Kit for detecting hereditary hearing loss related genes based on hybrid capture method, hybrid capture method and application

Abstract

According to the kit for detecting the hereditary hearing loss related genes based on the hybridization capture method, the hybridization capture method and the application, the kit comprises a probe pool of capture probes, and the capture probes are used for hybridizing and capturing the hereditary hearing loss related genes. The kit disclosed by the invention has the beneficial effects that when the kit is used for constructing a DNA library, the initial quantity of a template can be as low as 10ng, and enzyme digestion breaking, terminal filling and A tail adding are completed in one system, so that the consumed time is short, and the efficiency is high; secondly, the number of index pairs of the kit is up to 384, and 384 libraries can be constructed at the same time and can be used for sequencing on a machine together; in addition, according to a rapid hybridization system of the kit, the shortest hybridization time can reach 15 min, and the efficiency of common overnight hybridization can be achieved. The size of the finally obtained library fragment is about 300-500bp, and the library fragment can be compatible with various sequencing platforms of illumina for sequencing.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a kit for detecting hereditary deafness-related genes based on a hybridization capture method, a hybridization capture method and an application thereof. Background Art

[0002] The prevention and control of hereditary deafness is one of the main entry points for improving the hearing health level of the population, and genetic diagnosis plays an important role in the prevention and control of hereditary deafness.

[0003] Hereditary deafness refers to deafness caused by genetic variation, or deafness that occurs after exposure to certain environmental factors due to susceptibility to genetic variation. Hereditary deafness is divided into syndromic and non-syndromic types according to whether it is combined with other phenotypes. Syndromic deafness refers to functional or structural abnormalities of other organs or systems in addition to deafness; non-syndromic deafness is not accompanied by abnormalities of other organs or systems, but may be accompanied by tinnitus, dizziness, etc.

[0004] The phenotypic spectrum of hereditary deafness is wide, and the pathogenic genes have strong genetic heterogeneity. On September 16, 2020, the ClinGen Hearing Loss expert group reviewed and identified 112 genes with a grade of Moderate or above, of which 109 (excluding DSPP, GJB3, and GJB6) were recommended for the diagnosis of deafness. In addition, ClinGen's other disease expert group reviewed and identified four genes related to syndromic deafness, namely COL4A3, COL4A4, PTPN11, and COL1A1, which can also be used for the diagnosis of deafness.

[0005] The main methods for genetic diagnosis of hereditary deafness include gene chip, Sanger sequencing, targeted capture sequencing of target deafness genes (i.e., second-generation sequencing panels of known deafness genes), whole exome sequencing (WES), and whole genome sequencing (WGS). Gene chip is a hybridization sequencing method, that is, a method of determining nucleic acid sequences by hybridization with a set of nucleic acid probes of known sequences. It mainly detects hotspot pathogenic variants of known deafness genes. Its main advantages are rapidity, efficiency, and automation, but its detection range is limited. Sanger sequencing, the first generation of DNA sequencing technology, is recognized as the gold standard for genetic testing. It has a long read length and high accuracy, but its single base sequencing cost is high and its throughput is low. Targeted capture sequencing of target deafness genes, WES, and WGS are all based on next generation sequencing (NGS) technology. Compared with the first generation sequencing, NGS has greatly reduced the sequencing cost and sequencing time of a single base, while maintaining a high accuracy and detection throughput.

[0006] In the genetic diagnosis of more than 100 deafness patients reported in the literature: (1) Targeted capture sequencing of deafness genes, including 59 to 181 genes, can clearly diagnose the molecular cause in 38.8% to 39.3% of sporadic deafness patients, 33.5% to 47.6% of sporadic and familial deafness patients, and 48% to 56% of familial deafness patients; (2) After excluding patients with deafness caused by GJB2 or excluding patients with deafness caused by GJB2, SLC26A4, and mitochondrial DNA m.1555A>G, targeted capture sequencing of deafness genes, including 79 to 213 genes, can clearly diagnose the molecular cause in 15.5% to 31% of deafness patients; Therefore, taking into account both cost-effectiveness and detection rate, it is an economical and efficient strategy to use targeted capture sequencing of deafness genes with clear clinical significance for genetic diagnosis of deafness patients. Unfortunately, the current examination of genes related to hereditary deafness is usually carried out using direct sequencing methods, but this method is limited in clinical application due to its cumbersome operation, long time consumption, low throughput and low hybridization efficiency.

[0007] Therefore, there is an urgent need for a kit and a detection method that can quickly detect genes related to hereditary deafness. Summary of the invention

[0008] In view of this, the present invention proposes a kit, a hybridization capture method and an application for detecting hereditary deafness-related genes based on a hybridization capture method, so as to solve the problems of cumbersome operation, time-consuming and low throughput of current hereditary deafness-related gene detection methods.

[0009] In order to solve the above problems, the technical solution adopted by the present invention is:

[0010] One of the purposes of the present invention is to provide a kit for detecting hereditary deafness-related genes based on a hybridization capture method, characterized in that the kit comprises a probe pool containing capture probes, and the capture probes are used for hybridization capture of hereditary deafness-related genes.

[0011] As a preferred embodiment of the present application, the hereditary deafness-related gene comprises a molecule or a fragment thereof selected from the following group: ABHD12, ACTG1, ADGRV1, AIFM1, ATP6V1B1, BCS1L, BSND, CABP2, CACNA1D, CDC14A, CDH23, CEACAM16, CEP78, CHD7, CIB2, CISD2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL11A2, COL1A1, COL4A3, COL4A4, COL4A5, DIAPH1, DIAPH3, DMXL2, DNMT1, DSPP, EDN3, EDNRB, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FGF3, GATA3, GIPC3, GJB2, GJB3, GJB6, GPSM2, GRHL2, GRXCR1, GRXCR2, GSDME, HGF, HOMER2, HSD17B4, ILDR1, KCNE1, KCNQ1 , KCNQ4, LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, M YO3A, MYO6, MYO7A, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, POU3F4, POU4F3, PRPS1, PT PN11, PTPRQ, RDX, RIPOR2, S1PR2, SERPINB6, SIX1, SLC17A8, SLC26A4, SLC52A2, SLC52A3, SLITRK6, SMPX, SOX10, ST RC, SYNE4, TBC1D24, TECTA, TIMM8A, TMC1, TMIE, TMPRSS3, TPRN, TRIOBP, TUBB4B, USH1C, USH1G, USH2A, WFS1, WHRN.

[0012] As a preferred embodiment of the present application, the probe pool includes at least one of the capture probes having base sequences of SEQ ID NOs 1 to 500 as shown in Table 1:

[0013] Table 1

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] As a preferred embodiment of the present application, the probe pool includes at least 300 capture probes having base sequences as shown in Table 1 as SEQ ID NOs 1 to 500.

[0032] As a preferred embodiment of the present application, the probe pool includes all of the capture probes having base sequences as shown in Table 1 as SEQ ID NOs 1 to 500.

[0033] A second object of the present invention is to provide a hybrid capture method, characterized in that it comprises:

[0034] constructing a targeted sequencing library containing genes associated with hereditary deafness; and

[0035] The kit is used to perform hybridization capture on the hereditary deafness-related genes in the targeted sequencing library.

[0036] As a preferred embodiment of the present application, the step of constructing a targeted sequencing library containing genes related to hereditary deafness comprises:

[0037] Obtaining a DNA sample containing a hereditary deafness-related gene, and isolating the hereditary deafness-related gene DNA from the DNA sample;

[0038] Construct a DNA sample library containing genes related to hereditary deafness;

[0039] Mixing multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries;

[0040] Using a probe pool to perform a hybridization capture reaction on the hereditary deafness-related gene in the library pool to obtain a target complex; the probe pool comprises a capture probe for capturing the hereditary deafness-related gene;

[0041] After hybridization, the target complex is amplified by PCR;

[0042] The PCR amplification products of the target complex were purified to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp.

[0043] As a preferred embodiment of the present application, a DNA sample library containing genes related to hereditary deafness is constructed, including:

[0044] The hereditary deafness-related gene DNA is fragmented and phosphorylated to obtain the hereditary deafness-related gene;

[0045] Connectors are added to both ends of the hereditary deafness-related gene, and the connection products obtained by connecting the hereditary deafness-related gene and the connector are PCR amplified and purified to obtain a DNA sample library containing the hereditary deafness-related gene.

[0046] As a preferred embodiment of the present application, the genomic DNA fragmentation is obtained by using an enzyme digestion method to interrupt the hereditary deafness-related gene DNA in the whole blood sample.

[0047] As a preferred embodiment of the present application, the dosage of each DNA sample library in the DNA sample library is calculated according to the DNA mass concentration in each DNA sample library to ensure that the total amount of DNA in the library pool is 1.5-4 μg.

[0048] As a preferred embodiment of the present application, the step of adding linkers at both ends of the hereditary deafness-related gene comprises:

[0049] Adding Universal adapters to both ends of the gene associated with hereditary deafness; and

[0050] A step of adding UDI adapters at both ends of the gene associated with hereditary deafness.

[0051] As a preferred embodiment of the present application, after adding Universal adapters to both ends of the hereditary deafness-related gene, the ligation product is PCR amplified and purified.

[0052] As a preferred embodiment of the present application, after adding UDI connectors to both ends of the hereditary deafness-related gene, the ligation product is PCR amplified and purified.

[0053] As a preferred embodiment of the present application, the purification method for the PCR amplification product of the target complex is a magnetic bead enrichment method.

[0054] As a preferred embodiment of the present application, the amplified product obtained by PCR amplification of the connection product obtained by connecting the hereditary deafness-related gene to the connector is purified by a magnetic bead enrichment method.

[0055] The third object of the present invention also provides the use of the kit or the hybridization capture method in preparing a kit for genetic diagnosis of hereditary deafness.

[0056] The present invention only requires a trace amount of genomic DNA, and constructs a library pool by enzyme cutting and interruption, and adding a linker, and then uses 116 personalized capture probes for hybrid capture of hereditary deafness genes to perform hybrid capture and enrichment on the library pool, and the construction of the targeted library can be quickly completed. Through a simple and easy-to-use synthesis and NGS sequencing solution, all coding regions of 116 hereditary deafness-related genes can be detected. The variation results of 116 hereditary deafness-related genes can be quickly obtained in one test. At the same time, this kit is easy to operate, time-saving, and easy to realize automated detection. It is suitable for large sample size related screening and diagnosis, and can discover new pathogenic variations with high specificity and sensitivity.

[0057] Compared with the prior art, the present invention has the following technical effects: first, the template starting amount can be as low as 10ng when constructing a DNA library in the kit of the present application, and enzyme cutting, end filling and A-tailing are completed in one system, which is time-saving and efficient; second, the index of the kit is up to 384 pairs, which can realize the simultaneous construction and sequencing of 384 libraries; in addition, the rapid hybridization system of the kit can achieve the efficiency of general overnight hybridization with a hybridization time as short as 15min. The final library fragment size is about 300-500bp, which can be compatible with various sequencing platforms of Illumina for sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The present invention is a flowchart of constructing a targeted sequencing library containing genes associated with hereditary deafness in Example 1 of the present application.

[0059] Figure 2 This is the fragment analysis result of the targeted sequencing library constructed in Example 1 of the present application. DETAILED DESCRIPTION

[0060] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0061] The present invention will be further described below in conjunction with the embodiments.

[0062] Implementation 1

[0063] S1 Construction of a DNA sample library containing genes related to hereditary deafness

[0064] 1.0 DNA was sheared by enzyme digestion to shear the hereditary deafness-related genomic DNA in the whole blood sample, and the fragmented hereditary deafness-related genomic DNA was repaired and A-tailed (amplified region). The specific operation is as follows:

[0065] 1.0.1 Turn on the PCR instrument and set the temperature of the PCR cover to 70°C. The starting amount of DNA library construction is 10-100 ng. Mix thoroughly, centrifuge, and place on ice for later use.

[0066] 1.0.2 Go to the reagent preparation area, take a new 1.5mL centrifuge tube, and prepare the enzyme digestion reaction mixture. After melting 10xFragmentation Buffer and 5xFragmentationEnzyme (Twist Bioscience) on ice, prepare the mixture according to Table 2, mix and centrifuge, divide the enzyme digestion reaction mixture into eight tubes, and then transfer it to the amplification area through the transfer window. (At the same time, the DNA purification magnetic bead solution used for the next step of purification can be taken out of the refrigerator, shaken and mixed thoroughly, and then transferred to the amplification area together to equilibrate at room temperature for at least 30 minutes);

[0067] Table 2

[0068] Reagents Single reaction volume 10x Fragmentation Buffer 5μL 5x Fragmentation Enzyme 10μL Total volume 40μL

[0069] 1.0.3 Take 35 μL of each DNA sample to be tested and add them to the eight-tube strip containing the enzyme digestion reaction mixture. Gently shake and mix. Centrifuge for a moment and then quickly put it into the PCR instrument. Make sure the temperature of the PCR instrument's hot cover is 70°C, and then amplify using the program shown in Table 3:

[0070] Table 3

[0071] temperature time 32℃ 22min 65℃ 30min 4℃ HOLD

[0072] 1.1 Connect to Universal adapter

[0073] 1.1.1 After the above steps are completed, add 5 μL of Universal adapter (Twist Bioscience) to each reaction system, shake gently to mix, centrifuge and place on ice for later use.

[0074] 1.1.2 Go to the reagent preparation area and prepare the ligation reaction mixture. After melting DNA Ligation Buffer and DNA Ligation Mix (Twist Bioscience) on ice, prepare the mixture according to Table 4, mix well, centrifuge, place on ice, and transfer to the amplification area for use.

[0075] Table 4

[0076]

[0077]

[0078] 1.1.3 Add 45 μL of the ligation reaction mixture prepared in step 1.1.2 to each sample prepared in step 1.0.3. Gently shake and mix, centrifuge and quickly put into the PCR instrument, connect at 20℃ for 15 minutes (heat cover closed). After the end, take out and immediately start magnetic bead purification.

[0079] 1.2 First step magnetic bead purification (amplification area)

[0080] 1.2.1 Library purification magnetic beads (KAPA) should be taken out from 4°C before use, vortexed and mixed thoroughly, and equilibrated at room temperature for at least 30 minutes.

[0081] 1.2.2 Vortex the purified magnetic bead suspension (KAPA) equilibrated at room temperature for 1 min and dispense into 1.5 mL centrifuge tubes at a rate of 80 μL (0.8 times the sample volume) per sample.

[0082] 1.2.3 Transfer all the ligation products to the magnetic beads in the corresponding centrifuge tube and gently shake to mix to avoid bubbles.

[0083] 1.2.4 Leave the centrifuge tube at room temperature for 5 minutes, then place it on a magnetic rack for 5 minutes until the supernatant becomes clear. Carefully remove the supernatant.

[0084] 1.2.5 Keep the centrifuge tubes on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to each tube, let stand at room temperature for 1 min, and discard the supernatant.

[0085] 1.2.6 Repeat step 1.2.5. After a brief centrifugation, place the centrifuge tube back into the magnetic rack and use a 10 μL pipette to remove as much residual ethanol solution as possible from the bottom of the tube.

[0086] 1.2.7 Leave the centrifuge tube on the magnetic rack with the lid open for 5 minutes at room temperature to dry it so that all the ethanol evaporates. Avoid over-drying.

[0087] 1.2.8 Add 17 μL of enzyme-free water to each tube and mix up and down 10 times using a pipette. Incubate at room temperature for 5 min.

[0088] 1.2.9 Place each tube on a magnetic rack and let it stand for 5 minutes until the supernatant becomes clear.

[0089] 1.3UDI adapter primer PCR amplification

[0090] 1.3.1 During the rest period in step 1.2.9, go to the reagent preparation area and prepare eight tube strips on the ice box according to the number of samples. Take 25 μL KAPA HiFi HotStart ReadyMix (KAPA) and dispense it into each well of the eight tube strips.

[0091] 1.3.2 Add 10 μL of pre-set UDI primers with different indexes (Twist Bioscience) to each well of the eight-tube strip (UDI is a pair of primers, a total of 384 pairs, the sequences of these primers contain 8 bases (we call it index) which are different for distinguishing, except for these 8 bases, the other sequences of the 384 pairs of UDI are the same). Then transfer to the amplification area I through the transfer window.

[0092] 1.3.3 According to the set position order, pipette 15 μL of purified supernatant obtained in step 1.2.9 and add it to each well of the PCR reaction mixture that has been dispensed, cover the tube, shake gently, and centrifuge briefly to collect the liquid.

[0093] 1.3.4 Transfer the prepared PCR reaction tube to the amplification area through the transfer window for PCR reaction. Heat the cover at 105℃ and set the reaction program as shown in Table 5.

[0094] Table 5

[0095]

[0096] 1.4 The second step of magnetic bead purification (amplification area)

[0097] 1.4.1 Library purification magnetic beads (same as KAPA) should be taken out from 4°C before use, vortexed and mixed thoroughly, and equilibrated at room temperature for at least 30 minutes.

[0098] 1.4.2 Vortex the purified magnetic bead suspension equilibrated at room temperature for 1 min and dispense 50 μL (equal volume of sample) / sample into 1.5 mL centrifuge tubes.

[0099] 1.4.3 Take out the PCR reaction product, centrifuge for 30 seconds, transfer all the products to the magnetic beads in the corresponding centrifuge tube, and shake gently to avoid generating bubbles.

[0100] 1.4.4 After standing at room temperature for 5 minutes, place on a magnetic separation rack for 5 minutes until the supernatant is clear. Carefully remove the supernatant.

[0101] 1.4.5 Place on a magnetic separation stand, add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant.

[0102] 1.4.6 Repeat step 1.4.5 and try to remove all the residual ethanol solution.

[0103] 1.4.7 Leave the centrifuge tube on the magnetic rack with the lid open for 5 minutes at room temperature to dry, ensuring that all the ethanol evaporates.

[0104] 1.4.8 Add 22 μL of enzyme-free water, shake gently, and incubate at room temperature for 5 min.

[0105] 1.4.9 Place on the magnetic stand for 5 minutes, take 20 μL of supernatant (library) for the next step of library enrichment. Take 1 μL and dilute 10 times, use Qubit and fragment analyzer to measure library concentration and fragment size respectively. The average fragment length of the test library should be around 300-500 bp.

[0106] S2 mixes multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries;

[0107] 2.0.1 Calculate the amount of different DNA sample libraries and determine the mixing scheme of library pool DNA: The library pool often contains multiple different DNA sample libraries. Calculate the amount of each DNA sample library pool according to the concentration of the DNA sample library, and mix to obtain the library pool. The total amount of DNA in the library pool is 1.5-4μg. It is recommended to establish a library pool with samples of the same mass. Refer to Table 6 for the amount of DNA library:

[0108] Table 6

[0109] Number of mixed samples The amount of each library Total library volume per reaction 1 1000ng 1000ng 2 1000ng 2000ng 3 1000ng 3000ng 4 800ng 3200ng 8 400ng 3200ng

[0110] 2.0.2 Take samples according to the calculated amount of different DNA sample libraries, mix each DNA sample library in a 1.5mL centrifuge tube, shake gently, and centrifuge for standby use.

[0111] S3: using the kit or the hybridization capture method to perform hybridization capture on the hereditary deafness-related genes in the library pool, the hereditary deafness-related genes in the library pool are hybridized with the capture probe to generate a target complex, and after the hybridization is completed, PCR amplification is performed on the target complex;

[0112] 3.0.1 Design and synthesize capture probes and construct probe pools.

[0113] 3.0.2 Add the following pre-hybridization reagents to the mixed sample, mix well, centrifuge instantaneously, and try not to generate bubbles. The pre-hybridization reagents are configured according to Table 7. Capture probes, also known as XLID Panel probes, are personalized DNA probes designed to capture genetic deafness-related genes in genetic deafness-related genomes. The base sequence is at least one of SEQ ID NOs 1 to 500 shown in Table 1.

[0114] Table 7

[0115] Reagents volume Probe pool 4μL Universal Blockers(Twist Bioscience) 8μL Blocking Solution(Twist Bioscience) 5μL

[0116] 3.0.3 Open the cap of the pre-hybridization reagent tube, place the tube in a vacuum concentrator, balance it, and dry it at 45℃ until the solution in the tube is completely dry. The drying process takes about 30 minutes to 1 hour.

[0117] 3.0.4 When drying is almost finished, incubate Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min or until all precipitates are dissolved, and then set the PCR instrument program according to Table 8, with the heated cover at 85°C:

[0118] Table 8

[0119] temperature time 95℃ 5min 60℃ 15min~4h

[0120] 3.0.5 After drying, remove the centrifuge tube from the dryer, tear off the sealing film, quickly vortex Fast Hybridization Mix (Twist Bioscience) and add 20 μL to the tube to resuspend the sample (please do not let the hybridization solution return to room temperature), flick with your fingertips to mix, avoid bubbles, and let stand at room temperature for 5 minutes. Use a pipette to pipette up and down 10 times. After the DNA is completely dissolved, transfer all the solution in the tube to a new PCR tube as much as possible to avoid bubbles.

[0121] 3.0.6 Add 30 μL of paraffin oil to the tube to cover the surface of the reagent inside, and then centrifuge it instantly to remove bubbles.

[0122] 3.0.7 Place the PCR tube in the preheated PCR instrument for hybridization and incubate for 15 minutes to 2 hours. (During this period, you can go to the reagent preparation area to take out the Streptavidin Binding Beads (Twist Bioscience) used for the next capture from the refrigerator, shake and mix thoroughly, and then equilibrate to room temperature for at least 30 minutes).

[0123] S4 Purification of PCR amplification products of target complex

[0124] 4.0.1 Go to the reagent preparation area and dispense the enrichment reagent (reagent amount for a single enrichment reaction) into 1.5 mL centrifuge tubes according to Table 9, and then transfer each component and the balanced magnetic bead component (Streptavidin Binding Beads) to the amplification area through the transfer window.

[0125] Table 9

[0126] Components Required volume Fast Binding Buffer(Twist Bioscience) 900μL Fast Wash Buffer 1(Twist Bioscience) 450μL Wash Buffer 2(Twist Bioscience) 700μL

[0127] 4.0.2 Enter the amplification area and preheat 450μL Fast Wash Buffer 1 at 70℃ and 700μL Wash Buffer 2 at 48℃ in a thermostatic metal bath.

[0128] 4.0.3 Shake the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, and transfer 100 μL of magnetic beads to a 1.5 mL centrifuge tube;

[0129] 4.0.4 Add 200 μL of Fast Binding Buffer to the centrifuge tube and mix well by pipetting with a pipette;

[0130] 4.0.5 Place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant, and remove the centrifuge tube;

[0131] 4.0.6 Repeat the washing steps 4.0.4 to 4.0.5 twice, for a total of 3 times;

[0132] 4.0.7 After the third wash, add 200 μL of Last Binding Buffer and resuspend by vortexing to mix thoroughly.

[0133] 4.0.8 After the hybridization in step 3.0.6 is completed, open the cover of the PCR instrument and quickly transfer all the hybridization solution to the balanced magnetic bead solution. Note: Rapidly transferring the reagents in the PCR instrument to the magnetic beads is a key step. The sample must be loaded directly on the PCR instrument. Do not remove the hybridization tube to prevent the temperature of the hybridization solution from dropping.

[0134] 4.0.9 Close the cap of the centrifuge tube and seal the tube mouth with sealing film, then place it on the Biocate oscillator at 1200 rpm and mix thoroughly at room temperature for 30 minutes.

[0135] 4.0.10 Remove the centrifuge tube from the mixer, centrifuge quickly and place it on the magnetic rack for 1 minute, remove the supernatant, remove the tube, add 200μL preheated Fast Wash Buffer 1 (do not remove it from the metal bath), and gently shake to mix;

[0136] 4.0.11 Rapidly incubate the centrifuge tube at 66°C for 5 minutes;

[0137] 4.0.12 Centrifuge the tube and place it on a magnetic rack for 1 minute. Remove the supernatant, remove the tube, add 200 μL preheated Fast Wash Buffer 1 again, and gently shake to mix;

[0138] 4.0.13 Rapidly incubate the centrifuge tube at 66°C for 5 minutes;

[0139] 4.0.14 Centrifuge the centrifuge tube and transfer all the liquid in it to a new 1.5mL centrifuge tube; place it on the magnetic rack for 1 minute and remove the supernatant;

[0140] 4.0.15 Remove the tube, add 200 μL of preheated Wash Buffer 2 (do not remove it from the metal bath), and gently shake to mix;

[0141] 4.0.16 Rapidly incubate the centrifuge tube at 48°C for 5 minutes;

[0142] 4.0.17 After centrifugation, place the centrifuge tube on a magnetic rack for 1 minute, remove the supernatant and take out the tube;

[0143] 4.0.18 Repeat (steps 4.0.15 to 4.0.17) and wash twice, three times in total;

[0144] 4.0.19 After centrifugation, place the centrifuge tube on a magnetic rack and use a 10μL pipette tip to suck out the wash solution;

[0145] 4.0.20 Remove the centrifuge tube from the magnetic rack, add 45 μL of nuclease-free water, gently shake to mix, and incubate the solution on ice. If PCR amplification is not performed for the time being, the magnetic bead mixture can be stored at 20°C.

[0146] S5 PCR amplification of the target complex

[0147] 5.0.1 Enter the reagent preparation area, take a PCR tube, prepare the mixed solution, add (25μL KAPAHiFiHotStartReadyMix(KAPA)+2.5μL Amplification Primers(Twist Bioscience)=22.5μL) to the tube respectively (this Amplification Primer is unique, just like the Universal Adapter mentioned above, there is only one), and then transfer the PCR tube to the amplification area I through the transfer window. (Note: At this time, you can take out the DNA PurificationBeads(KAPA) from the refrigerator, shake and mix thoroughly, and transfer it to the amplification area together with the PCR tube to equilibrate at room temperature for at least 30 minutes)

[0148] 5.0.2 Take 22.5 μL of magnetic bead suspension obtained in step 5.0.1 and add it to the PCR tube, gently shake and mix, and centrifuge it instantly. The remaining 22.5 μL can be stored at -20℃ for later use.

[0149] 5.0.3 Place the PCR tube on the PCR instrument, heat the cover to 105°C, and amplify according to the reaction program in Table 10:

[0150] Table 10

[0151]

[0152] S6 uses magnetic beads to purify the PCR amplification products (amplification regions) of the target complex to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp

[0153] 6.0.1 Vortex and thoroughly mix the pre-equilibrated DNA purification magnetic beads (same as KAPA above), take a clean 1.5mL centrifuge tube, add 90μL (1.8*) DNA purification magnetic beads, and after the PCR is completed, centrifuge the PCR tube instantly, then transfer all the solutions in the PCR tube to a 1.5mL centrifuge tube and vortex to mix thoroughly;

[0154] 6.0.2 Incubate the centrifuge tube at room temperature for 5 minutes;

[0155] 6.0.3 Place the centrifuge tube on the magnetic rack for 1 minute and remove the supernatant after the solution becomes clear;

[0156] 6.0.4 Keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% ethanol wash once (2 times in total);

[0157] 6.0.5 After instant centrifugation, place the centrifuge tube on a magnetic rack and carefully remove the remaining ethanol with a 10μL pipette tip. Leave at room temperature for 5-10 minutes or until the magnetic beads are dry, but avoid cracking.

[0158] 6.0.6 Remove the tube from the magnetic rack and add 32 μL of nuclease-free water, gently shake to mix, and incubate at room temperature for 5 min;

[0159] 6.0.7 Place the centrifuge tube on the magnetic rack for 3 minutes or until the solution becomes clear;

[0160] 6.0.8 Transfer 30 μL of supernatant to a clean 1.5 mL centrifuge tube, label the library name and library construction date on the tube cap, and then transfer to the library quality inspection area. Note: If the enriched library cannot be sequenced immediately after purification, it can be stored below -20°C for 30 days.

[0161] 6.0.9 Take another 1 μL for use The library concentration was determined by dsDNAHS Assay Kit (KAPA), which should be greater than 0.5 ng / μL. The 2100 fragment analyzer (Agilent) was also used to detect the length distribution of the library fragments, and the average fragment length should be between 300 and 500 bp.

[0162] Example 2 Clinical sample detection

[0163] Three clinical whole blood samples with known positive gene mutations were collected to detect the genetic mutations related to hereditary deafness in these three samples. Genomic DNA of the three whole blood samples was extracted respectively, and then the library was constructed according to the hybridization capture method described in Example 1, and a targeted sequencing library was successfully constructed (see Figure 2 ), the targeted sequencing library was sequenced on an Illumina high-throughput sequencer, with sequencing parameters PE, 2×150. After quality control of the data, the data was analyzed using a self-built bioinformatics analysis process to obtain the genetic variation of each sample related to hereditary deafness, and then each site was verified by first-generation sequencing, with a compliance rate of 100%.

[0164] Table 11 Test results of patient 1

[0165] Gene Test results Zygote type Clinical significance SLC26A4 NM_000441.2: c.919-2A>G Homozygous Pathogenic variants

[0166] Table 12 Test results of patient 2

[0167] Gene Test results Zygote type Clinical significance GJB2 NM_004004.6:c.235del Heterozygous Pathogenic variants GJB2 NM_004004.6:c.299_300del Heterozygous Pathogenic variants

[0168] Table 13 Test results of patient 3

[0169] Gene Test results Zygote type Clinical significance MT-RNR1 m.1555A>G Heterogeneity Pathogenic variants

[0170] It can be seen from the test results that the kit of the present invention can detect genetic mutations related to hereditary deafness and can accurately screen for hereditary deafness. The DF Panel capture probe of the present invention can accurately capture 116 genes related to hereditary deafness. The detection of conventional specimens shows that the capture probe, hybridization capture method and kit of the present invention can detect genetic mutations related to hereditary deafness.

[0171] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention is not limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A kit for detecting genes related to hereditary deafness based on a hybridization capture method, characterized in that: The kit comprises a probe pool containing capture probes, and the capture probes are used for hybridization and capture of hereditary deafness-related genes.

2. The kit for detecting hereditary deafness-related genes based on hybridization capture method according to claim 1, characterized in that: The hereditary deafness-related gene comprises a molecule or a fragment thereof selected from the group consisting of ABHD12, ACTG1, ADGRV1, AIFM1, ATP6V1B1, BCS1L, BSND, CABP2, CACNA1D, CDC14A, CDH23, CEACAM16, CEP78, CHD7, CIB2, CISD2, CLDN14, CLIC5, CLPP, CLRN1, COCH, COL11A2, COL1A1, COL4A3, COL4A4, COL4A5, DIAPH1, DIAPH3, DMXL2, DNMT1, DSPP, EDN3, EDNRB, EPS8, EPS8L2, ESPN, ESRRB, EYA1, EYA4, FGF3, GATA3, GIPC3, GJB2, GJB3, GJB6, GPSM2, GRHL2, GRXCR1, GRXCR2, GSDME, HGF, HOMER2, HSD17B4, ILDR1, KCNE1, KCNQ1, KCNQ 4. LARS2, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR96, MITF, MSRB3, MT-RNR1, MT-TS1, MYH14, MYH9, MYO15A, MYO3A , MYO6, MYO7A, OSBPL2, OTOA, OTOF, OTOG, OTOGL, P2RX2, PAX3, PCDH15, PDZD7, PJVK, POU3F4, POU4F3, PRPS1, PTPN1 1. PTPRQ, RDX, RIPOR2, S1PR2, SERPINB6, SIX1, SLC17A8, SLC26A4, SLC52A2, SLC52A3, SLITRK6, SMPX, SOX10, STR C, SYNE4, TBC1D24, TECTA, TIMM8A, TMC1, TMIE, TMPRSS3, TPRN, TRIOBP, TUBB4B, USH1C, USH1G, USH2A, WFS1, WHRN.

3. The kit for detecting hereditary deafness-related genes based on hybridization capture method according to claim 1, characterized in that: The probe pool includes at least one of the capture probes having base sequences as shown in the following table:

4. A hybrid capture method, characterized in that: include: Construct a targeted sequencing library containing genes related to hereditary deafness; as well as The kit according to any one of claims 1 to 3 is used to perform hybridization capture on the hereditary deafness-related genes in the targeted sequencing library.

5. The hybrid capture method according to claim 4, characterized in that: Steps for constructing a targeted sequencing library containing genes related to hereditary deafness include: Obtaining a DNA sample containing a hereditary deafness-related gene, and isolating the hereditary deafness-related gene DNA from the DNA sample; Construct a DNA sample library containing genes related to hereditary deafness; Mixing multiple DNA sample libraries to establish a library pool containing multiple different DNA sample libraries; Using a probe pool to perform a hybridization capture reaction on the hereditary deafness-related gene in the library pool to obtain a target complex; the probe pool comprises a capture probe for capturing the hereditary deafness-related gene; After hybridization, the target complex is amplified by PCR; The PCR amplification products of the target complex were purified to obtain a targeted sequencing library with an average fragment length of 300 to 500 bp.

6. The hybridization capture method according to claim 5, characterized in that: Construction of a DNA sample library containing genes related to hereditary deafness, including: The hereditary deafness-related gene DNA is fragmented and phosphorylated to obtain the hereditary deafness-related gene; Connectors are added to both ends of the hereditary deafness-related gene, and the connection products obtained by connecting the hereditary deafness-related gene and the connector are PCR amplified and purified to obtain a DNA sample library containing the hereditary deafness-related gene.

7. The hybridization capture method according to claim 5, characterized in that: The genomic DNA fragmentation is obtained by using enzyme digestion to interrupt the hereditary deafness-related gene DNA in the whole blood sample.

8. The hybridization capture method according to claim 7, characterized in that: The amount of each DNA sample library in the DNA sample library is calculated according to the DNA mass concentration in each DNA sample library to ensure that the total amount of DNA in the library pool is 1.5-4 μg.

9. The hybridization capture method according to claim 1, characterized in that: The purification method for the PCR amplification product of the target complex is the magnetic bead enrichment method.

10. Use of a kit for detecting genes related to hereditary deafness based on a hybridization capture method according to any one of claims 1 to 3 or a kit for preparing a kit for genetic diagnosis of hereditary deafness using the hybridization capture method according to claim 4.

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