the sensitivity and accuracy of detection, reduces detection false positive rate.Therefore, the method for the present invention can be used for preparing kinds of tumors tracer or tumor screening kit, to meet all kinds of clinical and Research Requirements in field of biomedicine.
Description
Technical field
The present invention relates to biotechnology and DNA detection technique field more particularly to a kind of highly sensitive double-strand circulating tumors
DNA detection method and kit in particular to a kind of building dissociative DNA two generations sequencing library and carry out oncogene targeting
Detection kit made of the method and utilization the method for capture, moreover, it relates to above-mentioned detection method and kit
Application in biomedicine.
Background technique
Plasma DNA (cell free DNA, cfDNA), abbreviation circle nucleic acid (Circulating nucleic
Acid), refer to the body endogenous dna that extracellular Partial digestion is free in circulating.CfDNA earliest by Mandel and
Metais has found in nineteen forty-seven, due to lacking the experimental method of highly sensitive and high specific, cause its with it is disease associated
Research is made slow progress within the longer term, until the detection skill that dissociative DNA isolation technics and special fluorescent dye are combined with PCR
The appearance of art, above situation are just taken on a new look.With the fact that may include the identical gene mutation of DNA of tumor cell in cfDNA quilt
After it was found that, field of biomedicine increasingly increases its research interest.
Circulating tumor DNA (circulating tumor DNA, ctDNA), refers to and discharges into blood by tumour cell
DNA in the circulatory system is a kind of extracellular dna of cell-free state, is present in the body fluid such as blood, synovial fluid and cerebrospinal fluid,
It is mainly made of single-stranded or double-stranded DNA and the single-stranded mixture with double-stranded DNA, with DNA protein complex or dissociates
Two kinds of forms of DNA exist.CtDNA as it is a kind of have wide application prospect, hypersensitivity, high specific knubble biological label
Object, tracer and screening suitable for kinds of tumors.In addition, since ctDNA is mutated from tumor cell gene group, and its half-life period
It is shorter, when being used for neoplasm tracing and screening, compared with protide tumor marker, the vacation sun of ctDNA tumor marker analyte detection
Property rate is lower, and accuracy rate is higher.
Currently, researcher mainly carries out ctDNA detection, however this detection method using new-generation sequencing (NGS) technology
Still there are many limitations, such as: (1) NGS library construction needs larger amount of ctDNA, and usually researcher can be from sample
The experiment of acquisition is few with ctDNA amount, is not enough to support NGS library construction；(2) ctDNA property is extremely unstable, and it is extracted
Process will receive sample constituent, in sample ctDNA concentration, Sample preservation time, storage requirement, extraction process etc. it is a variety of because
The influence of element to cause the ctDNA quality difference for extracting acquisition significant, and then influences to build library quality stability；(3) NGS text
It is easily introduced artificial mutation (nucleotide variation generated during such as amplified library) in the building process of library and causes false positive, to drop
The accuracy of low ctDNA detection.To sum up, drawbacks described above greatly affected the sensitivity and reliability of ctDNA detection, into
And it is limited in the popularization and use of field of biomedicine.
To sum up, how from complex samples separation obtains micro ctDNA, and to NGS technology carry out necessary improvement with
Just the library NGS that more efficiently building quality is stable, artificial mutation is few, and then improve the sensitivity of ctDNA detection and accurate
Property, it is of great significance to improving its use value, expanding its suitable application area.
Summary of the invention
In order to overcome the defect of existing ctDNA detection technique, and new applicable neck is opened up for the exploration of ctDNA detection technique
Domain, the present invention establish a kind of completely new double-strand Circulating tumor DNA detection method, by each on the basis of testing repeatedly
Item testing conditions are screened and are optimized, and obtain higher library yield, effectively overcoming ctDNA quality difference leads to library
The defect of unstable quality fundamentally improves the sensitivity and accuracy of detection, greatly reduces detection false positive rate.This
Inventive method, which can be promoted, is suitable for preparation kinds of tumors tracer or tumor screening kit, each in field of biomedicine to meet
Class clinic and Research Requirements.
In a first aspect, the invention discloses a kind of highly sensitive double-strand Circulating tumor DNA detection method, this method includes blood
Two stages of building and oncogene targeted capture for starching dissociative DNA high-throughput sequencing library, specifically, the method includes under
State step:
(1) reparation of the end DNA is carried out to plasma DNA and tailing reacts；
(2) product of step (1) is attached with the adapter containing UMI and is reacted plus adapter connector；
(3) product of step (2) is purified with AMPure XP magnetic bead；
(4) qPCR is carried out with a part in step (3) product；
(5) remainder in step (3) product is carried out by library high-fidelity PCR amplification according to step (4) qPCR result；
(6) library after step (5) amplification is purified with AMPure XP magnetic bead and detects Library Quality；
(7) oncogene targeted capture is carried out to library after purification using oncogene targeted capture reagent.
In the present invention, the plasma DNA can be is separated using QIAamp MinElute ccfDNA kit
The cfDNA obtained with purifying.
Further, the end DNA described in the method for the present invention (1) step is repaired and tailings reactions are repaired using end
Reacting mixed enzyme for cfDNA fragment ends reparation with tailing is the flat end of 5 ' phosphorylations, and adds dA tail at 3 ' ends.
Further, above-mentioned end reparation and tailing reaction mixed enzyme in comprising Klenow segment (3 ' -5 ',
Exo-), T4DNA polymerase and T4 polynueleotide kinase.
In addition, the above-mentioned end DNA is repaired and tailings reactions are in a reaction tube while to carry out.
In the present invention, the adapter refer to one section short, chemically synthesized, double chain DNA molecule.The UMI also known as divides
Sub-barcode is one section of nucleotide chain containing 6-10 completely random base.Randomized bases are any in tetra- kinds of bases of A, T, C, G
One, due to the presence of four kinds of bases, it theoretically can produce 4 altogether10(about 1000000) plant UMI combination.Linking containing UMI
Son can add label to each DNA profiling, so as to track the source of amplified production.If two kinds of amplified productions are only single
Base difference and UMI sequence is different, then illustrate that this is a true mutation, conversely, illustrating to be source if UMI sequence is identical
In the amplified production of the same template, which is the false positive that PCR amplification introduces.The adapter of the design containing UMI can be with
False positive rate is substantially reduced, accuracy in detection is improved.
Further, the adapter described in the method for the present invention containing UMI includes T cohesive end, logical using T4DNA polymerase
The product for crossing the mode and step (1) of T/A connection is attached.
Preferably, the usage amount of adapter described in the method for the present invention and the weight ratio of initial plasma dissociative DNA dosage are
19 ﹕, 1~22 ﹕ 1.
It is highly preferred that the weight ratio of the adapter usage amount and initial plasma dissociative DNA dosage is 20 ﹕ 1.
Further, linking subsequence described in the method for the present invention is as shown in SEQ ID NO:1-2.
Further, the qPCR reaction mixture that qPCR described in the method for the present invention (4) step is used is SYBR
Green dyestuff, Taq archaeal dna polymerase, dNTPs mixed liquor, MgCl2, ROX dyestuff and reaction buffer mixed solution；For
The different plasma DNA sample of quality carries out qPCR accurately to obtain the content of plasma DNA, and reacts Ct according to qPCR
Value determines the cycle-index of follow-up library amplification PCR to guarantee that library yield is uniform and stable.
Further, the remainder in step (3) product is subjected to library height described in the method for the present invention (5) step
High fidelity PCR reaction mixture used in fidelity PCR amplification is the mixing of high-fidelity DNA polymerase and PCR reaction buffer
The cycle-index of solution, PCR determines that specific standards are as follows according to qPCR reaction Ct value:
(1) when qPCR reacts Ct value < 5, the cycle-index of PCR is Ct+1 times；
(2) when qPCR reaction Ct value is 5-8, the cycle-index of PCR is Ct+2 times；
(3) when qPCR reacts Ct value > 8, the cycle-index of PCR is Ct+3 times.
In addition, heretofore described library purifies the AMPure XP magnetic of magnetic bead preferred Beckman Coulter company used
Pearl.
Further, the probe combinations for including in oncogene targeted capture reagent described in the method for the present invention (7) step
For the targeted capture probe combinations designed for multiple cancer related genes, 127 cancers are directed to as employed in embodiment
The detection probe combination of related gene design or other functionally similar targeted capture probe combinations.
Moreover, it relates to which above-mentioned double-strand Circulating tumor DNA detection method is preparing neoplasm tracing or tumor screening
Application in kit.
On the other hand, the present invention provides a kind of highly sensitive double-strand Circulating tumor DNA detection kit, which can
It is used to prepare two generation of the plasma DNA sequencing library (NGS) and carries out oncogene targeted capture, the library through targeted capture can fit
High-flux sequence is carried out for Illumina microarray dataset to detect oncogene mutation that may be present in plasma DNA.
Specifically, including following component parts in detection kit of the present invention:
(1) end DNA is repaired and tailing reacts mixed enzyme and buffer；
(2) 6 kinds of adapters containing UMI；
(3) reaction mixture is connected；
(4) for purifying the AMPure XP magnetic bead of DNA；
(5) qPCR reaction mixture；
(6) high-fidelity PCR amplification reaction mixture；
(7) oncogene targeted capture reagent.
Further, above-mentioned double-strand Circulating tumor DNA detection kit, in which:
(1) DNA end described in repair and tailing reaction mixed enzyme be comprising Klenow segment (3 ' -5 ', exo-),
The mixed enzyme solution of T4DNA polymerase and T4 polynueleotide kinase；
(2) adapter described in containing UMI includes T cohesive end, and T4DNA polymerase can be used or similar products pass through
The mode of T/A connection is repaired with the end DNA and tailing reaction product is attached, sequence such as SEQ ID NO:1-2 institute
Show；
(4) for purifying the AMPure XP magnetic bead of the magnetic bead preferred Beckman Coulter company of DNA described in；
(5) qPCR reaction mixture described in is comprising SYBR Green dyestuff, Taq archaeal dna polymerase, dNTPs mixing
Liquid, MgCl2, ROX dyestuff and reaction buffer mixed solution；QPCR is carried out for the different plasma DNA sample of quality
Accurately to obtain the content of plasma DNA, and according to qPCR react Ct value determine the cycle-index of follow-up library amplification PCR with
Guarantee that library yield is uniform and stable；
(6) high-fidelity PCR amplification reaction mixture described in is to include high-fidelity DNA polymerase and PCR reaction buffer
Mixed solution, can be KAPA HiFi polymerase mix or similar products；
(7) reagent of oncogene targeted capture described in is the hybridization of oncogene targeted capture and elution reagent, wherein including needle
To the targeted capture probe combinations of multiple cancer related genes design, 127 cancer correlations are directed to as employed in embodiment
The detection probe combination of gene design or other functionally similar targeted capture probe combinations.
In summary it can be seen, compared with existing ctDNA detection technique, double-strand Circulating tumor DNA detection method of the present invention and
Kit has the advantage that and feature:
(1) high sensitivity and higher library yield.The adapter of special designing contains T cohesive end, dashes forward with containing A
The DNA fragmentation of end substantially increases the joint efficiency of adapter by T/A connection reaction out, thus obtains very high library
Yield；
(2) library stable yield is uniform.Connection product amount is measured by qPCR and provides guidance for follow-up library amplification, is solved
Different plasma DNA sample quality differences big problems；
(3) accuracy in detection is high.By the way that molecular label (UMI) is introduced linking subsequence, sequencing result analysis can be known
The false positive results that other PCR is generated, thus greatly reduce detection false positive rate.
Therefore, the method for the present invention, which can be promoted, is suitable for preparation kinds of tumors tracer or tumor screening kit, to meet life
All kinds of clinical and Research Requirements in object medical domain.
Detailed description of the invention
Fig. 1 is the qPCR testing result figure that adapter connects DNA fragmentation.
Fig. 2 is that Library Quality inspection is carried out before target gene targeted capture using Agilent TapeStation2100
The result figure of survey.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.
Unless otherwise defined, all technical and scientific terms and those skilled in the art of the present technique used in the present invention are usual
The meaning of understanding is identical.In addition to specific method, equipment, material used in the embodiment, according to those skilled in the art
Grasp and record of the invention to the prior art, can also use and method described in the embodiment of the present invention, equipment, material
Any method, equipment and the material of the similar or equivalent prior art realizes the present invention.
Material therefor, reagent etc., are commercially available unless otherwise specified in following embodiments.
Embodiment
(1) building of dissociative DNA high-throughput sequencing library
1. simulating the preparation of dissociative DNA
Make its fragmentation by human genome DNA through ultrasonication for simulation dissociative DNA clip size, after processing
The simulation dissociative DNA fragment length range 50-400bp arrived, peak value are 180bp or so.
The end 2.DNA is repaired to be connected with sequencing adapter
1) end reparation is carried out to simulation dissociative DNA in PCR plate or pipe and tailing reacts；
The end table 1DNA is repaired and tailing reaction solution
| Ingredient | Volume |
| Fragmentation double-stranded DNA (10ng) | XμL |
| Water | 12.5-XμL |
| End is repaired and tailing buffer | 1.75μL |
| End is repaired and tailings reactions enzyme mixation | 0.75μL |
| Total volume | 15μL |
Wherein: every end 1 μ L is repaired and tailings reactions enzyme mixation Klenow containing 2U segment (3 ' -5 ', exo-), 1U
T4DNA polymerase and 5U T4 polynueleotide kinase；
End is repaired and tailing buffer is 8.5x T4DNA polymerase buffer, includes 10mM ATP and 1mM dNTPs.
2) rapid centrifugation after of short duration vortex carries out in next step immediately；
3) it is put into thermal cycler, is incubated for according to following programs；
Table 2 is incubated for program
| Incubation temperature | Retention time |
| 20℃ | 30min |
| 65℃ | 30min |
| 4℃ | ∞ |
4) enter adapter after the completion of being incubated for connect, in the identical PCR plate or pipe of end reparation and tailings reactions, carry out
Connection reaction, assembles each adapter；
3 adapter of table connects reaction solution
| Ingredient | Volume |
| The product of step 3) | 15μL |
| Adapter (100ng/ μ L) | xμL |
| Sterile ultrapure water | (15-x)μL |
| Connect enzyme mixation | 30μL |
| Total volume | 60μL |
Wherein: linking subsequence is as follows:
3’-GTTCGTCTTCTGCCGTATGCTCTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGANNNNNNN
NNNAG--P7；
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNN
NNNTCT--P5；
Wherein N is any one in A, T, G, C.
Wherein: connection enzyme mixation T4DNA containing 2x polymerase buffer and 20U/ μ L T4DNA polymerase；
5) thoroughly mixing and of short duration centrifugation；
6) it is incubated for 15 minutes at 25 DEG C, closes heating lid, immediately enter in next step.
3. connection product purifies
1) the AmPure XP magnetic bead of 1 times of volume (60 μ L) is added；
2) it is sufficiently mixed by being vortexed；
3) 5 minutes are incubated at room temperature so that DNA to be integrated on magnetic bead；
4) plank/test tube is placed on magnetic frame to capture magnetic bead, is incubated for 3 minutes；
5) it carefully takes out and abandons supernatant；
6) plank/test tube is maintained on magnetic frame, 80% ethyl alcohol of the 200 fresh configurations of μ L is added；
7) plank/test tube is incubated for >=30 seconds on magnetic frame at room temperature；
8) it carefully draws and abandons ethyl alcohol；
9) step 6) -8 is repeated) once, and inhale and abandon remaining ethyl alcohol；
10) it is dried at room temperature for magnetic bead 3-5 minutes, or until all ethanol evaporations；
11) plank/test tube is removed from magnetic frame；
12) it is suspended again pearl with 30 μ L elution buffers；
13) plank/test tube is incubated at room temperature 2 minutes with from magnetic bead eluted dna；
14) plank/test tube is placed on magnetic frame to capture magnetic bead, is incubated for until liquid is limpid；
15) limpid supernatant is transferred in new plank/test tube, 30 μ L AmPure XP magnetic beads is added；
16) step 2) -14 is repeated again), and eluted with 20 μ L elution buffers.
4.qPCR is quantitative
1) qPCR reaction mixture is prepared, 3 repetitions are arranged in each sample；
Table 4qPCR reaction mixture
| Ingredient | Volume |
| Sterile ultrapure water | 7.4μL |
| PowerUp SYBR green mixed liquor | 10μL |
| 10 μM of amplified library primers 1 | 0.8μL |
| 10 μM of amplified library primer 2s | 0.8μL |
| The purified product of step 3 | 1μL |
| Total volume | 20μL |
Wherein, 1 sequence of amplified library primer is as follows:
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA-3'；
Amplified library primer 2 sequence is as follows:
5’-CAAGCAGAAGACGGCATACGAGATATCACGGTGACTGGAGTTCAGACG TGT-3’。
2) qPCR is run according to following programs；
Table 5qPCR runs program
3) qPCR is analyzed as a result, Ct threshold value is set as 0.5, calculate each sample Ct value.
5. gene library expands
1) each gene library amplified reaction is assembled；
Table 6PCR amplified reaction mixed liquor
| Ingredient | Volume |
| 2X KAPA HiFi polymerase mix | 50μL |
| 10 μM of amplified library primers 1 | 5μL |
| 10 μM of amplified library primer 2s | 5μL |
| The purified product of step 3 | 17μL |
| Water | 23μL |
| Total volume | 100μL |
2) it thoroughly mixes and is centrifuged；
3) PCR amplification is carried out according to following programs；
Table 7PCR amplification operation program
8 cycle-index X computational chart of table
| Ct value | <5 | 5-8 | >8 |
| PCR cycle number (secondary) | Ct+1 | Ct+2 | Ct+3 |
4) amplified library product uses 1 times of volume AmPure XP magnetic beads for purifying, and is eluted with 20 μ L water；
5) library yield is measured with Qubit and TapeStation.
6. experimental result and analysis
Using 10ng fragmentation double-stranded DNA, DNA sequencing library is constructed according to the method described above.For examination more of the present invention
The using effect of agent box and similar product KAPA Hyper sequencing library building kit (abbreviation KAPA), we utilize equal amount
DNA, be prepared for DNA sequencing library according to KAPA kit specification.
In order to both compare the efficiency for connecting reaction during building library, connection product after purification, use qPCR
Connection product is had detected, Ct threshold value is set as 0.5, as a result as shown in attached drawing 1 and table 9.
9 experimental group of table and control group Ct value
| Sample ID | Ct average value | Ct standard deviation value |
| CURA | 6.305 | 0.038 |
| KAPA | 8.353 | 0.103 |
As can be seen from the above table, kit (CURA) connection product qPCR detection Ct value of the present invention is 6.3, and KAPA connects
Both it is 8.3 that the object qPCR that practices midwifery, which detects Ct value, and higher Ct value shows containing less connection product, and the every difference 1 of Ct value illustrates
1 times of difference, the present invention detect Ct value ratio KAPA detected value few 2, illustrate that connection product efficiency is 4 times of KAPA.
Both Library PCR amplifications use 8 PCR cycles, are detected after amplified production is purified with Tapestation, tie
Fruit is as shown in Fig. 2.Sequencing library equally is prepared using 10ng DNA, library DNA amount (37.2ng/ μ L) prepared by the present invention is about
5 times that (7.48ng/ μ L) is measured in library are prepared for KAPA method.
The above results show that, to prepare same amount of library, the present invention needs less starting amount of DNA or less
PCR amplification cycle-index.Since dissociative DNA content is extremely low, inter-sample difference is very big, and lower starting amount of DNA requirement can be big
It is big reduce as initial dissociative DNA amount is very few and caused by detect failure.
(2) the general oncogene targeted capture sequencing of Horizon standard sample cfDNA
In order to examine this method to low frequency mutation detectability, we have purchased from Horizon Discovery company
CfDNA standard sample, and 1% frequency of mutation sample is diluted to 0.01% frequency of mutation sample using WT sample.
1. low frequency mutation cfDNA library construction
Text is constructed according to the method for embodiment (one) using 1 μ g Horizon cfDNA (WT and 0.01% frequency of mutation)
Library enters in next step after quality inspection is qualified.
2. the general oncogene targeted capture in library
1) following substance: 500ng DNA library and 5 μ L people Cot-1DNA (1 μ g/ μ L) is mixed in 1.7mL centrifuge tube；
2) the AMPure XP pearl of 2.5 times of volumes is added, be uniformly mixed and is being stored at room temperature 10 minutes to combine；
3) centrifuge tube is placed on magnetic frame and makes limpid (2 minutes)；
4) centrifuge tube is maintained on magnetic frame, takes out and abandons supernatant；
5) ethyl alcohol of the fresh configuration of 200 μ L 80% is added and inhales and makes a call to 3 times so that pearl is detached from tube wall, of short duration vortex is quick
Centrifugation, and pipe is placed on magnetic frame；
6) centrifuge tube is maintained on magnetic frame, takes out and abandons supernatant；
7) 5) -6 are repeated) once, and inhale and abandon remaining ethyl alcohol；
8) centrifuge tube is maintained on magnetic frame, opens lid and make it dry, while according to the form below configures hybrid mixed liquid；
10 hybrid mixed liquid of table
9) 15 μ L hybrid mixed liquid are added in the centrifuge tube on magnetic frame, pipette tips are blown and beaten 10 times, stand 3 minutes；
10) centrifuge tube is placed on magnetic frame and makes limpid (2 minutes)；
11) centrifuge tube of step 10 is denaturalized 10 minutes for 95 DEG C in the thermal cycler, is then taken out；
12) the general oncogene probe combinations of 4 μ L are added, then hybridize 4 hours under the conditions of 65 DEG C；
13) prepare washing buffer (Wash Buffer)；
11 washing buffer of table
| Reagent | Buffer | Water |
| 2x magnetic bead cleaning solution | 250μL | 250μL |
| 10x washing buffer 1 | 30μL | 270μL |
| 10x washing buffer 2 | 20μL | 180μL |
| 10x washing buffer 3 | 20μL | 180μL |
| 10x stringency wash buffer | 40μL | 360μL |
14) step 12) terminates first 30 minutes for Dynabeads M-270 Streptavidin magnetic beads
(streptavidin bead) takes out from refrigerator to be put to room temperature；
15) vortex magnetic bead 15 seconds；
16) 100 μ L magnetic beads are drawn into 1.7mL pipe；
17) it is placed on magnetic frame and makes limpid (2 minutes)；
18) it draws and abandons supernatant；
19) it is washed and is vortexed 10 seconds with 200 μ L magnetic bead cleaning solutions and mixed；
20) it is placed on magnetic frame and makes limpid (2 minutes)；
21) it draws and abandons supernatant；
22) step 19) -21 is repeated with 200 μ L magnetic bead cleaning solutions)；
23) 100 μ L magnetic bead cleaning solutions are added and mixture is transferred in 0.2mL PCR pipe；
24) it is placed on magnetic frame and makes limpid (2 minutes)；
25) it draws and abandons supernatant；
26) product of step 12) (about 19 μ L) is directly added into Streptavidin magnetic beads and blown and beaten 10 times up and down
Mixing；
27) by PCR pipe fast transfer to 65 DEG C thermal cycler 45 minutes, every 12 minutes take out and quickly with finger bullet
PCR pipe suspends magnetic bead again, puts back to thermal cycler rapidly；
28) after 45 minutes, the 100 μ L washing buffer 1 heated is added in PCR pipe；
29) mixture in PCR pipe is transferred to 1.7mL pipe；
30) it is placed on magnetic frame and makes limpid (2 minutes)；
31) it draws and abandons supernatant；
32) add the stringency wash buffer of 200 μ L heating；
33) it is incubated for 5 minutes in 65 DEG C of water-baths；
34) it is placed on magnetic frame and makes limpid (2 minutes)；
35) it draws and abandons supernatant (containing unbonded DNA)；
36) step 32) -35 is repeated with the stringency wash buffer of 200 μ L heating)；
37) 200 μ L washing buffers 1 are added and are vortexed 2 minutes (being vortexed 3 seconds within every 10 seconds)；
38) it is placed on magnetic frame and makes limpid (2 minutes)；
39) it draws and abandons supernatant；
40) 200 μ L washing buffers 2 are added and are vortexed 1 minute (being vortexed 3 seconds within every 10 seconds)；
41) it is placed on magnetic frame and makes limpid (2 minutes)；
42) it draws and abandons supernatant；
43) 200 μ L washing buffers 3 are added and are vortexed 30 seconds (being vortexed 3 seconds within every 10 seconds)；
44) it is placed on magnetic frame and makes limpid (2 minutes)；
45) it draws and abandons supernatant；
46) with 15 μ L H2Magnetic bead (pipettor move up and down piping and druming 10 times) is resuspended in O, by the mixed liquor containing magnetic bead directly into
Row is in next step (about 18 μ L)；
47) prepare KAPA PCR mixture；
Table 12KAPA PCR mixture
48) run PCR program (lid is heated to 105 DEG C)；
Table 13PCR runs program
49) with 75 μ L (1.5x volume) AMPure XP magnetic beads for purifying amplified production and with 20 μ L eluted dnas；
50) 2 μ L samples is taken to use Agilent D1000High Sensitivity DNA Tape measurement concentration and DNA points
Cloth region；
51) after quality inspection is qualified, sample carries out PE150 sequencing in Illumina HiSeqX microarray dataset.
3. sequencing result is analyzed
It is about 600G that total amount of data, which is sequenced, in sample.Average same label supports number 8 when merging same label sequence
Reads or more, average sequencing depth 45000X after merging.Detected Horizon standard sample cfDNA contains 8 site mutations, mutation
Frequency is 0.01%, detects wherein 7 kinds of mutation through this method sequencing, testing result is as shown in table 14 below, by table as it can be seen that we
Case can detect mutational site of most contents down to 0.01% in cfDNA.
14 sequencing result of table
| Chromosomal foci | Mutant designations | With reference to genotype | Mutated-genotype | Reference type read number | Saltant type read number | The frequency of mutation (%) |
| chr7:55181378 | EGFR-T790M | C | T | 53388 | 50 | 0.094 |
| chr7:55191822 | EGFR-L858R | T | G | 48343 | 61 | 0.126 |
| chr12:25245350 | KRAS-G12D | C | T | 54836 | 39 | 0.071 |
| chr1:114713915 | NRAS-A59T | C | T | 41060 | 92 | 0.224 |
| chr1:114713909 | NRAS-Q61K | G | T | 41711 | 17 | 0.041 |
| chr3:179218303 | PIK3CA-E545K | G | A | 42547 | 10 | 0.024 |
| chr7:55174771 | EGFR-E746_A750del | AGGAATTAAGAGAAGC | A | 31819 | 8 | 0.025 |
The preferred embodiments of the disclosure and embodiment are explained in detail above, but the present invention is not limited to
The above-described embodiment and examples can also not depart from the present invention within the knowledge of those skilled in the art
Various changes can be made under the premise of design.
Claims (13)
1. a kind of high sensitivity double-strand Circulating tumor DNA detection method, it is characterised in that: the method includes plasma DNA
Two stages of building and oncogene targeted capture of high-throughput sequencing library, specifically, the method includes the following steps:
(1) reparation of the end DNA is carried out to plasma DNA and tailing reacts；
(2) product of step (1) is attached with the adapter containing UMI and is reacted plus adapter connector；
(3) product of step (2) is purified with AMPure XP magnetic bead；
(4) qPCR is carried out with a part in step (3) product；
(5) remainder in step (3) product is carried out by library high-fidelity PCR amplification according to step (4) qPCR result；
(6) library after step (5) amplification is purified with AMPure XP magnetic bead and detects Library Quality；
(7) oncogene targeted capture is carried out to library after purification using oncogene targeted capture reagent.
2. double-strand Circulating tumor DNA detection method as described in claim 1, it is characterised in that the end DNA described in (1) step
End is repaired and tailings reactions are that end reparation and tailing reaction mixed enzyme is utilized to repair DNA fragmentation end for 5 ' phosphorylations
Flat end, and dA tail is added at 3 ' ends.
3. double-strand Circulating tumor DNA detection method as claimed in claim 2, the end is repaired and tailing reaction mixing
It include Klenow segment 3 ' -5 ', exo-, T4DNA polymerase and T4 polynueleotide kinase in enzyme.
4. double-strand Circulating tumor DNA detection method as claimed in claim 2, the end DNA is repaired and tailings reactions are one
It is carried out simultaneously in a reaction tube.
5. double-strand Circulating tumor DNA detection method as described in claim 1, it is characterised in that contain UMI described in (2) step
Adapter include T cohesive end, connected by product of the T4DNA polymerase in the way of T/A connection with step (1)
It connects.
6. double-strand Circulating tumor DNA detection method as claimed in claim 5, wherein the usage amount of the adapter and starting blood
The weight ratio for starching dissociative DNA dosage is 19 ﹕, 1~22 ﹕ 1.
7. double-strand Circulating tumor DNA detection method as described in claim 1, wherein the adapter sequence such as SEQ ID NO:
Shown in 1-2.
8. double-strand Circulating tumor DNA detection method as described in claim 1, it is characterised in that qPCR described in (4) step
The qPCR reaction mixture used is SYBR Green dyestuff, Taq archaeal dna polymerase, dNTPs mixed liquor, MgCl2, ROX dyestuff
With the mixed solution of reaction buffer；QPCR is carried out for the different plasma DNA sample of quality accurately to obtain blood plasma trip
Content from DNA, and Ct value is reacted according to qPCR and determines the cycle-index of follow-up library amplification PCR to guarantee that library yield is uniform
Stablize.
9. double-strand Circulating tumor DNA detection method as described in claim 1, it is characterised in that will step described in (5) step
Suddenly it is high-fidelity that the remainder in (3) product, which carries out High fidelity PCR reaction mixture used in the high-fidelity PCR amplification of library,
The cycle-index of the mixed solution of archaeal dna polymerase and PCR reaction buffer, PCR is determined according to qPCR reaction Ct value, specific to mark
It is quasi- as follows:
(1) when qPCR reacts Ct value < 5, the cycle-index of PCR is Ct+1 times；
(2) when qPCR reaction Ct value is 5-8, the cycle-index of PCR is Ct+2 times；
(3) when qPCR reacts Ct value > 8, the cycle-index of PCR is Ct+3 times.
10. double-strand Circulating tumor DNA detection method as described in claim 1, it is characterised in that cancer base described in (7) step
Because the probe combinations for including in targeted capture reagent are the targeted capture probe combinations designed for multiple cancer related genes.
11. as the described in any item double-strand Circulating tumor DNA detection methods of claim 1-10 are preparing neoplasm tracing or tumour
Application in kit for screening.
12. a kind of high sensitivity double-strand Circulating tumor DNA detection kit, it is characterised in that: comprising following in the kit
Component part:
(1) end DNA is repaired and tailing reacts mixed enzyme and buffer；
(2) 6 kinds of adapters containing UMI；
(3) reaction mixture is connected；
(4) for purifying the AMPure XP magnetic bead of DNA；
(5) qPCR reaction mixture；
(6) high-fidelity PCR amplification reaction mixture；
(7) oncogene targeted capture reagent.
13. double-strand Circulating tumor DNA detection kit as claimed in claim 12, in which:
(1) DNA end described in is repaired and tailing reaction mixed enzyme is to include Klenow segment 3 ' -5 ', exo-, T4DNA
The mixed enzyme solution of polymerase and T4 polynueleotide kinase；
(2) adapter described in containing UMI includes T cohesive end, and sequence is as shown in SEQ ID NO:1-2；
(5) qPCR reaction mixture described in be comprising SYBR Green dyestuff, Taq archaeal dna polymerase, dNTPs mixed liquor,
MgCl2, ROX dyestuff and reaction buffer mixed solution；
(6) high-fidelity PCR amplification reaction mixture described in is mixed comprising high-fidelity DNA polymerase and PCR reaction buffer
Close solution；
(7) reagent of oncogene targeted capture described in is the hybridization of oncogene targeted capture and elution reagent, wherein comprising for more
The targeted capture probe combinations of a cancer related gene design.
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