CN109652525A - Pulmonary thromboembolism gene panel kit and its application - Google Patents
Pulmonary thromboembolism gene panel kit and its application Download PDFInfo
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Abstract
The present invention relates to biomedicine technical fields, and the liquid phase gene panel (Panel) for specifically providing a kind of detection pulmonary thromboembolism related gene captures kit and its application, and it includes capture probe which, which captures kit, covers 92 genes.The design synthesis of gene panel capture kit of the invention pulmonary embolism is multi-level, various inherent causes, covering is comprehensive, its target acquistion has obvious specific aim, Genetic Detection can be carried out to the gene of larger flux simultaneously, efficiently concentrating is carried out by the target gene exon 1 domain dna to people, by subsequent sequencing, the sequencing depth and coverage of gene can be effectively improved, is provided safeguard to obtain more accurate gene genetic information and result.
Description
Technical field
The present invention relates to biomedicine technical fields, specifically provide a kind of liquid phase gene for detecting pulmonary thromboembolism gene
Panel (Panel) captures kit and its application.
Background technique
Pulmonary thromboembolism (pulmonary thromboembolism, PTE) refers to the blood from venous system or the right heart
Bolt blocks disease caused by pulmonary artery or its branch, using pulmonary circulation and respiratory dysfunction as main clinical manifestation and pathologic, physiologic
Feature.PTE is a kind of disease of multifactor initiation, and pathogenic factors includes disturbance of blood coagulation in venous system, environment and inherent cause.
It is mainly shown as blood circulation system thrombosis.It has been reported that the disease incidence of the U.S. DVT and PTE are in cardiovascular disease
Third position is accounted for, coronary heart disease and hypertension are only second to.Currently, China's PTE epidemiology lacks large sample statistics, but according to one
The item discovery of clinical data retrospective analysis in 32 years, China's PTE disease incidence are rising year by year, and have familial aggregation.Without controlling
The PTE mortality for the treatment of is about 30%, and after correct diagnoses and treatment, case fatality rate is 3% or so.The treatments such as anticoagulant and thrombolysis are most
It establishes after PTE generation, drug therapy is easy to recur there is also drug resistance or bleeding risk, and after being discontinued.Due to PTE clinic
Symptom specificity is unobvious, and disease prenatal diagnosis rate is lower than 50%.Therefore finding in time and correctly diagnose and treat can be effectively
Reduce mortality risk.More accurately the new method of medical diagnosis on disease or detection has important practical significance for exploitation.
PTE inherent cause accounts for 60% or more, and blood coagulation system genetic predisposition is pulmonary embolism pathogenic factors the most main
One of.PTE thrombosis and the genetic mutations such as blood coagulation or anticoagulant gene PROC, SERPINs, PROS are closely related, in Chinese
There is the genetic mutation site of specificity in group.Heredity deficiency of anticoagulant proteins is the Etiological of PTE.With the depth of research
Enter, a large amount of genetic virulence gene has been reported the close phase of onset risk, clotting defect or drug therapy of confirmation and PTE
It closes.
The detection of current conventional pulmonary embolism risk or pathogenic factors is largely by Elisa (blood coagulation four etc.), base
Because the genotyping techniques such as detection chip or PCR detect, detection speed is slow, low efficiency, at high cost, and the flux of detection is lower,
Lots of genes can not be detected simultaneously, while being detected various indexs and being needed through multiple technologies means.
Therefore a kind of high throughput, fast and accurately the genetic virulence factor of detection pulmonary embolism and drug resistance etc. are developed
The method of factor has important science and realistic meaning.High throughput, accurately and effectively Genetic Detection means simultaneously, can be PTE
The individual character chemoattractant molecule diagnosis and treatment of genetic mutation individual, anticoagulant or thrombolysis medication provide guidance, improve disease cured rate, reduce morbidity
Rate, mortality risk.
Summary of the invention
The embodiment of the present invention is designed to provide a kind of pathogenic gene, clotting defect base for detecting pulmonary thromboembolism
Liquid phase gene panel (panel) the capture kit of cause, clinical drug-resistant gene, can be used for the essence of pulmonary thromboembolism inherent cause
Quasi- detection.Method of the invention is based on target area paramagnetic particle method liquid phase capture systems and full exon two generations sequencing technologies, obtains
The full exon sequence information of target gene for pulmonary thromboembolism is obtained, gene panel carries out hereditary information through the invention
Capture and be sequenced on a large scale, the gene panel can be used for the pulmonary embolism group of familial and high risk genetic risk assessment,
Disease prevention, genetic counselling, and reference is provided for diagnosis and clinical application.
The liquid phase gene panel capture kit of detection pulmonary thromboembolism provided in an embodiment of the present invention includes following mesh
Mark the corresponding probe of gene:
(1) gene relevant to thrombotic risk factor
(2) gene relevant to blood coagulation
(3) gene relevant to drug resistance
, wherein each gene at least has, there are two corresponding probe sequences.
Preferably, the probe includes at least SEQ ID NO 1-SEQ ID NO 184.
It is further preferred that the liquid phase gene panel of detection pulmonary thromboembolism provided in an embodiment of the present invention captures reagent
Box, is liquid-phase chip, which includes the probe and Streptavidin of corresponding biotin labeling (in 5 ' end labels)
Magnetic bead.
Mentioned reagent box provided in an embodiment of the present invention can be used for detecting the gene mutation of gene panel member, for clinic
Diagnosis and medication provide reference and guidance.
Implementation of the invention includes the pre-treatment experiment skill of gene panel chip capture cartridge system and sample
The platform of art and subsequent data analysis, the detection genetic mutation information obtained can be used for disease prevention, genetic counselling, instruct
Clinical application selection and dosage determine.
The present invention is based on the inherent cause researchs to pulmonary embolism group to utilize biology in conjunction with mutational site has been reported at present
The capture probe of bioinformatics analysis, design object gene, and pass through Integrated DNA Technologies platform, screening
The genetic virulence gene order of pulmonary embolism high risk is obtained, entire capture chip and gene panel are designed.The panel can be to lung
The relevant risk genes F5 of embolism, PROTEIN C, Protein S and a series of coagulation factor full coding exon region carry out mesh
Mark capture, obtains target gene sequence, then carries out the sequencing of two generations to the target area sequence of acquisition, and utilize bioinformatics
The genetic mutation site for analyzing its carrying annotates its genetic locus and causes a disease, the correlation of clinical application.
Gene panel of the invention combines the inherent cause of the multi-level aspect of pulmonary embolism, and covering is comprehensive, target acquistion
The sequencing depth of gene can be significantly improved, is provided safeguard to obtain more accurate sequencing result.Meanwhile two generation sequencing technologies
Panel platform is mature, can be used as Pulmonary Embolism Patients and carries out hereditary inspection, and the clinical treatment of risk assessment and drug resistance crowd refer to
It leads.
Detailed description of the invention
Fig. 1 is that amplification library DNA analyzes peak figure.
The detection kit that Fig. 2 is the gene panel of pulmonary thromboembolism provided in an embodiment of the present invention examines certain patient
The verification result that gene Sanger is sequenced after survey, wherein SERPINE2 has mutation.
Fig. 3 is the detection kit of the gene panel of pulmonary thromboembolism provided in an embodiment of the present invention to another patient
The verification result that gene Sanger is sequenced after detection, wherein SERPINE2 is without mutation.
Fig. 4 is the detection kit of the gene panel of pulmonary thromboembolism provided in an embodiment of the present invention to another patient
The verification result of gene backward sequencing after detection, wherein PROC has mutation.
Fig. 5 is the detection kit of the gene panel of pulmonary thromboembolism provided in an embodiment of the present invention to another patient
The verification result of gene backward sequencing after detection, wherein PROC is without mutation.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Accompanying drawings and embodiments, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.
The embodiment of the present invention provides a kind of liquid-phase chip capture reagent for detecting the gene panel of pulmonary thromboembolism
Box, mainly for detection of 92 genes as described above, these genes form said gene panel.
Specifically, above-mentioned detection includes the steps that hybridizing target gene by probe, that is, the kit includes
The corresponding probe of above-mentioned 92 genes, each gene at least correspond to 2 probes, and probe sequence is at least 1-SEQ of SEQ ID NO
ID NO 184。
Oligonucleotide probe is marked, synthesizes at 5 ' ends using biotin, and probe passes through base pair complementarity and piece
The genome dna library of sectionization hybridizes, then pure with Streptavidin MagneSphere (Dynabeads Myone Streptavidin T1)
Change, which pulls down target area, to be enriched with.Detecting probe information based on biotin labeling passes through the base of patients with pulmonary thromboembolism
Because the liquid phase magnetic capture system of mutation effectively captures the target area of target gene, it is sequenced by subsequent two generation, it can
For detecting the genetic mutation of gene in Panel.
What the concentration of above-mentioned probe and the content of Streptavidin MagneSphere were well known to those skilled in the art, strepto- is affine
Biscuit porcelain pearl is commercially available.
Above-mentioned 92 genes are gene panel of the invention essential.
Gene panel is an assortment of genes, and all codings of the detected gene of gene panel are used in genetic test
Genetic locus, than round pcr detection gene information more comprehensively, completely, comparatively, the gene information amount of acquisition is bigger.People
Intracorporal gene has the gene of a coding protein more than 20,000, although also there is not coding protein, people disease occur and
The base quantity of the gene to play a significant role in inborn potential, the gene of people is up to 6,400,000,000, is carrying out gene with gene panel
When detection, core point is how to select in the assortment of genes and disease risks assessment, diagnosis and treatment-related gene and base
Because how information and each clinical, diagnosis and treatment factor correlation illustrate.
The detection probe of said gene panel (panel) provided by the invention can be lacked by discovery gene mutation and gene
It falls into, the clinic for being consequently for carrying out genetic counselling, risk assessment and drug resistance crowd for Pulmonary Embolism Patients high-risk patient is controlled
It treats and Genetic Detection information is provided, instruct clinical diagnosis and treatment etc., these purposes are mainly by liquid phase gene of the invention
Panel kit effectively captures target gene, and testing result carries out analysis acquisition.
Gene Panel of the present invention is calculated by independent research carries out probe design, in total 92 target genes, includes 1142
A target area, region segments size 205.1kbp；It amounts to probe and is greater than or equal to 184, each gene at least designs two
Probe preferably covers 99.5% region；The recommendation minimum sequencing data amount that the present invention is applied to sample sequencing is 57Mbp/ sample
Product.
In kit of the invention probe design based on human data library H.sapiens (UCSC hg19, GRCh37,
February 2009)；Target area probe and location information are based on database: CCDS, Ensembl, Gencode, RefSeq.
Detecting probe information is as shown in table 1 below:
1 probe of table designs relevant information
Inventor captures and is sequenced using above 92 gene panel, the specific steps are as follows:
1 genome DNA sample of embodiment extracts
Venous blood is carried out to subject using QIAamp DNA blood mini kit (QIAGEN, Cat:Y5-51106)
The extraction and purifying of DNA, the specific steps are as follows:
1. extracting vein blood, 1000g is centrifuged 15min under room temperature, draws the haemocyte of 1ml, adds DNA lysate 3ml, vortex shake
It swings after instrument mixes well, marks name and date.
2. drawing 20 μ l Proteinase Ks to the bottom of 1.5ml centrifuge tube.
200 μ l blood samples are added into centrifuge tube, add 200 μ l Buffer AL into sample, vortex oscillation 15s
It mixes.
3. rapid centrifugation, removal remains in the drop in 1.5ml centrifuge tube lid after 56 DEG C of incubation 10min.
4. the dehydrated alcohol of 200 μ l is added, vortex oscillation 15s is mixed.After oscillation, rapid centrifugation turns mixture
QIAamp Mini centrifugation tubing string is moved to, 6000g is centrifuged 1min.
5. QIAamp Mini centrifugal column is put into a new clean 2ml reception pipe, the receipts that filtrate was used in connection with
Collector abandons.
6. carefully opening QIAamp Mini centrifugal column, 500 μ l Buffer AW1,6000g are added and are centrifuged 1min.It repeats to walk
Rapid 5.
7. opening QIAamp Mini centrifugal column, 500 μ l Buffer AW2,20000g are added and are centrifuged 3min.
8. QIAamp Mini centrifugal column is transferred to a new 1.5ml collecting pipe, 200 μ l Buffer AE are added.Room
Temperature is incubated for 1min, and then 6000g is centrifuged 1min.
9. Concentration Testing: purity is generally advisable with OD260/280=1.8~2.0.
The full exon group capture of 2 target gene of embodiment, library construction
Probe sequence is designed and synthesized, as shown in SEQ ID NO.1 to SEQ ID NO.184, and is marked with biotin, is made
Standby gene Panel liquid-phase chip.
By sample acquired in embodiment 1 through full exon genes panel liquid-phase chip system acquisition of the invention, inspection
It surveys, obtains the target area library result captured before sequencing.
Using gene panel liquid-phase chip system of the invention, to the full exon 1 of the target gene DNA of embodiment 1
Domain dna carries out efficiently concentrating, and high-throughput, high depth sequencing is then carried out on Illumina Hiseq platform.Build library and capture
Experiment uses gene panel liquid-phase chip system of the invention, selected matched reagent and consumptive material to be provided by corresponding to supplier,
Operation is carried out by following experiment flow and specification adjustment suitable scheme using being grasped after experiment flow step is optimized
Make.
It is following (note: following English name is the title on specification) to test basic procedure:
One .DNA sample preparation:
Start Covaris and be crushed instrument, chiller temperature is set between 2 DEG C to 5 DEG C, it is ensured that the temperature reading in water-bath
It is shown as 5 DEG C.It detects and determines gDNA sample concentration.With 1x Low TE Buffer in 1.5-mL LoBind pipe, by 200ng
It is 50 μ L that gDNA, which is diluted to total volume, and micro-pipe is fixed in the bracket, and instrument parameter is arranged by table 2, gDNA is cut into length
For the segment of 150-200bp.
2 Covaris of table shearing setting (SonoLab software v7)
Two, library constructions:
It repairs 2.1 ends: preparing end and repair mix reagent, as shown in table 3, whole process operates on ice, vortex mixer
Upper mixing.To guarantee reaction sufficiently, every step reaction should all prepare excess reagent.
The preparation of 3 end of table reparation mix reagent
Every hole is added 52 ends μ L and repairs mix reagent and 50 μ L DNA shearing segment in 2.2 PCR plates, mixes well.
PCR plate is incubated for 30 minutes at 20 DEG C.
2.3 Sample Purification on Single: it after so that magnetic bead is incubated at room temperature 30 minutes, mixes, is added in the DNA repaired through end, PCR
180 μ L are added in the every hole of plate, draw suction 10 times and mix well.Incubation at room temperature after five minutes, then by plate is put into 5 in magnetic sorting apparatus
Minute.Plate is transferred on magnetic bracket, 70% ethyl alcohol that 200 μ L newly match is added in each sample, after standing 1 minute, is gone
Except ethyl alcohol.The step of addition ethyl alcohol and removal ethyl alcohol, is repeated once.Sample is maintained at 37 by the drying sample on thermo cycler
DEG C, continue 3 to 5 minutes, until residual ethanol evaporating completely, the water for adding 32 μ L nuclease frees (nuclease-free) is arrived
It in each sample, mixes, is incubated for 2 minutes at room temperature.Plate is placed in magnetic bracket, is placed 3 minutes, until solution clarification is
Only.Then supernatant (about 30 μ L) is moved into fresh PCR plate, discards magnetic bead.
2.4 end DNA fragmentation 3' polyadenylations: preparing Adenylation master mix using table 4 on ice, vortex
It is mixed on mixer, to the end DNA fragmentation 3' polyadenylation.
The preparation of 4 Adenylation Master mix of table
Adenylation Master is added in the DNA sample after purification repaired through end obtained into step 2.3
Mix, every 20 μ L of hole are incubated for 30 minutes for 37 DEG C in thermo cycler.
2.5 repeat step 2.3 once.
2.6 connection two end connectors of DNA fragmentation: the end 3' of A, B connector is connected to DNA fragmentation both ends by ligase.A, B connects
Head is all the double stranded oligonucleotide of 44bp, and wherein B connector 5' has the probe sequence of biotin, for purifying in next step.It presses
The dilution proportion of nuclease-free water:Oligo Mix=1:10, it is ready-to-use, as table 5 prepares connection master on ice
Mixed liquor (Ligation master mix) mixes on vortex mixer.
The preparation of 5 Ligation master mix of table
Ligation master mix is added in the DNA sample after purification of the addition A tail obtained into step 2.5, often
37 μ L of hole is incubated for 15 minutes for 20 DEG C in thermo cycler.
2.7 repeat step 2.3 once.
Three amplified libraries:
3.1 prepare pre-capture PCR reaction mixture as shown in table 6, and whole process is operated on ice, mixed on vortex mixer.
To guarantee reaction sufficiently, every step reaction should prepare excess reagent.
6 pre-capture PCR reaction mixture of table
Addition pre-capture PCR reaction mixture, every 20 μ L of hole, by table in DNA sample after purification are obtained into step 2.7
7 operation pre-capture PCR thermocycling programs.
7 pre-capture PCR thermocycling program of table
3.2 to repeat steps 2.3 primary, collect supernatant for capturing, hybrid experiment.
3.3 Library Qualities and quantity assessment: sample is carried out using 2100Bioanalyzer and 1000 instrument of DNA
Product analysis.Result is analyzed as shown in Figure 1, verifying electrophorogram shows that DNA fragmentation size is about 225
There is peak value when to 275bp.The concentration of peak lower integral method detection library DNA.
Four, hybridize and capture
DNA sample is hybridized to probe by 4.1, preparation capture library:
Amplified library products therefrom is used for subsequent probes hybridization reaction.Hybridization reaction needs sample about 750ngDNA.
4.1.1 it is higher than the pretreatment library of 221ng/L for DNA concentration, is diluted to 221ng/ μ L, takes 3.4 μ L volumes standby
With.
4.1.2 it is lower than the pretreatment library of 221ng/L for DNA concentration, it is using vacuum concentrator that sample is dense at 45 DEG C
Contracting, then it is molten with the water weight of nuclease free, make its ultimate density 221ng/L.After being mixed on vortex mixer, in centrifuge
Rotation 1 minute.
4.1.3 the library gDNA sample (750ng) of each 3.4 μ L is transferred to 96 orifice plates, is put on ice for protecting after sealing
It deposits.
4.1.4 the hybridization buffer of capture probe (is provided) to rewarming by supplier in advance.If there is precipitating, will hybridize
Buffer rewarming 5 minutes in 65 DEG C.Hybridization buffer is placed at room temperature using preceding.
4.1.5 each reagent is mixed as shown in table 8, preparation closing mixed liquor.The solution prepared is put on ice using preceding
It sets.
The preparation of the closing mixed liquor of table 8
4.1.6 96 orifice plates containing the library gDNA sample ready in step 4.1.3 are taken out, it is mixed that closing is added in every hole
Close 5.6 μ L of liquid.Suction pipe mixes up and down.
4.1.7 then it will be transferred to thermo cycler after 96 pore plate by sealing, 95 DEG C incubation 5 minutes in thermo cycler, then
5 minutes or more are kept the temperature in 65 DEG C.
4.1.8 appropriate diluted confining liquid is prepared according to the size in capture library.To guarantee reaction sufficiently, in preparation process
In, prepare excessive RNase confining liquid.Confining liquid is using being preceding put on ice for saving.
4.1.9 capture library mixed liquor is prepared according to table 9, saved backup at room temperature, middle probe synthesis is complete by supplier
At, and the probe provided is uniform concentration.
9 probe of table captures the preparation of library mixed liquor
4.1.10 Library hybridization mixed liquor will be captured to be added in 96 orifice plates in step 4.1.7,25 μ L are added in every hole.With suction
Pipe aspirates 8 to 10 mixings.
4.1.11 by after 96 pore plate by sealing, 65 DEG C are incubated for 16 to 24 hours.
4.2. the capture of hybrid product Streptavidin MagneSphere, purifying
The preparation of Streptavidin coating magnetic bead:
4.2.1 in circulator bath, 65 DEG C of preheating Wash Buffer 2 are spare.
4.2.2 Dyabead MyOne Streptavidin T1 magnetic bead is mixed energetically on turbine mixer.
4.2.3 using in new PCR plate, dosage is determined according to Hybridization samples number, each every hole of sample is added 50 μ L and is resuspended
Floating pearl.
4.2.4 it cleans magnetic bead: 200 μ l Binding Buffer is added, suction mixes, until pearl suspends again completely,
PCR plate is put into concentration equipment.After solution clarification, takes out and abandon supernatant.It is repeated two more times, washs 3 times in total.
4.2.5 magnetic bead is precipitated again in the Binding Buffer of 200 μ L.
4.3. hybrid dna is captured with Streptavidin coating pearl
4.3.1 estimate and record the volume of remaining hybridization solution after incubation in 24 hours.
4.3.2 the whole volume (about 25 to 29 μ L) of each hybridization mixture is transferred in use multi-channel pipette and is contained
When the PCR plate for the Streptavidin pearl for there are 200 μ L washed, PCR plate is maintained at 65 DEG C, slowly suction up and down, Zhi Daozhu
Son is thoroughly mixed.
4.3.3 it after sealing PCR plate, mixes and is incubated on 96 orifice mixers, be vigorously mixed at room temperature for (1400-
1800rpm) 30 minutes, it is ensured that sample is sufficiently mixed.
4.3.4 after simple centrifugation, plate is put into concentration equipment and collects magnetic bead.After solution clarification, takes out and abandon supernatant
Liquid.
4.3.5 the beads spun again in the Wash Buffer 1 of 200 μ L.Suction up and down, until pearl hangs again completely
It is floating.
4.3.6 after sample being incubated at room temperature 15 minutes, it is primary to repeat step 4.3.4.
4.3.7 magnetic bead is cleaned with Wash Buffer 2: magnetic is resuspended in the Wash Buffer 2 for the 200 μ L that 65 DEG C preheat
Pearl.Suction is until pearl suspends completely up and down.65 DEG C of samples of incubation plates after ten minutes, are put into concentration equipment on thermo cycler.
After equal solution are limpid, supernatant is removed.It washs 3 times altogether, it is ensured that Wash Buffer is completely removed in last operation.
4.3.8 30 μ L nuclease-free waters are added in each sample well, suction mixes, and magnetic bead is resuspended, and is placed in spare on ice (use
In step 5.1.5).
Five, sample Indexing and capture after the library PCR
5.1 libraries captured with indexing primers amplification
In this step, DNA library is amplified in PCR reaction, includes the indexing for being suitble to each sample in PCR reaction
primer。
5.1.1 index appropriate is distributed for each sample.Obtain the 8bp for DNA amplification library in this step
The partial sequence of indexing primers of the Indexes from A01 to H12.Using different on same lane
Each sample is sequenced in indexing primers.
5.1.2 the PCR reaction mixture for preparing proper volume is prepared on ice as shown in table 10, is mixed on vortex mixer
It closes.
The preparation of PCR reaction mixture after table 10 captures
5.1.3 in new PCR plate, PCR reaction mixture after 31 μ L capture is added in each sample well.
5.1.4 different indexing primer is chosen, 5 μ L are added in every hole into the PCR plate in step 5.1.3.
5.1.5 DNA library sample is added in PCR reaction: is taken out from ice and contains 30 μ L enriched target DNA Magnetic bead samples
PCR plate (being obtained in step 4.3.8), will each DNA sample up and down aspirate until pearl suspension it is uniform, then by the sample of 14 μ L
Product are transferred in the PCR plate containing PCR reaction mixture and indexing primer, are mixed.Remaining Magnetic bead sample is stored
At -20 DEG C in case using in the future.
5.1.6 PCR plate is transferred to thermo cycler, and PCR amplification program shown in table 11.
PCR amplification program after table 11 captures
5.1.7 after PCR EP (end of program), PCR plate is simply rotated.
5.2 Sample Purification on Single: after so that magnetic bead is incubated at room temperature 30 minutes, mixing, each hole for containing 50 μ L DNA amplification samples
90 μ L of middle addition clean magnetic bead, aspirate 10 times and mix well.Incubation at room temperature after five minutes, until solution is limpid, discards in every hole
Upper layer clear liquid.Plate is put into magnetic sorting apparatus 5 minutes again.Plate is transferred on magnetic bracket, is added in each sample
Enter 70% ethyl alcohol that 200 μ L newly match, after standing 1 minute, removes ethyl alcohol.The step of addition ethyl alcohol and removal ethyl alcohol, is repeated once.
Sample is maintained at 37 DEG C, continues 3 to 5 minutes by the drying sample on thermo cycler, until residual ethanol evaporating completely, then plus
Enter the water of 32 μ L nuclease frees (nuclease-free) into each sample, mix, is incubated for 2 minutes at room temperature.Plate is placed on magnetic
Property bracket in, place 3 minutes, until solution clarification until.Then supernatant (about 30 μ L) is moved into fresh PCR plate, discards magnetic
Pearl.
5.3 Library Qualities and quantity assessment, operating process such as step 3.3
5.4 adjust the amount of sample cells and ultimate density to proper states.
Six, upper machine sequencing
Library inspection is qualified, carries out the sequencing of Illumina Hiseq platform according to the effective concentration in library and data output demand.
Seven, data analysis and processing
It is analyzed by comparing, bioinformatics sequence, individuation data filtering and interpretation；It is final to determine target gene
In there is pathogenic SNP site, including risk site, with drug therapy dosage related locus etc..
Eight, the accuracy of Sanger sequence verification Panel testing result
The positive site that detection is sequenced after capturing by gene panel is chosen, detects lung thrombus bolt as Panel of the present invention
Plug disease hereditary information effective is as a result, and design the reliability in corresponding primer combined authentication site.
3 Application Example of embodiment
Using pathogenic sites information embodiment 2 gene panel liquid-phase chip system acquisition and detected, prepare corresponding
Primer combination, verification result.
The following table 12 is one corresponding to the pathogenic sites information that gene panel is captured and is detected in embodiment of the present invention
Group primer sequence:
12 primer sequence of table
The design of above-mentioned primer is that those skilled in the art are manageable using existing tool.It is tested using primer pair
The DNA of person carries out amplification and transfers corresponding gene order and be sequenced.Gene magnification and gene order sequencing are art technology
Personnel are known.The gene mutation situation of patient is analyzed according to sequencing result, the capture of target area is sequenced in verifying confirmation Panel
As a result accuracy.Fig. 2-5 is shown using kit (for the gene panel of pulmonary thromboembolism) of the invention to patient
Part sequence verification after corresponding gene magnification is as a result, it is mutational site that wherein arrow, which is directed toward site,.Wherein Fig. 2 is shown
SERPINE2 (gene relevant to thrombotic risk factor) has the case where mutation, and Fig. 3 is shown what SERPINE2 was not mutated
Situation；Backward sequencing is shown by Fig. 4 as a result, wherein PROC (gene relevant to blood coagulation) has mutation；Fig. 5 is shown reversely
Sequencing as a result, wherein PROC is not mutated.
The present invention provides a kind of gene panel kits (liquid-phase chip system) for pulmonary thromboembolism, utilize
Liquid phase gene panel capture kit can capture the exon region of target gene, can use capture systems acquisition
The hereditary information of gene completes the risk assessment of gene mutation, clotting defect then by sequencing analysis gene mutation situation
The hereditary information of genetic virulence assessment and medication related gene detects, and provides reference for genetic counselling, risk profile and medication
With guidance.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. a kind of liquid phase gene panel for detecting pulmonary thromboembolism captures kit, the probe including being directed to following target gene
Sequence:
(1) gene relevant to thrombotic risk factor
(2) gene relevant to blood coagulation
(3) gene relevant to drug resistance
,
Wherein at least there are two corresponding probe sequences for each gene.
2. liquid phase gene panel according to claim 1 captures kit, which is characterized in that the probe sequence at least wraps
Include 1-SEQ ID NO 184 of SEQ ID NO.
3. liquid phase gene panel according to claim 1 captures kit, which is characterized in that the kit is liquid phase core
Piece, the liquid-phase chip further include Streptavidin MagneSphere.
4. liquid phase gene panel according to claim 1 captures kit, which is characterized in that the end of probe 5 ' is marked with
Biotin.
5. the liquid phase gene panel that 1-SEQ ID NO 184 of probe sequence SEQ ID NO is used to prepare detection pulmonary thromboembolism
Kit purposes.
6. purposes according to claim 5, which is characterized in that the end of probe sequence 5 ' is marked with biotin.
7. purposes according to claim 5, which is characterized in that the kit is liquid-phase chip, and the liquid-phase chip is also
Including Streptavidin MagneSphere.
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| CN114410766A (en) * | 2021-11-24 | 2022-04-29 | 广州知力医学诊断技术有限公司 | Detection panel for thrombus and hemorrhagic coagulation diseases and application thereof |
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