Kit and method for detecting X chromosome linked hereditary retinal degeneration disease virulence gene

Abstract

The invention relates to a kit and a method for detecting pathogenic genes of hereditary retinal degeneration diseases linked with X chromosomes, the kit comprises a library hybridization reagent, the reagent comprises a probe group for detecting XLIRDs pathogenic genes, and the probe group comprises capture probes capable of simultaneously and specifically capturing RPGR, RP2, RS1 and CHM pathogenic genes; the capture probe for capturing the RPGR pathogenic gene comprises a probe with a sequence shown as SEQ ID NO.1-96, and a capture probe for a high GC region of the RPGR gene, and the sequence of the capture probe is shown as SEQ ID NO.24-55. Aiming at the four pathogenic genes, variation spectrums and sequence characteristics of the four pathogenic genes are comprehensively analyzed, an amplification region range is optimized, and a high-density capture probe group containing exons, introns, UTR regions and upstream and downstream 20kb regions is designed. Particularly aiming at a high-GC-content region of a No.15 exon of the RPGR gene, a probe sequence and a capture strategy are adjusted to realize efficient capture of the high-GC-content region; in a library PCR enrichment stage, amplification enzymes in the system are screened and optimized, and an amplification enzyme combination most suitable for the gene system is finally optimized by combining a probe group and adopting a plurality of amplification enzyme systems.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene diagnosis, and particularly relates to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. Background Art

[0002] X-linked Inherited Retinal Dystrophy (XLIRD) is a rare retinal degenerative disease caused by pathogenic variants of genes on the X chromosome. The core manifestation of this disease is progressive visual loss, and patients may ultimately progress to legal blindness or even complete blindness. As an important subtype of Inherited Retinal Dystrophies (IRDs), compared with other autosomal dominant / recessive IRDs, XLIRDs have unique characteristics. Through detailed family history information, combined with ophthalmic clinical examinations, the clinical diagnosis obtained through comprehensive analysis is usually clearer than that of other IRDs, significantly narrowing the range of candidate genes. At this time, molecular detection is urgently needed for further diagnosis. Currently, breakthrough progress has been made in gene therapy research for XLIRDs, and a number of clinical trials have been carried out at home and abroad. Effective therapeutic drugs will bring good news to many patients, and accurate molecular diagnosis has become the core prerequisite for patient enrollment screening, efficacy prediction, and treatment plan formulation. The rapid and accurate detection of XLIRD pathogenic genes will lay a solid foundation for the subsequent development of clinical precision diagnosis and treatment.

[0003] Mutations in the RPGR gene can cause various XLIRDs such as X-linked retinitis pigmentosa (XLRP), cone dystrophy, cone-rod dystrophy, or syndromic XLRP. Patients with RPGR gene mutations account for a high proportion among RP patients, more than 70% of XLRP. Approximately 60% of RPGR pathogenic mutations are located in open reading frame 15 (ORF15), and its alternatively spliced products are mainly expressed in the retina and are involved in maintaining the transport of proteins essential for photoreceptor cells. The high GC content in the ORF15 region of the RPGR gene results in extremely low coverage of this region by currently commonly used high-throughput sequencing technologies.

[0004] Mutations in the RP2 gene are another cause of XLRP. Mutations in the RP2 gene account for 15% to 20% of XLRP patients, and approximately 90% of XLRP patients are caused by mutations in both the RPGR and RP2 genes. The RP2 protein in the retina may act as a molecular chaperone to help fold and transport other proteins, thereby maintaining the normal function of photoreceptor cells. Mutations in the RP2 gene will interfere with this process, ultimately leading to the degeneration and death of retinal cells and the loss of vision in patients.

[0005] The RS1 gene is the only pathogenic gene of X-linked retinoschisis (XLRS) discovered so far. The RS1 gene encodes a retinoschisin (RS1) protein in the retina, which is crucial for maintaining the structure and function of the retina. XLRS can occur as early as two or three years old, with varying degrees of visual impairment, manifested as splitting of the inner retina, spoke-like changes caused by macular splitting, and peripheral retinal splitting. It can be complicated by vitreous hemorrhage and retinal detachment, and both the structure and function of the retina are severely damaged.

[0006] The CHM gene is currently considered the only pathogenic gene of choroideremia (CHM). The course of CHM is severely progressive, causing significant visual impairment to patients, and in severe cases, blindness can occur in middle age. The CHM gene expresses Rab escort protein (REP) 1, which plays an important role in the process of intracellular vesicle transport. Pathogenic variants of the CHM gene can lead to the complete absence or non-function of REP1, ultimately causing progressive atrophy of the retinal pigment epithelium, photoreceptor cells, and choroidal capillaries.

[0007] Currently, gene therapy drugs targeting the above four genes have entered the clinical / clinical pre-trial stage. Among them, clinical trials of gene therapy targeting RPGR and RS1 have been carried out in China. A clear molecular diagnosis of XLIRD patients is an essential prerequisite for treatment. For the above four pathogenic genes of XLIRD, there is currently a lack of economic, rapid, targeted, and accurate detection methods that cover all pathogenic variant types. Only through rapid targeted detection can timely gene therapy or other effective treatment means be provided for patients, thereby delaying the progression of the disease and improving the quality of life of patients. At the same time, it helps doctors provide timely genetic counseling and reproductive guidance for patients, reducing the transmission risk of genetic diseases. Therefore, conducting this detection is of great significance for the diagnosis and treatment of XLIRDs.

[0008] In addition, the diagnosis of XLIRDs mainly relies on gene sequencing technology. The preferred molecular genetic tests can include targeted detection of gene exon regions (single gene and multi-gene), whole exome sequencing (WES), and whole genome sequencing (WGS).

[0009] For RPGR gene detection, due to the high GC content in the ORF region, the capture efficiency of high GC regions by conventional high-throughput sequencing detection technology is relatively poor, and the ORF15 region cannot be completely captured, resulting in false negatives in the detection results. When using first-generation sequencing technology to detect the ORF15 sequence, the limitations in sequencing length (700 - 800bp) directly affect the detection of rare structural variations. At the same time, first-generation sequencing technology has the obvious defect of low throughput.

[0010] For the CHM gene, approximately 75% of CHM patients have deletions / insertions, missense mutations, nonsense mutations, and splice site mutations within the gene, while approximately 25% of CHM patients have deletions or duplications of exons or the entire gene. At this time, whole-spectrum mutations cannot be detected in one step by WES or WGS, and the accuracy is limited. After conventional WES / WGS screening, deletion / duplication analysis of target genes is required to infer structural variations of exons or the entire gene. At the same time, further applications including quantitative polymerase chain reaction (PCR), long-fragment PCR, multiplex ligation-dependent probe amplification technology, and gene-targeted microarrays for detecting single exon deletions or duplications are needed for verification, which have obvious practical problems such as long detection time and low efficiency.

[0011] There are also pathogenic variant types such as copy number variations and structural variations in RP2 and RS1 genes, and existing detection methods are difficult to meet the requirements of one-step detection.

[0012] In summary, it is necessary to design effective kits and corresponding detection methods to solve the following technical problems:

[0013] 1) Difficulty in amplifying gene regions: The ORF15 region of the RPGR gene has a high GC content and many repetitive sequences, and traditional amplification methods are prone to problems such as low amplification efficiency and poor library uniformity, resulting in incomplete coverage or large deviations in sequencing data, affecting the reliability of detection results.

[0014] 2) Difficulty in accommodating the diversity of variant types: Traditional gene detection methods usually require different technologies to detect point mutations, small insertions / deletions, copy number variations, and structural variations respectively. The process is complex and inefficient, increasing costs and time.

[0015] 3) Time-consuming verification of detection results: The preliminary results generated by high-throughput sequencing often need to be confirmed by low-throughput verification methods, which prolongs the detection cycle and increases the consumption of samples and reagents, restricting the wide application of the technology in clinical practice.

[0016] 4) Insufficient applicability: Existing technologies lack optimized amplification schemes during library construction, making it difficult to balance the detection requirements of full-length gene coverage and key regions (such as UTR regions and upstream and downstream regulatory regions), affecting the detection rate of certain pathogenic variants. Summary of the Invention

[0017] In order to solve the above technical problems, the object of the present invention is to provide a probe set, a kit and a detection method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The present invention is based on an efficient detection system optimized based on the mutation spectrum and sequence characteristics of target genes, aiming to comprehensively and accurately detect various types of mutations in four pathogenic genes, namely RPGR, RP2, RS1, and CHM, of X-chromosome-linked hereditary retinal degenerative diseases (XLIRDs).

[0018] The object of the present invention is achieved by the following technical solutions:

[0019] A probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, the probe set includes capture probes that can specifically capture pathogenic genes of RPGR, RP2, RS1, and CHM at the same time, and the probe set includes high-density capture probes for exons, introns, UTR regions, and 20 kb regions upstream and downstream; 20 kb upstream and downstream are used to cover large fragment deletions and duplications of genes; among them, the capture probe for specifically capturing the pathogenic gene of RPGR includes probes with sequences shown in SEQ ID NO.1-96, and the capture probe for specifically capturing the pathogenic gene of RPGR includes capture probes corresponding to the high GC region of the RPGR gene, and its sequence is shown in SEQ ID NO.24-55.

[0020] The capture probe for specifically capturing the pathogenic gene of RS1 includes probes with sequences shown in SEQ ID NO.97-149; the capture probe for specifically capturing the pathogenic gene of RP2 includes probes with sequences shown in SEQ ID NO.150-193; the capture probe for specifically capturing the pathogenic gene of CHM includes probes with sequences shown in SEQ ID NO.194-282. The sequences of the capture probes for pathogenic genes of RPGR, RP2, RS1, and CHM are shown in the probe sequence list for details.

[0021] The present invention comprehensively analyzes the mutation spectrum and sequence characteristics of four pathogenic genes, namely RPGR, RP2, RS1, and CHM, optimizes the amplification region range, and designs high-density capture probes including exons, introns, UTR regions, and 20 kb regions upstream and downstream. Especially for the high GC content region of exon 15 (ORF15 region) of the RPGR gene, by adopting the capture strategy of adjusting the probe dosage (probe gradient experiment) and overlapping and encrypting probes, efficient capture of the high GC region is achieved, overcoming the problems of low detection efficiency and uneven coverage in traditional technologies.

[0022] Use of the said probe set in the preparation of a kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases.

[0023] A kit for detecting pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases, which comprises reagents for library hybridization, and the reagents for library hybridization include the probe set for detecting pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases.

[0024] The reagents for library hybridization further include hybridization buffer, hybridization enhancer, universal blocking sequence mixture, and NF water (Nuclease-Free Water); the specific composition of the 19 μL hybridization mixture prepared by mixing each reagent for library hybridization is shown in the following table:

[0025]

[0026] The kit further includes amplification reaction reagents for library PCR enrichment after library hybridization capture, and the amplification reaction reagents include a hot start amplification reaction solution and an amplification primer premix; wherein, the hot start amplification reaction solution contains an amplification enzyme, and the amplification enzyme is one of KAPA HiFi HotStart Ready Mix (2X) (abbreviated as KAPA amplification enzyme), Watchmaker Equinox Amplification Master Mix (2X) (abbreviated as Watchmaker amplification enzyme), and TIANGEN 2x HiFi PCR Master Mix (abbreviated as TIANGEN amplification enzyme).

[0027] The 50 μL PCR reaction system established by the amplification reaction reagents is as follows:

[0028]

[0029] The DNA suspension with magnetic beads is obtained by capture after library hybridization.

[0030] Preferably, the amplification enzyme is TIANGEN 2x HiFi PCR Master Mix.

[0031] The inventor has verified through experiments that not all amplification enzymes can match the probes designed in the present invention, that is, not all amplification enzymes are effective. The inventor screened numerous amplification enzymes (the above 3 enzymes are only several of the amplification enzymes listed in the present invention. During the experimental process of optimizing and screening the amplification enzymes, there are far more than these 3 amplification enzymes for experimental optimization). Among them, Takara Ex Premier DNA (referred to as Takara amplification enzyme) polymerase has the lowest pre-library yield under the same number of amplification cycles (7 cycles) and cannot perform final library enrichment; among the three amplification enzymes of KAPA, Watchmaker, and TIANGEN, the TIANGEN amplification enzyme has the highest amplification efficiency under the same conditions. Therefore, the present invention selects the TIANGEN amplification enzyme as the amplification enzyme in the amplification reaction.

[0032] The present invention has screened and optimized the amplification enzyme of the amplification reaction reagent. In combination with the designed capture probe, the present invention adopts a variety of amplification enzyme systems and screens through quality parameters such as sequencing data coverage, uniformity, and accuracy, and finally optimizes the amplification enzyme most suitable for the gene system selected in the present invention, significantly improving the amplification efficiency and detection accuracy.

[0033] The described kit further includes pre-library construction reagents, and the pre-library construction reagents include enzyme digestion and fragmentation, end repair and A-tailing working solution, adapter ligation reagent, and pre-library amplification reagent;

[0034] The enzyme digestion and fragmentation, end repair and A-tailing working solution includes end repair enzyme and end repair buffer, and the specific composition of 10 μL of the enzyme digestion and fragmentation, end repair and A-tailing working solution is shown in the following table:

[0035]

[0036] The described adapter ligation reagent includes ligation premix and universal short adapter;

[0037] The described pre-library amplification reagent includes dual-end tag primers and library amplification mixed reaction solution, and the pre-library amplification system established by the pre-library amplification reagent is as follows:

[0038]

[0039] Among them, the purified product after adapter ligation is the product obtained by purifying the DNA sample, performing enzyme digestion and fragmentation, end repair, A-tailing, adapter ligation, and purification.

[0040] The library amplification reaction mixture is the amplification reaction solution, and the amplification reaction solution includes an amplification enzyme. The present invention lists the use of amplification reaction solutions containing four amplification enzymes for pre-library amplification. The four amplification enzymes are: ① KAPA HiFi HotStart Ready Mix (2X) (abbreviated as KAPA amplification enzyme), ② Watchmaker Equinox Amplification Master Mix (2X) (abbreviated as Watchmaker amplification enzyme), ③ Takara Ex Premier DNA Polymerase (abbreviated as Takara

[0041] amplification enzyme), and ④ TIANGEN 2x HiFi PCR Master Mix (abbreviated as TIANGEN amplification enzyme). Through experiments, it is preferred that the concentration of the PCR product of the TIANGEN amplification enzyme is the highest.

[0042] The inventor verified through experiments that not all amplification enzymes are effective. Among them, Takara Ex Premier DNA Polymerase (abbreviated as Takara amplification enzyme) has too low a concentration of amplification products under the same conditions and does not meet the requirements for continued library construction, so it is excluded. The concentration of the PCR product of the TIANGEN amplification enzyme is the highest, and it is superior to the KAPA amplification enzyme and the Watchmaker amplification enzyme in terms of amplification effect. Therefore, the TIANGEN amplification enzyme is selected as the amplification enzyme in the pre-library amplification reagent of the kit of the present invention.

[0043] Use of the described kit in detecting pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases.

[0044] For the use described above, the method for detecting pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases using the kit is as follows:

[0045] Fragment the genomic DNA of the detection object by enzymatic digestion, perform end modification, ligation of adapters, and PCR amplification, and simultaneously introduce dual-index sequences for sample identification when multiple samples are detected in parallel; then hybridize and capture the pre-library with a specific capture probe designed for the target region, specifically bind to the target DNA fragment in the genome according to the principle of base complementary pairing of nucleic acid molecules, and use streptavidin magnetic beads to capture the target region DNA fragment bound to the probe. Subsequently, enrich it by PCR amplification to form a final library, and perform a sequencing reaction using the reversible terminator sequencing method. The sequencing data is analyzed by bioinformatics and genetically interpreted to determine whether there are germline mutations with clinical significance in the test sample.

[0046] The capture probe includes probes capable of specifically capturing the pathogenic genes of RPGR, RP2, RS1, and CHM simultaneously, and the probe set includes high-density capture probes for exons, introns, UTR regions, and 20 kb regions upstream and downstream; among them, the capture probes for specifically capturing the pathogenic gene of RPGR include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes for specifically capturing the pathogenic gene of RPGR include capture probes corresponding to the high GC regions of the RPGR gene, and their sequences are as shown in SEQ ID NO.24-55.

[0047] The method for detecting pathogenic genes of X chromosome-linked hereditary retinal degeneration diseases using the kit includes the following specific steps:

[0048] (1) Extract genomic DNA of the detection object as a DNA sample;

[0049] (2) Pre-library construction: The purified DNA sample is fragmented by enzyme digestion, end-repaired, A-tailed, then ligated with adapters and purified, followed by amplification. After that, the amplification product is purified and quality inspected to obtain a pre-library.

[0050] (3) Library hybridization: The pre-libraries are mixed in equal amounts to form a mixed library; the capture probes are used to hybridize the target regions of the mixed library.

[0051] (4) Post-hybridization capture: React the capture beads with the hybridization mixture obtained after the hybridization reaction, then rinse and elute to obtain a DNA suspension with beads; then use the amplification reaction reagents for library PCR enrichment, followed by purification and quality inspection to form a final library.

[0052] (5) DNA sequencing reaction: Perform a sequencing reaction using the reversible terminator sequencing method.

[0053] (6) Data analysis.

[0054] Compared with the prior art, the advantages of the present invention are as follows:

[0055] 1. The present invention belongs to the technical field of genetic testing for genetic diseases, and specifically relates to an efficient detection system optimized based on the mutation spectrum and sequence characteristics of target genes, aiming to comprehensively and accurately detect various types of mutations of four pathogenic genes, RPGR, RP2, RS1, and CHM, of X chromosome-linked hereditary retinal degeneration diseases (XLIRDs). The specific technical innovation points are as follows:

[0056] 1) Precise design for the mutation spectrum of target genes: For the four pathogenic genes, RPGR, RP2, RS1, and CHM, the present invention comprehensively analyzes their mutation spectra and sequence characteristics, optimizes the amplification region range, and designs a high-density capture probe set including exons, introns, UTR regions, and 20 kb regions upstream and downstream.

[0057] 2) Specifically for the high-GC-content region of exon 15 (ORF15 region) of the RPGR gene, the probe sequences and capture strategies were adjusted to achieve efficient capture of high-GC regions, overcoming the problems of low detection efficiency and uneven coverage in traditional technologies.

[0058] 3) In the library PCR enrichment stage, the amplification enzymes in the PCR reaction system were screened and optimized: combined with the above probe sets, multiple amplification enzyme systems were used, and through screening of quality parameters such as sequencing data coverage, uniformity, and accuracy, the amplification enzyme combination most suitable for this gene system was finally optimized, significantly improving the amplification efficiency and detection accuracy.

[0059] 4) One-step precise detection of multiple mutation types: Using the optimized kit and high-throughput sequencing technology, point mutations, small insertions / deletions, large copy number variations, and complex structural variations (such as breakpoints) can be detected simultaneously.

[0060] 5) Compared with traditional technologies, the present invention realizes high-throughput, full-coverage, and high-precision mutation detection, greatly improving the detection efficiency, simplifying the process, and reducing the cost.

[0061] 2. Through in-depth analysis and optimization of the mutation spectrum and sequence characteristics of the target gene, the present invention designed an efficient and precise amplification system, and combined with high-throughput sequencing technology, achieved comprehensive detection of multiple types of mutations in XLIRDs pathogenic genes, significantly improving the detection efficiency and accuracy. Compared with the existing technologies, the present invention has the following outstanding advantages:

[0062] 1) Simplify the detection process and reduce the experimental cost;

[0063] 2) Improve the mutation detection rate and coverage uniformity, especially achieving breakthroughs in the amplification and detection of high-GC regions;

[0064] 3) Have the potential for flexible and personalized applications, providing a powerful tool for the precise diagnosis and personalized treatment of hereditary retinal diseases.

[0065] Specifically reflected in:

[0066] Compared with whole-genome sequencing (WGS): 1) For the specific detection requirements of XLIRDs pathogenic genes, it avoids data redundancy in WGS detection and reduces reagent and sequencing costs; 2) Through high-depth sequencing of specific regions, this kit significantly improves the detection rate of point mutations and small variations, and is superior to WGS in the detection accuracy of high-GC regions such as RPGR ORF15.

[0067] Compared with whole exome sequencing (WES): 1) This kit covers non-coding region variations and structural variations that cannot be detected by WES, especially the accurate positioning of the breakpoints of structural variations; 2) In the detection of complex variations in XLIRDs, this kit can provide comprehensive gene mutation analysis, filling the blind spot of WES technology.

[0068] Compared with Sanger sequencing: 1) This kit has the advantages of high throughput and high efficiency, can detect multiple genes and variation types simultaneously, and avoids the low-throughput limitation of Sanger sequencing. 2) In the diagnosis and differential diagnosis of XLIRDs, it provides more comprehensive genetic evidence.

[0069] 3. The kit of the present invention innovatively integrates the collaborative optimization strategy of amplification enzymes, probes and library construction process, successfully breaks through the technical bottlenecks of low long-fragment amplification efficiency and uneven coverage of complex regions that have long existed in the field of gene detection for ophthalmic diseases, and finally realizes the highly uniform coverage and high-fidelity amplification of target genes in NGS detection. In addition, the kit of the present invention is a complete system that can achieve one-step detection of multiple types of variations, and the probe set is a high-density probe set for the whole genes (including high GC regions) of four pathogenic genes and the 20 kb regions upstream and downstream. Brief Description of the Drawings

[0070] Figure 1 It is the average sequencing depth corresponding to different probe usage amounts of the present invention.

[0071] Figure 2 It is a comparison chart of the capture and amplification effects of three amplification enzymes, Watchmaker, KAPA, and TIANGEN, combined with probes of the present invention for high GC regions.

[0072] Figure 3 It is an IGV screenshot of single-site base mutation detection in Example 1 of the present invention.

[0073] Figure 4 It is a comparison chart of the detection results of other NGS in Example 1 of the present invention.

[0074] Figure 5 It is an IGV screenshot of base microdeletion mutation detection in Example 2-1 of the present invention.

[0075] Figure 6 It is a comparison chart of the detection results of Sanger sequencing in Example 2-1 of the present invention.

[0076] Figure 7 It is an IGV screenshot of base microdeletion mutation detection in Example 2-2 of the present invention.

[0077] Figure 8 It is a comparison chart of the detection results of Sange sequencing in Example 2-2 of the present invention.

[0078] Figure 9 It is a screenshot of IGV for detecting large fragment deletion mutations in Example 3-1 of the present invention.

[0079] Figure 10 It is a graph comparing the detection results of other NGS sequencing in Example 3-1 of the present invention.

[0080] Figure 11 It is a screenshot of IGV for detecting large fragment deletion mutations in Example 3-2 of the present invention.

[0081] Figure 12 It is a graph comparing the detection results of qPCR in Example 3-2 of the present invention.

[0082] Figure 13 It is the result of comparing the capture of high GC regions by three amplification enzymes in WES and WGS in Example 4 of the present invention. Detailed implementation manners

[0083] The content of the present invention will be described in detail below in conjunction with the specification drawings and embodiments:

[0084] The reagents (preferred) and alternative reagents (optional) used in the present invention are shown in the following reagent table:

[0085] Reagent table

[0086]

[0087] The present invention provides a probe set for detecting pathogenic genes of X chromosome-linked hereditary retinal degeneration diseases. The probe set includes capture probes (hereinafter named: XLIRDs gene capture probes) that can specifically capture pathogenic genes of RPGR, RP2, RS1, and CHM simultaneously, and the probe set includes high-density capture probes for exons, introns, UTR regions, and 20 kb regions upstream and downstream; among them, the capture probe for specifically capturing the pathogenic gene of RPGR includes probes with sequences as shown in SEQ ID NO.1-96, and the capture probe for specifically capturing the pathogenic gene of RPGR includes capture probes corresponding to the high GC region of the RPGR gene, and its sequence is as shown in SEQ ID NO.24-55.

[0088] The capture probe for specifically capturing the pathogenic gene of RS1 includes probes with sequences as shown in SEQ ID NO.97-149; the capture probe for specifically capturing the pathogenic gene of RP2 includes probes with sequences as shown in SEQ ID NO.150-193; the capture probe for specifically capturing the pathogenic gene of CHM includes probes with sequences as shown in SEQ ID NO.194-282. The probe sequences of the capture probes for pathogenic genes of RPGR, RP2, RS1, and CHM are shown in the following probe sequence table.

[0089] Probe Sequence List

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] A kit for detecting the pathogenic gene of X chromosome-linked hereditary retinal degeneration diseases, comprising a pre-library construction reagent, a reagent for library hybridization, a reagent for post-hybridization capture, and an amplification reaction reagent for library PCR enrichment;

[0100] The pre-library construction reagent includes an enzyme digestion fragmentation, an end repair and A-tailing working solution, a linker ligation reagent, and a pre-library amplification reagent;

[0101] The enzyme digestion fragmentation, end repair and A-tailing working solution includes an end repair enzyme and an end repair buffer, and the specific composition of 10 μL of the enzyme digestion fragmentation, end repair and A-tailing working solution is shown in the following table:

[0102]

[0103] The linker ligation reagent includes a ligation premix and a universal short linker;

[0104] The pre-library amplification reagent includes dual-end tag primers and a library amplification mixed reaction solution, and the pre-library amplification system established by the pre-library amplification reagent is as follows:

[0105]

[0106] Among them, the purified product after linker ligation is the product obtained by purifying the DNA sample, performing enzyme digestion fragmentation, end repair, A-tailing, linker ligation and purification.

[0107] The reagent for library hybridization includes the probe set for detecting the pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases, namely the XLIRDs gene capture probe.

[0108] The reagent for library hybridization also includes hybridization buffer, hybridization enhancer, universal blocking sequence mixture, and NF water; the specific composition of the 19 μL hybridization mixture prepared by mixing each reagent for library hybridization is shown in the following table:

[0109]

[0110] The amplification reaction reagent includes a hot start amplification reaction solution and an amplification primer premix; among them, the hot start amplification reaction solution contains an amplification enzyme, and the amplification enzyme is one of KAPA HiFi HotStart Ready Mix (2X), Watchmaker Equinox Amplification Master Mix (2X), Takara Ex Premier DNA Polymerase, and TIANGEN 2x HiFi PCR Master Mix. After screening and optimization, the amplification enzyme used in the present invention is TIANGEN 2x HiFi PCR Master Mix.

[0111] The 50 μL PCR reaction system established by the amplification reaction reagent is as follows:

[0112]

[0113] The DNA suspension with magnetic beads is obtained by capture after library hybridization.

[0114] The method for detecting the pathogenic genes of X chromosome-linked hereditary retinal degenerative diseases using the kit of the present invention includes the following specific steps:

[0115] The sample detected by this kit is human EDTA anticoagulated whole blood. It is recommended that sample collection, genomic DNA extraction, and sample transportation and storage be carried out according to the following methods:

[0116] (1) Sample type: Human EDTA anticoagulated whole blood.

[0117] (2) Requirements for DNA sample purification: Suitable for library construction of 50 ng - 200 ng of human genomic DNA and targeted capture and enrichment of gene fragments in its target region. For DNA samples with a higher degree of degradation, it is recommended to use an initial amount of not less than 100 ng for library construction. The sample concentration can be accurately quantified using a fluorescent dye method (such as Qubit, etc.), and the absorbance method at 260 nm is not recommended.

[0118]

Inspection method

[0119] 1. Pre-library construction

[0120] 1.1 Preparation before pre-library construction

[0121] 1.1.1 Purified magnetic beads and deionized (NF) water are left to stand at room temperature for 30 min in advance; the DNA is mixed and centrifuged briefly; the end repair enzyme and end repair buffer reagents are taken out and thawed on ice for standby;

[0122] 1.1.2 Prepare fresh 80% ethanol.

[0123] 1.2 DNA template purification

[0124] 1.2.1 Arrange the DNA samples in the order of the library construction task list, and sequentially add NF water and 50 ng - 200 ng DNA samples to the PCR 8-well strip tubes. The total volume of the sample and NF water is 30 μL.

[0125] 1.2.2 Purification process:

[0126] 1) Add 54 μL of purified magnetic beads (1.8×) to each sample respectively. After sealing, vortex and mix well, and let it stand for 5 min.

[0127] 2) After centrifuging briefly, place it on the magnetic rack. Wait until the solution is clear, and discard the supernatant, retaining the magnetic beads.

[0128] 3) Add 200 μL of freshly prepared 80% ethanol, let it stand for 30 s, and discard the supernatant.

[0129] 4) Repeat step 3) twice.

[0130] 5) After sealing, centrifuge briefly, place it on the magnetic rack. Wait until the solution is clear, and use a small-range pipette tip to discard the remaining liquid.

[0131] 6) Let it stand at room temperature until the magnetic beads are dry (about 5 min).

[0132] 7) Add 42 μL of NF water, seal it, vortex and mix well, and let it stand at room temperature for 5 min.

[0133] 1.2.3 Place the above PCR 8-well strip tubes on the magnetic rack, let it stand until the solution is clear, and transfer 40 μL of the supernatant to a new tube.

[0134] 1.3 Enzyme digestion fragmentation, end repair, and A-tailing

[0135] After thawing, the end repair enzyme and end repair buffer reagents are mixed, centrifuged briefly, and placed on an ice box. Take out a 1.5 mL EP tube, label it, and place it on ice. According to the number of samples, prepare the enzyme digestion fragmentation, end repair, and A-tailing working solution according to the following table ratio:

[0136]

[0137] Gently flick or invert the working solution to mix well 5 - 10 times. After brief centrifugation, take 10 μL and add it to the 40 μL purified sample prepared in the previous step. The total volume of the reaction system is 50 μL. After tightly covering the tube cap, vortex to mix well and centrifuge instantaneously, then place it in a PCR instrument to run the following enzyme digestion fragmentation and end repair plus A-tailing reaction program:

[0138]

[0139] For high-quality genomic DNA, it is recommended to set the enzyme digestion time according to the following table:

[0140]

[0141] 1.4 Adapter Ligation

[0142] Take the ligation premix and the universal short adapter and thaw them on an ice box. Invert the thawed ligation premix to mix well, and use a vortex oscillator to mix the universal short adapter for 10 s, then centrifuge briefly. Add 5 μL of the universal short adapter to each 50 μL sample after end repair plus A-tailing, cover the tube cap, vortex to mix well and centrifuge briefly. Add 20 μL of the ligation premix to the above system, cover the tube cap, vortex to mix well and centrifuge briefly. The total volume of the reaction system is 75 μL. Place it in a PCR instrument to run the following adapter ligation program:

[0143]

[0144] 1.5 Purification after Adapter Ligation

[0145] Add 60 μL of purified magnetic beads (0.8×) to each 75 μL sample after adapter ligation, seal and mix well by oscillation, and let it stand at room temperature for 5 min. Purify according to steps 2) - 6) in 1.2.2. The purified sample is eluted with 17 μL of NF water. After separating the magnetic beads, take 15 μL of the supernatant and transfer it to a new tube, which is the purified product.

[0146] 1.6 Pre-library Amplification

[0147] The library amplification reaction mixture is the amplification reaction solution, and the amplification reaction solution includes an amplification enzyme (i.e., DNA polymerase). In the present invention, amplification reaction solutions containing four amplification enzymes are respectively listed for pre-library amplification. The four amplification enzymes are: ① KAPA HiFi HotStart Ready Mix (2X) (abbreviated as KAPA amplification enzyme), ② Watchmaker Equinox Amplification Master Mix (2X) (abbreviated as Watchmaker amplification enzyme), ③ Takara Ex Premier DNA Polymerase (abbreviated as Takara amplification enzyme), and ④ TIANGEN 2x HiFi PCR Master Mix amplification enzyme (abbreviated as TIANGEN amplification enzyme). Through experiments, TIANGEN amplification enzyme is preferably used, and the concentration of the PCR product of TIANGEN amplification enzyme is the highest.

[0148] Prepare the following reaction on ice:

[0149]

[0150] Note: Record the label primer numbers corresponding to each sample. Only add one label primer to each reaction tube, and the label primers used for detection in the same batch cannot be repeated.

[0151] After preparation, cover the tube cap, vortex and mix well, and centrifuge briefly. Place it in a PCR instrument and run the following PCR amplification program:

[0152]

[0153] Recommended table for the number of amplification cycles for pre-library construction:

[0154]

[0155] 1.7 Purification of the amplification product

[0156] Add 50 μL of purification magnetic beads (1×) to each tube of the amplification product, seal and mix well by shaking, and let stand at room temperature for 5 min. Purify according to steps 2)-6) in 1.2.2. The purified sample is eluted with 62 μL of NF water and can be stored at 20°C for 1 month.

[0157] 1.8 Quality inspection of the pre-library

[0158] 1.8.1 Qubit quantification

[0159] Use a nucleic acid quantification reagent, such as Qubit dsDNA HS Assay Kit and the supporting instrument, to measure the concentration of the sample DNA library. The total library amount calculated should be greater than 300 ng. Otherwise, the library construction sample does not meet the requirements and should be rebuilt.

[0160] 1.8.2 Fragment quality inspection

[0161] Use capillary electrophoresis reagents and supporting instruments for fragment quality control. The library fragments should be between 300 - 500 bp, without obvious small - fragment and large - fragment heterozygous peaks. Otherwise, the library construction samples do not meet the requirements and should be reconstructed.

[0162] 1.9 Pre - library amplification results:

[0163] Use Takara, KAPA, Watchmaker, and TIANGEN four kinds of amplification enzymes for pre - library amplification respectively. Among them, the amplification product concentration obtained by Takara amplification enzyme under the same conditions is too low to meet the requirements for continued library construction. Among the remaining three kinds of amplification enzymes, the PCR product concentration of TIANGEN amplification enzyme is the highest (as shown in Table 1 - 5 specifically), and it is superior to the other two enzymes in terms of amplification effect.

[0164] Table 1. Statistical analysis results of pre - library concentration

[0165]

[0166] Table 2. PCR product concentration of Takara amplification enzyme used for pre - library construction

[0167]

[0168] Table 3. PCR product concentration of KAPA amplification enzyme used for pre - library construction

[0169]

[0170] Table 4. PCR product concentration of TIANGEN amplification enzyme used for pre - library construction

[0171]

[0172] Table 5. PCR product concentration of Watchmaker amplification enzyme used for pre - library construction

[0173]

[0174] 2 Library hybridization

[0175] 2.1 Making library mixing task list

[0176] When including genetic pedigree samples, members of the same pedigree are in the same mixed library, and the Index of the same mixed library has no repetition. The number of hybridization samples is 12. The total amount of pre - library is 3.6 μg and they are mixed in equal mass.

[0177] 2.2 Library mixing

[0178] The pooling operation requires double-checking by two people. According to the library pooling task sheet, transfer the pre-libraries to new 1.5 mL centrifuge tubes in sequence, and record the total volume of each pooled library. Thaw the blocking agent in advance, add 5 μL of the blocking agent to each pooled library, vortex and mix well, and then perform a brief centrifugation for subsequent purification steps.

[0179] 2.3 Preparation of Hybridization Mix

[0180] 1) Optimization test of probe dosage: To determine the optimal probe dosage, the inventors conducted an optimization test on the probe dosage:

[0181] Sample information: 7 positive, 3 negative, 2 NA12878, a total of 12 samples. Probe gradient test: 1 μL, 2 μL, 4 μL. Loading volume: 0.5 Gb.

[0182] The experimental results showed that: for different probe dosages, the fragment size of the final library was not different, and the total amount increased with the increase of the probe dosage.

[0183] The sequencing results indicated that: for different probe dosages, there were no significant differences in the various indicators of data quality control. There were significant differences in the average coverage depth in the target region (bed) file, that is, the higher the probe dosage, the higher the coverage depth ( Figure 1 , Table 6-8).

[0184] Table 6. Sequencing Data Quality Control

[0185]

[0186] Table 7. Different Gradient Probe Tests:

[0187]

[0188] Table 8. Statistical Analysis Results of Probe Gradient Tests

[0189]

[0190] Based on the above optimization test results, the experimental determination of the usage amount of the XLIRDs gene capture probe is 4 μL.

[0191] 2) Preparation of hybridization mix: Label a 0.2 mL PCR tube, and prepare the hybridization mix by mixing the pre-thawed hybridization buffer, hybridization enhancer, universal blocking sequence mix, and XLIRDs gene capture probe according to the following table:

[0192]

[0193] Place the prepared hybridization mix on ice for subsequent use.

[0194] 2.4 Purification before Hybridization

[0195] 1) After the purification magnetic beads are equilibrated at room temperature for 30 min, add 3 volumes of the purification magnetic beads to a centrifuge tube containing the mixed library and the blocking agent, vortex to mix well, and let stand at room temperature for 5 min.

[0196] 2) After transient centrifugation, place it on a magnetic stand. Wait until the solution is clear, discard the supernatant and retain the magnetic beads.

[0197] 3) Add 200 μL of freshly prepared 80% ethanol, let stand for 30 s, and discard the supernatant.

[0198] 4) Add 200 μL of freshly prepared 80% ethanol for the second time, let stand for 30 s, and discard the supernatant.

[0199] 5) After transient centrifugation, place it on a magnetic stand. Wait until the solution is clear, and discard the ethanol residue.

[0200] 6) Let stand at room temperature until the magnetic beads are dry (about 10 min).

[0201] 7) Add 19 μL of the hybridization mixture prepared in step 2.3, vortex to mix well, and let stand for 5 min.

[0202] 8) Place it on a magnetic stand, let stand until the solution is clear, and transfer all the supernatant (the volume should be no less than 17 μL) to a new 0.2 mL PCR tube.

[0203] 9) Place the above PCR tube on a PCR instrument and prepare to run the hybridization reaction program.

[0204] 2.5 Running the hybridization reaction

[0205] Run the following hybridization reaction program on a PCR amplifier:

[0206]

[0207] Note: The hybridization time generally should not exceed 16 h at most. Keep the sample on the PCR instrument and the temperature at 65 °C before proceeding to the subsequent steps.

[0208] 3 Post-hybridization capture

[0209] 3.1 Preparation of washing working solution

[0210] Thaw the magnetic bead elution buffer and the washing buffer in advance, and equilibrate the capture magnetic beads to room temperature (30 min). According to the number of capture libraries, prepare the magnetic bead elution buffer working solution and the washing buffer working solution according to the following table:

[0211]

[0212] Note: If necessary, Wash Buffer 1 can be heated at 65°C. The prepared Wash Working Solution can be stored at room temperature (15 - 25°C) for 4 weeks.

[0213] The usage amounts and temperatures of the Wash Buffer 1 Working Solution and the Stringent Wash Buffer Working Solution are as follows in the table:

[0214]

[0215] Note: After preparing the Wash Buffer 1 Working Solution and the Stringent Wash Buffer Working Solution, they need to be immediately preheated at 65°C, and the remaining buffers can be placed at room temperature.

[0216] 3.2 Target Fragment Capture

[0217] 1) Vortex the captured magnetic beads balanced in 3.1 for 15 s. Add 80 μL of magnetic beads to each library in a 0.2 mL PCR tube (for each captured library), and pipette to mix well.

[0218] 2) After brief centrifugation, place on the magnetic stand until clear (~1 min), discard the supernatant, taking care not to aspirate the magnetic beads.

[0219] 3) Add 200 μL of the Magnetic Bead Elution Buffer Working Solution, vortex thoroughly for 10 s, after brief centrifugation, place on the magnetic stand until clear (~1 min), discard the supernatant, taking care not to aspirate the magnetic beads.

[0220] 4) Repeat step 3) once.

[0221] 5) Add 100 μL of the Magnetic Bead Elution Buffer Working Solution, vortex thoroughly for 10 s, after brief centrifugation, place on the magnetic stand until clear (~1 min), discard the supernatant, taking care not to aspirate the magnetic beads. After brief centrifugation, use a small-volume pipette tip to completely remove the supernatant.

[0222] 6) Add the hybridization mixture after the overnight hybridization reaction in step 2.5 (operate on a PCR instrument at 65°C as much as possible), and pipette to mix well 10 times.

[0223] 7) Place the 0.2 mL PCR tube containing the captured magnetic beads and the hybridization mixture at 65°C for 45 min. During the reaction, vortex and resuspend once every 15 min to avoid magnetic bead precipitation and splashing onto the PCR tube lid.

[0224] Note: After completing this step, the next step should be carried out immediately.

[0225] 3.3 Post-Capture Rinsing and Elution

[0226] 1) Add the above reaction product to a 0.2 mL PCR tube containing 100 μL of the working solution of Wash Buffer 1 preheated to 65 °C, and pipette thoroughly to mix (preferably operate on a metal bath at 65 °C).

[0227] 2) After brief centrifugation, place it on a magnetic stand until clear (~1 min), discard the supernatant, and be careful not to aspirate the magnetic beads.

[0228] 3) Add 200 μL of the working solution of stringent wash buffer preheated to 65 °C, pipette and mix 10 times, and let it stand at 65 °C for 5 min.

[0229] 4) After brief centrifugation, place it on a magnetic stand until clear (~1 min), discard the supernatant, and be careful not to aspirate the magnetic beads.

[0230] 5) Repeat steps 3) - 4) once.

[0231] 6) Add 200 μL of the working solution of Wash Buffer 1, pipette and mix 10 times, and shake at 2000 rpm at room temperature for 2 min.

[0232] 7) After brief centrifugation, place it on a magnetic stand until clear (~1 min), discard the supernatant, and be careful not to aspirate the magnetic beads.

[0233] 8) Add 200 μL of the working solution of Wash Buffer 2, pipette and mix 10 times, and shake at 2000 rpm at room temperature for 1 min.

[0234] 9) After brief centrifugation, place it on a magnetic stand until clear (~1 min), discard the supernatant, and be careful not to aspirate the magnetic beads.

[0235] 10) Add 200 μL of the working solution of Wash Buffer 3, pipette and mix 10 times, and then shake at 2000 rpm at room temperature for 30 s.

[0236] 11) After brief centrifugation, place it on a magnetic stand until clear (~1 min), discard the supernatant, and be careful not to aspirate the magnetic beads.

[0237] 12) Remove the centrifuge tube from the magnetic stand, briefly centrifuge and then place it on the magnetic stand for ~30 s, and use a 10 μL pipette to remove the residual liquid as much as possible.

[0238] 13) Add 20 μL of NF water, vortex thoroughly to mix, and perform PCR with the magnetic beads after brief centrifugation.

[0239] Note: After washing with the working solution of Wash Buffer 2 and the working solution of Wash Buffer 3, when placed on the magnetic stand, it can be observed that the magnetic beads are diffusely distributed. If not, it is necessary to review whether there are errors in the reagents and operations. Basically, if this phenomenon is not observed, subsequent experiments will fail and the final library yield will be low.

[0240] 3.4 Library PCR Enrichment and Purification

[0241] 3.4.1 Library PCR Enrichment

[0242] To meet the specific requirements of gene detection related to ophthalmic diseases, the present invention focuses on the full-length detection of 4 key target genes, etc. These genes exhibit unique sequence characteristics, including GC content bias, repetitive sequences, and complex secondary structures. A typical example is the RPGR gene, which is rich in high-GC regions and long fragment repetitive sequences. However, when traditional NGS general amplification enzymes encounter such regions, they often show bottlenecks in amplification efficiency and fidelity. Therefore, it is urgent to discover specific PCR amplification enzymes suitable for the system of the present invention through systematic screening, so as to significantly improve the amplification uniformity and fidelity of target genes. Especially for the detection application scenarios with limited target genes but significant sequence characteristics, the advantages are more prominent. The specific screening process of different amplification enzymes in the present invention is as follows:

[0243] 1) Optimization experiments of different amplification reaction enzymes:

[0244] The amplification reaction solution in Reagent I and the amplification reaction solution in Reagent III of the present invention are the same component. The present invention uses KAPA HiFi HotStart Ready Mix (2X) (abbreviated as KAPA amplification enzyme), Watchmaker Equinox Amplification Master Mix (2X) (abbreviated as Watchmaker amplification enzyme), Takara Ex Premier DNA Polymerase (abbreviated as Takara amplification enzyme), and TIANGEN 2x HiFi PCR Master Mix (abbreviated as TIANGEN amplification enzyme) as candidate amplification enzymes in the amplification reaction solution (i.e., the hot-start DNA polymerase described in the reagent table). Select 20 positive samples and 4 negative samples, and operate according to the "Product Instruction Manual of XLIRDs Gene Mutation Detection Kit (Reversible Termination Sequencing Method)". The reaction systems and amplification conditions of the four enzymes are the same as those in the instruction manual.

[0245] Take the hot-start amplification reaction solution and the amplification primer premix reagent to thaw at room temperature, and prepare the PCR reaction system in a 0.2 mL PCR tube according to the following table:

[0246]

[0247] Run the following program on a PCR amplifier:

[0248]

[0249] Note: The PCR program needs to be preheated in advance.

[0250] The experimental results show that there are significant differences in the total amount of the final library for different amplification enzymes, as shown in Tables 9 and 10. In addition, the amplification products were sequenced, and the sequencing data were analyzed. It was found that the data generated by TIANGEN amplification enzyme had higher accuracy, and TIANGEN amplification enzyme had the highest amplification efficiency for regions with high GC content (Tables 11 - 12).

[0251] Table 9: Total amount of the final library for different amplification enzymes (mass concentration ng / ul)

[0252]

[0253] The statistical results show that for the libraries constructed with different amplification enzymes, among which Takara Ex Premier DNA polymerase had the lowest pre-library yield at the same amplification cycle number (7 cycles) and could not be enriched to the final library; among the three amplification enzymes of KAPA, Watchmaker, and TIANGEN, TIANGEN amplification enzyme had the highest amplification efficiency under the same conditions, and there were significant statistical differences compared with the other two amplification enzymes (P value < 0.05).

[0254] Table 10: Statistical analysis of the total amount of the final library for different amplification enzymes:

[0255]

[0256] The results of the sequencing data show that there are no obvious differences in the QC indicators of the sequencing data, but there are significant differences in the average sequencing depth and the capture of high GC regions. For example, in the region of exons 14 to 15 of the RPGR gene, the coverage of the library constructed with TIANGEN 2x HiFi PCR Master Mix in this region was significantly improved, and the average sequencing depth was higher than that of the other two enzymes ( Figure 2 ). Therefore, we preferably selected TIANGEN amplification enzyme as the amplification enzyme for this kit. Among them, Figure 2 is a comparison of the capture and amplification effects of the probes combined with the three amplification enzymes of Watchmaker, KAPA, and TIANGEN on high GC regions.

[0257] Table 11. Comparison of Q30 of the sequencing data of the three amplification enzymes under the condition of an average sequencing depth of 500×

[0258]

[0259] The statistical analysis results show that the data generated by TIANGEN amplification enzyme have higher accuracy.

[0260] Table 12. Comparison of the capture and amplification efficiency of the high GC region of exon 15 of the RPGR gene under the condition of an average sequencing depth of 500×.

[0261]

[0262] The comparison results show that TIANGEN amplification enzyme has the highest amplification efficiency for regions with high GC content.

[0263] 3.4.2 Library Purification

[0264] 1) Add 50 μL (1×) DNA purification magnetic beads to the PCR reaction product in step 3.4.1, vortex to mix well, and let stand at room temperature for 5 min.

[0265] 2) After centrifuging briefly, place on the magnetic stand. Wait until the solution is clear, then discard the supernatant and retain the magnetic beads.

[0266] 3) Add 200 μL of freshly prepared 80% ethanol, let stand for 30 s, and discard the supernatant.

[0267] 4) Add 200 μL of freshly prepared 80% ethanol for the second time, let stand for 30 s, and discard the supernatant.

[0268] 5) Centrifuge briefly, place on the magnetic stand. Wait until the solution is clear, and use a small-volume pipette tip to discard the ethanol residue.

[0269] 6) Let stand until the magnetic beads are dry (about 5 min).

[0270] 7) Add 32 μL of NF water, vortex to mix well, and let stand for 5 min.

[0271] 8) Place on the magnetic stand and wait until the solution is clear. Transfer 30 μL of the supernatant to a new 0.2 mL PCR tube, make a mark, and this is the prepared capture library.

[0272] 3.5 Final Library Quality Inspection

[0273] 3.5.1 Qubit Quantification

[0274] Use a nucleic acid quantification reagent, such as the Qubit dsDNA HS Assay Kit and the supporting instrument, to measure the concentration of the final library. The total amount of the final library should be greater than 10 ng, otherwise it does not meet the requirements and the library construction should be redone.

[0275] 3.5.2 Fragment Quality Inspection

[0276] Use capillary electrophoresis reagents and the supporting instrument for fragment quality control, such as Agilent 4150 and Qsep100. The library fragments should be between 300 - 500 bp, without obvious small-fragment and large-fragment heterozygous peaks. Otherwise, the library construction sample does not meet the requirements and the library construction should be redone. If the library is not sequenced in time, it can be stored at -20 °C for 1 month.

[0277] 4 DNA Sequencing Reaction

[0278] The amount of data uploaded for each sample is not less than 0.2 Gb (500X).

[0279] 5 Data Analysis

[0280] This kit is based on high-throughput sequencing technology and uses an open-source bioinformatics analysis process. After data splitting, Sentieon-bwa is used to align the raw data with the reference genome hg19. Picard is used to perform quality control, deduplication, and sorting on the aligned bam file. Qualimap is used to perform quality control on the aligned file. For the filtered bam file, Sentieon-GATK is used to analyze SNVs and small indels, and ISoGTools is used for locus function annotation. For CNV copy number variation analysis, the CNVexon algorithm is used to analyze the copy number of exonic bases. For pseudogenes and genes in homologous regions, a self-developed Misalignment homologous region optimization algorithm is used for CNV detection.

[0281] The data quality control link consists of the following two parts:

[0282] Link 1: Sample traceability quality control, including sample gender determination, that is, the actual gender of the tested sample is determined by the ratio of the coverage of the X and Y chromosomes in the high-throughput sequencing data.

[0283] Link 2: Overall quality control of high-throughput sequencing, meeting the requirements that the Q30 of the raw sequencing data is ≥90%, the effective depth of the target region is ≥200X, the coverage at 20X is ≥99%, the capture efficiency is ≥80%, and there is no deviation in the conversion transversion rate, heterozygous to homozygous ratio, and SNV / Indel locus count between library samples in different batches.

[0284] 6 Specific Detection Examples

[0285] To prove the effectiveness of the kit described in the present invention, the present invention uses the above method to detect 20 cases of XLIRD probands and carriers. Through the above technical solution, DNA library construction is carried out and sequencing is performed. The analysis of the sequencing results shows that this kit can effectively detect gene mutation types such as single-site base mutations, microdeletions / duplications, and large deletions.

[0286] Example 1. The IGV screenshot of the single-site base mutation detected by using the kit described in the present invention is as Figure 3 shown: R_17:RS1:chrx:18660182:NM_000330.4:c.617G>A(p.Trp206*), hemizygous mutation.

[0287] Comparison with WES detection:

[0288] Basic process of WES: After extracting the genomic DNA to be tested, construct a library using the Illumina standard protocol, perform exome target capture using the IDT xGenExome Research Panel v2, and after library preparation, perform paired-end 150bp (PE150) sequencing on the NovaSeq platform (Illumina). Perform quality control and analysis on the data downloaded from the machine to ensure that 96% of the regions reach a sequencing depth of 20x, and perform alignment analysis with the human reference genome GRCh37 / hg19. The results of WES are as Figure 4 shown.

[0289] Comparison conclusion: This representative example confirms that this kit can detect single-site base mutations with high efficiency and high precision. Based on the sequencing depth, it can be higher than that of ordinary WES, so its accuracy is better than that of WES; at the same time, the detection range of this kit for the target gene includes exons, intron regions and gene flanking sequences. Therefore, the comprehensive performance of this kit is better than that of WES and Sanger sequencing.

[0290] Example 2-1. The IGV screenshot of the base microdeletion mutation detected by using the kit of the present invention is as Figure 5 shown: R_09: chrx:46696544-46696546:RP2:NM_006915.3:c.14_16del(p.Phe5del) hemizygous variation.

[0291] Comparison with Sanger sequencing

[0292] Basic process of Sanger sequencing: Design multiple primer sequences for the region of the gene to be tested to ensure coverage of the entire gene length, and use the dideoxy chain termination method (Sanger et al., 1977) to sequence the target DNA fragment. Sequencing reaction: Add the purified DNA template, specific sequencing primer (0.5 μM), and BigDye Terminator v3.1 reagent (containing fluorescently labeled ddNTPs, DNA polymerase, and buffer) to a 20 μL reaction system, and perform the amplification program in an ABI Veriti thermal cycler (pre-denaturation at 96 °C for 2 min; 25 cycles × 96 °C for 10 s, 50 °C for 5 s, 60 °C for 4 min). After purifying the PCR product, perform electrophoresis separation using an ABI 3730xl sequencer, and automatically identify the base signals through the Sequencing Analysis v6 software.

[0293] The detection results are as Figure 6 shown.

[0294] Example 2-2. The IGV screenshot of the base microdeletion mutation detected by using the kit of the present invention is as Figure 7 shown:

[0295] R_13: hrx: 38145521 - 8145522: RPGR: NM_001034853.2: c.2730_2731delGG(p.Glu911Glyfs*167) hemizygous variant.

[0296] Compared with Sanger sequencing detection (the basic process is as described above), the results are as Figure 8 shown.

[0297] Conclusion of Example 2 comparison: This representative example confirms that this kit can detect base micro - deletion mutations with high efficiency and high precision, especially for micro - variations in high - GC regions. The amplification enzyme optimized by this kit has the advantages of strong uniformity in the enrichment of high - GC regions, so its detection of mutations in high - GC regions is superior to WES; at the same time, the detection range of this kit for the target gene includes exons, intron regions and gene flanking sequences. Therefore, the comprehensive performance of this kit is superior to WES and Sanger sequencing, and it can achieve full coverage of the pathogenic gene mutation types of XLIRDs.

[0298] Example 3 - 1. The IGV screenshot of the large - fragment deletion mutation detected by using the kit of the present invention is as Figure 9 shown: R_04: chrx: 85096185 - 85161914 hemizygous deletion, including exons 10 - 15 of the CHM gene.

[0299] Compared with WES detection

[0300] WES detection process for large - fragment deletions: The processes of genome library construction sequencing and obtaining raw data are the same as described above. For CNV analysis, ExomeDepth and CODEX tools are combined to analyze the change in coverage. The filtering threshold for the deleted fragment usually requires that the coverage of ≥3 consecutive exons drops by more than 50%, or a continuous low - coverage area with a span of >50kb. The results are as Figure 10 shown.

[0301] Example 3 - 2. The IGV screenshot of the large - fragment deletion mutation detected by using the kit of the present invention is as Figure 11 shown: R_19: chrx: 18670779 - 18682478 hemizygous deletion, including exons 2 - 3 of the RS1 gene.

[0302] Compared with qPCR detection

[0303] Basic process of qPCR: Specific primers are designed for the target gene sequence. After obtaining the genomic DNA to be tested, it is detected simultaneously with a template-free negative control and a known normal sample. A 20 μL reaction system is prepared, including 10 μL of premix (containing SYBR Green I dye, hot-start DNA polymerase, dNTPs, and optimized buffer), 1 μL of forward and reverse primers (final concentration 0.2 μM), 2 μL of DNA template (about 10 ng / μL), and 7 μL of sterilized ultrapure water. Subsequently, amplification is performed using a real-time fluorescence quantitative PCR instrument, and the program is set as follows: pre-denaturation at 95°C for 30 seconds; cycling steps of 95°C for 5 seconds and 60°C for 30 seconds (a total of 40 cycles); finally, melting curve analysis (from 60°C to 95°C, continuous heating, rate 0.1°C / second) is performed to verify the product specificity. Fluorescence signals are collected in real time through the SYBR Green channel, and ROX dye is used as an internal reference to correct the fluorescence fluctuations between wells. The amplification efficiency is verified by the standard curve method (R 2 > 0.98). The ΔΔCt method is used to compare the Ct value differences between the target region and the internal reference gene. If the Ct value of the target region is significantly higher than that of the control sample (such as ΔCt difference ≥ 1), it indicates a copy number deletion in this region, and the relative quantification value (RQ) is calculated by comparing with the normal control. The results are as Figure 12 shown.

[0304] Conclusion of Comparative Experiment in Example 3: This representative example confirms that this kit can efficiently and comprehensively detect large fragment deletion mutations of genes, especially the breakpoints in intron regions. WES only has a high sensitivity to exon-level CNV, and qPCR is not the preferred method for routine clinical detection. The performance of this kit is significantly better than WES and qPCR sequencing, and it can achieve full coverage of the pathogenic gene mutation types of XLIRDs.

[0305] Example 4 (6 random samples). Effective capture and amplification results of the high GC region of exon 15 of the RPGR gene detected by the kit described in the present invention

[0306] The inventor also replaced the TIANGEN amplification enzyme in the kit with KAPA and Watchmaker amplification enzymes for comparative experiments, and at the same time compared the capture effects of the high GC region by the detection method established by the kit of the present invention with WES and WGS methods. Among them, the capture results of the three amplification enzymes for the high GC region compared with WES and WGS are as Figure 13 shown.

[0307] Conclusion of Comparison in Example 4: Capturing and amplifying high-GC regions in high-throughput sequencing is a challenge in sequencing. The capture and amplification efficiency of WES in high-GC regions is low, and the uniformity of the overall data is poor; WGS is also limited in high-GC regions, with low accuracy and uniformity. However, the kit obtained by using the TIANGEN amplification enzyme optimized in this patent in combination with an effective probe set can efficiently amplify and enrich high-GC regions, and the mutation detection in high-GC regions is significantly better than that of WES and WGS( Figure 13 In the sample names in the following, they represent the results of library construction and sequencing using TIANGEN, KAPA, and Watchmaker amplification enzymes, and WES and WGS for capturing and sequencing high-GC regions).

[0308] Table 13: Detection Results of 20 Positive Samples

[0309] Experiment_ID Gender Mutation Gene Chr_Location Results R_01 M Yes RPGR X：38146015-38146016 NM_001034853.2:c.2236_2237delGA(p.Glu746Argfs*23), Hemi R_02 F Yes RPGR X：38146015-38146016 NM_001034853.2:c.2236_2237delGA(p.Glu746Argfs*23), Hete R_03 M Yes CHM X：85218827 NM_000390.4:c.545del(p.Cys182Leufs*15), Hemi R_04 M Yes CHM X：85096185-85161914 chrX:85096185 - 85161914del, CHM:exon10 - 15del, Hemi R_05 M Yes CHM X：85212923 NM_000390.4:c.877C>T(p.Arg293*), Hemi R_06 M Yes RS1 X：18662583 NM_000330.4:c.489del(p.Trp163*), Hemi R_07 M Yes RP2 X：46715956-46723515 chrX:46715956 - 46723515del, RP2:exon3del, Hemi, R_08 F Yes RP2 X：46715176-46725075 chrX:46715176 - 46725075del, RP2:exon3del, Hete R_09 M Yes RP2 X：46696544-46696546 NM_006915.3:c.14_16del(p.Phe5del), Hemi R_10 F Yes RP2 X：46696544-46696546 NM_006915.3:c.14_16del(p.Phe5del), Hete R_11 M Yes RP2 X：46713155 NM_006915.3:c.347A>G(p.Gln116Arg), Hemi R_12 F Yes RP2 X：46713155 NM_006915.3:c.347A>G(p.Gln116Arg), Hete R_13 M Yes RPGR X：38145521-38145522 NM_001034853.2:c.2730_2731delGG(p.Glu911Glyfs*167), Heml R_14 F Yes RPGR X：38145521-38145522 NM_001034853.2:c.2730_2731delGG(p.Glu911Glyfs*167)Hete R_15 M Yes RPGR X：38164016 NM_001034853.2:c.806G>A(p.Gly269Glu), Hemi R_16 F Yes RPGR X：38164016 NM_001034853.2:c.806G>A(p.Gly269Glu), Hete R_17 M Yes RS1 X：18660182 NM_000330.4:c.617G>A(p.Trp206*), Hemi R_18 F Yes RS1 X：18660182 NM_000330.4: c.617G>A(p.Trp206*), Hete R_19 M Yes RS1 X：18670779-18682478 chrX: 18670779-18682478del, RS1: exon2-3del, Hemi R_20 F Yes RS1 X：18670779-18682478 chrX: 18670779-18682478del, RS1: exon2-3del, Hete

[0310] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A probe set for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, characterized in that: The probe group includes capture probes that can simultaneously and specifically capture RPGR, RP2, RS1, and CHM pathogenic genes, and the probe group includes high-density capture probes for exons, introns, UTR regions, and 20kb upstream and downstream regions; wherein, the capture probes that specifically capture the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes that specifically capture the RPGR pathogenic gene include capture probes corresponding to high GC regions of the RPGR gene, and their sequences are shown in SEQ ID NO.24-55.

2. Use of the probe set as claimed in claim 1 in preparing a kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases.

3. A kit for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases, characterized in that: It comprises a reagent for library hybridization, wherein the reagent for library hybridization comprises the probe set for detecting the pathogenic gene of X-chromosome-linked hereditary retinal degeneration disease as claimed in claim 1.

4. The kit according to claim 3, characterized in that: The library hybridization reagents also include hybridization buffer, hybridization enhancer, universal blocking sequence mixture and NF water; the specific composition of 19 μL hybridization mixture prepared by mixing the library hybridization reagents is shown in the following table:

5. The kit according to claim 3, characterized in that: It also includes an amplification reaction reagent for library PCR enrichment after library hybridization capture, and the amplification reaction reagent includes a hot start amplification reaction solution and an amplification primer premix; wherein the hot start amplification reaction solution contains an amplification enzyme, and the amplification enzyme is TIANGEN 2x HiFi PCR Master Mix.

6. The kit according to claim 3, characterized in that: It also includes pre-library construction reagents, which include enzyme fragmentation, end repair and A-tail working solution, adapter connection reagents and pre-library amplification reagents; The enzyme fragmentation, end repair and A-tailing working solution includes an end repair enzyme and an end repair buffer, and the specific composition of 10 μL of the enzyme fragmentation, end repair and A-tailing working solution is shown in the following table: The linker connection reagent includes a connection premix and a universal short linker; The pre-library amplification reagent includes a double-ended label primer and a library amplification mixed reaction solution, and the pre-library amplification system established by the pre-library amplification reagent is as follows: The purified product after adapter ligation is a product obtained after the DNA sample is purified, enzyme fragmented, end-repaired, A-tailed, adapter-ligated and purified.

7. Use of the kit according to any one of claims 3 to 6 in detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases.

8. The use according to claim 7, characterized in that: The method for detecting the pathogenic gene of X-chromosome linked hereditary retinal degenerative disease using the kit is as follows: The extracted genomic DNA of the test object is subjected to enzyme fragmentation, end modification, adapter ligation and PCR amplification, and a double-end tag sequence is introduced at the same time for the identification of samples when multiple samples are tested in parallel; then, the pre-library is hybridized and captured using a specific capture probe designed for the target region, and the target DNA fragments in the genome are specifically bound according to the principle of complementary base pairing of nucleic acid molecules, and the target region DNA fragments bound to the probe are captured using streptavidin magnetic beads, and then PCR amplification and enrichment are performed to form a final library, and the sequencing reaction is performed using the reversible terminal termination sequencing method. The sequencing data is subjected to bioinformatics analysis and genetic interpretation to determine whether there are clinically significant germline mutations in the sample to be tested; The capture probes include probes that can specifically capture RPGR, RP2, RS1, and CHM pathogenic genes at the same time, and the probe group includes high-density capture probes for exons, introns, UTR regions, and 20kb upstream and downstream regions; wherein, the capture probes that specifically capture the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO.1-96, and the capture probes that specifically capture the RPGR pathogenic gene include capture probes corresponding to high GC regions of the RPGR gene, and their sequences are shown in SEQ ID NO.24-55.

9. The use according to claim 8, characterized in that: The method for detecting the pathogenic gene of X-chromosome-linked hereditary retinal degeneration disease using the kit comprises the following specific steps: (1) extracting genomic DNA of the test subject as a DNA sample; (2) Prelibrary construction: The purified DNA sample is fragmented by enzyme digestion, end-repaired, and A-tailed, then ligated with adapters and purified before amplification. The amplified product is then purified and quality tested to obtain a prelibrary. (3) Library hybridization: pre-libraries of equal quality are mixed to form a mixed library; Hybridize the target region of the mixed library using capture probes; (4) Post-hybridization capture: The capture magnetic beads are reacted with the hybridization mixture obtained after the hybridization reaction, and then rinsed and eluted to obtain a DNA suspension with magnetic beads; then the library is PCR enriched using an amplification reaction reagent, and then purified and quality tested to form a final library; (5) DNA sequencing reaction: using reversible terminal termination sequencing method for sequencing reaction; (6) Data analysis.

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