CN114410766A - Detection panel for thrombus and hemorrhagic coagulation diseases and application thereof - Google Patents

Abstract

The application discloses a detection panel for thrombus and hemorrhagic coagulation diseases and application thereof. The detection panel for the thrombus and the hemorrhagic coagulation diseases comprises a specific hybridization probe for detecting or hybridizing 86 genes related to the thrombus and the hemorrhagic coagulation diseases. The thrombus and procoagulant disease detection panel can be used for detecting various gene structures related to thrombus and procoagulant, is helpful for explaining molecular mechanisms of thrombus and procoagulant disease, and provides a comprehensive framework and basis for developing researches on procoagulant disease functionality, hereditary family verification, pathogenesis molecular mechanisms and the like. The gene information covered by the panel is comprehensive, various thrombi and coagulation diseases can be directly subjected to combined detection, the panel is applied to the companion diagnosis of the coagulation diseases, and the obtained accurate target sequencing result can accurately evaluate the genetic risk, the recurrent risk, the disease risk and the like of the thrombi and the coagulation diseases.

Description

Detection panel for thrombus and hemorrhagic coagulation diseases and application thereof

Technical Field

The application relates to the field of gene detection, in particular to a detection panel for thrombus and hemorrhagic coagulation diseases and application thereof.

Background

High throughput sequencing technology (NGS), also known as Next Generation sequencing. The sequence determination can be carried out on hundreds of thousands to millions of DNA molecules at a time, and the core idea is that the sequence is determined by capturing the label of the newly synthesized end while synthesizing and sequencing. The method has the advantages of high output, high resolution and the like, can quickly read the sequence, provides abundant genetic information, and can also play a role in shortening the sequencing time. NGS technology can provide the scalability, speed and resolution needed to meet the needs of evaluating targeted genes of interest, can simultaneously evaluate multiple genes in many samples, can run multiple independent analyses, thus saving time and reducing cost. In addition, patch-targeted gene sequencing generates less data, is easier to manage, and has low detection cost, and thus is easier to analyze, compared to a broader range of methods, such as whole genome sequencing.

Gene sequencing is mainly applied to scientific research, clinical research, hospital diagnosis and biological medicine scenes. The scientific research still belongs to the mainstream application scene of gene sequencing, and the market proportion reaches more than 55 percent; the ratio of the three application fields of clinical research, hospital diagnosis and biological medicine is respectively 17%, 14% and 10%, and the total ratio of the three fields is up to 41%. NGS is mainly applied to the fields of susceptible gene detection, companion diagnosis, personalized medicine application and the like since the emergence of the core technology of second-generation gene sequencing, and is a main development direction of companion diagnosis industry.

The number of the death of cardiovascular and cerebrovascular diseases such as cerebral thrombosis, cerebral infarction, myocardial infarction, coronary heart disease, atheroma and the like reaches 1200 thousands, which is close to one fourth of the total death number in the world, and the cardiovascular and cerebrovascular diseases become the number one killer which surpasses malignant tumors. The main pathogenesis of cardiovascular and cerebrovascular diseases is thrombus.

Although the coagulation index is an intuitive index of human health, the index cannot be used as a unique target point in treatment if the disease source is not in the coagulation system, such as coagulation function problems caused by leukemia, trauma, infection and other factors.

The research finds that the gene variation plays an important determining role in thrombosis, and the statistics of the gene detection results of 912 cases of patients with the blood coagulation diseases in 2017 to 2020 attached to the first hospital of Suzhou university show that: 395 patients confirmed the genetic defect, confirmed the rate of diagnosis 43.3%, found the new pathogenic gene locus 51 cases, totally 66 loci. NGS is of great significance in diagnosis, typing, risk stratification, treatment target screening, curative effect judgment and blood disease minimal disease residual (MRD) monitoring of blood diseases.

22 Von Willebrand Disease (VWD) patients were collected, and NGS were performed on peripheral blood samples and clinical and laboratory examination data of the patients, among which 11 patients were simultaneously sequenced on the VWF gene by Sanger method. The results showed 4 cases of VWD type 1, 1 cases of VWD type 2A, 2 cases of VWD type 2, 3 cases of VWD type 2M, and 12 cases of VWD type 3 among 22 patients. By comprehensively analyzing sequencing data of the NGS detection and the Sanger method, 25 VWF gene mutations were found out of 22 VWD patients, and 8 of them were novel gene mutations that were not reported. Patients with VWD type 1 and 2 predominate heterozygous mutations, while patients with VWD type 3 predominantly exhibit heterozygous mutations. Moreover, NGS detection discovers 14 VWF gene mutations in 10 VWD patients, and the detection rate is 91%.

Research proves that NGS provides an effective laboratory detection means for analyzing a single large gene, the convenience and the accuracy of the NGS are greatly improved, and the NGS has important significance for improving a VWD gene diagnosis strategy. However, no specific Panel exists that can be used for detecting thrombus and hemorrhagic coagulation diseases.

Disclosure of Invention

The object of the present application is to provide a new panel with full coverage and in particular for the detection of thrombotic and hemorrhagic diseases and its applications.

The following technical scheme is adopted in the application:

one aspect of the application discloses a thrombus and bleeding blood coagulation disease detection panel, which comprises a specific hybridization probe for detecting or hybridizing and capturing thrombus and bleeding blood coagulation disease related genes; the related genes of thrombus and hemorrhagic coagulation diseases comprise 86 genes.

The detection panel for the thrombus and the hemorrhagic coagulation diseases covers related genes such as an anticoagulation system, a hemostasis system, a fibrinolysis system, platelet related genes, personalized medicine genes and the like, and can comprehensively cover the existing thrombus and the hemorrhagic coagulation diseases. The thrombus and go out coagulation disease detection panel of this application through the next generation sequencing, can be accurate, comprehensive, effectual detection coagulation disease relevant gene and the mutation condition, and then realize the diagnosis and the risk assessment of hemorrhage and thrombus disease, is the pioneering that thrombus and go out coagulation disease detected, has filled the blank in thrombus and the second generation sequencing detection field of going out coagulation disease.

It should be noted that the blood clot and bleeding blood coagulation disease detection panel of the present application can be used for detecting various gene structures related to blood clot and bleeding blood, and helps explain molecular mechanisms of blood clot and bleeding blood coagulation diseases. Moreover, gene information included in the panel is comprehensive, various thrombi and coagulation-causing diseases can be directly subjected to combined detection, the panel is applied to the concomitant diagnosis of the coagulation-causing diseases, and the obtained accurate target sequencing result can accurately evaluate genetic risks, recurrent risks, disease risks and the like of the thrombi and the coagulation-causing diseases.

Preferably, the 86 genes of the present application include A2, ABCB, ABCG, ABO, ACE, ACTN, ADAMTS, ADGRB, ADTRP, ANKRD, ANO, ANXA, AP3B, AP3D, APOH, APOM, ARPC1, ATOH, B3GAT, BAZ1, BDKRB, BLOC1S, BLZF, C4BPB, CADM, CALR, CBS, CES, CETP, CFB, CFD, CFH, CFP, CHST, COL1A, COL28A, COL3A, COL5A, CPB, CYCS, CYP2C, CYP3A, CYP4F, CYP4V, DIA, DTETP, EDEM, KRV, FGF, GFF, FGF, FENBF, FGF, FENBNA, FENBGP, FENBF 1, FENBNA, FENBF, FEF, FENBF, FEF, and FLF.

Preferably, the thrombus and hemorrhagic coagulation disease detection panel of the present application has specific hybridization probes covering gene coding regions, non-coding regions, promoter regions, 3 'UTR regions and 5' URT regions of genes associated with thrombus and hemorrhagic coagulation diseases.

Preferably, the thrombus and hemorrhagic coagulation disease detection panel of the present application has a specific hybridization probe covering gene mutations of genes related to thrombus and hemorrhagic coagulation disease, and the gene mutations include SNV, CNV, Indel and gene fusion.

It should be noted that, the specific hybridization probe of the present application can fully cover 86 genes as required, i.e., the coverage rate is 100%; depending on the application, only 100% coverage of known mutations in 86 genes can be performed.

Preferably, the thrombus and hemorrhagic blood coagulation disease detection panel of the present application has specific hybridization probes consisting of 12000 probes.

It should be noted that 12000 probes in the present application are probes designed to cover 100% of 86 genes by using the probe design principle and software, and the 12000 probes can be used to comprehensively and effectively capture and detect the 86 genes. It can be understood that 12000 probes in the present application are designed according to a conventional scheme, and the specific sequence can also be changed according to different parameter settings, as long as the 86 genes can be covered by 100%; therefore, the specific sequence is not specifically limited herein.

The application also discloses application of the blood thrombus and blood coagulation-causing disease detection panel in preparation of a reagent for detecting blood thrombus and blood coagulation-causing disease.

The thrombus and hemorrhagic coagulation disease detection panel of the present application is composed of a specific hybridization probe of 86 genes, and therefore can be used as a reagent for detecting thrombus and hemorrhagic coagulation disease.

The application further discloses a gene chip, a device or a kit for detecting the thrombus and the hemorrhagic coagulation diseases, which comprises the thrombus and the hemorrhagic coagulation disease detection panel.

The gene chip including the thrombus and hemorrhagic blood coagulation disease detection panel of the present invention may be a solid phase chip or a liquid phase chip, and the specific requirements are determined according to the use requirements. The device comprising the blood clot-associated disease detection panel of the present application can be used, for example, to immobilize the specific hybridization probe in the blood clot-associated disease detection panel of the present application in a reaction device, and the reaction device can be used to capture or detect the hybridization between the blood clot and the gene associated with the blood clot-associated disease. The kit for detecting a blood coagulation disease and a thrombus according to the present invention may be a kit simply composed of specific hybridization probes for 86 genes, or a hybridization capture chip on which the specific hybridization probes are immobilized, and is not particularly limited herein.

The beneficial effect of this application lies in:

the detection panel for the thrombus and the hemorrhagic coagulation diseases comprises a specific hybridization probe for detecting or hybridizing 86 genes related to the thrombus and the hemorrhagic coagulation diseases, can be used for detecting various gene structures related to the thrombus and the hemorrhagic coagulation, is helpful for explaining molecular mechanisms of the thrombus and the hemorrhagic coagulation diseases, and provides a comprehensive framework and basis for developing functional science of the hemorrhagic coagulation diseases, genetic family verification and pathogenic molecular mechanism research. The gene information covered by the panel is comprehensive, various thrombi and coagulation diseases can be directly subjected to combined detection, the panel is applied to the companion diagnosis of the coagulation diseases, and the obtained accurate target sequencing result can accurately evaluate the genetic risk, the recurrent risk and the disease risk of the thrombi and the coagulation diseases.

Detailed Description

The method carries out the design of the specific disease related gene probe on the basis of searching a large amount of data such as databases, documents, consensus of disease treatment experts and the like, and has wide coverage genes; specifically, the design of the application realizes the complete coverage of 86 disease-related genes, including related genes such as an anticoagulation system, a hemostasis system, a fibrinolysis system, platelet-related genes and personalized medicine genes. In order to realize 100% full coverage of 86 genes, 12000 probes are designed aiming at 10000 sites in total in gene coding region, non-coding region, promoter region, 3 'UTR region and 5' URT region of 86 genes, and SNV, CNV, Indel, gene fusion and the like of 86 genes can be detected based on mutation types.

In an implementation manner of the application, the thrombus and hemorrhagic coagulation disease detection panel can capture a specific region of a specific gene, and can perform targeted sequencing, wherein the sequencing depth is large, the data validity is high, the signal-to-noise ratio is high, the detection sensitivity is 1 per mill, and 1 read in a target region can detect the gene variation type; moreover, the redundant data brings convenience to bioinformatics analysis, and the data accuracy is correspondingly improved.

The detection panel for the thrombus and the hemorrhagic coagulation diseases is the pioneer of detecting related gene mutation of the thrombus and the hemorrhagic coagulation diseases by applying second-generation sequencing at present; the diagnosis and risk assessment of thrombotic and hemorrhagic diseases based on the panel of the present application was also pioneering. The thrombus and the bleeding blood coagulation disease detection panel are adopted to detect thrombus and the bleeding blood coagulation disease, and a large amount of accumulated sequencing data is helpful for building a bleeding blood coagulation disease related gene variation database with independent intellectual property rights. In addition, the detection panel for the thrombus and the hemorrhagic coagulation diseases is also beneficial to strengthening individualized medication and improving the curative effect of the medicine and reducing adverse reactions, thereby generating better social value and economic value.

In this application, detecting panel is a term of art for high throughput gene detection and gene sequencing, and refers to a combination of specific probes for several genes, which can be designed on a plurality of capture chips to capture target DNA and used for subsequent gene detection or sequencing. Based on the detection of panel, the simultaneous detection of multiple loci and multiple genes can be realized. The present invention relates to 86 gene probe hybridization capture scheme, and compared with the whole genome detection, the present invention relates to the detection of thrombus and coagulation diseases panel, the panel can only capture and detect a small amount of target region.

The present application will be described in detail with reference to specific examples. The following examples are intended to be illustrative of the present application and should not be construed as limiting the present application.

Examples

In this example, a blood clot-inducing disease detection panel was prepared, and 95 samples of von Willebrand disease were examined using the prepared detection panel, to verify the effect of detecting the blood clot-inducing disease detection panel. The method comprises the following specific steps:

preparation of panel for detecting thrombus and hemorrhagic coagulation diseases

1. Screening of genes associated with thrombotic and procoagulant diseases

The embodiment collects and collates the data of the prior database, literature, consensus of disease therapists and the like related to the thrombus and the hemorrhagic coagulation diseases; finally, 86 gene detection target genes are obtained by screening and are used for constructing the panel of the embodiment. The 86 genes include A2, ABCB, ABCG, ABO, ACE, ACTN, ADAMTS, ADGRB, ADTRP, ANKRD, ANO, ANXA, AP3B, AP3D, APOH, APOM, ARPC1, ATOH, B3GAT, BAZ1, BDKRB, BLOC1S, BLZF, C4BPA, C4BPB, CADM, CALR, CBS, CES, CETP, CFB, CFD, CFH, CFP, CHST, COL1A, COL28A, COL3A, COL5A, CPB, CYCS, CYP2C, CYP3A, CYP4F, CYP4V, DIAPH, DTP, ETEM, EDN, CFV, KRF, GCF, FGF 13, GFF, FENFG, FLF, FTGP, FTNA, FTF, and FLFFNNA.

2. Detection target area selection

Based on 86 genes obtained by screening genes related to thrombus and hemorrhagic coagulation diseases 1, the target region selection is carried out aiming at the gene coding region, the non-coding region, the promoter region, the 3 'UTR region and the 5' URT region of the 86 genes, and the specific screening standard is as follows: (1) keeping the locus with the frequency less than or equal to 0.02 in the genome database of thousands of people; (2) the synonymous mutations reported in the literature are retained; (3) the site with mutation frequency of more than 5 and mutation frequency of more than 0.3 is reserved.

In this example, 10000 loci in total were finally obtained from the gene coding region, non-coding region, promoter region, 3 'UTR region and 5' URT region of 86 genes.

3. Probe design for detecting panel

In this example, 12000 probes were designed using probe design software for 10000 sites obtained by "2. detection target region selection". The length of the probe is 120 nt/strip, the total coverage of the coding region, the non-coding region, the promoter region, the 3 'UTR region and the 5' URT region of the 86 genes is more than 99 percent, the coverage uniformity is more than 99 percent, and the coverage depth is 1X for each site.

4. Detection of panel Synthesis

12000 designed probes were synthesized and fixed on a slide glass to prepare a blood clot and hemorrhagic blood coagulation disease detection panel of this example.

Second, application of detecting panel for thrombus and hemorrhagic coagulation diseases

The blood samples from 95 von Willebrand diseases were tested using the test panel prepared in this example as follows:

1. genomic DNA extraction

This example uses the TIANAmp Blood DNA Kit for DNA extraction from Blood samples. The method comprises the following specific steps:

(1) pretreatment

200 μ L Whole blood DNA extraction: aspirate 200. mu.L of blood into a correspondingly labeled 1.5mL EP tube directly for the next proteinase K treatment.

500 μ L whole blood DNA extraction: the method comprises the steps of sucking 500 mu L of blood into a corresponding marked 1.5mL EP tube, adding 500 mu L of cell lysate CL, reversing and mixing the mixture evenly, centrifuging the mixture for 1min at 10,000rpm (11,500 Xg), sucking the supernatant, leaving a cell nucleus precipitate (if the cell nucleus precipitate is not completely lysed, repeatedly lysing once), adding 200 mu L of buffer GS into the cell nucleus precipitate, shaking the mixture till the mixture is completely mixed evenly, and then carrying out the next experiment.

(2) Proteinase k treatment

Prepare a premix according to the amount of 200. mu.L buffer GB and 20. mu.L protease K for each sample, mix well, and centrifuge instantaneously.

220. mu.L of the above premix was added to the sample pretreated in (1).

Incubation at 56 deg.C and 1000rpm for 10min until the solution becomes clear, and if not clear, the incubation time can be prolonged appropriately.

(3) Cross post

Standing at room temperature for 2-5min, adding 350 μ L buffer BD, mixing thoroughly, and centrifuging instantaneously.

The sample was transferred to the adsorption column, centrifuged at 12,000rpm (-13, 400 Xg) for 30s and the effluent discarded.

(4) Washing and drying

The column was washed with 500. mu.L of buffer GDB, 600. mu.L of PWB in sequence, and centrifuged at 12,000rpm (. about.13,400 Xg) for 30s each time.

Finally, the column was dried by air-separation at 12,000rpm (. about.13,400 Xg) for 2 min.

The adsorption column was transferred to a new 1.5mL centrifuge tube, and the column was left open for 2min at room temperature to air dry.

(5) Elution is carried out

50 μ L of elution buffer TB was added dropwise to the middle of the adsorption membrane, left at room temperature for 2min, and centrifuged at 12,000rpm (13,400 Xg) for 2 min. Repeating the steps once

The eluate was transferred to a low adsorption centrifuge tube and stored at-20 ℃ for further use.

Preparation of DNA library

(1) Genomic DNA fragmentation treatment

This example used a Covaris EM80 sonicator to fragment the extracted DNA sample. Dilute the sample with TE to 6ng/μ L in a 130 μ L interrupt tube to a total volume of 55 μ L; break time 50 s.

The ultrasonic interrupt instrument was turned on, the instrument parameters were set and examined as follows:

Peak Incident Power(W)50、Duty Factor 20％、Cycle per Burst 200、Treatment Time(s)50、Temperature(c)6。

and after interruption, uniformly mixing the interruption products, centrifuging for a short time, and transferring all the interruption products to a centrifugal tube for temporary storage on ice or storing in a refrigerator at the temperature of-20 ℃.

(2) End repair and addition of "A"

A. The XP beads were removed from the refrigerator, the following reagents were removed from the sureselect (pre pcr) kit, and prepared as required. Placing the AgencourtAmpure XP Kit (abbreviated as XP magnetic bead) at room temperature; unfreezing the End Repair and A labeling Buffer on ice, and then placing the mixture on ice; thawing the Ligation Buffer on ice, and placing the mixture on ice; placing the End Repair & A labeling Enzyme Mix on ice; t4 DNA Ligase on ice; after thawing the Adaptor Oligo Mix on ice, it was placed on ice.

B. And taking out the broken sample to be detected, mixing uniformly after dissolving, centrifuging, and placing on ice.

C. The PCR machine was opened and the hot lid was confirmed to be opened. The program is suspended immediately after the program is started.

End repair & add a procedure: 15min at 20 ℃, 15min at 72 ℃ and finally standby at 4 ℃.

D. And (4) taking a proper centrifugal tube, and preparing a joint connection mixed solution.

The total volume of adaptor ligation mix (Lmix) was 25. mu.L, including: ligation Buffer (bottled) 23. mu.L and T4 DNA Ligase (blue cap) 2. mu.L.

E. The adaptor-ligated mixture (Lmix) was vortexed at high speed for 15s, mixed thoroughly, and centrifuged instantaneously. Keeping the temperature at room temperature for 30-45 minutes.

F. Taking a proper centrifuge tube, marking Emix, and preparing a mixed solution with a repaired tail end and a 3' end on an ice box.

End repair & 3' end add a mix (Emix) total volume 20 μ L, including: 16. mu.L of End Repair & A labeling Buffer (bottled) and 4. mu.L of End Repair & A labeling Enzyme Mix (orange lid).

G. And (3) adding the mixed solution A (Emix) at the end of the terminal repair and the end of the terminal repair, sufficiently and uniformly mixing the mixture for 5 to 10 seconds by high-speed vortex, carrying out instantaneous centrifugation, and placing the mixture on ice.

H. Add 20. mu.L of end repair & A mix (Emix) to the 3' end separately, cover the tube lid, vortex for 5-10s, mix well, and centrifuge instantaneously to remove air bubbles.

I. The PCR program was checked and it was confirmed that the "end repair & Add A program" step 1 residual time was 15min and the module temperature was 20 ℃. And (3) placing the sample tube on a PCR instrument, pressing a 'continue' starting program under the condition of opening the cover of the PCR instrument, covering the cover of the PCR instrument after the reaction is finished at 20 ℃ for 15min, and continuing the reaction at 72 ℃ for 15 min.

J. When the temperature of the PCR program is reduced to 4 ℃, the sample is taken down and placed on ice for standby.

(3) Connecting joint

After the end repair & Add A procedure was completed, the ligation adapter procedure was selected, and the check procedure was as follows, confirming that the hot lid was closed and the reaction volume was 100. mu.L. The program is suspended immediately after the program is started.

And (3) connecting joint program: 20 ℃ for 30min, and 4 ℃ for standby.

Add 5. mu.L of Adaptor Oligo Mix to the PCR tube of the 70. mu.L product from the previous step, cover the tube, vortex 5-10s, and flash centrifuge.

To each sample tube 25. mu.L of adaptor ligation mix was added at room temperature, the tube cap was closed, vortexed for 5-10s, and centrifuged instantaneously.

The PCR program was checked and the ligation linker program was confirmed to have a residual time of 30min and a temperature of 20 ℃. The sample tube was placed in the PCR instrument and the program was run "on" with the lid of the PCR instrument opened.

And (3) taking out the reaction tube when the temperature of the PCR instrument is reduced to 4 ℃, carrying out instantaneous centrifugation, and standing at 4 ℃ for later use or standing at-20 ℃ overnight.

(4) Ligation product purification

The XP magnetic beads are taken out of the refrigerator 30min ahead of time and balanced to the room temperature, and fresh 80% ethanol (preparation method, NF water: absolute ethanol is 2: 8) is prepared according to the amount of 450 mu L of each sample and is fully mixed.

Fully oscillating the XP magnetic beads for not less than 1min to fully mix the XP magnetic beads.

Weighing a 1.5mL centrifuge tube according to the number of samples, marking Ln, and sucking 80 mu L of the uniformly mixed magnetic beads into the marked 1.5mL centrifuge tube.

The ligation products (about 100. mu.L) were transferred to a centrifuge tube containing 80. mu.L of magnetic beads, vortexed for 5-10s and incubated at room temperature for 5 min.

The sample tube was centrifuged instantaneously and placed on a magnetic stand for 5min until the solution was completely clear.

Carefully aspirate the supernatant with a pipette to ensure that the tip does not touch the beads.

Add 200. mu.L of fresh 80% ethanol, cover the centrifuge tube, rotate the centrifuge tube on the magnetic frame rapidly clockwise 8 times, 45 degrees each time, until it is rotated back to the original position, and rapidly aspirate all supernatants.

The above step was repeated and washed once with 200. mu.L of 80% ethanol for a total of two washes.

The 1.5mL centrifuge tube was removed from the magnetic frame, placed back on the magnetic frame after transient centrifugation, and the remaining liquid in the tube was removed using a 10. mu.L/20. mu.L pipette.

Keeping the centrifugal tube on a magnetic frame, opening a tube cover, standing and drying the magnetic beads at room temperature without reflecting light and cracks.

The sample tube was removed from the magnetic frame, 35. mu.L NF water was added, the tube cap was closed, vortexed until the beads were thoroughly mixed, and incubated at room temperature for 5 min.

And (5) performing instantaneous centrifugation, putting the 1.5mL centrifuge tube back to the magnetic frame, and standing for 5min at room temperature until the solution is completely clear.

Prepare a new 0.2mL PCR tube for sample size and transfer 17.25. mu.L of supernatant to the labeled PCR tube twice to ensure that no magnetic beads are aspirated. The samples were kept on ice or at 4 ℃.

(5) Library amplification

The reagents in the kit are taken out in advance and prepared as required.

Herc μ lase II Fusion DNA Polymerase on ice, 5 × Herc μ lase II Reaction Buffer on ice, 100mM dNTP Mix on ice, Forward Primer on ice, SureSelect XT Low Input Index Primers on ice.

The PCR cycle number program for the sample was confirmed, and the control program was as follows, confirming that the hot lid was opened and the reaction volume was 50. mu.L. The program is suspended immediately after the program is started.

PCR reaction procedure: 98 ℃ for 2min, then enter 6 cycles: 30s at 98 ℃, 30s at 60 ℃ and 1min at 72 ℃, 5min at 72 ℃ after circulation is finished, and finally standby at 4 ℃.

And taking out the purified ligation product, flicking, uniformly mixing, then performing instantaneous centrifugation, and placing on ice for later use.

Each sample was assigned a different Index Primer.

And (4) taking a proper centrifuge tube, marking Ymix, and preparing a pre-library amplification mixed solution on ice. Vortex for 5-10s, mix well and centrifuge instantaneously.

The pre-library amplification mix (Ymix) was 13.5 μ L in total volume and included: 5 × Herc μ lase II Reaction Buffer (clear cover) 10 μ L, Forward Primer 2 μ L (brown cover), 100mM dNTP Mix (green cover) 0.5 μ L, Herc μ lase II Fusion DNA Polymerase (red cover) 1 μ L.

Add 13.5. mu.L of pre-library amplification mix to the sample tube.

And adding 2 mu L of Index Primer corresponding to the distribution number in the step D into the sample tube, covering the tube cover tightly, vortexing for 5s, mixing uniformly, and performing instantaneous centrifugation to collect liquid and remove air bubbles in the tube.

The PCR program was checked to confirm that the remaining time of the first step of the PCR reaction program was 2min, the temperature was 98 ℃ and the number of cycles was correct. And (4) putting the sample tube into the PCR instrument, covering the PCR instrument, and continuing to operate the program.

And (4) after the reaction of the PCR instrument is finished, taking out the reaction tube, and instantly and centrifugally placing the reaction tube on ice for later use.

(6) Amplification product purification

A. The XP magnetic beads are taken out of the refrigerator in advance, are placed at room temperature for 30min in a balanced way, are prepared into fresh 80% ethanol according to the amount of 450 mu L of each sample (the preparation method is that NF water and absolute ethanol are 2: 8), and are fully mixed.

B. Fully oscillating the XP magnetic beads for not less than 1min to fully mix the XP magnetic beads.

C. Weighing a 1.5mL centrifuge tube according to the number of samples, and sucking 50 μ l of the uniformly mixed XP magnetic beads into the 1.5mL centrifuge tube.

D. The pre-library amplification products (about 50. mu.L) were all transferred to a centrifuge tube containing 50. mu.L of magnetic beads, vortexed for 5-10s, and incubated at room temperature for 5 min.

E. The sample tube was centrifuged instantaneously and placed on a magnetic stand for 5min until the solution was completely clear.

F. Carefully aspirate the supernatant with a pipette to ensure that the tip does not touch the beads.

G. Add 200. mu.L of fresh 80% ethanol, cover the centrifuge tube lid, rotate the centrifuge tube clockwise on the magnetic frame 8 times, 45 degrees each time, until rotating back to the original position, aspirate all supernatants to avoid aspirating magnetic beads.

H. The above step was repeated and washed once with 200. mu.L of 80% ethanol for a total of two washes.

I. The 1.5mL centrifuge tube was removed from the magnetic frame, placed back on the magnetic frame for 30s after transient centrifugation, and the remaining liquid in the tube was removed with a 10. mu.L/20. mu.L pipette.

J. Keeping the centrifuge tube on the magnetic frame, opening the tube cover, and standing at room temperature to dry the magnetic beads. (drying criteria: beads are not reflective, no cracks. excessive drying affects elution efficiency.)

K. The sample tube was removed from the magnetic frame, 15 μ L NF water was added, the tube cap was capped, vortexed until the beads were completely mixed, centrifuged instantaneously, and incubated at room temperature for 5 min.

L. put 1.5mL centrifuge tube on magnetic rack, stand for 2-3min at room temperature until the solution is completely clear.

Transfer about 15 μ L of supernatant to a new PCR tube. Mixing and instantaneous centrifugation. All samples were kept on ice or at 4 ℃ for future reaction, and if the next step is not performed, the product was kept at-20 ℃.

(7) The panel cross

The reagents in the kit are taken out in advance and prepared as required.

SureSelect XT HS and XT Low Input Block Mix, SureSelect RNase Block on ice, and SureSelect Fast Hybridization Buffer at room temperature.

The PCR instrument was turned on, the hybridization procedure was selected, and the control procedure was as follows, confirming that the hot lid was opened and the reaction volume was 30. mu.L. The program is suspended immediately after the program is started.

Hybridization procedure: 95 ℃ 5min, 65 ℃ 10min, 65 ℃ 1min, where the addition of reagents is suspended and then 60 cycles are entered: 1min at 65 ℃ and 3s at 37 ℃, and standby at 65 ℃ after circulation is finished.

And taking out the purified pre-library, mixing uniformly, performing instantaneous centrifugation, and placing on ice for later use.

The total loading amount of the pre-library is 500-1000 ng, and the volume is 12 mu l. The pre-library addition volume was calculated from the pre-library concentration and made up to 12 μ L with NF water.

The calculation formula is as follows:

the amount of mixing is the amount of data on the sample x 1000/sigma on the machine

Mixing volume is mixing amount/concentration

And (3) computer data amount: the panel 1000X depth data size is about 0.7G

For example, sample 1 concentration is 200 ng/. mu.L, mix volume is 667ng, mix volume is 3.34. mu.L; sample 2 concentration was 250 ng/. mu.L, mix volume was 167ng, mix volume was 0.67. mu.L; sample 3 concentration was 100 ng/. mu.L, mix volume was 167ng, mix volume was 1.67. mu.L; NF Water 6.32. mu.L.

Add 5. mu.L of SureSelect XT HS and XT Low Input Block Mix to each tube, cover the tube, vortex 5s Mix, centrifuge for a short period of time to collect the liquid and remove the bubbles in the tube.

The PCR instrument program was checked and it was confirmed that the remaining time of step 1 of the hybridization program was 5min at a temperature of 95 ℃ and the sample was placed in the PCR instrument to start the program.

The tube was removed, labeled as system and mixed with hybridization mix (Zmix) at room temperature and centrifuged instantaneously.

Hybridization mix (Zmix) was included in a total volume of 13. mu.L: NF water 1.5. mu.L, SureSelect RNase Block 0.5. mu.L, SureSelect Fast Hybridization Buffer 6. mu. L, panel 5. mu.L.

Of these, 1.5. mu.L of NF water and 0.5. mu.L of SureSelect RNase Block, 25% RNase Block.

When the PCR program is run to step 3: pressing pause key at 65 deg.C for 1min, keeping the sample on PCR instrument, adding 13 μ L hybridization mixture (Zmix) into each tube, sucking up and down slowly for 8-10 times, and mixing thoroughly.

And covering a tube cover, carrying out short-time centrifugation after short-time vortex, collecting reaction liquid to the bottom of the tube, removing bubbles, and immediately putting the sample tube back to the PCR instrument to continue to run the PCR program.

(8) Capture hybridization libraries

The following reagents and T1 magnetic beads were removed from the kit and prepared as required.

The SureSelect Binding Buffer is reversed and mixed evenly, and placed at room temperature; mixing the SureSelect Wash Buffer1 by reversing, and standing at room temperature; mixing the SureSelect Wash Buffer2 by reversing, and standing at room temperature; dynabeads MyOne Streptavidin T1, left to equilibrate for 30min at room temperature, and kept at room temperature for further use.

The T1 magnetic beads were vortexed for more than 1min, mixed well, and loaded into an appropriate centrifuge tube at 50. mu.L/reaction, and the T1 magnetic beads were washed as follows.

Adding 200 mu L of SureSelect Binding Buffer into each 50 mu L T1 magnetic bead, vortexing for 5-10s, mixing well, placing the centrifuge tube on a magnetic rack for 5min, and absorbing and removing supernatant after the solution is clarified.

Repeating the above step for 2 times, and washing for 3 times.

For each reaction (50. mu.L of stock solution of magnetic beads), 200. mu.L of SureSelect Binding Buffer was added and the beads were resuspended thoroughly by vortexing for more than 1 min.

Prepare 1.5mL or 0.6mL centrifuge tubes for each sample, and label Cn. And 200. mu.L of each tube of the resuspended T1 magnetic beads was dispensed into Cn tubes.

Prepare SureSelect Wash Buffer2, and preheat:

SureSelect Wash Buffer2 was dispensed from 0.5mL centrifuge tubes, and 3 tubes of 450. mu.L each were prepared for each sample. The dispensed SureSelect Wash Buffer2 was placed on a PCR instrument programmed at 70 ℃ for 4h, hot-capped at 105 ℃ and the reaction volume was 200. mu.L. The PCR instrument program was started to preheat SureSelect Wash Buffer2 and was kept on the 70 ℃ PCR instrument for use.

After the hybridization step was completed and the PCR instrument reached 65 ℃, the samples were removed and centrifuged instantaneously. All samples were immediately transferred to a magnetic bead tube containing 200. mu. L T1. Shaking and mixing, placing on a constant temperature mixing instrument, and mixing at the room temperature of 1400 ℃ and 1800rpm for 30 min.

A0.2 mL PCR tube was prepared for the number of samples.

After mixing at room temperature for 30min, the sample was removed from the thermostatic mixer and centrifuged instantaneously.

And (4) placing the sample tube on a magnetic frame, and removing the supernatant after the solution is clarified.

And (3) taking the sample tube off the magnetic frame, adding 200 mu L of SureSelect Wash Buffer1 into each tube, sucking and beating for 15-20 times or vortexing for 5-10s, uniformly mixing, transferring to a PCR tube marked with Cn, placing the PCR tube on the magnetic frame for 1min, and removing the supernatant after the solution is clarified.

Taking down the sample tube from the magnetic frame, and washing the magnetic beads according to the following steps:

a. add 200. mu.L of preheated SureSelect Wash Buffer2 to the sample tube, vortex the tube with the lid and mix for 8s, and centrifuge the tube instantaneously.

b. The sample tube was incubated on a PCR instrument at 70 ℃ for 10 min.

c. And taking out the sample, placing the sample on a magnetic frame, standing the sample for 1min at room temperature, and sucking and removing the supernatant after the solution is clarified.

d. The a to c steps were repeated 5 more times for a total of 6 times.

J. The sample tube was centrifuged briefly and placed on a magnetic stand for 1min, and the remaining liquid was aspirated off with a 10 μ L pipette.

K. Add 25. mu.L NF water to each sample tube, vortex 8s with the tube lid, mix, and centrifuge instantaneously. The samples were placed on ice until use.

(9) Amplification of hybrid captured libraries

The following reagents were removed from the sureselect (post pcr) kit and prepared as required.

Herc μ lase II Fusion DNA Polymerase, gently mixing, and standing on ice; thawing 5 XHerc μ lase II Reaction Buffer on ice, mixing by vortex, centrifuging for a short time, and standing on ice; thawing 100mM dNTP Mix on ice, mixing evenly by vortex, centrifuging for a short time, and placing on ice; after thawing on ice, vortex and Mix, centrifuge for a short time, and place on ice.

The PCR instrument was turned on, the program for the number of cycles corresponding to PCR was selected, the check program was pressed, and the reaction volume was set: 50 μ L, confirming opening of the hot lid. And pause immediately after the program is started.

PCR reaction procedure: 98 ℃ for 2min, then enter 6 cycles: 30s at 98 ℃, 30s at 60 ℃ and 1min at 72 ℃, 5min at 72 ℃ after circulation is finished, and finally standby at 4 ℃.

And (4) taking a proper centrifuge tube, marking Pmix, and preparing a PCR mixed solution after capture on ice. Vortex for 5-10s, mix well and centrifuge instantaneously.

The post capture PCR mix was in a total volume of 25 μ Ι _, including: 12.5. mu.L NF water, 10. mu.L 5 × Herc. mu.laser II Reaction Buffer, 0.5. mu.L 100mM dNTP Mix, 1. mu. L, Herc. mu.L Sureselect Post-Capture Primer Mix, 1. mu.L DNA Polymerase.

Add 25. mu.L of post-capture PCR cocktail (Pmix) to about 25. mu.L of sample product Cn, pipette up and down 10-15 times and mix the sample well.

The PCR program was checked to confirm that the remaining time in step 1 of the PCR reaction program was 2min, the temperature was 98 ℃ and the number of cycles was correct. And putting the sample tube into the PCR instrument, covering the PCR instrument, and continuously operating the PCR program.

(10) Sequencing library purification

Preparation before magnetic bead purification:

A. the XP magnetic beads are taken out of the refrigerator in advance, are placed at room temperature for 30min in a balanced way, are prepared into fresh 80% ethanol according to the amount of 450 mu L of each sample (the preparation method is that NF water and absolute ethanol are 2: 8), and are fully mixed.

B. Fully oscillating the XP magnetic beads for not less than 1min to fully mix the XP magnetic beads.

C. A1.5 mL centrifuge tube was prepared for the number of samples and labeled PCn. Add 50. mu.L of the mixed XP beads to the PCn centrifuge tube.

Magnetic bead purification:

A. after the PCR reaction of the PCR instrument is finished and the temperature is reduced to 4 ℃, taking out the reaction tube and carrying out instantaneous centrifugation.

B. And (3) placing the sample tube on a magnetic frame, standing for 2min, transferring all supernatants into a PCn centrifugal tube filled with XP magnetic beads after the solution is clarified, uniformly mixing by swirling for 5-10s, and incubating for 5min at room temperature.

C. The sample tube was centrifuged instantaneously and placed on a magnetic stand for 5min until the solution was completely clear.

D. Carefully aspirate the supernatant with a pipette to ensure that the tip does not touch the beads.

E. Add 200. mu.L of fresh 80% ethanol, cover the centrifuge tube lid, rotate the centrifuge tube clockwise on the magnetic frame 8 times, 45 degrees each time, until rotating back to the original position, aspirate all supernatants to avoid aspirating magnetic beads.

F. The previous step was repeated with one more 200 μ L80% ethanol wash, for a total of two washes, ensuring that all supernatants were aspirated at each wash step.

G. The 1.5mL centrifuge tube was removed from the magnetic frame, placed back on the magnetic frame for 30s after transient centrifugation, and the remaining liquid in the tube was removed with a 10. mu.L/20. mu.L pipette.

H. Keeping the centrifuge tube on the magnetic frame, opening the tube cover, and standing at room temperature to dry the magnetic beads. The drying standard is that the magnetic beads are not reflective and have no cracks. Excessive drying can affect elution efficiency.

I. And (3) taking down the sample tube from the magnetic frame, adding 20 mu L of NF water, covering the tube cover, whirling until the magnetic beads are completely mixed, performing instantaneous centrifugation, and incubating at room temperature for 5 min.

J. Preparing a new 1.5mL low-adsorption centrifuge tube according to the number of samples, marking a tube cover with Lib-n-month day, and marking a tube body with a corresponding library number, concentration, library builder and detection batch.

K. And (3) placing the sample tube on a magnetic frame, and standing for 2-3min at room temperature until the solution is completely clear.

Transfer approximately 20 μ L of supernatant to a centrifuge tube labeled Lib-n-month day. All samples were kept on ice or at 4 ℃ for future use, and the product was stored at-20 ℃ without further manipulation.

3. Sequencing

The captured target region was sequenced using the Illumina 550 sequencer in this example.

4. Analysis of sequencing results

In the process of establishing the biological information analysis flow, necessary quality control standards including reads quality, GC content, comparison rate, reads comparison quality, minimum sequencing depth, minimum mutation rate and the like need to be established. Thresholds are established for these criteria based on the characteristics of the detection method and the intended use. Each quality control criterion may establish a different level threshold, such as highly trusted (pass), alarm (warning), untrusted (fail). In the analysis process, the accuracy of the analysis result can be ensured only by performing subsequent analysis on the basis of meeting the threshold value of the quality control standard. The method comprises the following specific steps: q30 > 85%; PF is more than 95 percent; FL170-230k/mm²The coverage is more than 95%, the uniformity is more than 99%, and the target-hitting rate is more than 95%.

In this example, 95 blood samples obtained from hemophilia vasculosa were tested and analyzed according to the above method using a designed pancel for the detection of thrombotic and hemorrhagic disease; the results show that the thrombus and hemorrhagic coagulation disease detection panel of the embodiment can directly carry out combined detection on a plurality of thrombus and hemorrhagic coagulation diseases, and the detection results are consistent with the actual diagnosis conditions. The blood coagulation and hemorrhagic disease detection panel can be used for detecting various gene structures related to blood coagulation and thrombosis, is helpful for explaining the molecular mechanism of the blood coagulation and hemorrhagic disease, and provides a comprehensive framework and basis for developing the researches on blood coagulation disease functional science, hereditary family verification, pathogenesis molecular mechanism and the like; and the method can be applied to the companion diagnosis of the coagulation diseases, and the obtained accurate target sequencing result can accurately evaluate the genetic risk, the recurrent risk, the disease risk and the like of the thrombus and the coagulation diseases.

The foregoing is a detailed description of the present application in connection with specific embodiments thereof, and implementations of the present application are not to be considered limited to those descriptions. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the basic inventive concepts herein.

Claims (8)

1. A thrombus and hemorrhagic clotting disease detection panel, characterized by: the panel comprises a specific hybridization probe for detecting or hybridizing the capture thrombus and the gene related to the hemorrhagic clotting diseases; the genes related to the thrombus and the hemorrhagic coagulation diseases comprise 86 genes.

2. The blood clotting and blood clotting disease detection panel of claim 1, wherein: the 86 genes include A2, ABCB, ABCG, ABO, ACE, ACTN, ADAMTS, ADGRB, ADTRP, ANKRD, ANO, ANXA, AP3B, AP3D, APOH, APOM, ARPC1, ATOH, B3GAT, BAZ1, BDKRB, BLOC1S, BLZF, C4BPA, C4BPB, CADM, CALR, CBS, CES, CETP, CFB, CFD, CFH, CFP, CHST, COL1A, COL28A, COL3A, COL5A, CPB, CYCS, CYP2C, CYP3A, CYP4F, CYP4V, DIAPH, DTP, EDEM, EDN, KRV, F, FGF, GFF 13, GFF, GFGP, GFNA, FLF, FTNA, and FLFNBNA.

3. The blood clotting and blood clotting disease detection panel of claim 1, wherein: the specific hybridization probe covers the gene coding region, the non-coding region, the promoter region, the 3 'UTR region and the 5' URT region of the genes related to the thrombus and the hemorrhagic coagulation diseases.

4. The blood clotting and blood clotting disease detection panel of claim 3, wherein: the specific hybridization probe covers the gene mutation of the thrombus and the genes related to the hemorrhagic clotting diseases, and the gene mutation comprises SNV, CNV, Indel and gene fusion.

5. The blood clotting and blood clotting disease detection panel of claim 4, wherein: the specific hybridization probe covers 100% of the genes related to the thrombus and the hemorrhagic coagulation diseases.

6. The blood clotting and blood clotting disease detection panel according to any one of claims 1-5, wherein: the specific hybridization probe consisted of 12000 probes.

7. Use of the blood clot and blood clot disease detection panel according to any one of claims 1 to 6 in the manufacture of a reagent or device for detecting blood clot and blood clot disease.

8. A gene chip, device or kit for detecting thrombus and hemorrhagic coagulation diseases is characterized in that: a panel comprising the thrombus and bleeding blood coagulation disease detection panel according to any one of claims 1 to 6.