CN109234393A - It is a kind of for detecting the gene probe composition and kit of metastatic castration-resistant prostate cancer - Google Patents
It is a kind of for detecting the gene probe composition and kit of metastatic castration-resistant prostate cancer Download PDFInfo
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Abstract
It is a kind of for detecting the gene probe composition of metastatic castration-resistant prostate cancer, be made of 50 gene probes such as ATM gene probe, ATR gene probe, AR gene probe, BARD1 gene probe, BRCA1 gene probe, BRCA2 gene probe, BRIP1 gene probe, CDH1 gene probe, CDK12 gene probe, CHEK2 gene probe, EPCAM gene probe, ERCC2 gene probe, ERCC3 gene probe, ERCC4 gene probe.The present invention also provides a kind of for detecting the kit of metastatic castration-resistant prostate cancer, contains above-mentioned gene probe composition.The present invention is designed by probe, can be provided for clinical treatment decision with reference to letter with a variety of germline mutations of one-time detection and a variety of somatic mutation types, including SNV, Indel, CNV, Gene Fusion etc., comprehensive interpretive analysis target gene relevant to drug.
Description
Technical field
The invention belongs to bioengineering field, it is related to a kind of gene probe and kit, it is specifically a kind of for examining
Survey the gene probe composition and kit of metastatic castration-resistant prostate cancer.
Background technique
NGS technology: high throughput sequencing technologies once carry out sequencing, high pass to millions of DNA moleculars to hundreds of thousands
(Next Generation Sequencing, NGS) also known as next-generation sequencing technologies are measured, reflect its epoch-making change, simultaneously
High-flux sequence to carry out the analysis of careful overall picture to the transcript profile and genome of species, so claimed again
For deep sequencing (Deep Sequencing).Illumina platform is two generation microarray datasets of present mainstream, and occupation rate of market exists
90% or more.
Compared with early gene detection technique: fluorescence in situ hybridization (FISH) though do not lose highly sensitive operation difficulty compared with
Greatly;Conventional polymeric enzyme chain reaction (PCR) has much advantage in ease-to-operate and rapidity, but technical principle limits it
One or several genes can only be detected;Although biochip technology can detecte lots of genes, but can only detect known, false
Positive rate is high, and accuracy is insufficient;NGS then has the advantages that flux is high, accuracy is high, cost is acceptable, both can disposably examine
It surveys lots of genes (including known and unknown gene), accuracy rate is higher than genetic chip again, while sequencing cost is not again relatively high.From
In the long run, with the continuous decrease of sequencing cost and be sequenced process successive optimization, gene sequencing to genetic chip etc. other
The substitution of technique of gene detection will become trend of the times;But current each technology branch is according to different clinical demand and intrinsic advantage
Still have and specifically segments market.
Target sequence capture sequencing: target sequence capture sequencing is that interested genome area is passed through capture kit
The research strategy being sequenced again after being enriched with can be obtained by superelevation using less data amount according to different applications
Sensitivity and accuracy, can quickly screen variant sites.The targeting technology of present mainstream, is mainly based upon multiplex PCR
Technology or probe hybrid capture technology.
Liquid biopsy: liquid biopsy (Liquid Biopsy) has rapid, convenient, damage compared with traditional tissue biopsy
The small equal many merits of wound property.Clinician can monitor reaction of the tumour to treatment with it, predict tumor recurrence.From long-range angle
From the point of view of degree, liquid biopsy can also help doctor to find the tumour of initial stage when patient any symptom does not occur.From blood
The genomic information obtained in the DNA fragmentation to dissociate in liquid is even it can be pointed out that the position that in-vivo tumour rises.
In recent years attention of the liquid Biopsy of tumour by industry and capital, the technology are to find to fall off in peripheral blood
Or the Circulating tumor DNA (ctDNA) or circulating tumor cell (CTC) or excretion body (exosome) of apoptosis release carry out gene survey
Sequence, target are to develop the sieving and diagnosis technology of a kind of " general cancer ".
Compared with traditional tumor tissues sampling, the major advantage of liquid Biopsy includes: noninvasive sampling (blood inspection
Survey), overcome the Tumor Heterogeneity genetic mutation information of patient tumors cell entirety (reflection), Real Time Monitoring (such as ctDNA
The progress of concentration and tumour is highly relevant);The critical function that the technology may be implemented include: tumour early diagnosis, auxiliary by stages, prognosis
Assessment, recurrence monitoring, medication guide etc..Liquid Biopsy not upstart in fact, is based on inspection as NIPT principle
The cfDNA (foetal DNA to dissociate in such as maternal blood, the ctDNA to dissociate in individual blood) to dissociate in peripheral blood is surveyed, or
Detect the fetal cell/tumour cell CTC to dissociate in peripheral blood.This technology is equally built upon the basis of NGS technological break-through
On, it may be implemented to carry out trace amount DNA pinpoint accuracy, highly sensitive detection, to reach corresponding testing goal.
Metastatic castration-resistant prostate cancer: prostate cancer (PCa) is that male genitourinary system is most common pernicious
Tumour, disease incidence occupy the first in American male cancer patient, and the death rate is only second to lung cancer;Chinese disease incidence is relatively
It is low, but with aging of population, westernization and detection means the progress of life style, the disease incidence of PCa is in significantly rise
Gesture, and high-risk, progressivity, metastatic PCA Proportion of patients are big.Endocrine therapy be metastatic patient (metastatic PCa,
MPCa), the standard of the adjuvant treatment of high-risk or advanced age PCA patient (> 75 years old) and local treatment (including operation and radiotherapy) is controlled
Treatment scheme, however most patients translate into metastatic castration-resistant after the initial validity of endocrine therapy
Prostate cancer (mCRPC).Such patient's poor prognosis, and Tumor Heterogeneity is extremely obvious, treats rather intractable.Approval is answered at present
Drug for mCRPC mainly includes endocrine therapy of new generation, chemotherapy, radionuclide therapy, immunization therapy etc..
With a variety of therapeutic agents approval be applied to mCRPC treatment, how Accurate Prediction mCRPC patient's difference treatment side
The prognosis of case becomes the hot spot of current PCa area research with guiding treatment.Due to lacking the target of specific prediction curative effect to refer to
The selection of therapeutic scheme is led, therefore how the formulation therapeutic scheme of individuation benefits to be to grind both at home and abroad at present to reach best existence
The hot spot and difficult point studied carefully.Clinically also there is an urgent need to have one can be with full forecast mCRPC patient prognosis and its to different treatments
The detection method of scheme sensibility.
It may be that the accurate targeted therapy of CRPC mention that the genomics research about CRPC external at present, which has become future,
For the research direction of theoretical basis.It is multinomial the study found that main genetic mutation includes androgen receptor (Androgen in CRPC
Receptor, AR) exception, TMPRSS2-ERG fusion, PTEN missing, TP53 mutation, RB gene delection, C-MYC amplification with
And DNA repair signal access (such as BRCA2 missing, ATM gene mutation) catastrophe.In recent years, basic research prompts TP53
Indication CRPC patient is lacked simultaneously with RB gene with neuroendocrine differentiated, prompts patient resistance to for endocrine therapy of new generation
Medicine.Meanwhile it is multinomial researches show that AR amplification also prompt patient for endocrine therapy unsatisfactory curative effect of new generation, poor prognosis.Simultaneously
In recent years, effect of the DNA-repair gene in PCa patient also increasingly obtains the concern of domestic and foreign scholars.Pritchard equal part
The embryonal system for having analysed 20 DNA-repair genes relevant to autosomal dominant tumor susceptibility syndrome in 692 mPCa patients is prominent
Become situation, finds in 82 patients there are 16 DNA-repair genes, the germline mutation in totally 84 mutational sites, account for total number of persons
11.8%;Wherein, the most common mutational site is that (5.3%) 37, account for BRCA2, other mutation include: ATM, cell cycle inspection
Make an inventory of 2 gene of kinases (Checkpoint kinase 2, CHEK2), BRCA1, recombinase RAD51D (DNA repair RAD51-
Like protein D) and breast cancer susceptibility gene GAP-associated protein GAP 2 (PALB2).However, in cancer gene group map (TCGA)
There are 23 patients (23/499,4.6%) in 499 localized prostate cancer patients there are the germline mutation of DNA-repair gene, because
This, carries out correlative study for the DNA-repair gene site of these mutation, is expected to make the control strategy of PCa to change.
Recently, more and more researchs confirm that PARP inhibitor has definite curative effect to CRPC patient.TOPARP-A's is more
Center II phase clinical research discovery, the total effective rate (overall response rate, ORR) that olaparib treats CRPC are
32.7%, and in the specific sub-group of patients with DNA-repair gene defect (such as BRCA2 and ATM), ORR is then up to 87.5%.
Boysen etc. the study found that adjust DNA double chain fracture restoration SPOP (Speckle-type POZ gene mutation can lead to CRPC
Patient gene's is unstable;The mutation of SPOP gene can increase CRPC patient to polyadenosine diphosphate ribose polymerase
The sensibility of (poly ADP ribose polymerase, PARP) inhibitor.It therefore, to be pointedly suitable in clinic
Patient select PARP inhibitor for treating, just can more effectively predict its curative effect.So needing at present through genomics to PCA
Accurate parting is expected to be obviously improved patient's prognosis in this way to instruct precisely to treat.But mCRPC patient is mostly based on Bone tumour, former
Position stove due to after previously-accepting endocrine therapy tumor load it is smaller, cause tumor tissues materials it is more difficult, operation wound is big, because
This also brings the difficulty of genomics research tumor tissues materials.To sum up, carrying out individuation gene point to mCRPC patient
Analysis and carried out by a kind of detection means of relative noninvasive be current prostate cancer area research trend, this also will greatly have
Conducive to the selection for instructing maximally efficient personalized treatment drug.
The appearance of two generation sequencing technologies is that the tumor susceptibility gene detection of tumour, adjoint diagnosis, personalized medicine etc. provide more
Good technical support and selection, the tumour polygenes detection panel for being based especially on NGS accelerate field detection can more
It is fast and cheap, achieve the purpose that while having detected several genes and displacement point.Currently, domestic produce for prostate cancer detection
Product mainly include several aspects on clinical meaning: the early sieve product such as PCA3 detection, AR of heredity prostate cancer, prostate cancer
Drug resistance such as AR-V7 detection, chemotherapy medicinal products, targeted drug product etc.;In terms of technology platform selection, mainly have NGS platform,
PCR platform, CTC platform etc.;In detection sample type, there are tumor tissues, peripheral blood, urine etc..
The main problem for detecting metastatic castration-resistant prostate cancer in the prior art is:
1) existing detection method cannot analyze androgen receptor (AR) signal path and DNA damage reparation (DRR) phase comprehensively
Correlation gene;
2) existing detection method detection heredity relating to prostate cancers because germline mutation, it is not smart enough to the detection of CNV
Really, and CNV is important to (mCRPC);
3) existing detection method lacks the concern of the genetic analysis closely related to immunotherapeutic;
4) lack can be in the method for supporting tissue and blood ctDNA liquid biopsy for existing product, or the spirit of most detections
Sensitivity is not high;
5) clinically lack a comprehensive, high sensitivity, the metastatic castration of liquid biopsy at low cost, compatible to resist
Property prostate cancer (mCRPC) polygenes testing product.
Summary of the invention:
The purpose of the present invention is to provide a kind of for detecting the gene probe group of metastatic castration-resistant prostate cancer
Object and kit are closed, described is this for detecting the gene probe composition and reagent of metastatic castration-resistant prostate cancer
Not high, the inaccurate technology of sensitivity that box will solve to detect metastatic castration-resistant prostate cancer in the prior art is asked
Topic.
The present invention provides a kind of for detecting the gene probe composition of metastatic castration-resistant prostate cancer, by
ATM gene probe, ATR gene probe, AR gene probe, BARD1 gene probe, BRCA1 gene probe, BRCA2 gene probe,
BRIP1 gene probe, CDH1 gene probe, CDK12 gene probe, CHEK2 gene probe, EPCAM gene probe, ERCC2 base
Because probe, ERCC3 gene probe, ERCC4 gene probe, ERCC5 gene probe, ESR1 gene probe, FAM175A gene are visited
Needle, FANCA gene probe, FANCD2 gene probe, FOXA1 gene probe, GEN1 gene probe, HDAC2 gene probe,
HOXB13 gene probe, IDH1 gene probe, MLH1 gene probe, MLH3 gene probe, MRE11A gene probe, MSH2 gene
Probe, MSH6 gene probe, MUTYH gene probe, NBN gene probe, NCOR1 gene probe, NCOR2 gene probe, PALB2
Gene probe, PMS1 gene probe, PMS2 gene probe, POLE gene probe, POLD1 gene probe, PTEN gene probe,
RAD50 gene probe, RAD51 gene probe, RAD51B gene probe, RAD51C gene probe, RAD51D gene probe, SPOP
Gene probe, STK11 gene probe, TP53 gene probe, XRCC4 gene probe, ZBTB16 gene probe and RB1 gene probe
Composition.
The present invention also provides a kind of for detecting the kit of metastatic castration-resistant prostate cancer, containing above-mentioned
Gene probe composition.
Gene in above-mentioned each gene probe is described in detail below:
In said gene, the relevant gene of endocrine therapy are as follows: AR gene, BARD1 gene, BRIP1 gene, CDH1 base
Cause, EPCAM gene, ESR1 gene, FAM175A gene, FOXA1 gene, GEN1 gene, IDH1 gene, MUTYH gene needle, NBN
Gene, NCOR1 gene, NCOR2 gene, PTEN gene, SPOP gene, STK11 gene, ZBTB16 gene.
Gene relevant to NE differentiation is TP53 gene and RB1 gene.
Gene relevant to DNA reparation are as follows: ATM gene, ATR gene, BRCA1 gene, BRCA2 gene, BRIP1 gene are visited
Needle, CDK12 gene, CHEK2 gene, ERCC2 gene, ERCC3 gene, ERCC4 gene, ERCC5 gene, FANCA gene,
FANCD2 gene, HDAC2 gene, MLH1 gene, MLH3 gene, MRE11A gene, MSH2 gene, MSH6 gene, PALB2 base
Cause, PMS1 gene, PMS2 gene, POLD1 gene, POLE gene, RAD51 gene, RAD51B gene, RAD51C gene, XRCC4
Gene.
Gene relevant to heredity prostate cancer are as follows: ATM gene, ATR gene, BRCA1 gene, BRCA2 gene,
BRIP1 gene, CHEK2 gene, FAM175A gene, GEN1 gene, HOXB13 gene, MLH1 gene, MRE11A gene, MSH2
Gene, MSH6 gene, NBN gene, PALB2 gene, PMS2 gene, RAD50 gene, RAD51 gene, RAD51C gene,
RAD51D gene.
The present invention is sequenced using hybrid capture+NGS, can disposable Parallel testing metastatic castration-resistant prostate cancer
(mCRPC) medication, relevant 50 genes of drug resistance are targeted.
Present invention is specifically directed to (mCRPC), clinically mainly solve following problems:
(1) novel endocrine therapy (the Ah ratio of mCRPC patient's receiving is probed by analyzing androgen receptor (AR) signal path
Special miscellaneous Shandong amine of dragon/grace etc.) drug resistance reason, with reach in time adjustment therapeutic scheme.
(2) by analysis DNA damage reparation (DDR) related gene, adjuvant clinical doctor selects platinum-based chemotherapy or addition
PARP inhibitor such as olaparib.
(3) by analyzing the closely related gene of immunotherapeutic (MMR family and CDK12), so that patient controls in two wires
After treatment, judge whether using immunologic test point inhibitor.
(4) by analysis heredity relating to prostate cancers because germline mutation, analyze related family members and accomplish to prevent in advance
Accurate screening.
(5) Plantago fengdouensis of gene is detected by ctDNA, dynamic detection medication curative effect carries out full range management.
The present invention is compared with prior art, its technical effect is that actively and apparent.The present invention is designed by probe, can be with
The a variety of germline mutations of one-time detection and a variety of somatic mutation types, including SNV, Indel, CNV, Gene Fusion etc. solve comprehensively
It reads to analyze target gene relevant to drug;The present invention supports ctDNA to detect, and detection sensitivity is up to 0.1%, can be compatible with blood
The liquid biopsy such as liquid, urine, Pleural effusions provides reference information for clinical treatment decision;Probe Panel of the invention can be multiple
Sample mixing, testing cost is low, and detection cycle is very fast.The present invention can be used for metastatic castration-resistant prostate cancer
(mCRPC) the accurate treatment and diagnosis of patient, supports liquid biopsy, integration solves analysis of drug use, and carries out full range management.
Detailed description of the invention
Fig. 1 shows the process using kit detection metastatic castration-resistant prostate cancer of the invention.
Fig. 2 is information analysis flow diagram of the invention.
Specific embodiment
Embodiment 1
1) probe designs: by searching for the associated guideline and text for detecting and treating metastatic castration-resistant prostate cancer
It offers, finds out the accurate molecule parting of mCRPC and prognosis to curative effect and judge relevant gene;50 above-mentioned genes have been selected,
Guarantee all genetic mutation types of a performance detection, including SNV, InDel, CNV, Gene Fusion;
2) probe source: 50 gene probes that the present invention uses are customized from Integrated Device Technology, Inc. of the U.S., can specifically be joined
The method preparation of the prior art is examined, this is the ordinary skill in the art, and details are not described herein.
3) together by 50 probe combinations, cooperation third party builds library kit, forms one for detecting metastatic
The kit of castration-resistant prostate cancer gene mutation.
The main agents that following embodiments use are as follows:
2 cfDNA/FFPEgDNA of embodiment is extracted:
The sample that the present invention uses can be patient tissue white tiles (FFPE) (5-10 piece) or 10ml peripheral blood.
Tissue samples or dissociative DNA (cfDNA) sample are extracted to leucocyte gDNA: related using Qiagen company of Germany
Kit extracts (article No. 56404,55114 etc.) or other equivalent agent boxes.
CfDNA total amount is greater than 30ng;FFPE DNA total amount is greater than 500ng;It compares gDNA total amount and is greater than 500ng.
Steps are as follows for the library NGS preparation manipulation:
Swift company of the present invention to be compatible with Illumina platform build library kit for.
1.1.FFPE with gDNA sample since interrupting (cfDNA sample builds library since 1.2):
1.1.1. 500ng is taken according to qubit concentration, water is added to mend to 100 μ l, 130 μ l of covaris is added and interrupts in pipe, if
Set program: 50W, 20%, 200cycles, 330s. take 1 μ l, qsep100 to detect segment distribution, main peak 150- after interrupting
200bp.
1.1.2. product will be interrupted to be transferred in new 1.5ml centrifuge tube, the AMPure beads of 1.4 times of volumes is added,
Sufficient vortex mixes, and is incubated at room temperature 5min.
1.1.3. centrifuge tube is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Clearly.
1.1.4. it keeps centrifuge tube on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh is added, be incubated at room temperature 30sec.
1.1.5. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.1.6. step 7.1.3-7.1.4 primary (cleaning twice in total) is repeated.
1.1.7. centrifuge tube to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.1.8. 52.5 μ l water are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min. at room temperature
1.1.9. it is placed on magnetic frame, (about 2min) takes 50 μ l into 200 μ l PCR pipes after supernatant clarification.
1.1.10. 1 μ l is taken to useDsDNA HS Assay Kit is quantitative.
1.2.Repair I and purifying: (cfDNA sample builds library since 1.2)
1.2.1. it takes 100ng to interrupt gDNA or 30-50ng cfDNA after purification into PCR pipe, is mended with low TE to 40 μ
Following reagent is added in l, is vortexed and mixes.
Component | Volume |
gDNA/cfDNA | 40μl |
Low EDTA TE | 13μl |
Buffer W1 | 6μl |
Enzyme W2 | 1μl |
Total volume | 60 |
1.2.2. setting following procedure is reacted in PCR instrument:
1.2.3.AMPure beads purifying:
1.2.3.1. magnetic bead, which is vortexed, mixes, and takes 108 μ l to be added in the PCR pipe after above-mentioned reaction, sufficient vortex mixes, room temperature
It is incubated for 5min.
1.2.3.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.2.3.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.2.3.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.2.3.5. step 1.2.3.3 and 1.2.3.4 primary (cleaning twice in total) is repeated.
1.2.3.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.3.RepairⅡ:
1.3.1. following reagent is prepared, is vortexed to mix and 7.2.3 is added after of short duration centrifugation and dry in magnetic bead, mixing is played in suction:
1.3.2. setting following procedure is reacted in PCR instrument:
1.3.3.AMPure beads purifying:
1.3.3.1. plus in the PCR pipe after 82.5 μ l PEG/NaCl to above-mentioned reaction, sufficient vortex is mixed, incubation at room temperature
5min。
1.3.3.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.3.3.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.3.3.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.3.3.5. step 7.3.3.3 and 7.3.3.4 primary (cleaning twice in total) is repeated.
1.3.3.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.4. connector connection I:
1.4.1. following reagent is prepared, is vortexed to mix and 1.3.3 is added after of short duration centrifugation and dry in magnetic bead, 5 μ l phases are added
Index reagent Reagent Y2 is answered, mixing is played in suction:
Component | Volume |
Low EDTA TE | 20μl |
Buffer Y1 | 3μl |
Enzyme Y3 | 2μl |
Total volume | 25μl |
1.4.2. setting following procedure is reacted in PCR instrument:
Temperature | Time |
25℃ | 15min |
4℃ | ∞ |
1.4.3.AMPure beads purifying:
1.4.3.1. plus in the PCR pipe after 49.5 μ l PEG/NaCl to above-mentioned reaction, sufficient vortex is mixed, incubation at room temperature
5min。
1.4.3.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.4.3.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.4.3.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.4.3.5. step 1.4.3.3-1.4.3.4 primary (cleaning twice in total) is repeated.
1.4.3.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.5. connector connection II:
1.5.1. following reagent is prepared, is vortexed to mix and 1.4.3 is added after of short duration centrifugation and dry in magnetic bead, mixing is played in suction:
Component | Volume |
Low EDTA TE | 30μl |
Buffer B1 | 5μl |
Reagent B2/B2-MID | 2μl |
Reagent B3 | 9μl |
Enzyme B4 | 1μl |
Enzyme B5 | 2μl |
Enzyme B6 | 1μl |
Total volume | 50μl |
1.5.2. setting following procedure is reacted in PCR instrument:
Temperature | Time |
40℃ | 10min |
25℃ | ∞ |
1.5.3.AMPure beads purifies (non-MID connector):
1.5.3.1. plus in the PCR pipe after 82.5 μ l PEG/NaCl to above-mentioned reaction, sufficient vortex is mixed, incubation at room temperature
5min。
1.5.3.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.5.3.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.5.3.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.5.3.5. step 1.5.3.3 and 1.5.3.4 primary (cleaning twice in total) is repeated.
1.5.3.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.5.3.7. 22.5 μ l low TE are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min. at room temperature
1.5.3.8. it is placed on magnetic frame, (about 2min) takes 20 μ l into new PCR pipe after supernatant clarification.
1.5.4.AMPure beads purifies (MID connector):
1.5.4.1. plus in the PCR pipe after 60 μ l PEG/NaCl to above-mentioned reaction, sufficient vortex is mixed, incubation at room temperature
5min。
1.5.4.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.5.4.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.5.4.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.5.4.5. step 1.5.4.3 and 1.5.4.4 primary (cleaning twice in total) is repeated.
1.5.4.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.5.4.7. 50 μ l low TE are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min at room temperature.
1.5.4.8. plus in the PCR pipe after 60 μ l PEG/NaCl to above-mentioned reaction, sufficient vortex is mixed, incubation at room temperature
5min。
1.5.4.9. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.5.4.10. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec.
1.5.4.11. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.5.4.12. step 1.5.4.10 and 1.5.4.11 primary (cleaning twice in total) is repeated.
1.5.4.13. PCR pipe is removed from magnetic frame, centrifugation is placed on magnetic frame to draw remaining ethyl alcohol and do
Only, it uncaps at room temperature and dries magnetic bead, not overdrying.
1.5.4.14. 22.5 μ l low TE are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min at room temperature.
1.5.4.15. it is placed on magnetic frame, (about 2min) takes 20 μ l into new PCR pipe after supernatant clarification.
1.6 amplified library
1.6.1 following reaction system is prepared, be vortexed mixing and of short duration centrifugation:
Component | Volume |
2×KAPA HiFi HotStart ReadyMix | 25μl |
Reagent R1 | 5μl |
Connector connects purified product | 20μl |
Total volume | 50μl |
1.6.2 setting following procedure is reacted in PCR instrument:
1.6.3AMPure beads purifying:
1.6.3.1 plus in the PCR pipe after the magnetic bead to above-mentioned reaction of 90 μ l vortex mixing, sufficient vortex is mixed, and room temperature is incubated
Educate 5min.
1.6.3.2 PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.6.3.3 it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh is added, incubation at room temperature is at least
30sec.
1.6.3.4 ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.6.3.5 step 1.6.3.3 and 1.6.3.4 primary (cleaning twice in total) is repeated.
1.6.3.6 PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.6.3.7 25 μ l low TE are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min at room temperature.
1.6.3.8 it is placed on magnetic frame, (about 2min) takes 23 μ l into new PCR pipe after supernatant clarification.
1.7 library QC
1.7.3Qubit quantitative
1 μ l is taken to useDsDNA HS Assay Kit is quantitative.
Library total amount requires to reach 200ng or more.
1.7.4Qsep100 fragment analysis:
1 μ l is taken to be diluted to 0.1-1ng/ μ l, MB1-MA2 is that marker detects fragment size distribution.Library size requirements symbol
It closes and is expected.
1.7.5 library is placed in -20 DEG C and saves or carry out next step Library hybridization.
3 50 gene panel hybrid capture of embodiment
1.1. compatible Illumina platform experiment flow Library hybridization: is recommended using Integrated Device Technology, Inc. of the U.S.;
1.1.1. following reagent is added in 1.5ml low adsorption centrifuge tube, 45 DEG C are drained, and the sample after draining can continue
Hybridization or left at room temperature over night.
Component | Amount |
Mix library | 500ng |
Cot-1DNA | 5μg |
xGen Universal Blockers-TS Mix | 2μl |
1.1.2. probe hybridizes
1.1.2.1. following reagent is added into the centrifuge tube drained, is placed at room temperature for 5-10min, suction is gone to after playing mixing
In 0.2ml PCR pipe:
Component | Volume |
2×Hybridization Buffer | 8.5μl |
Hybridization Buffer Enhancer | 2.7μl |
H2O | 1.8μl |
1.1.2.2. be placed in PCR instrument, 95 DEG C of 10min, after removed from PCR instrument immediately and at once be added 4 μ l visit
Needle (50 gene Panel), is vortexed and centrifuged, and is placed in 65 DEG C of hybridized overnights in PCR instrument, and hot lid temperature is set as 75 DEG C.
1.2. reagent and magnetic bead prepare:
1.2.1. reagent prepares:, can be most in being placed at room temperature for according to following table (1 sample) by reagent dilutions to 1x working solution
4 weeks.
1 × Wash, I Buffer dispenses out 100 μ l and 1 × Stringent Wash Buffer with preceding in 65 DEG C of heat
It is at least placed on block 2 hours.
1.2.2. magnetic bead prepares:
1.2.2.1.M270 magnetic bead takes out from 4 DEG C of refrigerators, equilibrium at room temperature 30min.
1.2.2.2. be vortexed mix after take in 100 μ l/capture to 1.5ml low adsorption centrifuge tubes (one pipe can at most wash
600 μ l), it is placed on magnetic frame, until solution is clarified completely in pipe, carefully removes supernatant.
1.2.2.3. 200 μ l 1 × Bead Wash Buffer are added, suction is played mixing, is placed on magnetic frame, until in pipe
Solution is clarified completely, carefully removes supernatant;It is repeated once, cleans twice altogether.
1.2.2.4. 100 μ l/capture 1 × Bead Wash Buffer are added, score 100 μ l/ after mixing are made in suction
In capture to 0.2ml PCR pipe, be placed on magnetic frame, until solution is clarified completely in pipe, carefully remove supernatant, immediately into
Row.
1.3. it captures and cleans
1.3.1. the hybrid product in PCR instrument is shifted into the magnetic bead of previous step, and 10 mixings are played in suction, are placed in PCR instrument
65 DEG C of 45min (hot lid temperature is set as 75 DEG C) are every 12min vortex 3sec magnetic bead is resuspended.
1.3.2. plus 1 × Wash, I Buffer of 65 DEG C of 100 μ l preheatings is into above-mentioned PCR pipe, is vortexed and centrifuged, is transferred to
It in 1.5ml low adsorption centrifuge tube, is vortexed, is placed on magnetic frame until solution is clarified completely in pipe, carefully removes supernatant.
1.3.3. plus 65 DEG C of 200 μ l preheating 1 × Stringent Wash Buffer, suction beat mix not have bubble,
5min is placed on 65 DEG C of heat block, is placed on magnetic frame until solution is clarified completely in pipe, carefully removes supernatant.
1.3.4. it is primary to repeat 1.3.3.
1.3.5. plus 200 1 × Wash of μ l room temperature I Buffer, vortex 2min, it is placed on magnetic frame until solution is complete in pipe
Clarification, carefully removes supernatant.
1.3.6. plus 200 1 × Wash of μ l room temperature II Buffer, vortex 1min, it is placed on magnetic frame until solution is complete in pipe
Full clarification, carefully removes supernatant.
1.3.7. plus 200 1 × Wash of μ l room temperature III Buffer, vortex 30sec, it is placed on magnetic frame until solution is complete in pipe
Full clarification, carefully removes supernatant.
1.3.8. centrifuge tube is removed from magnetic frame, 20 μ l H is added2Mixing is played in O, suction.
1.4. library PCR is enriched with
1.4.1. following reaction system is prepared, mixing is played in suction, guarantee that magnetic bead is evenly dispersed in the solution:
1.4.2. setting following procedure is reacted in PCR instrument:
1.4.3.AMPure beads purifying:
1.4.3.1. plus in the PCR pipe after 75 μ l AMPure beads to above-mentioned reaction, sufficient vortex is mixed, and room temperature is incubated
Educate 5min.
1.4.3.2. PCR pipe is placed on magnetic frame, wait 3-5min until in pipe solution clarify completely, carefully remove
Supernatant.
1.4.3.3. it keeps PCR pipe on magnetic frame, 80% ethyl alcohol of 200 μ l Fresh, incubation at room temperature is added
30sec。
1.4.3.4. ethyl alcohol is carefully drawn and lost, magnetic bead is not encountered.
1.4.3.5. 1.4.3.3 and 1.4.3.4 primary (cleaning twice in total) is repeated.
1.4.3.6. PCR pipe to be removed from magnetic frame, centrifugation, which is placed on magnetic frame, draws remaining ethyl alcohol completely,
It uncaps at room temperature and dries magnetic bead, not overdrying.
1.4.3.7. 25 μ l low TE are added into centrifuge tube, magnetic bead is resuspended in sufficient vortex, places 2min at room temperature.
1.4.3.8. it is placed on magnetic frame, (about 2min) takes 23 μ l into new centrifuge tube after supernatant clarification.
1.5. library QC
1.5.1.Qubit quantitative
1 μ l is taken to useDsDNA HS Assay Kit is quantitative.
1.5.2.Qsep100 fragment analysis:
1 μ l is taken to be diluted to 0.1-1ng/ μ l, MB1-MA2 is that marker detects fragment size distribution.
1.5.3. library is placed in -20 DEG C of preservations.
4 Illumina sequencing of embodiment, information analysis, genetic counselling
1. sequencing:
ILLumina Nextseq500 platform is recommended to be sequenced, sequencing process refers to Illumina official manual;Tumour
Tissue samples recommend 2G clean data/ sample, and cfDNA sample recommends 4clean data/ sample, and blood check sample is recommended
0.5G clean data/ sample.Though the present invention is tested based on Illumina platform, its probe combinations and kit forms, can
To pass through the change of corresponding platform joint sequence, compatible multiple microarray datasets, the ion torrent system including ThermoFisher
Arrange (PGM, Proton, S5/S5XL etc.), Hua Da BGISEQ, MGISEQ etc.;
2. information analysis process (Fig. 2)
1) tumour and check sample initial data fastq after splitting to lower machine carry out connector respectively and go low quality sequence
Column analysis, using Trimmomatic default parameters, generates the fastq file of clean, and to original fastq and cleanfastq
The parameters such as reads and bases number and Q20 and Q30 are counted respectively;
2) the clean fastq file of tumour and control is compared using BWA mem method, file is distinguished after comparison
According to UMI information in fastq, UMI tag is added, and bam after comparison is ranked up and is constructed index with samtools;
3) bam file carries out PCR according to UMI tag and repeats after sorting, and the use of software is picard, PCR repetitive sequence
Directly excision counts the reads repetitive rate in sequencing sample without retaining, and comparison rate, on target rate is averagely covered
The quality control indexs such as lid depth and homogeneity;
4) tumour and check sample data file after completing duplicate removal carry out the copy number of somatic and germline respectively
The detection of variation, using software be GATK4CNV detection module, using database be identical experiment method and microarray dataset processing,
The baseline database that identical raw letter analysis method is handled;
5) bam file after going PCR to repeat using GATK3.7 carry out indel it is local compare again with base mass calibration, and
The statistics of the indexs such as intubating length and base error rate is carried out to the bam file after calibration;
It 6) is to be carried out using the Mutect2 function of GATK3.7 to the bam file of tumour and control after the completion of correction
The detection in the mutational site somatic and the HaplotypeCaller function of GATK3.7 carry out the inspection in the mutational site germline
It surveys.
7) to base quality correction after control bam file using GATK3.7 UnifiedGenotyper and
The genotype that HaplotypeCaller function carries out specific chemotherapy site determines.
8) CAVA, annovar are used respectively to the mutational site the somatic and germline message file vcf detected
And self-built background database is annotated, then the screening of frequency and depth etc., result after screening are carried out to each mutational site
It can carry out medicine interpretation.
3. data are interpreted:
Analyze many-sided clinical meaning such as AR drug resistance/targeted drug/chemotherapeutics/hereditary tumor/dynamic monitoring;Specifically
Process is as follows:
Mutation is divided into detrimental mutation and the unknown mutation two major classes of meaning in 3.1 raw letter comment files.
Detrimental mutation includes:
A) frameshift mutation of tumor suppressor gene, stop mutation, Loss-of-function point mutation and indel, there is clear physiology to anticipate
The missing of justice;
B) the function activation type point mutation of proto-oncogene and indel, the amplification for having clear physiologic meaning.Wherein frameshit and
The information that stops mutation can be obtained from the column of the ExonicFunc.refGene in comment file, be needed through internal database, CKB
Database, CIVic database, consulting literatures determine the afunction or function activation information of point mutation and indel.
3.2 for detrimental mutation, needs to carry out relevant clinical meaning parsing.Mutation unknown for meaning, needs basis
The clinical research of gene-correlation corresponding portion in report prompts.
3.3 pairs detect mutation and carry out clinical meaning parsing
Gene relevant to abiraterone, the miscellaneous Shandong amine curative effect of grace has:
AR | ATM | BRCA2 | TP53 | SPOP |
Gene relevant to PARPi curative effect has:
Gene relevant to platinum-based chemotherapy drug has:
ATM | ERCC3 | MLH3 | POLD1 |
ATR | ERCC4 | MRE11A | POLE |
BARD1 | ERCC5 | MSH2 | RAD50 |
BRCA1 | FAM175A | MSH6 | RAD51 |
BRCA2 | FANCA | NBN | RAD51B |
BRIP1 | FANCD2 | PALB2 | RAD51C |
CHEK2 | GEN1 | PMS1 | RAD51D |
ERCC2 | MLH1 | PMS2 |
Genetic correlation gene has:
AR | BRCA2 | EPCAM | ESR1 | MLH1 |
ATM | BRIP1 | ERCC2 | FAM175A | MSH2 |
ATR | CDH1 | ERCC3 | FANCA | MSH6 |
BARD1 | CDK12 | ERCC4 | FANCD2 | PMS2 |
BRCA1 | CHEK2 | ERCC5 | HOXB13 |
Gene involved in immunity
CDK12 | MLH1 | PMS2 |
MSH2 | MSH6 | EPCAM |
4. generating report: providing genetic counselling and interpret report.
Embodiment 5
Objects, technical solutions and advantages in order to better illustrate the present invention, it is special for example: an example mCRPC patient, nothing
Method operation obtains tumor tissues, to carry out the accurate molecule parting of patient to instruct therapeutic regimen to select, using of the invention
Kit is detected: process as shown in Figure 1, with specific reference to the above embodiments,
1. acquiring peripheral blood carries out the biopsy of ctDNA liquid, ctDNA total amount 30ng;
2. building library, it is sequenced, analysis is sequenced by Illumina platform, is interpreted, is obtained as follows by raw letter analysis and heredity
Data result:
Other genes are feminine gender.
By genetic test, we expand according to AR, and TP53/RB1 exists simultaneously systematic mutation prompt patient, and to receive AR short of money
Anti-agent curative effect is poor;BRCA1 heterozygous lacks germline mutation, and patient can benefit from PARP inhibitor such as olaparib;
Since patient's BRCA1 heterozygous missing is germline mutation, there are still the risks for suffering from other tumours by he or she.
By analyzing above;Kit of the invention has the following technical effect that:
1. sensitivity is sufficiently high (0.1%), the patient population for that can not obtain tissue can also have an opportunity to obtain gene
It detects (liquid biopsy);
2. known or unknown mutation can be detected simultaneously, all possible mutation type is covered;
3. first is directed to the polygenes PANEL of mCRPC, integration solves analysis of drug use, and carries out full range management.
4. carrying out comprehensive analysis based on the variations all kinds of for gene of ctDNA technology, it is based especially on multiple noise reduction technology
Copy number variation (CNV) analysis;
5. one-time detection can have AR drug resistance/targeted drug/chemotherapeutics/hereditary tumor/dynamic monitoring etc. more simultaneously
Aspect clinical meaning, may to patient's benefit as much as possible.
Claims (2)
1. a kind of for detecting the gene probe composition of metastatic castration-resistant prostate cancer, it is characterised in that: by ATM base
Because of probe, ATR gene probe, AR gene probe, BARD1 gene probe, BRCA1 gene probe, BRCA2 gene probe, BRIP1
Gene probe, CDH1 gene probe, CDK12 gene probe, CHEK2 gene probe, EPCAM gene probe, ERCC2 gene are visited
Needle, ERCC3 gene probe, ERCC4 gene probe, ERCC5 gene probe, ESR1 gene probe, FAM175A gene probe,
FANCA gene probe, FANCD2 gene probe, FOXA1 gene probe, GEN1 gene probe, HDAC2 gene probe, HOXB13
Gene probe, IDH1 gene probe, MLH1 gene probe, MLH3 gene probe, MRE11A gene probe, MSH2 gene probe,
MSH6 gene probe, MUTYH gene probe, NBN gene probe, NCOR1 gene probe, NCOR2 gene probe, PALB2 gene
Probe, PMS1 gene probe, PMS2 gene probe, POLE gene probe, POLD1 gene probe, PTEN gene probe, RAD50
Gene probe, RAD51 gene probe, RAD51B gene probe, RAD51C gene probe, RAD51D gene probe, SPOP gene
Probe, STK11 gene probe, TP53 gene probe, XRCC4 gene probe, ZBTB16 gene probe and RB1 gene probe group
At.
2. a kind of for detecting the kit of metastatic castration-resistant prostate cancer, it is characterised in that: contain claim 1 institute
The gene probe composition stated.
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CN114364816A (en) * | 2020-03-23 | 2022-04-15 | 皇家飞利浦有限公司 | Prediction of radiotherapy response to prostate cancer subjects based on DNA repair genes |
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