Primer probe, kit and application for detecting copy number of GJB2 gene in non-syndromic hearing loss patient by using droplet digital PCR

Abstract

The application particularly relates to a primer probe, a kit and application for detecting the GJB2 gene copy number of a non-syndromic hearing loss patient by using microdroplet digital PCR. The primer probe combination provided by the application comprises a target gene detection primer pair, a target gene detection probe, an upstream primer of a reference gene, a downstream primer of the reference gene and a reference gene probe, and the sequence information is shown as SEQ ID NO. 6-11; and the application further provides a kit for detecting the GJB2 gene copy number of a non-syndromic hearing loss patient. The primer probe combination and the kit provided by the application are applied to the detection of the GJB2 gene copy number variation of a non-syndromic hearing loss patient, and the accuracy of the detection of genetic hearing loss is significantly improved; according to the determination result, it is determined whether the GJB2 gene expression is abnormal or not, the disease prognosis is evaluated, potential therapeutic drugs are screened, genetic counseling services are provided or individualized medical schemes are formulated, and the application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of gene diagnostic technology, specifically relating to primers, probes, kits, and applications for detecting the copy number of the GJB2 gene in non-syndromic deafness patients using droplet digital PCR. Background Technology

[0002] Hearing loss is a common sensorineural disorder, ranging from mild to moderate, severe, and profound, with severe or profound hearing loss typically defined as deafness. The causes of deafness are extremely complex, broadly categorized into environmental and genetic factors, with genetic factors contributing over 60%. Based on the presence or absence of abnormalities in other systems or organs, deafness can be further subdivided into syndromic hearing loss (SHL, approximately 30%) and non-syndromic hearing loss (NSHL, approximately 70%). Hereditary deafness is largely a single-gene inherited disease, exhibiting high genetic heterogeneity. Based on their mode of inheritance, hereditary deafness can be classified into several categories, including autosomal dominant deafness (DFNA), autosomal recessive deafness (DFNB), X-linked deafness (DFNX), Y-linked deafness (DFNY), and mitochondrial inheritance. Among these types of hereditary deafness, DFNA accounts for approximately 20%, DFNB for approximately 70%, DFNX for approximately 1%, mitochondrial inheritance for approximately 1%, while DFNY has only been reported in a few isolated cases.

[0003] Currently, over 100 genes have been confirmed to be associated with non-syndromic hearing loss, while reports indicate that over 400 genes are closely related to syndromic hearing loss. Among all hereditary deafness, non-syndromic DFNB is the most common. Because it follows an autosomal recessive inheritance pattern, it often manifests as deafness in the child, but the parents have normal hearing and no family history of deafness. For these children with deafness, obtaining a definitive genetic diagnosis is crucial. This not only provides genetic evidence for the child's rehabilitation treatment but also offers key guidance for the parents' decision to have another child. In my country, the GJB2 gene is the most common cause of non-syndromic DFNB (deafness-induced hearing loss). Homozygous or compound heterozygous mutations in this gene can lead to deafness, accounting for 20-30% of congenital hearing loss cases. Common mutation forms include small insertions / deletions (c.235delC, c.299_300delAT, c.176_191del) and single nucleotide variants (SNVs) (c.109G>A). Besides small insertion / deletion mutations and SNVs, copy number variations (CNVs) are also common types of gene mutations associated with deafness. Statistics show that approximately 20% of hearing loss cases may be caused by CNVs.

[0004] Currently, the main methods for detecting copy number variations (CNVs) include next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and digital PCR. Among these, NGS suffers from long detection cycles and high costs, and has stringent requirements for target gene design coverage and bioinformatics analysis, making it difficult to achieve rapid and convenient clinical testing. MLPA-based methods have specific sample size requirements and are also costly, leading to several limitations in clinical applications. In contrast, digital PCR offers advantages such as lower cost, less stringent sample size requirements, and shorter detection cycles, typically providing results within 3-5 hours.

[0005] In the process of researching the genetic factors of deafness, the inventors of this invention employed a tiered detection strategy, including high-throughput sequencing technologies such as whole-exome sequencing, to conduct in-depth analysis of deaf patients. They found that approximately 70% of patients had identifiable genetic factors, with pathogenic or potentially pathogenic single nucleotide variants (SNVs) or small insertion / deletion variants (indels) detectable in known deafness genes. However, the molecular etiology of the remaining considerable number of patients remains unknown. Of particular note is that some patients, after undergoing whole-exome sequencing, were found to carry a single heterozygous variant of the GJB2 gene. However, this finding was insufficient for a definitive diagnosis and was not accompanied by the presence of other suspicious genes or mutations. Considering the high carrier rate of pathogenic GJB2 gene variants in the Chinese population, the inventors of this invention hypothesize that the cause may involve a novel, currently unidentified deafness gene, or the existence of an undetected pathogenic GJB2 gene variant.

[0006] The GJB2 gene, short for gap junction protein beta-2, encodes a protein called connexin 26 (Cx26). This protein is a key component of gap junction proteins, primarily expressed in the supporting cells of the inner ear, and is essential for maintaining normal inner ear function. The GJB2 gene plays a crucial role in maintaining normal inner ear function, and its mutation is one of the major causes of deafness. When the GJB2 gene mutates, the expression, structure, and function of the Cx26 protein may be affected, leading to abnormal intercellular communication. This, in turn, affects signal transmission between sensory epithelial cells and neurons in the inner ear, causing hearing loss. GJB2 gene mutations can lead to congenital deafness or late-onset deafness, with patients experiencing varying degrees of hearing loss, accompanied by symptoms such as tinnitus and vertigo. In China, approximately 20% of patients with congenital deafness carry GJB2 gene mutations. For deafness caused by GJB2 gene mutations, genetic testing can be performed to determine the presence of the GJB2 gene mutation. Reports indicate that deletions in the upstream regulatory region of the GJB2 gene may affect its transcriptional activity, thereby impacting its expression level.

[0007] In nomenclature for autosomal dominant inherited deafness (DFNA) and autosomal recessive inherited deafness (DFNB), the numbers following the nomenclature indicate the chronological order of gene locus location, such as DFNA1, DFNA2, and DFNB1. The DFNB1 locus contains two genes, GJB2 and GJB6, encoding Cx26 and Cx30 respectively, which are major connective proteins in the cochlea. GJB2 and GJB6 work synergistically in the inner ear, sharing regulatory sequences. Because the transcription direction of GJB2 and GJB6 genes is opposite to the genomic direction, their regulatory sequences are as follows: Figure 1The downstream portion shown extends as far as part or all of the CRYL1 gene. Current research indicates that six deletion types of the upstream regulatory region of the GJB2 gene have been identified in European populations: del (GJB6-D13S1830, 309kb), del (GJB6-D13S1854, 232kb), del (179kb), del (131kb), del (>920kb), and del (101kb). Recent research in China has also identified three deletion types of the upstream regulatory region of the GJB2 gene, the most common being del (125kb), and the relatively rare being (GJB6-D13S1854, 232kb) and del (34kb). Based on reports from both domestic and international sources regarding different deletion types of the upstream regulatory region of the GJB2 gene, researchers have found a common minimum region in these deletions, 34kb in length, encompassing exons 4-6 of the CRYL1 gene. Therefore, the copy number of exon 4-6 of the CRYL1 gene can be used as an approximation of the copy number of the GJB2 gene (exons and regulatory regions), and it can cover almost all known deletion types of the upstream regulatory regions of the GJB2 gene.

[0008] Therefore, it is of great significance to develop a more efficient and economical method for detecting copy number variations of the GJB2 gene in patients with non-syndromic hearing loss based on a digital PCR platform, so as to achieve large-scale clinical application, improve the accuracy of hereditary hearing loss detection, and reduce the burden on patients. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention aims to provide primers, probes, kits, and applications for detecting the copy number of the GJB2 gene in patients with non-syndromic hearing loss using droplet digital PCR. Based on droplet digital PCR detection technology, this invention provides primer and probe combinations and kits for detecting the copy number of the GJB2 gene in patients with non-syndromic hearing loss. Using the primer and probe combinations and kits provided by this invention significantly improves the accuracy of hereditary hearing loss detection.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The first objective of this invention is to provide a primer-probe combination for detecting the copy number of the GJB2 gene in non-syndromic deafness patients using droplet digital PCR, comprising a target gene detection primer pair, a target gene detection probe, an upstream primer for an internal reference gene, a downstream primer for an internal reference gene, and a probe for an internal reference gene; wherein:

[0012] The sequence information of the target gene detection primer pair is as follows: the sequence information of the upstream primer of GJB2 is shown in SEQ ID NO. 6; the sequence information of the downstream primer of GJB2 is shown in SEQ ID NO. 7; the sequence information of the target gene detection probe is shown in SEQ ID NO. 8;

[0013] The internal reference gene is the CFTR gene; the sequence information of the upstream primer of the internal reference gene is shown in SEQ ID NO.9; the sequence information of the downstream primer of the internal reference gene is shown in SEQ ID NO.10; and the sequence information of the probe of the internal reference gene is shown in SEQ ID NO.11.

[0014] Preferably, the GJB2 gene copy number includes the copy number of the coding region and the regulatory region of the GJB2 gene.

[0015] Preferably, the target gene detection probe and the internal reference gene probe contain fluorescent groups and quenching groups.

[0016] Preferably, the target gene detection probe is labeled with a FAM fluorescent group at 5' and a BHQ1 quencher group at 3'; the internal reference gene probe is labeled with a CY5 fluorescent group at 5' and a BHQ2 quencher group at 3'.

[0017] Another object of the present invention is to provide a kit for detecting the copy number of the GJB2 gene in patients with non-syndromic hearing loss using droplet digital PCR, the kit comprising the primer-probe combination described above.

[0018] Another object of the present invention is to provide the application of the primer-probe combination or kit described above in detecting the copy number of the GJB2 gene in patients with non-syndromic hearing loss for non-diagnostic and therapeutic purposes.

[0019] Another object of the present invention is to provide a method for detecting the copy number of the GJB2 gene in patients with non-syndromic hearing loss for non-diagnostic and therapeutic purposes using the above-described primer-probe combination or kit, characterized by comprising the following steps:

[0020] S1. Extract genomic DNA from the biological sample to be tested to obtain a droplet digital PCR reaction template;

[0021] S2. Use the primer-probe combination or kit described above to perform droplet digital PCR detection.

[0022] Preferably, the droplet digital PCR detection in step S2 includes detection using a digital PCR biochip analyzer and data analysis using biochip reader software; based on the detection results of the 2D scatter plot and positive control, a suitable positive region is delineated; based on the number of positive droplets detected in the delineated positive region and the Poisson distribution principle, the copy number of the fluorescent channels of the target gene and the internal reference gene is calculated; based on the ratio of the copy number of the GJB2 gene to the copy number of the internal reference gene CFTR gene, the change in the copy number of the GJB2 gene is calculated.

[0023] The digital PCR biochip analyzer is a Chip Reader R1 model manufactured by Xinyi Manufacturing Technology (Beijing) Co., Ltd.

[0024] Preferably, the copy number of the GJB2 gene is calculated as follows:

[0025] The copy number of the target gene is calculated as the ratio of the copy numbers of the GJB2 gene to the internal reference gene CFTR * 2. A copy number between 0 and 0.1 is considered 0; between 0.4 and 0.6 is considered 0.5; and between 0.8 and 1.2 is considered 1. If the value falls within these ranges and cannot be determined, the experiment must be repeated.

[0026] Preferably, the method further includes: based on the determination results of the copy number change of the GJB2 gene, further analyzing the association between the copy number variation of the GJB2 gene and non-syndromic deafness.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] First, this invention provides a primer-probe combination and kit for detecting the copy number of the GJB2 gene in patients with non-syndromic deafness using droplet digital PCR detection technology. The primer-probe combination and kit provided by this invention significantly improve the accuracy of hereditary deafness detection.

[0029] Secondly, the primer-probe combination and kit provided by this invention can also be applied to detect GJB2 gene copy number variations in patients with non-syndromic hearing loss. Based on the detection of GJB2 gene copy number variations, it can help determine whether there is an abnormality in GJB2 gene expression, thereby assessing disease prognosis, screening potential therapeutic drugs, providing genetic counseling services, or developing personalized medical plans. It can be seen that this invention has broad application prospects. Attached Figure Description

[0030] Figure 1 A schematic diagram showing the missing control area of ​​GJB2;

[0031] Figure 2Sanger sequencing image of a sample with a 125kb deletion in the regulatory region of the GJB2 gene;

[0032] Figure 3 Primer pair design and results;

[0033] Figure 4 The results of the GJB2 gene double copy number deletion detection;

[0034] Figure 5 The results of the single copy number deletion detection of the GJB2 gene;

[0035] Figure 6 The result indicates that the GJB2 gene copy number is normal. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0039] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0040] 1. Reagents and materials

[0041] The blood genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0042] The universal reagent kit for microdroplet detection (10002) was purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd.

[0043] 2. Equipment and Instruments

[0044] The digital PCR biochip analyzer was purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd., model ChipReader R1.

[0045] The biochip reader software was purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd., model BBAIH.

[0046] The spectrophotometer was purchased from IMPLEN, model NanoPhotometer NP50;

[0047] The microdroplet generator was purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd., model Drop Maker M1;

[0048] The universal reagent kit for microdroplet detection and the matching biochip analyzer were purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd., model 7.0;

[0049] The digital PCR analysis system software was purchased from Xinyi Manufacturing Technology (Beijing) Co., Ltd., model Chip ReaderR1 V1.0.2.

[0050] 3. Study population:

[0051] All deaf patients and their family members in this invention were collected from the Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University, and informed consent was obtained from all of them.

[0052] Based on the above findings, the inventors of this invention used the human reference genome sequence (GRCh37 version) and the Primer 3 primer design tool to design two pairs of common primers in the exon 4-6 region of the CRYL1 gene to detect large copy number deletions in the regulatory region of the GJB2 gene. Subsequently, the inventors used these two pairs of primers to perform PCR and first-generation sequencing on nucleic acid samples from families of deaf patients with undetermined etiologies. Analysis of the breakpoints in the sequencing results revealed that large copy number deletions in the regulatory region of the GJB2 gene also exist in families of deaf patients in China. The specific steps include the following:

[0053] (1) Specific methods of PCR and first-generation sequencing:

[0054] Based on domestic and international reports on deletion types in the upstream regulatory region of the GJB2 gene, the position range of the CRYL1 gene sequence information in the GRCh38.p14 reference sequence version is determined as follows: >NC_000013.11:c20525857-20403669Homo sapiens chromosome 13, GRCh38.p14 Primary Assembly (Reference URL: https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000013.10?from=20977808&to=21099996&report=fasta&strand=true).

[0055] Two pairs of common primers for detecting large copy number deletions were designed within the CRYL1 gene region, as shown in Table 1 (SEQ ID NO. 1-4).

[0056] Table 1. Information on Common Primers with Copy Number Deletion

[0057]

[0058] DNA samples from deaf patients and their families at the Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University were used as test samples. Primers at the upstream and downstream breakpoints were added to the PCR amplification tubes, along with the test sample DNA, 2×Taq Master Mix, and DEPC water. The reaction system configuration is shown in Table 2 below. The PCR amplification reagent, 2×Taq Master Mix, was purchased from Nanjing Novizan Biotechnology Co., Ltd. (catalog number P111).

[0059] Table 2. Standard PCR reaction system

[0060]

[0061] After configuring the PCR amplification system, place the PCR tubes on a standard PCR instrument for PCR amplification. Set the amplification conditions as shown in Table 3 below (temperature increase / decrease rate set to 1.5℃ / s, amplification volume set to 25μL). Once set, save the file and run the reaction program.

[0062] Table 3 Standard PCR Reaction Procedure

[0063]

[0064] After the PCR reaction was completed, 1% agarose gel electrophoresis was performed. After confirming the presence and correct size of bands under UV light, Sanger sequencing of the PCR products was commissioned to Sangon Biotech Co., Ltd. The Sanger sequencing image of the sample with a 125kb deletion in the GJB2 gene regulatory region is shown below. Figure 2 As shown.

[0065] (2) Breakpoint analysis method:

[0066] Primer pair design and results are as follows Figure 3As shown, for the samples to be tested, PCR amplification was performed using primer pairs 125kb up F and 125kb down R, primer pairs 125kb up F and 125kb up R, and primer pairs 125kb down F and 125kb down R, according to the methods in Tables 2 and 3. The PCR products were analyzed by 1% agarose gel electrophoresis. Homozygous deletion patients only showed a single, bright, and correctly sized band in primer pairs 125kb up F and 125kb down R, and no bands were observed in primer pairs 125kb up F and 125kb up R, and primer pairs 125kb down F and 125kb down R. Heterozygous deletion patients showed bands in all three primer pairs. In the normal control, bands were amplified in primer pairs 125kb up F and 125kb up R, and primer pairs 125kb down F and 125kb down R, respectively, but no bands were observed in primer pairs 125kb up F and 125kb down R. These PCR products were then subjected to Sanger sequencing (Sangon Biotech Co., Ltd.). The sequences obtained were compared with the reference sequences using SnapGene software to determine whether the patient contained a deleted fragment in this region, and to identify the site of the deleted fragment.

[0067] (3) Criteria for collecting and screening family pedigree samples of deaf patients:

[0068] The collection and screening criteria for family pedigree samples from deaf patients must adhere to the following principles: 1) The family must include at least one proband with prelingual deafness confirmed by clinical audiology examination; 2) Cases of hearing loss caused by environmental factors (such as noise exposure, history of ototoxic drugs) or acquired diseases (such as otitis media, trauma) must be excluded; 3) Peripheral venous blood will be collected for genomic DNA extraction, and clinical phenotypic data (hearing threshold, age of onset, accompanying symptoms, etc.) will be compiled simultaneously; 4) All participants must sign informed consent forms. Known pathogenic variants carried by the proband will be screened through whole-exome sequencing.

[0069] (4) Verification of experimental results:

[0070] Based on the above information, the inventors of this invention decided to use high-throughput sequencing methods (such as whole-exome sequencing) commonly used in clinical practice to detect single nucleotide variants (SNVs) and small insertion / deletion (indel) mutations to analyze copy number variations in exons related to the CRYL1 gene. If a CNV is found in the exon region of the CRYL1 gene in a clinical case, specifically manifested as heterozygous or homozygous deletion, the inventors will further use digital PCR technology to detect the upstream regulatory region of the GJB2 gene to confirm whether a CNV is indeed present. Simultaneously, the presence or absence of pathogenic variants in the exon region of the GJB2 gene will be used for auxiliary analysis of the case. By integrating this information, the molecular etiology of clinical cases can be more accurately determined, thus providing solid data support and a theoretical foundation for subsequent in-depth research. This strategy not only helps improve the diagnostic accuracy of specific genetic information but also provides the possibility of developing more effective treatment strategies.

[0071] Therefore, this invention aims to provide a detection reagent based on a digital PCR platform, utilizing digital PCR to detect copy number variations in the regulatory region of the GJB2 gene, enabling large-scale clinical application, improving the accuracy of hereditary deafness detection, and reducing the burden on patients. Specifically, it includes the following:

[0072] Example 1: Design of Primer-Probe Combinations

[0073] To improve detection efficiency and clinical applicability, this invention is based on the human reference genome sequence (GRCh37 version), and also references the gnomAD and ExAC databases. Based on this, the Primer 3 primer design tool was used to design a primer-probe combination for the CRYL1 gene and the internal reference gene (CFTR gene) based on digital PCR.

[0074] For the CRYL1 gene, the primer design region is located within a shared deletion region of 34kb in length. This region was determined based on domestic and international reports on deletion types in the upstream regulatory region of the GJB2 gene. The primer sequence design region is Chr13:20994200-20994800, and its sequence information is shown in SEQ ID NO.5.

[0075] (SEQ ID NO.5).

[0076] In the process of primer design, the following three principles should be followed:

[0077] 1. Avoid polymorphism interference: The primer and probe design regions should avoid population polymorphism (SNP / Indel) sites (population frequency > 0.1%) as much as possible to avoid interference from some polymorphic sites on the results and improve the detection accuracy.

[0078] 2. Matching amplification efficiency: making the amplification efficiency of the internal reference gene as close as possible to the amplification efficiency of the target gene;

[0079] 3. Optimize amplification length and annealing temperature: The amplification length of the target gene and the internal reference gene is between 100bp and 200bp, and the annealing temperature is designed to be 58℃. At the same time, different fluorescent labels are placed on the target gene and the internal reference gene to facilitate differentiation and detection during the digital PCR process.

[0080] After extensive screening and experimental verification, the primer and probe sequences designed in this invention are shown in Table 4 below. The target gene probe is labeled with FAM fluorescence at 5' and BHQ1 quencher at 3'. The internal reference gene probe is labeled with CY5 fluorescence at 5' and BHQ2 quencher at 3'.

[0081] Table 4 Primer base sequence listing

[0082]

[0083] Detection method:

[0084] The primer and probe combinations designed in Table 4 above were configured into corresponding reaction systems and detected on a digital PCR microarray analyzer. After detection, data analysis was performed using microarray reader software. During the analysis, a suitable positive region was delineated mainly based on the 2D scatter plot and the detection results of the positive control (generally, the positive region was delineated at the center of the area where the FAM fluorescence signal (GJB2 gene) and VIC fluorescence signal (CFTR gene) were concentrated).

[0085] After delineating the positive regions, the copy numbers of the target gene (GJB2 gene) and the internal reference gene (CFTR gene) fluorescence channels were calculated using the detected positive droplet count and the Poisson distribution principle. Then, based on the ratio of the GJB2 gene copy number to the CFTR gene copy number, the change in the GJB2 gene copy number was calculated. The formula for calculating the GJB2 gene copy number is: Target gene copy number = Ratio of GJB2 gene copy number to CFTR gene copy number * 2.

[0086] Result interpretation:

[0087] Theoretically, the ratio of GJB2 gene copy number change to CFTR gene copy number has the following relationship:

[0088] When a double copy of the GJB2 gene is deleted, the copy number ratio of the GJB2 gene to the CFTR gene should be 0; when a single copy of the GJB2 gene is deleted, the copy number ratio of the GJB2 gene to the CFTR gene should be 0.5; when the copy number of the GJB2 gene is normal, the copy number ratio of the GJB2 gene to the CFTR gene should be 1. However, considering experimental and detection errors, the ratios for double copy deletion, single copy deletion, and normal copy number are set to 0~0.1, 0.4~0.6, and 0.8~1.2, respectively, as shown in Table 5 below.

[0089] Table 5. Correspondence between copy number ratio and copy number variation in GJB2

[0090]

[0091] The above process can accurately and quickly detect changes in the copy number of the GJB2 gene.

[0092] Example 2: PCR amplification of the test sample using the primer-probe combination designed in this invention.

[0093] (1) Sample processing and nucleic acid extraction

[0094] Sample selection: Clinical blood samples were selected from three patients with non-syndromic hearing loss who were verified by first-generation sequencing to have single copy deletion of GJB2 gene, double copy deletion of GJB2 gene, and normal copy number of GJB2 gene (without deletion). Sample numbers: 3313151-1, 3313151-2, and 3313715-1.

[0095] Nucleic acid extraction: Using a DNA extraction kit, extract nucleic acid from the selected samples according to the instructions.

[0096] Concentration determination: The concentration of extracted DNA was determined using a spectrophotometer. The DNA concentration of the sample to be tested should be within the range of 5 ng / μL to 100 ng / μL to ensure the smooth progress of subsequent PCR amplification reactions.

[0097] (2) Configure the amplification reaction system

[0098] Primer and probe sets for the upstream primers of the internal reference gene were added to the PCR amplification tubes, along with the DNA of the sample to be tested, 4×Accumix, and DEPC water. The configuration of the reaction system is shown in Table 6 below. 4×Accumix is ​​a nucleic acid amplification reagent purchased from Xinyi Manufacturing (Beijing) Technology Co., Ltd.

[0099] Table 6 Composition of the reaction system

[0100]

[0101] (3) Preparation of microdroplets

[0102] The sample preparation process was conducted using a microdroplet generator provided by Xinyi Manufacturing Technology (Beijing) Co., Ltd. A 30 μL pre-prepared reaction mixture was transferred to the sample wells of the microdroplet preparation chip, followed by the addition of 180 μL of microdroplet preparation oil to the oil wells. The microdroplet preparation chip containing the reaction mixture and preparation oil was then placed in the sample preparation device, and microdroplet generation was performed according to the instrument's operating instructions. After droplet generation, two processing options were available: 1) Immediately initiate PCR amplification to ensure experimental continuity; 2) Store the chip containing the microdroplets at 4°C, but begin the amplification reaction within one hour to prevent sample degradation or contamination.

[0103] (4) PCR amplification reaction

[0104] After the microdroplets are prepared, remove the sealed 8-tube array containing stable microdroplets from the microdroplet preparation instrument and place it on a PCR instrument for PCR amplification. The amplification conditions are set as shown in Table 7 below (temperature increase / decrease rate set to 1.5℃ / s, amplification volume set to 100μL). After setting, save the file and run the reaction program.

[0105] Table 7 Reaction Procedure

[0106]

[0107] (5) Droplet detection

[0108] Droplet detection was performed using the Universal Microdroplet Detection Kit from Xinyi Manufacturing Technology (Beijing) Co., Ltd., and the accompanying Xinyi Biochip Analyzer. For specific operating procedures, please refer to the instruction manual for the Universal Microdroplet Detection Kit.

[0109] (6) Results Analysis

[0110] After the chip detection was completed, data analysis was performed using digital PCR analysis system software. Positive areas were delineated based on the 2D scatter plot results. Then, the copy number ratio of the GJB2 gene to the CFTR gene was calculated. The copy number, copy number ratio, and GJB2 gene copy number detection results are interpreted as shown in Table 8 below. The results were interpreted according to Table 8. Simultaneously, the fluorescence scatter plots of the three samples are shown below. Figure 4 , Figure 5 and Figure 6 As shown. Figure 4 The test results for the double copy number deletion sample showed that only the CFTR gene in the VIC fluorescence channel had positive droplets, while the GJB2 gene in the FAM fluorescence channel had no positive droplets. Figure 5 The test results for a single copy number deletion sample showed positive droplets for the CFTR gene in the VIC fluorescent channel and positive droplets for the GJB2 gene in the FAM fluorescent channel. Figure 6 For samples with normal copy number, positive droplets were found in the CFTR gene of the VIC fluorescent channel and in the GJB2 gene of the FAM fluorescent channel.

[0111] Table 8. Correspondence table of three types of GJB2 copy number variations

[0112]

[0113] Therefore, the results obtained using the method described in this invention show a high degree of consistency with the results verified by first-generation sequencing. This not only verifies the accuracy and reliability of the method of this invention, but also further demonstrates its effectiveness and application value in detecting the GJB2 gene copy number in patients with non-syndromic hearing loss. The method of this invention can quickly and accurately obtain gene copy number information from patients.

[0114] Example 3: Application of the primer-probe combination designed in this invention in non-syndromic hereditary deafness

[0115] (1) Sample preparation

[0116] Five clinical sample families (a total of 11 clinical samples) were selected, and DNA was extracted from the samples using nucleic acid extraction reagents. The sample and family information is shown in Table 9 below.

[0117] (2) Digital PCR detection

[0118] According to the method described in Example 2, digital PCR chip detection was performed on family samples. After detection, data analysis was performed using digital PCR analysis system software. Positive regions were delineated based on the detection results of the 2D scatter plot, and then the copy number ratio of the GJB2 gene to the CFTR gene was calculated.

[0119] (3) GJB2 gene copy number analysis

[0120] The copy number ratio was used to determine the variation in the GJB2 gene copy number. The results are shown in Table 9 below. As can be seen from the actual clinical phenotype, the digital PCR chip detection of the GJB2 gene is highly consistent with the actual clinical phenotype of the patients, verifying the accuracy and applicability of the results of this invention.

[0121] Table 9. Sample Information and GJB2 Gene Copy Number Variation

[0122]

[0123] In summary, the method described in this invention demonstrates excellent accuracy and applicability in GJB2 gene copy number detection.

[0124] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The primer probe combination for detecting the GJB2 gene copy number of non-syndromic hearing loss patients by using droplet digital PCR in the detection of the GJB2 gene copy number of non-syndromic hearing loss patients for non-diagnostic and therapeutic purposes, characterized in that, The primer-probe combination includes a target gene detection primer pair, a target gene detection probe, an upstream primer for the internal reference gene, a downstream primer for the internal reference gene, and a probe for the internal reference gene; wherein: The sequence information of the target gene detection primer pair is as follows: the sequence information of the upstream primer of GJB2 is shown in SEQ ID NO.6; the sequence information of the downstream primer of GJB2 is shown in SEQ ID NO.7; the sequence information of the target gene detection probe is shown in SEQ ID NO.8; The internal reference gene is the CFTR gene; the sequence information of the upstream primer of the internal reference gene is shown in SEQ ID NO.9; the sequence information of the downstream primer of the internal reference gene is shown in SEQ ID NO.10; the sequence information of the probe of the internal reference gene is shown in SEQ ID NO.

11. The GJB2 gene copy number includes the copy number of the coding region and the regulatory region of the GJB2 gene.

2. Use according to claim 1, characterized in that, The target gene detection probe and the internal reference gene probe contain fluorescent groups and quenching groups.

3. Use according to claim 1, characterized in that, The target gene detection probe is labeled with FAM fluorescent group at 5' and BHQ1 quencher group at 3'; the internal reference gene probe is labeled with CY5 fluorescent group at 5' and BHQ2 quencher group at 3'.

4. The use of the kit for detecting the GJB2 gene copy number in non-syndromic hearing loss patients by microdroplet digital PCR for the purpose of non-diagnostic and therapeutic detection of the GJB2 gene copy number in non-syndromic hearing loss patients, characterized by, The kit includes the following primer-probe combination: The target gene detection primer pair, the target gene detection probe, the upstream primer of the internal reference gene, the downstream primer of the internal reference gene, and the probe of the internal reference gene; among which: The sequence information of the target gene detection primer pair is as follows: the sequence information of the upstream primer of GJB2 is shown in SEQ ID NO.6; the sequence information of the downstream primer of GJB2 is shown in SEQ ID NO.7; the sequence information of the target gene detection probe is shown in SEQ ID NO.8; The internal reference gene is the CFTR gene; the sequence information of the upstream primer of the internal reference gene is shown in SEQ ID NO.9; the sequence information of the downstream primer of the internal reference gene is shown in SEQ ID NO.10; the sequence information of the probe of the internal reference gene is shown in SEQ ID NO.

11. The GJB2 gene copy number includes the copy number of the coding region and the regulatory region of the GJB2 gene.

5. Use according to claim 4, characterized in that, The target gene detection probe and the internal reference gene probe contain fluorescent groups and quenching groups.

6. Use according to claim 4, characterized in that, The target gene detection probe is labeled with FAM fluorescent group at 5' and BHQ1 quencher group at 3'; the internal reference gene probe is labeled with CY5 fluorescent group at 5' and BHQ2 quencher group at 3'.

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