Probe set and kit for detecting single-gene genetic disease variation and application

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a probe set for detecting single-gene genetic disease variation, a kit and application. The invention provides a more comprehensive detection gene coverage and complete detection scheme for gene detection of more than 700 kinds of single-gene genetic diseases, supplementation is carried out for carrier screening of single diseases clinically at present, meanwhile, the detection period is shortened, and the genetic disease risk of fertility people can be rapidly and accurately determined clinically.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and specifically relates to a probe set, a kit and an application for detecting single gene genetic disease mutations. Background Art

[0002] Genetic monogenic diseases are one of the important causes of birth defects in newborns. Birth defects can bring great pain and burden to children and their families. About 30% of children with birth defects die before the age of 5, and 40% are disabled for life. It is a global public health problem that affects the quality of the newborn population. . According to epidemiological statistics, monogenic diseases affect 1% of live births, causing 10% of infant mortality and 20% of pediatric hospitalization rates. In recent studies, 36.7%-50% of critically ill infants were diagnosed with Mendelian genetic diseases by rapid clinical exome sequencing or whole genome sequencing. Heterozygous carriers of autosomal recessive genetic diseases generally have no clinical symptoms. If both men and women carry the same pathogenic gene for an autosomal recessive genetic disease, or if women carry pathogenic variants for X-linked recessive diseases, the risk of giving birth to offspring with monogenic diseases increases. According to the OMIM database, as of December 31, 2023, there are 8,590 clear monogenic diseases, of which more than 1,300 are clear autosomal and sex chromosome recessive genetic diseases.

[0003] Carrier screening refers to the identification of carriers of autosomal or X-linked recessive genetic diseases before or during early pregnancy. In recent years, many domestic and international studies have shown that pre-pregnancy carrier screening for recessive genetic diseases with high carrier rates is an important primary prevention measure to prevent birth defects. Initial carrier screening is mainly targeted at individuals with a family history of specific genetic diseases or individuals from specific high-risk groups.

[0004] Expanded carrier screening is a screening technology for the reproductive population. It can fully understand the genetic background of the couple before or during early pregnancy to assess the risk of their offspring suffering from multiple genetic diseases. According to the "Expert Consensus on Carrier Screening Laboratory and Clinical Practice for Reproductive Populations", carrier screening has evolved from a pre-pregnancy screening technology that was originally only targeted at specific populations and specific diseases to a screening for the general population and multiple diseases, showing significant clinical utility.

[0005] At present, the target diseases for clinical carrier screening are mainly limited to autosomal recessive or X-linked genetic diseases, with priority given to diseases of moderate severity or above, and the use of high-throughput targeted sequencing technology for multi-disease joint screening. For some special variants, other technologies may need to be supplemented, such as Sanger sequencing. Compared with WES, Panel testing targets a small range of genetic testing and has a higher sequencing depth, which can ensure the sensitivity of the detection range.

[0006] The single-gene genetic disease carrier screening products on the market mainly use NGS technology, which can efficiently detect multiple genes at the same time, covering dozens or even hundreds of genetic diseases, greatly improving screening efficiency and reducing costs.

[0007] However, the current single-gene genetic disease carrier screening products have the following limitations: 1. There are many types of single-gene diseases, and the types of disease mutations are equally complex. It is difficult for carrier screening products on the market to cover all known single-gene diseases, and they can usually only screen for common or high-risk diseases in specific populations; 2. There is no unified consensus on the types of diseases included in the screening products. The large number of testing items has led to longer screening cycles and increased costs, which to a certain extent limits the popularization and application of screening technology; 3. Whole genome and whole exome sequencing are relatively accurate and comprehensive, but the sequencing depth is low, which may lead to the missed detection of some low-frequency mutations, and some VUS sites are difficult to interpret; 4. Certain special genetic diseases or mitochondrial genetic diseases may require specific detection technologies, which are difficult to accurately detect with conventional NGS technology. Summary of the invention

[0008] Based on this, an embodiment of the present application provides a probe set, a kit and an application for detecting single gene genetic disease mutations.

[0009] On the one hand, the present application provides a probe set for detecting single gene genetic disease variation.

[0010] The probe set can detect the exon regions of 532 single-gene genetic disease-related pathogenic gene mutations in the human genome, the intron regions 25 bp upstream and downstream of the exons, and the pathogenic copy number variations (CNVs) of high-incidence diseases;

[0011] The detected variant types include one or more of the following (1) to (5);

[0012] (1) Single nucleotide variations, insertions and deletions;

[0013] (2) copy number variation of ≥2 consecutive exons in DMD;

[0014] (3) exon 7 of SMN1 is deleted;

[0015] (4) Loss of -α3.7; -α4.2; --SEA of HBA1 / HBA2;

[0016] (5) SLC25A13 gene ins3kb and ins6kb, CFTR c.1210-12T[5] special mutation.

[0017] The GRCh38 version of the human nuclear genome and the rCRS NC_012920 version of the human mitochondrial genome are used as standard reference genomes, and the probe set includes 611 probes.

[0018] On the other hand, the present application provides a kit for detecting a single gene genetic disease, wherein the kit comprises the probe set.

[0019] In some embodiments, the kit further comprises a hybridization reaction reagent.

[0020] In some embodiments, the hybridization reaction reagents include one or more of magnetic beads, blocking solution and hybridization buffer.

[0021] On the other hand, the present application provides a method for detecting a target region, comprising: using the probe composition or the kit to capture the target region.

[0022] In some embodiments, the method comprises the steps of:

[0023] Extracting genomic DNA from the sample to be tested and preparing a genomic pre-library;

[0024] Adding the probe set according to claim 1 or the kit according to any one of claims 2 to 4 to the genomic pre-library to perform hybridization capture on the target gene region;

[0025] The hybridization captured product is detected to determine the gene mutation of the single gene genetic disease in the sample to be tested.

[0026] In some of these embodiments, the method for detecting the hybridization captured product comprises high-throughput sequencing.

[0027] In some embodiments, the sample to be tested is a blood sample.

[0028] In some embodiments, the blood sample comprises a peripheral blood sample.

[0029] In some of the embodiments, the steps of preparing the genomic pre-library include: fragmenting the genomic DNA, performing end-repair and A-tailing and adapter ligation treatments, and purifying the adapter-ligated product.

[0030] The purified product was amplified by PCR to prepare a genomic pre-library.

[0031] On the other hand, the present application provides the use of the probe set or the kit in preparing a product for detecting a single gene genetic disease.

[0032] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0034] Figure 1 This is a flow chart of a method for detecting a single gene genetic disease in one embodiment of the present application;

[0035] Figure 2 This is the quality inspection result of the library in one embodiment of the present application;

[0036] Figure 3 For one embodiment of the present application, the DMD gene is detected to have an Exon48-53 deletion;

[0037] Figure 4 For one embodiment of the present application, the DMD gene is detected to have an Exon48-53 deletion;

[0038] Figure 5 For one embodiment of the present application, SMN1 Exon7 heterozygous deletion is detected;

[0039] Figure 6 For one embodiment of the present application, SMN1 Exon7 heterozygous deletion is detected;

[0040] Figure 7 For one embodiment of the present application, the HBA genotype αα / -α4.2 is detected;

[0041] Figure 8 For one embodiment of the present application, the HBA genotype αα / -α3.7 is detected;

[0042] Fig. 9 For one embodiment of the present application, the HBA genotype αα / --SEA is detected;

[0043] Fig.10 For one embodiment of the present application, the insertion variation of gene SLC25A13 was detected: IVS16ins3Kb (detected in all 6 cases);

[0044] Fig.11 This is a sequencing mutation result diagram of an embodiment of the present application, the mutation type is RDH12 c.437T>Ap.Val146Asp;

[0045] Fig.12 This is a sequence mutation result diagram of an embodiment of the present application, the mutation type is GJB2 c.235delp.Leu79Cysfs*3;

[0046] Fig.13 Negative sample type. DETAILED DESCRIPTION

[0047] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0049] the term

[0050] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0051] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0052] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0053] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0054] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0055] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0056] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0057] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0059] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0060] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0061] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0062] On the one hand, the present application provides a probe set for detecting variations in single-gene genetic diseases, using the GRCh38 version of the human nuclear genome and the rCRS NC_012920 version of the human mitochondrial genome as standard reference genomes, and the probe set includes 611 probes. On the other hand, the present application provides a kit for detecting single-gene genetic diseases, including the above-mentioned probe set.

[0063] In some embodiments, hybridization reaction reagents are also included.

[0064] In some embodiments, the hybridization reaction reagents include one or more of magnetic beads, blocking solution and hybridization buffer.

[0065] On the other hand, the present application provides a method for detecting a target region, comprising: using the above-mentioned probe composition, or the above-mentioned kit, to capture the target region.

[0066] In some embodiments, the process includes extracting genomic DNA from a sample to be tested and preparing a genomic prelibrary.

[0067] The above probe set or the above kit is added to the genomic pre-library to perform hybridization capture on the target gene region, and the hybridization capture product is detected to determine the gene mutation of the single gene genetic disease in the sample to be tested.

[0068] In some of these embodiments, the method for detecting the hybridization captured product comprises high-throughput sequencing.

[0069] In some embodiments, the sample to be tested is a blood sample.

[0070] In some embodiments, the blood sample includes a peripheral blood sample.

[0071] In some of the embodiments, the steps of preparing the genomic pre-library include: fragmenting the genomic DNA, performing end-repair and A-tailing and adapter ligation treatments, purifying the adapter-ligated products, and performing PCR amplification on the purified products to prepare the genomic pre-library.

[0072] On the other hand, the present application provides the use of the above-mentioned probe set and kit in the preparation of products for detecting single gene genetic diseases.

[0073] This application provides more comprehensive gene coverage and complete testing solutions for more than 700 single-gene genetic diseases, supplementing the current carrier screening for single diseases in clinical practice, while shortening the testing cycle, and can quickly and accurately determine the genetic disease risks of the reproductive population in clinical practice.

[0074] Compared with traditional single-gene testing solutions, the following effects are included:

[0075] 1. More comprehensive coverage of single gene genetic diseases

[0076] The panel covers more than 700 diseases associated with more than 500 genes.

[0077] 2. High coverage of target sites

[0078] The coverage of the target region (CDS±25bp) ≥30X is as high as 99%, the average depth of the fixed site is as high as 500X or more, and the coverage of the fixed site at 30X is as high as 99%.

[0079] 3. Stable detection system

[0080] The experimental system is stable, and the CV of library results between samples is less than 10%; it relies on DA500, DA5000 and DNBSEQ-T7 platforms for detection, and the operation is stable.

[0081] 4. Lower cost and higher efficiency

[0082] This product can be matched with an automated data analysis platform, and a single sample can be completed within 3-4 hours from data download to report issuance.

[0083] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0084] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0085] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0086] Example 1

[0087] This embodiment provides a method for capturing gene mutations or fragment deletions associated with different single-gene genetic diseases using the probe set of the present application.

[0088] This probe set can detect 532 pathogenic genes related to single-gene genetic diseases and special genetic diseases DMD, HBA, SMA, as well as special mutations of the SLC25A13 gene.

[0089] The detected variant types include one or more of the following (1) to (5);

[0090] (1) Single nucleotide variations, insertions and deletions;

[0091] (2) copy number variation of ≥2 consecutive exons in DMD;

[0092] (3) exon 7 of SMN1 is deleted;

[0093] (4) Loss of -α3.7; -α4.2; --SEA of HBA1 / HBA2;

[0094] (5) SLC25A13 gene ins3kb and ins6kb, CFTR c.1210-12T[5] special mutation; the specific detection is shown in Table 1 below:

[0095] Table 1

[0096]

[0097]

[0098] 71 peripheral blood samples with known mutation information were selected, and the specific categories were as follows: 8 Duchenne muscular dystrophy (DMD) samples, DMD ≥ 2 consecutive exon deletions; 28 HBA samples; 19 spinal muscular atrophy (SMA) samples, all of which showed deletion of exon 7 of the SMN1 gene; 6 IVS16ins3Kb samples of the SLC25A13 gene; 10 SNV / INDEL samples, and the test results are shown in Table 11. DNA was extracted from the blood of positive patients and controls, and the library was constructed using this kit, probe hybridization capture and high-throughput sequencing (detailed steps are as follows).

[0099] Step 1: Pre-capture library construction

[0100] 1. Genomic DNA fragmentation and end repair with A tailing

[0101] Prepare the reaction system according to Table 2 below, perform pre-amplification on a PCR instrument according to the conditions shown in Table 3, and fragment peripheral blood samples of different mutation types:

[0102] Table 2: DNA fragmentation and end repair plus A-tailing system

[0103]

[0104] Table 3: DNA fragmentation and end-repair plus A-tailing reaction procedures

[0105]

[0106] 2. Adapter ligation reaction

[0107] Prepare the adapter ligation system according to the reagent sequence in Table 4 and mix well. Close the thermal cover of the PCR instrument, incubate at 22°C for 15 minutes, and store at 4°C.

[0108] Table 4: Connector connection system

[0109]

[0110]

[0111] 3. Sorting of connector products

[0112] (1) Vortex DNA purification magnetic beads (Ampure XP beads, A63882) for 30 seconds to mix and equilibrate at room temperature for 30 minutes. Aspirate slowly when using. Prepare fresh 80% ethanol solution for later use.

[0113] (2) Add 29 μL of magnetic beads to the 110 μL reaction system after the previous step, vortex thoroughly to mix, and let stand at room temperature for 5 minutes.

[0114] (3) Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify.

[0115] (4) Keep the PCR tube on the magnetic rack and carefully transfer 130 μL of supernatant into a new PCR tube.

[0116] (5) Add 16 μL of magnetic beads to the supernatant, vortex thoroughly to mix, and let stand at room temperature for 5 minutes.

[0117] (6) Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clear. Keep the PCR tube on the magnetic rack and carefully discard the supernatant.

[0118] (7) Keep the PCR tube on the magnetic rack, add 150 μL of 80% ethanol solution, let it stand at room temperature for 30 seconds, and discard the supernatant.

[0119] (8) Repeat the previous step.

[0120] (9) Cover the tube, centrifuge briefly, reinsert the PCR tube into the magnetic rack for 1 to 2 minutes, and use a 10 μL pipette to remove the ethanol remaining at the bottom.

[0121] (10) Keep the PCR tube on the magnetic rack and leave it at room temperature to allow the ethanol to evaporate completely.

[0122] (11) Remove the PCR tube from the magnetic rack, add 22 μL of nuclease-free water, vortex thoroughly to mix, and let stand at room temperature for 2 minutes.

[0123] (12) Place the PCR tube on a magnetic rack for 2 minutes. Wait for the solution to become clear and pipette 20 μL of the supernatant into a new PCR tube.

[0124] 4. PCR Amplification before Capture

[0125] Prepare the reaction system according to the reagent sequence in Table 5, mix well and amplify according to the procedure in Table 6.

[0126] Table 5: Pre-capture PCR reaction system

[0127]

[0128] Table 6: Pre-capture PCR amplification program

[0129]

[0130]

[0131] 5. Purification of PCR products before capture

[0132] (1) Vortex DNA purification magnetic beads (Ampure XP beads, A63882) for 30 seconds to mix and equilibrate at room temperature for 30 minutes. Aspirate slowly when using. Prepare fresh 80% ethanol solution for later use.

[0133] (2) Remove the PCR tubes from the gene amplification instrument, add 50 μL of DNA purification magnetic beads to each PCR tube, vortex thoroughly to mix, and let stand at room temperature for 5 minutes.

[0134] (3) Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clear. Keep the PCR tube on the magnetic rack and carefully discard the supernatant.

[0135] (4) Keep the PCR tube on the magnetic rack, add 150 μL of 80% ethanol solution, let it stand at room temperature for 30 seconds, and discard the supernatant.

[0136] (5) Repeat the previous step.

[0137] (6) Cover the tube, centrifuge briefly, reinsert the PCR tube into the magnetic rack for 1 to 2 minutes, and use a 10 μL pipette to remove the remaining ethanol at the bottom (be careful not to suck up the magnetic beads).

[0138] (7) Keep the PCR tube on the magnetic rack and leave it at room temperature to allow the ethanol to evaporate completely (be careful not to over-dry. If cracks appear on the magnetic beads, cover the tube and proceed to the next experimental operation immediately).

[0139] (8) Remove the PCR tube from the magnetic rack, add 30 μL of nuclease-free water, vortex thoroughly to mix, and let stand at room temperature for 2 minutes.

[0140] (9) Place the PCR tube on a magnetic rack for 2 minutes. Wait for the solution to become clear. Pipette 28 μL of the supernatant into a new EP tube and mark it.

[0141] 6. Quality control of the library before capture

[0142] Library concentration was measured. 1 μL of library was taken and quantified using Qubit dsDNA HS Assay Kit. The sample concentration should be ≥20 ng / μL and the library yield should be ≥500 ng. Only when both conditions were met were the criteria for the assay to be considered qualified.

[0143] Step 2: Hybridization capture

[0144] 1. Probe hybridization

[0145] Prepare the hybridization reaction system according to the reagent sequence in Table 7. After mixing, mix the 8 libraries with different label primers. Take 500ng of each library and add it to the PCR tube. Add DNA purification magnetic beads (Ampure XPbeads, A63882) to each PCR tube (DNA purification magnetic beads: total library volume = 1.8:1), mix and incubate at room temperature for 5 minutes, then place on the magnetic stand for 3 minutes, and discard the supernatant. Wash twice with 80% ethanol according to the magnetic bead manual, add 26μL of hybridization reaction system, gently blow or vortex at low speed to mix, and let stand at room temperature for 3 minutes. Place on the magnetic stand until the solution is clear, draw 26μL of supernatant into a new PCR tube with the corresponding number, add 2μL of blocking solution 2 (Aijitaikang Biotechnology (Beijing) Co., Ltd., catalog number: C80532) and 2μL of probe, gently blow or vortex at low speed to mix, and centrifuge briefly. Set the parameters of the gene amplification instrument as shown in Table 8, place the PCR tube on the gene amplification instrument, run the program, and incubate overnight (hybridization 12 hours to 18 hours).

[0146] Table 7: Hybridization reaction system

[0147]

[0148] Table 8: Probe hybridization program

[0149]

[0150] 2. Wash the capture beads

[0151] (1) Remove the capture magnetic beads in advance, vortex mix and place at room temperature for equilibration for 30 minutes before use. Prepare fresh 80% ethanol solution for use.

[0152] (2) Prepare and label PCR tubes, add 50 μL of capture beads to the tubes, place the tubes on a magnetic rack, wait for the solution to clear, and discard the supernatant.

[0153] (3) Remove the PCR tube from the magnetic rack, add 180 μL of Wash Buffer 1 and gently pipette several times to mix, resuspend the magnetic beads, place the PCR tube on the magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0154] (4) Repeat step (3) twice, washing the magnetic beads three times in total.

[0155] (5) Remove the PCR tube from the magnetic stand, add 180 μL of Wash Buffer 1, and gently pipette to resuspend the magnetic beads for later use.

[0156] 3. Target region DNA library enrichment

[0157] Keep the hybridization product on the gene amplification instrument, add the resuspended 180μL capture beads to the hybridization product, and mix gently with a pipette. Place the PCR tube on a rotating mixer at 10 rpm, combine at room temperature for 30 minutes, adsorb on a magnetic rack and discard the supernatant; add 150μL of wash buffer 2, mix well, place on a rotating mixer for 15 minutes, adsorb on a magnetic rack and discard the supernatant; add 150μL of 50℃ preheated wash buffer 3, mix well, incubate at 50℃ for 10 minutes, adsorb on a magnetic rack and discard the supernatant (wash with wash buffer 3: repeat 2 more washes + discard the supernatant, a total of 3 times). Add 150μL 80% ethanol solution, let stand for 30 seconds, then completely remove the ethanol solution, dry at room temperature, and add 24μL nuclease-free water to mix gently.

[0158] 4. Post-capture PCR amplification

[0159] Prepare the post-capture PCR amplification system according to the reagent sequence in Table 9. Add 24 μL of the magnetic bead product from the previous step and mix gently, then place in a gene thermal cycler and run the program according to the system in Table 10.

[0160] Table 9: Post-capture PCR amplification system

[0161]

[0162] Table 10: Post-capture PCR reaction system

[0163]

[0164] 5. Post-capture PCR product purification

[0165] Take out the PCR tube from the previous step, add 55μL DNA purification magnetic beads, mix thoroughly and let stand for 5 minutes, place on a magnetic rack and discard the supernatant. Wash twice with 150μL 80% ethanol solution, wait for the ethanol to evaporate completely, add 25μL nuclease-free water, mix thoroughly and let stand for 2 minutes. Place on a magnetic rack until the solution is clear, and pipette 23μL supernatant into a new EP tube.

[0166] 6. Post-capture library quality control

[0167] Library concentration was measured. 1 μL of library was taken and quantified using Qubit dsDNA HS Assay Kit. The library concentration was approximately ≥6 ng / μL.

[0168] Step 3: Library on the machine

[0169] The single-stranded library was circularized according to the instructions of the universal circularization reaction kit and sequenced using the DA5000 / DA500 platform.

[0170] Through analysis, the main DNA band is clear and ≥10kb, the DNA concentration is ≥10ng / uL, the total sample volume is ≥1ug, and there is no protein and salt ion contamination, indicating that the quality of genomic DNA meets the standard. The quality of the library before and after capture meets the requirement that the main peak is in the range of ~400bp ( Figure 2 Some results are listed). The data quality control results of high-throughput sequencing also showed high data quality: with an average data volume of 0.4G, the nuclear genome coverage depth reached an average of 150×, and the 30× coverage reached 99% (see Table 11). 8 cases of DMD, 28 cases of HBA, 19 cases of SMA, and 6 cases of SLC25A13 special mutations were detected (partial results are shown in Table 12, Figure 3-13 ).

[0171] Table 11: Quality control of high-throughput sequencing data of some samples (30 cases)

[0172]

[0173]

[0174] Table 12: Test results of some samples (30 cases)

[0175]

[0176]

[0177] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A probe set, characterized in that: The probe set can detect the exon regions of 532 single-gene genetic disease-related pathogenic gene mutations in the human genome, the intron regions 25 bp upstream and downstream of the exons, and the pathogenic copy number variations (CNVs) of high-incidence diseases; The detected variant types include one or more of the following (1) to (5); (1) Single nucleotide variations, insertions and deletions; (2) copy number variation of ≥2 consecutive exons in DMD; (3) exon 7 of SMN1 is deleted; (4) Loss of -α3.7; -α4.2; --SEA of HBA1 / HBA2; (5) SLC25A13 gene ins3kb and ins6kb, CFTR c.1210-12T[5] special mutation; The 532 single-gene genetic disease-related pathogenic genes in the human genome include: CRB1, CSF2RA, CSF3R, CTNS, CTSD, CTSF, CTSK, CYBA, CYBB, CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP1B1, CYP21A2, CYP27A1, CYP27B1, CYP7B1, DAG1, DBT, DCLRE1C, DGKE, DGUOK, DHCR7, DHDDS, DKC1, DLD, DLL3, DMD, DMP1, DNAH5, DNAI1, DNAI2, DNMT3B, DOK7, DPYD, DUOX2, DY NC2H1, DYSF, EDA, EFL1, EIF2AK3, EIF2B5, ELP1, EMD, ENPP1, ERCC2, ERCC6, ERCC8, ESCO2, ETFA, ETFB, ETFDH, ETHE1, EVC, EVC2, EYS, F11, F7, F8, F9, FA H, FAM161A, FANCA, FANCC, FANCD2, FANCG, FANCI, FGF23, FH, FHL1, FKRP, FKTN, FMO3, FMR1, FOXP3, G6PC1, G6PC3, G6PD, GAA, GALC, GALE, GALK1, GALM, GA LNS, GALT, GAMT, GBA1, GBE1, GCDH, GCH1, GCK, GCM2, GDAP1, GFER, GFM1, GH1, GHR, GHRHR, GJB2, GJB6, GLA, GLB1, GLDC, GLE1, GLIS3, GMPPB, GNE, GNPTAB , GNPTG, GNRHR, GNS, GPR143, GRHPR, GRIP1, GUCY2D, GUSB, HADH, HADHA, HADHB, HARS1, HAX1, HBA1, HBA2, HBB, HCFC1, HES7, HEXA, HEXB, HFE, HGD, HGSNAT , HLCS, HMGCL, HMGCS2, HNF1A, HOGA1, HPD, HPS1, HPS3, HSD17B3, HSD17B4, HSD3B2, HSD3B7, HYLS1, IDS, IDUA, IER3IP1, IGF1, IGSF1, IL17RD, IL2RG, IL7 R, INS, ITGA2B, ITGB3, IVD, JAK3, KCNJ10, KCNJ11, KRT14, KRT5, L1CAM, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAP1, LEP, LEPR, LFNG, LHX3, LIFR,<h2 style=";text-align:left;direction:ltr">LIPA、LMBRD1、LMNA、LOXHD1、LPL、LRMDA、LRP2、LRP5、LRPPRC、LYST、LZTR1、 MAN2B1、MAT1A、MCCC1、MCCC2、MCEE、MCOLN1、MCPH1、MED17、MEFV、MESP2、MF SD8, MID1, MKS1, MLC1, MMAA, MMAB, MMACHC, MMADHC, MMUT, MOCS1, MPI, MPL, MPV17, MTHFR, MTM1, MT-ND1, MT-ND4, MT-ND6, MTR, MT-RNR1, MTRR, MTTP, MVK MYH7, MYL3, MYO7A, NAGA, NAGLU, NAGS, NBN, NCF2, NDRG1, NDUFAF5, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NEB, NEFL, NPC1, NPC2, N PHP1、NPHP3、NPHS1、NPHS2、NR0B1、NR2E3、NTRK1、OAT、OCA2、OCRL、OPA1、OP A3、OTC、PAH、PC、PCBD1、PCCA、PCCB、PCDH15、PCDH19、PDHA1、PDHB、PDX1、PEX 1、PEX10、PEX12、PEX2、PEX6、PEX7、PFKM、PHGDH、PKHD1、PLA2G6、PLEC、PLP1 、PMM2、POLG、POMGNT1、POMT1、POMT2、PPT1、PRDX1、PRF1、PROP1、PRPS1、PSA P、PTF1A、PTH、PTS、PUS1、PYGM、QDPR、RAB23、RAG1、RAG2、RAPSN、RARS2、RDH 12、RFX6、RIPPLY2、RMRP、RNASEH2B、RP2、RPE65、RPGR、RPGRIP1、RPGRIP1L、R RM2B、RS1、RTEL1、SACS、SAMHD1、SBDS、SCN1B、SCN9A、SCO2、SDCCAG8、SDHA、 SDHAF1、SEPSECS、SERPINA1、SERPING1、SGCA、SGCB、SGCD、SGCG、SGSH、SH2D1 A, SLC12A3, SLC12A6, SLC17A5, SLC19A2, SLC19A3, SLC22A5, SLC24A5, SLC25A13, SLC25A15, SLC25A20, SLC25A3, SLC26A2, SLC26A3, SLC26A4, SLC34A3SLC35A3, SLC37A4, SLC39A4, SLC45A2, SLC4A11, SLC6A8, SLC7A7, SMARCAL1, SMN1, SMPD1, SPATA7, SPINK5, SPR, ST3GAL5, STAR, STX11, STXBP1, STXBP2, SUCLA2, SUMF1, SURF1, TACO1, TACR3, TAT, TBCE, TBX4, TCIRG1, TECPR2, TF, TFR2, TG, TGM1, TH, TJP2, TK2, TMEM216, TMEM67, TNXB, TPO, TPP1, TRHR, TRIM32, TRIM37, TRMU, TRPM6, TSFM, TSHB, TSHR, TTC8, TTPA, TUFM, TYMP, TYR, TYRP1, UNC13D, UQCRB, USH1C, USH2A, VPS13A, VPS13B, VPS45, VRK1, VSX2, VWF, WAS, WDR35, WFS1, WIPF1, WNT10A, XIAP, XPA, XPC, ZFP57, ZFYVE26 and special mutations; The specific variants include DMD, HBA, SMN1, CFTR c.1210-12T[5], SLC25A13 ins3kb and ins6kb.

2. The probe set according to claim 1, characterized in that: The human nuclear genome of the GRCh38 version and the human mitochondrial genome of the rCRSNC_012920 version are used as standard reference genomes. The probe set includes 611 probes, as follows:

3. A detection kit, characterized in that: The kit comprises the probe set according to claim 1; Optionally, the kit further comprises a hybridization reaction reagent.

4. The kit according to claim 3, characterized in that The hybridization reaction reagents include one or more of magnetic beads, blocking solution and hybridization buffer.

5. A method for detecting a target area, characterized in that: include: The target region is captured using the probe composition according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 4.

6. The method according to claim 5, characterized in that The steps include: Extracting genomic DNA from the sample to be tested and preparing a genomic pre-library; Adding the probe set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 4 to the genomic pre-library to perform hybridization capture on the target gene region; and, The hybridization captured product is detected to determine the gene mutation of the single gene genetic disease in the sample to be tested.

7. The method according to claim 6, characterized in that Methods for detecting hybridization captured products include high-throughput sequencing.

8. The method according to any one of claims 6 to 7, characterized in that: The sample to be tested includes a blood sample; Optionally, the blood sample comprises a peripheral blood sample.

9. The method according to any one of claims 6 to 7, characterized in that: The steps of preparing the genomic prelibrary include: fragmenting the genomic DNA, performing end repair and A-tailing and linker ligation treatments; purifying the adapter-ligated product; and, The purified product was amplified by PCR to prepare a genomic pre-library.

10. Use of the probe set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 4 in preparing a sequencing product for detecting a single gene genetic disease.

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