Application of senile deafness susceptibility gene locus, and mutation screening reagent and kit thereof

Abstract

The invention discloses application of senile deafness susceptibility gene loci, a mutation screening reagent and a kit thereof, and belongs to the technical field of biomedicine. The invention discloses application of more than thirty senile deafness susceptibility gene loci as markers in preparation of senile deafness screening reagents on the first aspect. The invention further discloses a senile deafness susceptibility gene mutation screening reagent. The senile deafness susceptibility gene mutation screening reagent comprises primers for detecting mutation states of all gene loci. On the third aspect, the invention discloses a multiplex PCR detection kit for senile deafness susceptibility gene mutation. The mutation sites of more than thirty senile deafness susceptibility genes are screened out for the first time, the multiple PCR detection kit for the mutation sites of the susceptibility genes of senile deafness patients is designed, and the kit is used for senile deafness screening and is low in cost, low in required quantity of samples to be detected, short in detection time and high in sensitivity. And the possibility of early discovery and early intervention of senile deafness can be greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of susceptibility gene loci for age-related hearing loss, and their mutation screening reagents and kits. Background Technology

[0002] Presbycusis refers to bilateral, symmetrical, progressive, and irreversible sensorineural hearing loss, starting with high-frequency hearing loss, in individuals aged 60 and over (defined by developing countries). Globally, over 58% of people aged 60 and over suffer from disabling hearing loss. Presbycusis is the leading cause of hearing disability (51.61%). While not fatal, presbycusis causes significant health burdens due to communication difficulties, potentially leading to a range of neuropsychiatric disorders. Currently, there are no specific drug treatments for presbycusis; intervention primarily relies on hearing aids and cochlear implants. However, due to cost and other factors, only 15% of eligible patients receive cochlear implants. Therefore, novel treatments for presbycusis, such as drug therapy, gene therapy, and stem cell transplantation, hold great promise.

[0003] Currently, numerous clinical studies have found that presbycusis is a complex disease caused by the combined effects of multiple factors, mainly including age, environmental factors (noise and exposure to ototoxic substances / drugs), genetic susceptibility (race, sex, and family-specific genetic characteristics), and health complications (hypertension, diabetes, stroke, smoking, etc.). Among these, genetic factors account for 5% to 75% of the pathogenesis of presbycusis. Most scholars believe that presbycusis is likely a disease caused by polygenic inheritance (Wang J, Puel JL. Presbycusis: An Update on Cochlear Mechanisms and Therapies. Journal of clinical medicine. 2020 Jan 14;9(1).). In the field of genetic research, Friedman et al. (Friedman RA, Van Laer L, Huentelman MJ, et al. GRM7 variantsconfer susceptibility to age-related hearing impairment. Human moleculargenetics. 2009 Feb 15;18(4):785-96.) first reported in 2009 that the metabolite glutamate receptor 7 gene (GRM7 gene) is associated with age-related hearing loss. They demonstrated a significant association between the SNP site (rs11928865) of the GRM7 gene and age-related hearing loss through a genome-wide association study (GWAS).

[0004] In summary, the occurrence and development of age-related hearing loss may involve thousands of genetic risk loci, which have a rich spectrum of gene mutations and show significant differences in different regions and ethnic groups (Li Yue, Huang Lihui, Zhao Xuelei, et al. Research progress on candidate pathogenic genes related to age-related hearing loss [J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery. 2022(12):1529-34.). Although existing literature has reported several gene mutation sites related to age-related hearing loss, the exploration of gene mutation sites is still insufficient, and there is currently a lack of specific detection methods for gene mutations related to age-related hearing loss. Therefore, in-depth exploration of susceptibility genes related to age-related hearing loss is of great significance for accurately predicting its occurrence and development, finding potential molecular targets, formulating scientific and reasonable prevention programs, and assessing prognosis. Summary of the Invention

[0005] One of the objectives of this invention is to provide the application of susceptibility gene loci for age-related hearing loss as biomarkers in the preparation of screening reagents for age-related hearing loss. This can enable gene diagnosis or auxiliary diagnosis of age-related hearing loss, and at the same time, it can also conduct genetic screening for families carrying pathogenic variants of age-related hearing loss, greatly increasing the possibility of early detection and early intervention for age-related hearing loss.

[0006] The second objective of this invention is to provide a screening reagent for gene mutations that predispose people to age-related hearing loss.

[0007] The third objective of this invention is to provide a multiplex PCR detection kit for gene mutations that predispose to age-related hearing loss.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses the application of age-related hearing loss susceptibility gene loci as biomarkers in the preparation of age-related hearing loss screening reagents, wherein the gene loci are a combination of the following SNP loci: MUC3A: rs9691167, TTLL8: rs738333, DCLK2: rs13152820, OR2AG1: rs2659879, FSCB: rs11621923, MUC6: rs201234174, SMPD1: rs1050228, FLG2: rs76514540, OR56A5: rs7114672, MUC22: rs34632463, ANGPT2: rs6559167, PRAM1: rs4804305, CDK11A, CDK11B: rs1137005, CFAP44: rs6768642, CDK11A, CDK11B: rs1059831, MUC3A: rs10258821, PLIN4: rs7256387, TTN: rs746578, PAWR: rs2307223, TUBA4B: rs3731892, KIAA1755: rs1205435, CENPJ: rs35498994, EEF1D: rs4874160, PTCHD3: rs2484180, FLG2: rs138997916, ARL14EPL: rs6880759, OR9G9, OR9G1: rs503776, TMEM92: rs6504642, PARP8: rs282547, ZNF214: rs1156525, FAT1: rs1280098, TRDN: rs2873479, CNGB1: rs413562, PTPN23: rs6789730.

[0009] The second aspect of this invention discloses a screening reagent for gene mutations that predispose to age-related hearing loss, comprising primers for detecting mutation status at all the aforementioned gene loci, wherein the primers for each gene locus are as follows: The upper and lower primer sequences of MUC3A and rs9691167 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; The upper and lower primer sequences of TTLL8 and rs738333 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; The upper and lower primer sequences of DCLK2 and rs13152820 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; or in SEQ ID NO:7 and SEQ ID NO:8; or in SEQ ID NO:9 and SEQ ID NO:10. The upper and lower primer sequences of OR2AG1 and rs2659879 are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively; The upper and lower primer sequences of FSCB and rs11621923 are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; The upper and lower primer sequences of MUC6, rs201234174 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; The upper and lower primer sequences of SMPD1 and rs1050228 are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. The upper and lower primer sequences of FLG2, rs76514540, and rs138997916 are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively. The upper and lower primer sequences of OR56A5 and rs7114672 are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively; The upper and lower primer sequences of MUC22 and rs34632463 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. The upper and lower primer sequences of ANGPT2 and rs6559167 are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; The upper and lower primer sequences of PRAM1 and rs4804305 are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively; The upper and lower primer sequences of CDK11A, CDK11B, rs1137005, and rs1059831 are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively. The upper and lower primer sequences of CFAP44 and rs6768642 are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively; or in SEQ ID NO:33 and SEQ ID NO:34; or in SEQ ID NO:35 and SEQ ID NO:36. The upper and lower primer sequences of MUC3A and rs10258821 are shown in SEQ ID NO:37 and SEQ ID NO:38, respectively; The upper and lower primer sequences of PLIN4 and rs7256387 are shown in SEQ ID NO:39 and SEQ ID NO:40, respectively. The upper and lower primer sequences of TTN and rs746578 are shown in SEQ ID NO:41 and SEQ ID NO:42, respectively; The upper and lower primer sequences of PAWR, rs2307223 are shown in SEQ ID NO:43 and SEQ ID NO:44, respectively; The upper and lower primer sequences of TUBA4B and rs3731892 are shown in SEQ ID NO:45 and SEQ ID NO:46, respectively. The upper and lower primer sequences of KIAA1755 and rs1205435 are shown in SEQ ID NO:47 and SEQ ID NO:48, respectively. The upper and lower primer sequences of CENPJ, rs35498994 are shown in SEQ ID NO:49 and SEQ ID NO:50, respectively; The upper and lower primer sequences of EEF1D and rs4874160 are shown in SEQ ID NO:51 and SEQ ID NO:52, respectively. The upper and lower primer sequences of PTCHD3 and rs2484180 are shown in SEQ ID NO:53 and SEQ ID NO:54, respectively. The upper and lower primer sequences of ARL14EPL and rs6880759 are shown in SEQ ID NO:55 and SEQ ID NO:56, respectively. The upper and lower primer sequences of OR9G9 / OR9G1 and rs503776 are shown in SEQ ID NO:57 and SEQ ID NO:58, respectively. The upper and lower primer sequences of TMEM92 and rs6504642 are shown in SEQ ID NO:59 and SEQ ID NO:60, respectively; The upper and lower primer sequences of PARP8 and rs282547 are shown in SEQ ID NO:61 and SEQ ID NO:62, respectively. The upper and lower primer sequences of ZNF214 and rs1156525 are shown in SEQ ID NO:63 and SEQ ID NO:64, respectively. The upper and lower primer sequences of FAT1 and rs1280098 are shown in SEQ ID NO:65 and SEQ ID NO:66, respectively. The upper and lower primer sequences of TRDN, rs2873479 are shown in SEQ ID NO:67 and SEQ ID NO:68, respectively; The upper and lower primer sequences of CNGB1 and rs413562 are shown in SEQ ID NO:69 and SEQ ID NO:70, respectively; The upper and lower primer sequences of PTPN23 and rs6789730 are shown in SEQ ID NO:71 and SEQ ID NO:72, respectively.

[0010] The third aspect of this invention discloses a multiplex PCR detection kit for susceptibility gene mutations in age-related deafness, comprising two sets of PCR primers, wherein the first set of PCR primers is a specific multiplex PCR primer set, including the primers shown in SEQ ID NO.1~SEQ ID NO.4, SEQ ID NO.11~SEQ ID NO.30, and SEQ ID NO.37~SEQ ID NO.72. And any one of the following three pairs of primers for detecting DCLK2, rs13152820: (1) SEQ ID NO:5 and SEQ ID NO:6, (2) SEQ ID NO:7 and SEQ ID NO:8; (3) SEQ ID NO:9 and SEQ ID NO:10; And any one of the following three pairs of primers for detecting CFAP44, rs6768642: (1) SEQ ID NO:31 and SEQ ID NO:32; (2) SEQ ID NO:33 and SEQ ID NO:34; (3) SEQ ID NO:35 and SEQ ID NO:36; The second set of primers are adapter primers.

[0011] The adapter primers described in this invention are based on conventional technology. In some embodiments of this invention, the MultipSeq® adapter kit manufactured by Aijitech Biotechnology (Beijing) Co., Ltd. is used.

[0012] In some embodiments of the present invention, the kit further includes a PCR amplification premixed solution.

[0013] In some embodiments of the present invention, the kit further includes double-distilled water.

[0014] In some embodiments of the present invention, the volumes of the PCR reaction system for the first set of primers are as follows: 9-x parts double-distilled water, 3.5 parts Enhancer buffer NB (1N), 2.5 parts Enhancer buffer M, 5 parts of the first set of primers, x parts of sample, and 10 parts of IGT-EM808 polymerase mixture; wherein the concentration of Enhancer buffer NB is 1N, the initial amount of sample DNA is 40 ng / reaction tube, and the concentration of the DNA is determined by Qubit. TM The fluorescence was measured using a Thermo Fisher Scientific. In some embodiments of the present invention, the volumes of the PCR reaction system for the second set of primers are as follows: 13.5 parts of the purified product from the first round of PCR, 2.5 parts of Enhancer buffer M, 2 parts of the second set of primers, 10 parts of IGT-EM808 polymerase mixture, and 2 parts of double-distilled water.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention identifies, for the first time, mutation sites of over thirty susceptibility genes for age-related hearing loss and designs a multiplex PCR detection kit for these mutation sites in patients with age-related hearing loss. This multiplex PCR detection kit is inexpensive, requires minimal sample volume, and has a short testing time for screening for age-related hearing loss. The kit can be applied clinically as a genetic diagnostic or auxiliary diagnostic tool for age-related hearing loss, and can also be used for genetic screening of families carrying pathogenic variants for age-related hearing loss, greatly increasing the possibility of early detection and intervention. Attached Figure Description

[0016] Appendix Figure 1 Quantile-quantile plot for whole exome association analysis (GWAS); x-axis: Expected–log 10 ( p ) represents the theoretical value – log 10 ( p ); y-axis: Observed-log 10 ( p ) represents the observed value – log 10 ( p ); Appendix Figure 2 This is a Manhattan plot of exome association analysis (GWAS); the horizontal axis represents chromosome location, and the vertical axis represents observed-log. 10 ( p ) represents the observed value – log 10 ( p ). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0018] Example 1 This invention collected clinical data, related audiological examinations, and blood samples from elderly patients diagnosed with hearing loss in the otolaryngology departments of multiple medical institutions in the Sichuan-Chongqing region. Peripheral blood whole-exome high-throughput sequencing was performed, and after genome-wide association study (GWAS) bioinformatics analysis, mutation sites of more than thirty susceptibility genes were screened. Details are as follows: I. Standard Analysis 1.1 Sequencing Depth and Coverage Statistics The valid sequencing data were aligned to the reference genome (B37) using BWA software to obtain the initial alignment results in BAM format. Then, the alignment results were sorted and duplicate reads were marked using Sambamba software. Finally, the alignment results with duplicate marking were used to perform statistical analysis on coverage, depth, etc.

[0019] Typically, sequencing reads from human samples can achieve an alignment rate of over 95%; when the base coverage depth (read depth) of a site reaches 10X or more, the SNP detected at that site is relatively reliable.

[0020] 1.2 Variation Detection Results Based on the initial alignment results (BAM file), SNP and InDel sites were identified using bcftools. The germline SNP and InDel filtering parameters were as follows: QUAL≥20; DV≥4; MQ≥30.

[0021] 1.3 Somatic cell variation detection Somatic cell-level SNV, InDel, and CNV were detected using Mutect, Strelka, and Control-FREEC, respectively. For somatic cell CNV detection, the officially recommended window = 0 was used as the filter parameter in the config folder.

[0022] 1.4 Notes ANNOVAR is a powerful software tool that uses the latest information to functionally annotate gene variations detected from multiple genomes. ANNOVAR was used to annotate previously obtained variant call formats (vcf).

[0023] The gene structure of variant sites is annotated using Refseq, including mRNA and non-coding RNA. The impact of non-synonymous mutations on disease is comprehensively assessed using methods such as SIFT, PolyPhen, and MutationTaster. Annotations are provided from databases including dbSNP, the 1000 Genomes SNP database, the COSMIC database of known tumor somatic mutations, and the esp6500 variant database, allowing for screening of any combination of variant results. Annotations also include functional annotation of the gene containing the mutation, using databases such as GO, KEGG, Reactome, Biocarta, and PID.

[0024] II. GWAS Analysis 1. Using the gnomAD East Asian population database as a control, locus association analysis was performed on 173 samples. gnomAD v2.1.1 was downloaded from http: / / gnomad-sg.org / downloads / (The gnomAD v2.1.1 dataset contains data from 125,748 exomes and 15,708 whole genomes, all mapped to the GRCh37 / hg19 reference sequence). The URL is: https: / / storage.googleapis.com / gcp-public-data--gnomad / release / 2.1.1 / vcf / exomes / gnomad.exomes.r2.1.1.sites.vcf.bgz, with the largest number of users being from East Asia (9435). 2. Association analysis of individual loci based on 173 case and control samples. Based on the SNP variants detected in each sample, all sample variants were pooled, and the mutation status at each locus was statistically analyzed. For each locus, the differences in mutation distribution between the case and control groups were compared, and the p-value and OR value for each locus were obtained using Fisher's two-sided test.

[0025] Perform the following standard filtering on the results: (1) Ensure case detection rate: Filter the genotyping rate in the case and retain the sites with case callrate > 0.9.

[0026] (2) Filtering sites located in the genomic repetitive region: retain sites annotated with "." by genomicSuperDups and Repeat.

[0027] (3) Filtering P-value and OR value. P<5e-7, OR>1. Explanation: Since the number of loci reaches the order of 100,000, the threshold for the correction P is set according to the principle of multiple test correction in association analysis, which is approximately 0.05 / 100,000=5e-7. (In GWAS studies, the number of loci across the entire genome is usually on the order of 1 million, and the significance level of GWAS is generally 0.05 / 1,000,000=5e-8). An OR greater than 1 prioritizes loci that have a high number of mutations in the case and are risk factors for the case.

[0028] (4) Filter out sites with a maf value (minor allele frequency) ≥ 0.05.

[0029] III. Analysis Results: Filtering P-value and OR (P<5e-8, 0R>1); Func: exonic, identified 34 gene mutations / variants associated with the pathogenesis of age-related hearing loss, all of which were newly discovered variants. In this invention, some SNP sites are located in gene overlap or highly homologous regions (such as CDK11A / CDK11B), and may be annotated to multiple genes in the preliminary association analysis. The final mutation site attribution is based on the functional validation results and the gene names in the primer sequences of the detection kit to ensure the accuracy and operability of clinical applications. Specific analysis results are as follows: Table 1

[0030] Note: The allele association P-value for the PTPN23 gene approaches 0 infinitely, which is less than the minimum value (i.e., lower bound) that the software can effectively represent using current methods. Therefore, in this table, following the numerical output convention, it is represented as "0.000E+00".

[0031] The quantile-quantile plots from whole exome association analysis (GWAS) are attached. Figure 1 As shown in the attached Manhattan plot of the whole exome association analysis (GWAS). Figure 2 As shown.

[0032] Example 2 This embodiment discloses primers for detecting the susceptibility gene locus for age-related deafness in this invention, as follows: Table 2

[0033] Example 3 This embodiment discloses a method for detecting gene mutations related to age-related hearing loss using a multiplex PCR reaction with the kit of the present invention. The reagents used in steps 1 and 3 of this embodiment were provided by Aijitech Biotechnology (Beijing) Co., Ltd., the primer sequences were synthesized by Aijitech Biotechnology (Beijing) Co., Ltd., and the purified magnetic beads were IGT™ PureBeads. Details are as follows: Step 1: First round of specific multiplex PCR reaction 1.1 Remove the Enhancer buffer NB (1N), Primer pool, and IGT-EM808 polymerase mix from the -20°C freezer beforehand, place them on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly before placing them on an ice box for later use.

[0034] 1.2 Remove Enhancer buffer M from the 4°C freezer and allow it to thaw at room temperature.

[0035] 1.3 Prepare the reaction system according to the table below on the ice box: Table 3 First-round PCR reaction system

[0036] The total concentration of primers in the primer pool was 47 μM, with each primer having the same concentration. The initial amount of sample DNA was 40 ng / reaction tube.

[0037] 1.4 First-round PCR amplification conditions: Run the PCR program: Heat the lid to 105℃ Table 4. Amplification conditions for the first round of PCR reaction.

[0038] Step 2. Purification 2.1. Prepare 80% ethanol in advance with anhydrous ethanol and nuclease-free water, and keep it at room temperature. Please use freshly prepared 80% ethanol for magnetic bead purification whenever possible.

[0039] 2.2. Remove the purified magnetic beads from the 4℃ refrigerator in advance, mix them well and let them equilibrate at room temperature for 30 min; vortex the purified magnetic beads that have been equilibrated to room temperature and set them aside.

[0040] 2.3. Add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system after step 1, mix by suction or vortexing, and let stand at room temperature for 5 min.

[0041] 2.4. Perform a brief centrifugation, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear.

[0042] 2.5. Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, mix well, and let stand at room temperature for 5 min.

[0043] 2.6. Briefly centrifuge the PCR tubes and place them on a DynaMag-96 Side magnetic rack for 3 min.

[0044] 2.7. Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let stand for 30 seconds.

[0045] 2.8. Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 s, and discard the supernatant.

[0046] 2.9. Cap the tube, centrifuge briefly to remove residual ethanol to the bottom, place the PCR tube on a magnetic rack, and carefully use a 10μL pipette to remove any residual ethanol from the bottom, being careful not to pick up the magnetic beads.

[0047] 2.10. Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow any residual ethanol to evaporate completely.

[0048] 2.11. Add 24 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 min.

[0049] 2.12. Perform a brief centrifugation, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0050] 2.13. Use a pipette to draw 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified multiplex PCR product. Label it and prepare for the reaction in step 3.

[0051] Step 3. Second round of adapter sequence PCR reaction 3.1 Reaction system: Prepare the reaction system according to the table below on an ice box: Table 5 Second round PCR reaction system

[0052] The PCR product mixture is the multiplex PCR product purified in the previous step.

[0053] UDI Primer is a pre-connector primer, with each primer having a concentration of 10 μM.

[0054] Second round PCR amplification conditions: Run the PCR program: Heat the lid to 105℃ Table 6. Amplification conditions for the second round of PCR reaction.

[0055] Step 4. Second round of purification 4.1. Prepare 80% ethanol in advance with anhydrous ethanol and nuclease-free water, and keep it at room temperature. Please use freshly prepared 80% ethanol for magnetic bead purification whenever possible.

[0056] 4.2. Remove the purified magnetic beads from the 4℃ refrigerator in advance, mix them well and let them equilibrate at room temperature for 30 min; vortex the purified magnetic beads that have been equilibrated to room temperature and set them aside.

[0057] 4.3. After completing step 3, add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system, mix by suction or vortexing, and let stand at room temperature for 5 min.

[0058] 4.4. Briefly centrifuge the PCR tubes on a magnetic rack for 3 minutes until the solution becomes clear.

[0059] 4.5. Thoroughly remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, mix well, and let stand at room temperature for 5 min.

[0060] 4.6. Briefly centrifuge the PCR tubes and place them on a DynaMag-96 Side magnetic rack for 3 min.

[0061] 4.7. Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let stand for 30 s.

[0062] 4.8. Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 s, and discard the supernatant.

[0063] 4.9. Cap the tube, centrifuge briefly to remove residual ethanol to the bottom, place the PCR tube on a magnetic rack, and carefully use a 10 μL pipette to remove any residual ethanol at the bottom, being careful not to pick up the magnetic beads.

[0064] 4.10. Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow any residual ethanol to evaporate completely.

[0065] 4.11. Add 24 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 min.

[0066] 4.12. Perform a brief centrifugation, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0067] 4.13. Use a pipette to draw 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

[0068] Step 5. Library Quantitative Analysis and Quality Control A 1 μL sample of the library was taken and its concentration was determined using a Qubit® 3.0 Fluorometer (Qubit dsDNA HS Assay Kit). The concentration was recorded, and the concentration was greater than 3 ng / μL, meeting the requirements. The library fragment length and purity were measured using the Qsep400 fully automated nucleic acid and protein analysis system. There was no primer dimer contamination in the pre-peak region and no high molecular weight tailing in the post-peak region. The peak morphology was normal, and the quality inspection was qualified.

[0069] Step 6. Analysis High-throughput sequencing was performed using a next-generation sequencing platform, and the sequencing data were statistically analyzed. The QC rate was >95%, the alignment rate was >95%, the coverage was 100%, the target hit rate was >70%, and the coverage at 0.2x average sequencing depth was >80%.

[0070] By comparing and analyzing the data, the gene mutations related to age-related hearing loss can be accurately identified.

[0071] Example 4 Blood samples from 17 elderly patients with hearing loss were tested using the method described in Example 3 (results are shown in Tables 7-1 to 7-17). The results showed that all samples had gene locus mutations of varying degrees, consistent with the main clinical diagnostic methods in the "Expert Consensus on Diagnosis and Intervention of Hearing Loss in the Elderly (2019)". The main diagnostic methods are: 1. Medical history inquiry; 2. Otolaryngological examination; 3. Basic audiological examinations: (1) pure tone audiometry, (2) acoustic impedance testing, and (3) speech audiometry. According to the World Health Organization (1997) hearing loss classification standard, a mean hearing threshold greater than or equal to 26 dBHL is considered mild hearing loss (Note: the mean hearing threshold refers to the average value of the air conduction hearing thresholds at four frequencies: 500, 1000, 2000, and 4000 Hz).

[0072] Table 7-1 Test results of Case 1

[0073] Table 7-2 Test results of Case 2

[0074] Table 7-3 Test results of Case 3

[0075] Table 7-4 Test results of Case 4

[0076] Table 7-5 Test results of Case 5

[0077] Table 7-6 Test results of Case 6

[0078] Table 7-7 Test results of Case 7

[0079] Tables 7-8 show the test results of Case 8.

[0080] Table 7-9 Test results of Case 9

[0081] Table 7-10 Test results of Case 10

[0082] Table 7-11 Test Results of Case 11

[0083] Table 7-12 Test Results of Case 12

[0084] Table 7-13 Test Results of Case 13

[0085] Table 7-14 Test Results of Case 14

[0086] Table 7-15 Test Results of Case 15

[0087] Table 7-16 Test Results of Case 16

[0088] Table 7-17 Test Results of Case 17

[0089] Functional experiments have verified that the rs1137005 / rs1059831 site mainly affects the function of the CDK11A gene, and therefore it is clearly attributed to CDK11A in this embodiment.

[0090] The embodiments described above are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of age-related hearing loss susceptibility gene loci as biomarkers in the preparation of age-related hearing loss screening reagents, characterized in that, The gene locus is a combination of the following SNP loci: MUC3A: rs9691167, TTLL8: rs738333, DCLK2: rs13152820, OR2AG1: rs2659879, FSCB: rs11621923, MUC6: rs201234174, SMPD1: rs1050228, FLG2: rs76514540, OR56A5: rs7114672, MUC22: rs34632463, ANGPT2: rs6559167, PRAM1: rs4804305, CDK11A, CDK11B: rs1137005, CFAP44: rs6768642, CDK11A, CDK11B: rs1059831, MUC3A: rs10258821, PLIN4: rs7256387, TTN: rs746578, PAWR: rs2307223, TUBA4B: rs3731892, KIAA1755: rs1205435, CENPJ: rs35498994, EEF1D: rs4874160, PTCHD3: rs2484180, FLG2: rs138997916, ARL14EPL: rs6880759, OR9G9, OR9G1: rs503776, TMEM92: rs6504642, PARP8: rs282547, ZNF214: rs1156525, FAT1: rs1280098, TRDN: rs2873479, CNGB1: rs413562, PTPN23: rs6789730.

2. A screening reagent for susceptibility gene mutations in age-related deafness, characterized in that, Includes primers for detecting the mutation status of all gene loci as described in claim 1, with the primers for each gene locus as follows: The upper and lower primer sequences of MUC3A and rs9691167 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; The upper and lower primer sequences of TTLL8 and rs738333 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; The upper and lower primer sequences of DCLK2 and rs13152820 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; or in SEQ ID NO:7 and SEQ ID NO:8; or in SEQ ID NO:9 and SEQ ID NO:

10. The upper and lower primer sequences of OR2AG1 and rs2659879 are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively; The upper and lower primer sequences of FSCB and rs11621923 are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; The upper and lower primer sequences of MUC6, rs201234174 are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively; The upper and lower primer sequences of SMPD1 and rs1050228 are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. The upper and lower primer sequences of FLG2, rs76514540, and rs138997916 are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively. The upper and lower primer sequences of OR56A5 and rs7114672 are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively; The upper and lower primer sequences of MUC22 and rs34632463 are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; The upper and lower primer sequences of ANGPT2 and rs6559167 are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; The upper and lower primer sequences of PRAM1 and rs4804305 are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively; The upper and lower primer sequences of CDK11A, CDK11B, rs1137005, and rs1059831 are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively. The upper and lower primer sequences of CFAP44 and rs6768642 are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively; or in SEQ ID NO:33 and SEQ ID NO:34; or in SEQ ID NO:35 and SEQ ID NO:

36. The upper and lower primer sequences of MUC3A and rs10258821 are shown in SEQ ID NO:37 and SEQ ID NO:38, respectively; The upper and lower primer sequences of PLIN4 and rs7256387 are shown in SEQ ID NO:39 and SEQ ID NO:40, respectively. The upper and lower primer sequences of TTN and rs746578 are shown in SEQ ID NO:41 and SEQ ID NO:42, respectively; The upper and lower primer sequences of PAWR, rs2307223 are shown in SEQ ID NO:43 and SEQ ID NO:44, respectively; The upper and lower primer sequences of TUBA4B and rs3731892 are shown in SEQ ID NO:45 and SEQ ID NO:46, respectively. The upper and lower primer sequences of KIAA1755 and rs1205435 are shown in SEQ ID NO:47 and SEQ ID NO:48, respectively. The upper and lower primer sequences of CENPJ, rs35498994 are shown in SEQ ID NO:49 and SEQ ID NO:50, respectively; The upper and lower primer sequences of EEF1D and rs4874160 are shown in SEQ ID NO:51 and SEQ ID NO:52, respectively. The upper and lower primer sequences of PTCHD3 and rs2484180 are shown in SEQ ID NO:53 and SEQ ID NO:54, respectively. The upper and lower primer sequences of ARL14EPL and rs6880759 are shown in SEQ ID NO:55 and SEQ ID NO:56, respectively. The upper and lower primer sequences of OR9G9 / OR9G1 and rs503776 are shown in SEQ ID NO:57 and SEQ ID NO:58, respectively. The upper and lower primer sequences of TMEM92 and rs6504642 are shown in SEQ ID NO:59 and SEQ ID NO:60, respectively; The upper and lower primer sequences of PARP8 and rs282547 are shown in SEQ ID NO:61 and SEQ ID NO:62, respectively. The upper and lower primer sequences of ZNF214 and rs1156525 are shown in SEQ ID NO:63 and SEQ ID NO:64, respectively. The upper and lower primer sequences of FAT1 and rs1280098 are shown in SEQ ID NO:65 and SEQ ID NO:66, respectively. The upper and lower primer sequences of TRDN, rs2873479 are shown in SEQ ID NO:67 and SEQ ID NO:68, respectively; The upper and lower primer sequences of CNGB1 and rs413562 are shown in SEQ ID NO:69 and SEQ ID NO:70, respectively. The upper and lower primer sequences of PTPN23 and rs6789730 are shown in SEQ ID NO:71 and SEQ ID NO:72, respectively.

3. A multiplex PCR detection kit for susceptibility gene mutations in age-related deafness, characterized in that, It includes two sets of PCR primers. The first set of PCR primers is a specific multiplex PCR primer set, including the primers shown in SEQ ID NO.1~SEQ ID NO.4, SEQ ID NO.11~SEQ ID NO.30, and SEQ ID NO.37~SEQ ID NO.

72. And any one of the following three pairs of primers for detecting DCLK2, rs13152820: (1) SEQ ID NO:5 and SEQ ID NO:6, (2) SEQ ID NO:7 and SEQ ID NO:8; (3) SEQ ID NO:9 and SEQ ID NO:10; And any one of the following three pairs of primers for detecting CFAP44, rs6768642: (1) SEQ ID NO:31 and SEQ ID NO:32; (2) SEQ ID NO:33 and SEQ ID NO:34; (3) SEQ ID NO:35 and SEQ ID NO:36; The second set of primers are adapter primers.

4. The multiplex PCR detection kit for susceptibility gene mutations in age-related deafness according to claim 3, characterized in that, The kit also includes a PCR amplification premix solution.

5. The multiplex PCR detection kit for susceptibility gene mutations in age-related deafness according to claim 3, characterized in that, The kit also includes double-distilled water.

6. The multiplex PCR detection kit for susceptibility gene mutations in age-related deafness according to claim 3, characterized in that, The volumes of the PCR reaction system for the first set of primers are as follows: 9-x parts double-distilled water, 3.5 parts Enhancer buffer NB, 2.5 parts Enhancer buffer M, 5 parts of the first set of primers, x parts of sample, and 10 parts IGT-EM808 polymerase mixture; wherein the concentration of Enhancer buffer NB is 1N, and the initial amount of sample DNA is 40 ng / reaction tube.

7. The multiplex PCR detection kit for susceptibility gene mutations in age-related deafness according to claim 3, characterized in that, The volumes of the PCR reaction system for the second set of primers are as follows: 13.5 parts of the purified product from the first round of PCR, 2.5 parts of Enhancer buffer M, 2 parts of the second set of primers, 10 parts of IGT-EM808 polymerase mixture, and 2 parts of double-distilled water.

Images