Protein C deficiency related gene capture probe group, kit, library construction method and application

Abstract

The invention discloses a protein C deficiency related gene capture probe group, a kit, a library construction method and application, and belongs to the technical field of gene detection. The capture probe group aiming at the protein C deficiency related gene PROC disclosed by the invention is composed of probes as shown in SEQ ID NO.1-SEQ ID NO.17, so that effective enrichment of a target gene is realized, and the detection cost is reduced. The gene is effectively captured through 17 probes, the detection result consistent with that of a Sanger sequencing method is achieved, and the gene has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a capture probe group, a kit, a library construction method and an application of protein C deficiency-related genes. Background Art

[0002] Protein C deficiency is characterized by recurrent deep vein thrombosis (DVT). Patients are often under 50 years old. Thrombosis can be spontaneous or secondary, with surgery, trauma, childbirth, and oral contraceptive use being common predisposing factors. Pulmonary embolism is the most serious and common complication.

[0003] Protein C deficiency is caused by mutations in the PROC gene and is inherited in an autosomal dominant manner. The incidence of asymptomatic heterozygous protein C deficiency is 1 / 200 to 1 / 500, while the incidence of symptomatic heterozygous protein C deficiency is 1 / 16,000 to 1 / 32,000. The incidence of homozygous protein C deficiency is 1 / 360,000 to 1 / 160,000. Symptoms in homozygous individuals are more severe, potentially leading to premature birth or the development of deep vein thrombosis in the first few days of life. However, some patients also present with clinical manifestations similar to those of heterozygous carriers.

[0004] The PROC gene, located at position q14.3 on chromosome 2, is 9546 bases long and contains nine exons. It encodes protein C, a vitamin K-dependent plasma glycoprotein. Under physiological conditions, protein C primarily exists as an inactive zymogen. It is activated by the thrombin and thrombomodulin complex on the endothelial cell surface to become activated protein C (APC). In the presence of the cofactor protein S (PS), activated protein C (APC) selectively degrades factors Va and VIIIa, inactivating them and thus exerting its anticoagulant effect. Its affinity for phospholipids increases, forming an APC-PS complex that blocks and reduces the binding of factors Va and VIIIa to the platelet membrane, thereby inhibiting coagulation. Studies have shown that PC deficiency predisposes to thrombosis, particularly during pregnancy and the postpartum period, and can lead to recurrent miscarriage. Therefore, testing for the PROC gene, which is associated with protein C deficiency, is essential for avoiding or preventing these conditions.

[0005] NGS (next-generation gene sequencing) detection technology has a much higher sensitivity than current traditional detection technologies. It can detect 0.03% point mutations and less than 0.1% gene fusions, and can perform multi-level detection of DNA and RNA to ensure detection accuracy. To sequence the genome sequence, in order to reduce costs, it is necessary to design a gene panel with certain diagnostic value. The commonly used methods for enriching target genes are multiplex PCR and probe hybridization capture. Hybridization capture requires the design of specific probes for the genomic region of interest. Using the principle of complementary hybridization of nucleic acid molecule bases, it is hybridized with genomic DNA to enrich DNA fragments in the target genomic region. The genomic DNA is then broken, and after adding sequencing adapters, it is hybridized with the probe to capture the target region of the genomic DNA, recover the target DNA fragments, and directly construct a high-throughput sequencing library for sequencing.

[0006] Sequencing technology can provide a more comprehensive understanding of genes associated with protein C deficiency and further differentiate patient genotypes. Testing for the PROC gene requires whole-genome sequencing or exome sequencing of the sample to be tested, which results in high sequencing costs and is not conducive to high-throughput sequencing analysis of the PROC gene. Therefore, selecting the appropriate target capture region is key to ensuring accurate sequencing results while reducing testing costs.

[0007] For example, patent document CN114807336A discloses a detection method for thrombosis-related gene chip, which requires detecting 62 pathogenic mutations in 21 genes. One of the thrombosis genes is PROC, which includes 12 pathogenic mutation sites: rs774572099, rs574949343, rs199469469, rs146922325, rs199469476, rs199469470, rs777486993, rs121918150, rs780456728, rs200721675, rs199469471, and rs571278160. Capture probes are designed with the above-mentioned pathogenic mutation sites as target areas, and a high-throughput sequencing library is constructed for sequencing.

[0008] As can be seen, existing disease detection panels require multiple pathogenic mutations, resulting in high testing costs and a large analytical workload. In light of this, the present invention provides a capture probe set targeting the protein C deficiency-associated gene PROC. This 17-probe set effectively captures the gene, achieving detection results consistent with Sanger sequencing. Summary of the Invention

[0009] One object of the present invention is to provide a capture probe group for protein C deficiency-related gene PROC, wherein the capture probe can effectively enrich the target gene; the present invention also provides a kit comprising the capture probe group, wherein the kit is used to construct a capture library for detecting protein C deficiency-related gene PROC; based on this, the present invention provides a method for constructing a capture library for protein C deficiency-related gene PROC, and by performing high-throughput sequencing analysis on the constructed capture library, the diagnosis or auxiliary diagnosis of protein C deficiency is achieved.

[0010] The present invention includes the following technical solutions:

[0011] In a first aspect, the present invention provides a capture probe group for capturing protein C deficiency-related gene PROC, characterized in that the capture probe group consists of probes shown in SEQ ID NO.1 to SEQ ID NO.17.

[0012] In a second aspect, the present invention provides a use of the capture probe set according to the first aspect of the present invention in at least one of the following:

[0013] (1) Application of hybridization to capture PROC, a gene related to protein C deficiency;

[0014] (2) Application in the preparation of products for hybrid capture of protein C deficiency-related genes PROC.

[0015] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0016] In a third aspect, the present invention provides a kit, characterized in that the kit comprises the capture probe group described in the first aspect of the present invention.

[0017] Furthermore, the kit also includes reagents commonly used in the art based on second-generation gene sequencing, including but not limited to adapters, pre-amplification primers, and tag primers.

[0018] In a specific embodiment of the present invention, the linker sequence is shown as SEQ ID NO.18 to SEQ ID NO.19.

[0019] In a specific embodiment of the present invention, the pre-amplification primers are shown as SEQ ID NO.20 to SEQ ID NO.21.

[0020] In a specific embodiment of the present invention, the tag primers are shown as SEQ ID NO.22 to SEQ ID NO.23.

[0021] Furthermore, the kit also includes a premix for fragmentation and repair, a ligation buffer, an amplification premix, a blocking agent, a hybridization solution, capture magnetic beads, a magnetic bead cleaning solution, and a washing solution. Unless otherwise specified in the present invention, the reagents can be obtained through commercial channels.

[0022] In a fourth aspect, the present invention provides a use of the kit according to the third aspect of the present invention in at least one of the following:

[0023] (1) Application in constructing PROC capture library of protein C deficiency-related genes;

[0024] (2) Application in the preparation of products for constructing PROC capture libraries of protein C deficiency-related genes;

[0025] (3) Application in the preparation of products for detecting mutations at the PROC site of genes associated with protein C deficiency;

[0026] (4) Application in the preparation of products for screening PROC-related genes related to protein C deficiency;

[0027] (5) Use in the preparation of products for diagnosing or assisting in the diagnosis of protein C deficiency.

[0028] The products include but are not limited to chips or detection platforms.

[0029] In a specific embodiment of the present invention, the method for constructing a protein C deficiency-related gene PROC capture library comprises the following steps:

[0030] (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation;

[0031] (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it;

[0032] (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library;

[0033] (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification. The amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes.

[0034] In a specific embodiment of the present invention, the method for detecting a mutation at the PROC site of a protein C deficiency-related gene comprises the following steps:

[0035] (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation;

[0036] (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it;

[0037] (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library;

[0038] (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain a PROC capture library of protein C deficiency-related genes;

[0039] (5) Sequencing the captured library and analyzing the sequencing results to obtain the PROC site mutation results of the protein C deficiency-related gene.

[0040] In a specific embodiment of the present invention, the method for diagnosing or assisting in the diagnosis of protein C deficiency comprises the following steps:

[0041] (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation;

[0042] (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it;

[0043] (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library;

[0044] (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain a PROC capture library of protein C deficiency-related genes;

[0045] (5) Sequencing the captured library and analyzing the sequencing results to obtain the PROC site mutation results of the protein C deficiency-related gene;

[0046] (6) To diagnose or assist in the diagnosis of protein C deficiency based on the mutation results of PROC sites of protein C deficiency-related genes.

[0047] In a fifth aspect, the present invention provides a method for constructing a protein C deficiency-related gene PROC capture library, the method comprising the following steps:

[0048] (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation;

[0049] (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it;

[0050] (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library;

[0051] (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification. The amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes.

[0052] In a sixth aspect, the present invention provides a method for detecting a mutation in a PROC site of a protein C deficiency-related gene for purposes other than disease diagnosis and treatment, comprising the following steps:

[0053] (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation;

[0054] (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it;

[0055] (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library;

[0056] (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain a PROC capture library of protein C deficiency-related genes;

[0057] (5) Sequencing the captured library and analyzing the sequencing results to obtain the PROC site mutation results of the protein C deficiency-related gene.

[0058] The technical solution provided by the present invention has the following technical advantages:

[0059] (1) The capture probe set of the protein C deficiency-related gene PROC provided by the present invention can capture the specific coding region of the gene and sequence the target gene accurately;

[0060] (2) The method for constructing a sequencing library for the protein C deficiency-related gene PROC provided by the present invention uses liquid phase hybridization sequence capture technology to enrich the target gene, thereby achieving effective enrichment of the target gene, accurately selecting the target detection region, and better controlling the sequencing cost;

[0061] (3) The protein C deficiency-related gene PROC provided by the present invention can conduct a more in-depth study of gene characteristics, including not only gene screening, but also further analysis of the gene's SNP (single nucleotide polymorphism) and Indel (base insertion and deletion), to achieve accurate diagnosis or auxiliary diagnosis of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Flowchart of library construction and sequencing for detection of PROC, a gene associated with protein C deficiency.

[0063] Figure 2Sanger sequencing results of sample number 2PROC chr2:128183757c.632G>A.

[0064] Figure 3 Sanger sequencing results of sample number 2PROC chr2:128186235c.1099G>A.

[0065] Figure 4 Sanger sequencing results of sample number 3PROC NM_000312.4:exon7:c.577_579del.

[0066] Figure 5 Sanger sequencing results of sample number 5PROC chr2:128183757c.632G>A.

[0067] Figure 6 Sanger sequencing results of sample number 5PROC chr2:128186235c.1099G>A. DETAILED DESCRIPTION

[0068] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0069] Example 1 Design of capture probe set for protein C deficiency-related gene PROC

[0070] The target genes captured in this example and their chromosome coordinate information are shown in Table 1:

[0071] Table 1 Details of the genes captured in the present invention and their chromosome coordinate information

[0072] Gene name Capture length chromosome Gene start site Gene termination site PROC 1769 chr2 128176003 128186818

[0073] The design rules of the capture probe set for capturing the target gene PROC provided by the present invention are as follows:

[0074] (1) The probe length is 120 bp;

[0075] (2) The probe adopts a flat-lay design;

[0076] (3) The probe reference is the positive strand of the gene;

[0077] (4) Probe design ensures probe specificity by comparing whole genome information;

[0078] (5) Design probes in the low GC content region.

[0079] Based on the above design rules, multiple capture probe groups of the present invention were obtained. After screening and optimization, an optimal capture probe group consisting of probes shown in SEQ ID NO.1 to SEQ ID NO.17 was obtained. The nucleotide information of the probes is shown in Table 2.

[0080] Table 2 Nucleotide sequence information of capture probe set targeting PROC gene

[0081]

[0082]

[0083] Example 2: Linker sequences for constructing capture libraries

[0084] The sequence information of the linker designed and synthesized in this example is shown in Table 3:

[0085] Table 3 Details of the linker sequences provided by the present invention

[0086] name sequence Serial number Adapter sequence Adapter-1 5'-phos-CTACACGACGCTCTTCCGATCTT-3' SEQ ID NO.18 Adapter sequence Adapter-2 5'-AGATCGGAAGAGCACACGTCTG-3' SEQ ID NO.19

[0087] Example 3 Pre-amplification primer sequences for constructing capture libraries

[0088] The sequence information of the pre-amplification primers designed and synthesized in this example is shown in Table 4:

[0089] Table 4 Details of the preamplification primer sequences provided by the present invention

[0090] name sequence Serial number Upstream primer / F 5'-CTACACGACGCTCTTCCG-3' SEQ ID NO.20 Downstream primer / R 5'-CAGACGTGTGCTCTTCCG-3' SEQ ID NO.21

[0091] Example 4: Tag Primer Sequences for Constructing a Capture Library

[0092] The sequence information of the tag primers designed and synthesized in this example is shown in Table 5:

[0093] Table 5 Details of the tag primer sequences provided by the present invention

[0094]

[0095] Example 5 Kit for detecting protein C deficiency-related gene PROC

[0096] The kit includes the following reagents:

[0097] Table 6 Composition information of each reagent in the kit

[0098] name Element Capture probe set Capture probe set consisting of SEQ ID NO.1 to SEQ ID NO.17 Interrupt the final repair premix Rapid fragmentation / end repair / A-tail addition module Adapter sequence Sequences shown in SEQ ID NO. 18 to SEQ ID NO. 19 Ligase Rapid DNA Ligase Module Ligation buffer Rapid DNA Ligation Module-Buffer Amplification master mix KAPA HiFi HotStart ReadyMix 2x Pre-amplification primers Sequences shown in SEQ ID NO.20 to SEQ ID NO.21 sealant Human Cot DNA Hybridization Solution A 2X Hybridization Buffer Hybridization Solution B Hybridization Buffer Enhancer Magnetic bead washing solution (2x) Working solution volume 300μL (stock solution 150μL + water 150μL), 2X Bead Wash Buffer Washing solution 1 (10x) Working solution volume 300μL (stock solution 30μL + water 270μL), 10X Stringent Wash Buffer Washing solution 2 (10x) Working solution volume 250 μL (stock solution 25 μL + water 225 μL), 10X Wash Buffer I Washing solution 3 (10x) Working solution volume 150μL (stock solution 15μL + water 135μL), 10X Wash Buffer II Washing solution 4 (10x) Working solution volume 150μL (stock solution 15μL + water 135μL), 10X Wash Buffer III Index primer sequence Sequences shown in SEQ ID NO. 22 to SEQ ID NO. 23 Capture magnetic beads Streptavidin magnetic beads

[0099] In the above kit, except for the capture probe set, other reagents can be purchased through commercial channels.

[0100] Example 6 Library construction and sequencing method

[0101] The library for detecting protein C deficiency-related genes PROC described in the present invention is constructed based on the 150PE sequencing platform of the BGI platform. The library construction and sequencing process are as follows:

[0102] Step 1: Genomic DNA fragmentation and end repair

[0103] 1.1. Extract the genomic DNA of the sample, centrifuge, and place on ice. Take 50 μL of genomic DNA, add 10 μL of the shearing and repair premix, vortex to mix, and centrifuge briefly.

[0104] 1.2. Start the reaction program on the PCR instrument: 4℃ for 1 min, 30℃ for 15 min, 72℃ for 20 min, hold at 4℃, set the reaction volume to 60 μL, and heat the lid at 75℃.

[0105] Step 2: Adapter ligation and purification

[0106] 2.1. Centrifuge the product from the previous step, place on ice, and add 30 μL of ligation buffer and 5 μL of ligase;

[0107] 2.2. Add 5 μL of adapters (Adapter-1 and Adapter-2) to the wall of the PCR tube, vortex to mix, and centrifuge briefly to ensure that all the reaction solution is at the bottom of the PCR tube;

[0108] 2.3. Start the reaction program on the PCR instrument: 20℃ for 15 min, 4℃ hold, reaction volume set to 100 μL, no heated cover required;

[0109] 2.4. Mix the purified magnetic beads by inversion and equilibrate at room temperature for 30 minutes. Add 75 μL of purified magnetic beads to the ligation reaction product in step 2.3, mix well, and incubate at room temperature for 5 minutes.

[0110] 2.5. Centrifuge the PCR tube briefly and place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0111] 2.6. Slowly add 150 μL of 80% ethanol solution along the side of the PCR tube, taking care not to disturb the magnetic beads. Let it stand for 30 seconds, and use a pipette to remove and discard the supernatant.

[0112] 2.7. Repeat step 2.6 once;

[0113] 2.8. Centrifuge the PCR tube briefly and place it on a magnetic rack. Use a 10 μL pipette tip to remove any remaining 80% ethanol solution.

[0114] 2.9. Open the PCR tube cap and let it stand at room temperature for about 2-3 minutes until the ethanol evaporates completely.

[0115] 2.10. Remove the PCR tube from the magnetic stand, add 25 μL of Nuclease Free Water to the PCR tube, mix thoroughly, and centrifuge briefly to remove the droplets from the tube cap. Let it stand at room temperature for 5-10 minutes.

[0116] 2.11. Place the reaction tube on a magnetic rack and allow to adsorb for 3 minutes. Pipette 23 μL of supernatant and place it in a new PCR tube.

[0117] Step 3: Prelibrary Amplification and Purification

[0118] 3.1. Place the purified product recovered in the previous step on ice, add 25 μL of amplification premix and 2 μL of preamplification primers (upstream primer / F and downstream primer / R), mix well, and centrifuge briefly to allow the entire reaction solution to settle at the bottom of the PCR tube;

[0119] 3.2. Place the PCR tube in a thermal cycler and start the following program: 98°C for 45 seconds; 11 cycles of (98°C for 15 seconds, 58°C for 30 seconds, 72°C for 30 seconds); 72°C for 1 minute, hold at 4°C, volume set to 50 μL, heated lid at 105°C.

[0120] 3.3. Purify the product from the previous step using purification magnetic beads. The purification steps are the same as 2.4-2.11. After purification, aspirate 33 μL of supernatant and place it in a new PCR tube. This is the pre-library.

[0121] Step 4: Quantification of the library

[0122] Use a fluorescence quantification instrument (such as QubitTM 3.0 Fluorometer) or quantitative PCR to quantitatively check the library. The library fragment distribution quality control was performed using fragment analysis instruments such as Agilent and Qsep100 (Bioptic).

[0123] Step 5: Hybridization of the library

[0124] 5.1. Take 1 to 8 purified prelibraries (500 ng each) and mix them. After the prelibrary mixture is completed to 60 μL with nuclease-free deionized water, add 5 μL of blocking agent.

[0125] 5.2. Mix the magnetic beads equilibrated at room temperature for 30 minutes by inversion. Add 75 μL of purified magnetic beads to the reaction tube from the previous step. Mix thoroughly, centrifuge briefly to remove droplets from the tube cap, and let stand at room temperature for 5-10 minutes.

[0126] 5.3. After standing, place the product on a magnetic rack and adsorb for 5 minutes, then remove the supernatant with a pipette;

[0127] 5.4. Slowly add 150 μL of 80% ethanol solution along the wall of the centrifuge tube, taking care not to disturb the magnetic beads. Let it stand for 30 seconds, and use a pipette to remove and discard the supernatant.

[0128] 5.5. Repeat step 5.4 once;

[0129] 5.6. After a brief centrifugation, place the tube on a magnetic rack and use a 10 μL pipette tip to remove a small amount of residual 80% ethanol solution, being careful not to absorb the magnetic beads.

[0130] 5.7. Let the magnetic beads dry at room temperature for 5 minutes until the ethanol evaporates completely.

[0131] 5.8. Remove the centrifuge tube from the magnetic stand and add 9.5 μL of hybridization solution A, 3 μL of hybridization solution B, and 6.5 μL of the capture probe set provided by the present invention;

[0132] 5.9. Vortex to mix the magnetic beads and hybridization reaction solution thoroughly, incubate at 65°C for 4 minutes, vortex to mix, centrifuge briefly, and let stand at room temperature for 4 minutes;

[0133] 5.10. Centrifuge the tube briefly and place it on a magnetic rack until the liquid is completely clear. Use a pipette to transfer 17 μL of the supernatant to a new PCR tube.

[0134] 5.11. Start the following hybridization program on the PCR instrument: 95°C for 2 minutes; 65°C for 12 hours, hold at 65°C, heat the cover at 105°C, and add 17 μL of the reaction system. The hybridization time should be no less than 12 hours and no more than 24 hours.

[0135] Step 6: Magnetic Bead Capture

[0136] 6.1. After the hybridization reaction, adjust the PCR instrument to the elution program: 65°C∞, heated cover 70°C, reaction volume: 34μL;

[0137] 6.2. Add the resuspended capture magnetic beads (washed according to existing techniques) to the hybridization system and mix by gently pipetting or vortexing;

[0138] 6.3. Incubate at 65°C for 45 minutes. Gently vortex every 15 minutes to ensure complete resuspending of the beads. The reaction temperature should be maintained at around 65°C throughout the entire operation.

[0139] Step 7: High temperature washing

[0140] 7.1. After incubation, remove the PCR tube from the PCR instrument and add 100 μL of preheated 65°C Wash Buffer 2 to the tube. Pipet and mix the hybridization system containing the magnetic beads.

[0141] 7.2. Remove the PCR tube from the magnetic rack and add 100 μL of preheated 65°C Wash Buffer 1. Mix thoroughly by gently pipetting 10 times. Incubate the tube at 65°C in a PCR instrument for 5 minutes. Then, place the tube on the magnetic rack for 1 minute until the solution is clear. Remove the supernatant and repeat the previous step twice.

[0142] Step 8: Wash at room temperature

[0143] 8.1. Remove the sample from the magnetic rack, add 150 μL of room temperature Wash Buffer 2, vortex mix for 30 seconds, let stand at room temperature for 30 seconds, vortex mix for 30 seconds, centrifuge the PCR tube briefly, and place it on the magnetic rack for 1 minute. After the liquid is completely clear, aspirate and discard the supernatant.

[0144] 8.2. Remove the sample from the magnetic rack, add 150 μL of room temperature Wash Buffer 3, vortex mix for 30 seconds, let stand at room temperature for 30 seconds, vortex mix for 30 seconds, centrifuge the PCR tube briefly, and place it on the magnetic rack for 1 minute. After the liquid is completely clear, aspirate and discard the supernatant.

[0145] 8.3. Remove the sample from the magnetic rack, add 150 μL of room temperature Wash Buffer 4 working solution, vortex mix for 30 seconds, let stand at room temperature for 30 seconds, vortex mix for 30 seconds, centrifuge the PCR tube briefly, and place it on the magnetic rack for 1 minute. After the liquid is completely clear, aspirate and discard the supernatant.

[0146] 8.4. Use a 10μL pipette tip to remove a small amount of residual Wash Buffer 4 working solution;

[0147] 8.5. Remove the PCR tube from the magnetic stand, add 20 μL of Nuclease-Free Water, and gently pipette 10 times to ensure even mixing.

[0148] Step 9: Post-capture PCR amplification

[0149] 9.1. Centrifuge 20 μL of the magnetic beads with captured DNA from the previous step, place on ice, add 25 μL of amplification premix and 5 μL of label primer, mix well, and centrifuge.

[0150] 9.2. Set the PCR instrument to the following program: 98°C for 45 s; 11 cycles (98°C for 15 s, 58°C for 30 s, 72°C for 30 s), 72°C for 1 min, 4°C for ∞, set the volume to 50 μL, heat the lid at 105°C, place the reaction tube on the heat exchanger, and start PCR amplification.

[0151] Step 10: Library purification

[0152] Use purification magnetic beads to purify the product from the previous step. The purification steps are the same as 2.4-2.11. After purification, aspirate 33 μL of supernatant and place it in a new EP tube.

[0153] Step 11: Quantification of the library

[0154] Use a fluorescence quantification instrument (such as QubitTM 3.0 Fluorometer) or quantitative PCR to perform quantitative quality control on the library. The library fragment distribution quality control was performed using fragment analysis instruments such as Agilent and Qsep100 (Bioptic).

[0155] Step 12: Sequencing

[0156] Using the final product of step 10, the captured region was sequenced with a 150 bp read length using the MGISEQ-2000 second-generation sequencer produced by Wuhan MGI Intelligent Manufacturing Technology Co., Ltd.

[0157] Step 13: Bioinformatics Analysis

[0158] The bioinformatics analysis process is as follows:

[0159] 13.1. Raw FASTQ data were quality controlled, adapter removed, and low-quality data filtered using fastp (version 0.23.2).

[0160] 13.2. Align the filtered FASTQ to the human reference genome (build hg19) using bwa-mem (version 2.2.1) to generate an alignment file.

[0161] 13.3. Alignment files were processed and quality controlled using samblaster (version 0.1.26), samtools (version 1.16.1), and mosdepth (version 0.3.3).

[0162] 13.4. The processed alignment files were used for variant detection using gatk (version 4.3.0.0);

[0163] 13.5. Variant detection results were filtered and quality-controlled using bcftools (version 1.16);

[0164] 13.6. Mutation detection results were annotated using snpeff (version 5.0) and annovar (version 2020-06-08).

[0165] Step 14: Report interpretation;

[0166] Step 15: Sanger sequencing verification.

[0167] Application Examples

[0168] According to the method provided by the present invention, PROC gene testing was performed on 10 patients clinically diagnosed with deep vein thrombosis. Five of the patients with deep vein thrombosis were found to carry PROC gene mutations, with a positive test rate of 50%. Three of these patients had compound heterozygous PROC gene mutations, and two patients had heterozygous PROC gene mutations. The statistical results are shown in Table 7, and the genetic testing results are shown in Table 8.

[0169] Table 7 Statistical results of genetic testing

[0170]

[0171] Table 8 PROC gene abnormality test results

[0172]

[0173]

[0174] Sanger validation was performed on samples numbered 2, 3, and 5. The results are shown in the attached Figures 1 to 5 It can be seen that the PROC gene detection results obtained using the kit and detection method provided by the present invention are consistent with the Sanger method detection results, indicating that the detection method provided by the present invention has accurate detection results and has the potential for clinical application development.

[0175] Capture probe set optimization process

[0176] (1) Selection of detection target area

[0177] Those skilled in the art know that the capture probe used to capture the target gene can fully cover the target gene as needed, that is, the coverage rate is 100%; or it can only cover the known mutations in the target gene 100%. Those skilled in the art can make specific choices based on detection requirements.

[0178] In a specific embodiment of the present invention, the exons, 3' and 5' URTs, promoter region, and known mutation sites of the target gene PROC are targeted regions.

[0179] (2) Probe design rules

[0180] The probe length is 120bp; the probe adopts a tiled design; the probe reference is the positive strand of the gene; the probe specificity is ensured by comparing the whole genome information during probe design; the probe is designed with encryption in the low GC content region.

[0181] The present invention uses probe design software to design multiple capture probe panels, compares and analyzes the designed probes, increases the probe coverage of genes with low coverage, and makes the coverage reach above 99.9%, the coverage uniformity is greater than 99%, and the depth is 1X for sequence synthesis.

[0182] Exemplarily, the present invention provides a set of capture probe groups, as shown in Table 9:

[0183] Table 9 Nucleotide sequence information of another capture probe set targeting PROC gene

[0184]

[0185]

[0186]

[0187] The difference between the capture probe set shown in Table 9 and the optimal capture probe set shown in Table 2 of the present invention is that the probes have different coverage ratios of the target region. The performance of the two capture probe sets was compared, and the results are shown in Table 10:

[0188]

[0189]

[0190] From the comparison results in the above table, it can be seen that even if the technology for designing capture probes for target genes is conventional in the art, the selection of target regions, probe design rules, and adjustment of target gene coverage will affect the final detection results of the capture probe set. The capture probe set provided by the present invention (Table 2) can achieve effective enrichment of the target gene, and ultimately obtain detection results consistent with the Sanger sequencing method.

[0191] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A capture probe set, wherein the capture probe set is used to capture the protein C deficiency-related gene PROC, characterized in that: The capture probe group consists of probes shown in SEQ ID NO.1 to SEQ ID NO.

17.

2. Use of the capture probe set according to claim 1 in at least one of the following: (1) Application of hybridization to capture PROC, a gene related to protein C deficiency; (2) Application in the preparation of a product for hybrid capture of protein C deficiency-related gene PROC; The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

3. A kit, characterized in that The kit comprises the capture probe set according to claim 1.

4. The kit according to claim 3, wherein The kit also includes reagents commonly used in the art based on second-generation gene sequencing, including adapters, pre-amplification primers, and tag primers.

5. Use of the kit according to claim 3 or 4 in at least one of the following: (1) Application in constructing PROC capture library of protein C deficiency-related genes; (2) Application in the preparation of products for constructing PROC capture libraries of protein C deficiency-related genes; (3) Application in the preparation of products for detecting mutations at the PROC site of genes associated with protein C deficiency; (4) Application in the preparation of products for screening PROC-related genes related to protein C deficiency; (5) Use in the preparation of products for diagnosing or assisting in the diagnosis of protein C deficiency.

6. The use according to claim 5, characterized in that The method for constructing a protein C deficiency-related gene PROC capture library comprises the following steps: (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation; (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it; (3) adding the capture probe set described in claim 1 to perform hybridization reaction on the purified pre-library; (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification. The amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes.

7. The use according to claim 5, characterized in that The method for detecting a mutation at the PROC site of a protein C deficiency-related gene comprises the following steps: (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation; (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it; (3) adding the capture probe set described in claim 1 to perform hybridization reaction on the purified pre-library; (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes; (5) Sequencing the captured library and analyzing the sequencing results to obtain the mutation results of the PROC site of the protein C deficiency-related gene.

8. The use according to claim 5, characterized in that The method for diagnosing or assisting in the diagnosis of protein C deficiency comprises the following steps: (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation; (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it; (3) adding the capture probe set described in claim 1 to perform hybridization reaction on the purified pre-library; (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes; (5) Sequencing the captured library and analyzing the sequencing results to obtain the PROC site mutation results of the protein C deficiency-related gene; (6) To diagnose or assist in the diagnosis of protein C deficiency based on the mutation results of the PROC site of the protein C deficiency-related gene.

9. A method for constructing a protein C deficiency-related gene PROC capture library, the method comprising the following steps: (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation; (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it; (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library; (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification. The amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes.

10. A method for detecting mutations at the PROC site of a protein C deficiency-related gene for purposes other than disease diagnosis and treatment, comprising the following steps: (1) Extracting DNA from the sample to be tested, fragmenting and end-repairing the DNA sample, and adding adapter sequences for adapter ligation; (2) adding pre-amplification primers for pre-amplification, obtaining a pre-library and purifying it; (3) adding the capture probe set described in the first aspect of the present invention to perform hybridization reaction on the purified pre-library; (4) After the hybridization reaction, magnetic beads are added for capture, and after capture, label primers are added for amplification, and the amplified products are purified to obtain the PROC capture library of protein C deficiency-related genes; (5) Sequencing the captured library and analyzing the sequencing results to obtain the mutation results of the PROC site of the protein C deficiency-related gene.

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