Primer group and kit for detecting genetic deafness gene locus polymorphism

Abstract

The invention discloses a primer group for detecting hereditary hearing loss gene locus polymorphism, a kit and a detection method thereof. The primer group comprises a multiple PCR amplification primer and a UEP primer used for a single base extension reaction, multiple PCR amplification is firstly carried out, SAP purification is then carried out, the extension reaction is then carried out, nucleic acid mass spectrometry detection is carried out on an extension product, and related genotype information is obtained. The method has the advantages of high accuracy, high flux, low cost and short period, 96 samples or 384 samples can be detected in one batch, only 8-10 hours are needed, the time is short, and the price is low. 27 hereditary deafness gene variation sites can be detected, 9 genes are involved, the coverage range is wider, and the misdiagnosis risk caused by incomplete detection sites is reduced. The method has high accuracy, sensitivity and repeatability, and the detection result has important guidance and research value for auxiliary diagnosis and treatment of hereditary hearing loss.

Description

Technical Field

[0001] The invention relates to a primer set and a kit for detecting polymorphism of hereditary deafness gene loci. Background Art

[0002] Hearing impairment is one of the most common birth defects in humans. Every year, there are 60,000 to 80,000 newborns with hearing loss in my country. The causes of hearing loss are complex, and more than 60% of hearing loss is caused by genetic factors.

[0003] Many of the genes related to hereditary deafness are autosomal recessive, and 90% of the parents of deaf children have normal hearing. The Hereditary Hearing Loss Homepage (http: / / hereditaryhearingloss.org / ) database shows that there are more than 80 known deafness-causing genes for non-syndromic hearing loss (NSHL), involving more than 1,000 pathogenic variants and 150 related gene regions. The prevalence of deafness gene variants in the Chinese population is about 6.3%, of which about 70% of the variants come from four hotspot genes, including GJB2, SLC26A4, mitochondrial DNA 12S rRNA and GJB3.

[0004] There are many pathogenic genes involved in deafness. So far, more than 100 gene regions and 46 pathogenic genes have been found, and there are a large number of deafness mutation sites in each gene, so there is a high genetic heterogeneity of genes and sites. Most of the existing technologies focus on the mutation of sites of four hotspot genes, such as GJB2, SLC26A4, mitochondrial DNA 12SrRNA and GJB3. However, there are still many deafness mutation sites such as KCNQ4, COCH, TMIE, and POU3F4 gene sites that are rarely detected. In order to more comprehensively cover high-frequency detection sites and improve the detection accuracy of mutation sites, it is necessary to develop detection methods for more susceptible gene mutation sites, simplify the detection steps, reduce costs, meet the needs of large-scale screening, and provide new technical means and support for early screening of deafness susceptibility genes. Summary of the invention

[0005] The purpose of the present invention is to provide a primer set and a kit for more comprehensive detection of polymorphisms of hereditary deafness gene loci, as well as a method for detecting mutations of hereditary deafness gene loci using the kit.

[0006] The technical solution adopted by the present invention is:

[0007] A primer set for detecting polymorphism of hereditary deafness gene loci, comprising multiple PCR amplification primers, the sequences of the multiple PCR amplification primers are shown in SEQ ID NOs: 1 to 54, and are used to amplify 27 hereditary deafness-related gene loci:

[0008]

[0009]

[0010] The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ ID NO:55 to SEQ ID NO:81:

[0011]

[0012] The present invention also provides an application of a primer set for detecting polymorphisms of genetic deafness loci, which is at least one of the following (1)-(2): (1) detecting polymorphisms of genetic deafness loci on a sample; (2) preparing a kit for detecting polymorphisms of genetic deafness loci on a sample.

[0013] Preferably, the above primer set is used to prepare a kit for detecting polymorphism of hereditary deafness gene loci.

[0014] Furthermore, the present invention also provides a kit for detecting polymorphism of hereditary deafness gene loci, the kit comprising the above-mentioned multiple PCR amplification primers and UEP primers.

[0015] Furthermore, the kit comprises a multiplex PCR amplification reaction system containing the multiplex PCR amplification primers, a shrimp alkaline phosphatase (SAP) purification reaction system, and a single base extension reaction system containing the UEP primers.

[0016] Furthermore, the multiplex PCR amplification reaction solution includes multiplex PCR amplification primers having base sequences as shown in SEQ ID NOs: 1 to 54, and the concentration of each primer is 0.1 to 1 μM (preferably 0.5 μM);

[0017] Preferably, the multiplex PCR amplification reaction solution comprises multiplex PCR amplification primers, PCR buffer, deoxynucleoside triphosphate mixture (dNTP), DNA polymerase, and MgCl2.

[0018] The SAP purification reaction system comprises SAP enzyme and SAP buffer.

[0019] SAP enzyme and SAP buffer can be purchased from Agena.

[0020] The single base extension reaction system includes UEP primers with base sequences as shown in SEQ ID NOs: 55 to 81, and the concentration of each primer is 0.1 to 1 μM (preferably 0.7 μM).

[0021] Preferably, the single base extension reaction system comprises UEP primer, iPLEX Buffer Plus, iPLEXTermination mix and iPLEX Pro Enzyme;

[0022] iPLEX Buffer Plus, iPLEX Termination mix, and iPLEX Pro Enzyme were purchased from Agena.

[0023] The present invention also provides application of the kit in detecting polymorphism of hereditary deafness gene loci.

[0024] The hereditary deafness genes include one or more of GJB2, GJB3, GJB6, SLC26A4, KCNQ4, COCH, POU3F4, TMIE, and MT-RNR1 (mtDNA).

[0025] Genetic polymorphisms of hereditary deafness include one or more of the following gene mutations:

[0026] GJB2: c.109G>A, c.167del, c.176_191de, c.235del, c.299_300del, c.35del;

[0027] GJB3: c.538C>T, c.547G>A;

[0028] GJB6: c.14C>T;

[0029] KCNQ4: c.827G>C;

[0030] COCH: c.151C>T;

[0031] POU3F4: c.652del, c.896del;

[0032] TMIE: c.274C>T;

[0033] SLC26A4: c.1174A>T, c.1226G>A, c.1229C>T, c.1707+5G>A, c.1975G>C, c.2027T>A, c.2162C >T, c.2168A>G, c.281C>T, c.589G>A, c.919-2A>G; MT-RNR1 (mtDNA): m.1494C>T, m.1555A>G.

[0034] The present invention also provides a method for detecting hereditary deafness gene polymorphism in vitro for non-diagnostic purposes using the kit, the method comprising the following steps:

[0035] (1) Multiplex PCR amplification reaction:

[0036] Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products;

[0037] The multiplex PCR amplification reaction system is a 5 μL reaction system, including the following components:

[0038] Ultrapure water 0.8μL, 10×PCRBuffer 0.5μL, 25mM MgCl2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCR Enzyme 0.2μL, DNA template 2μL;

[0039] The P mix is ​​a primer sequence of SEQ ID NO: 1 to SEQ ID NO: 54, and the concentration of each primer is 0.5 μM;

[0040] The reaction conditions of the multiplex PCR amplification reaction were 95°C for 2 min; 95°C for 30 s, 56°C for 30 s, 72°C for 60 s, 45 cycles; 72°C for 5 min; and insulation at 4°C.

[0041] (2) Shrimp alkaline phosphatase (SAP) purification reaction:

[0042] Purifying the multiplex PCR amplification products with SAP enzyme to obtain purified products;

[0043] The SAP purification reaction system includes the following components: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL amplification product, a total of 7 μL reaction system.

[0044] The reaction conditions for the purification reaction were 37°C for 40 min, 85°C for 5 min, and insulation at 4°C.

[0045] (3) Single base extension reaction

[0046] The purified product of step (2) is subjected to a single base extension reaction to obtain an extension product;

[0047] The single base extension reaction system includes the following components: ultrapure water 0.62μL, iPLEX Buffer Plus 0.2μL, iPLEX Termination mix 0.2μL, UEP Mix 0.94μL, iPLEX Pro Enzyme 0.04μL, purified product 7μL, a total of 9μL reaction system;

[0048] UEP Mix consisted of primer sequences from SEQ ID NO:55 to SEQ ID NO:81, with a concentration of 0.7 μM for each primer;

[0049] The reaction procedure of the single base extension reaction is 95° C. for 30 s; 95° C. for 5 s, {52° C. for 5 s, 80° C. for 5 s, 5 internal cycles}, 40 external cycles; 72° C. for 3 min; and insulation at 4° C.

[0050] (4) Nucleic acid mass spectrometry analysis

[0051] The extension products were detected by nucleic acid mass spectrometer to obtain the polymorphism information of the related sites of hereditary deafness gene.

[0052] The nucleic acid mass spectrometer is generally a MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer.

[0053] The nucleic acid mass spectrometer detector converts electrical signals into visible peak graphs. Each genotype has a unique molecular weight, and the software automatically analyzes the corresponding base type to obtain the final genotyping results.

[0054] The present invention firstly performs PCR amplification by using primers designed independently, then performs SAP purification, and finally uses probes designed independently to perform extension reaction of target bases, and the obtained extension products are subjected to nucleic acid mass spectrometry detection to finally obtain relevant genotype information.

[0055] The kit for detecting polymorphism of hereditary deafness gene loci provided by the present invention has the advantages of high accuracy, high throughput, low cost and short cycle. Compared with conventional Sanger sequencing, pyrophosphate sequencing and real-time fluorescence PCR technology, it has a significant high throughput advantage, and can detect 96 samples (96-well plate) or 384 samples (384-well plate) in one batch, and each sample can measure 27 gene variation sites; compared with the first generation sequencing, the present invention can complete a batch of samples in only 8-10 hours, which is short in time and improves the detection efficiency. The present invention only needs a trace amount of genomic DNA to obtain the test results.

[0056] The present invention can detect 27 genetic mutation sites of hereditary deafness, involving 9 genes, covering a wider range, reducing the risk of misdiagnosis due to incomplete detection sites, and better meeting the clinical diagnosis needs of deafness. The present invention has high accuracy, sensitivity and repeatability, and the test results have important guidance and research value for auxiliary diagnosis and treatment of hereditary deafness. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the mass spectrometry result of the GJB2 c.109G>A site of sample 85, and the result has been boxed.

[0058] Figure 2 This is the Sanger sequencing result of the GJB2 c.109G>A site of sample 85, and the result is boxed.

[0059] Figure 3 This is the mass spectrometry result of the GJB2 c.109G>A site of sample 86, and the result has been boxed.

[0060] Figure 4 This is the Sanger sequencing result of the GJB2 c.109G>A site of sample 86, and the result is boxed.

[0061] Figure 5 This is the mass spectrometry result of the SLC26A4 c.919-2A>G site of sample 85, and the result is boxed.

[0062] Figure 6 This is the Sanger sequencing result of the SLC26A4 c.919-2A>G site of sample 85, and the result is boxed.

[0063] Figure 7 This is the mass spectrometry result of the SLC26A4 c.919-2A>G site of sample 86, and the result is boxed.

[0064] Figure 8 This is the Sanger sequencing result of the SLC26A4 c.919-2A>G site of sample 86, and the result is boxed.

[0065] Fig. 9 This is the mass spectrometry result of the MT-RNR1 m.1555A>G site of sample 85, and the result is boxed.

[0066] Fig.10 This is the Sanger sequencing result of the MT-RNR1 m.1555A>G site of sample 85, and the result is boxed.

[0067] Fig.11 This is the mass spectrometry result of the MT-RNR1 m.1555A>G site of sample 86, and the result is boxed.

[0068] Fig.12 This is the Sanger sequencing result of the MT-RNR1 m.1555A>G site of sample 86, and the result is boxed. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0070] In the present invention, SEQ ID NO: 1 to SEQ ID NO: 81 were all commissioned to be synthesized by Shanghai Bio-Tech Co., Ltd.

[0071] Example 1

[0072] One whole blood sample was taken from each of the three volunteers to detect the 27 hereditary deafness gene mutation sites in these three samples. First, the whole blood genomic DNA of the three volunteers was extracted respectively, and then multiple PCR reactions, SAP purification and single base extension reactions were performed, and then the products were placed in the nucleic acid mass spectrometer for detection. Finally, the offline data was analyzed by relevant software to obtain the genotype of the 27 deafness gene mutation sites of each sample. At the same time, the mass spectrometry results were verified by the first-generation sequencing method. The verification results showed that the results of the method of the present invention and the first-generation sequencing method had a 100% consistency rate.

[0073] Detection method of the kit of the present invention: 1. Multiplex PCR amplification reaction

[0074] Single sample PCR amplification system: ultrapure water 0.8μL, 10xPCRBuffer 0.5μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), 25mM MgCl2 0.4μL, dNTP 0.1μL, P Mix 1μL, PCR Enzyme 0.2μL (PCR Reagent And Spectro CHIP Kit, purchased from Agena Bioscience, catalog number: 21327M), DNA template 2μL, a total of 5μL reaction system. The reaction conditions are 95℃2min; 95℃30s, 56℃30s, 72℃60s, 45cycles; 72℃5min; 4℃ insulation. The primers of P Mix are the sequences shown in SEQ ID NO:1 to SEQ ID NO:54 in Table 1, and the concentration of each primer is 0.5μM.

[0075] The DNA template was extracted using the CWE960 Blood DNA Kit from Kangwei Century. The multiplex PCR primer sequences are shown in Table 1:

[0076] Table 1

[0077]

[0078]

[0079] 2. Shrimp alkaline phosphatase (SAP) purification reaction

[0080] 2 μL of SAP mixed solution was added to the above amplification reaction wells, including: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), to obtain a SAP purification system: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, 5 μL of the first step PCR amplification product, a total of 7 μL reaction system. The reaction conditions of the purification reaction were 37°C for 40 min, 85°C for 5 min, and 4°C insulation.

[0081] 3 Single base extension reaction

[0082] After the purification reaction was completed, 2 μL of iPLEX reaction system was added to the reaction space, and the extension reaction system for each well was: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), 0.2 μL of iPLEX Termination mix (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), and 7 μL of the second step purified product, a total of 9 μL of reaction system. The extended UEP Mix sequence is the primer sequence shown in SEQ ID NO: 55 to SEQ ID NO: 81 in Table 2, and the concentration of each primer is 0.7 μM. The reaction conditions were 95°C for 30 s; 95°C for 5 s, {52°C for 5 s, 80°C for 5 s, 5 cycles}, 40 cycles; 72°C for 3 min; and insulation at 4°C.

[0083] Table 2

[0084]

[0085]

[0086] 4. Nucleic acid mass spectrometry analysis

[0087] Add 41 μL of deionized water to the extension product. Place the sample in the MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometer, and set the detection parameters: Wells to Process is set to Automatic, Start Disoense Condition is set to 1000, Resin Volume is set to 13, Shot is set to 30, Maximum Acquisitions is set to 9, Minimum Good Spectra and Maximum Good Spectra are both set to 5, and 5 items need to be checked, including TumOffHyAfterAnalysis, Transfer Resin to MTP, Dispense Samples onto Chips, Transferchips toAnalyzer, and AnalyzeChips. Then the mass spectrometer spotter spots the extension product onto the Spectro CHIP (chip) for detection. After the detection, the detector converts the electrical signal into a visible peak graph. Each genotype has a unique molecular weight. The software automatically analyzes the corresponding base type to obtain the final genotyping result. The results of the three test samples are shown in Tables 3 to 5.

[0088] Tables 3 to 5 are the test results of 3 samples respectively:

[0089] Table 3

[0090]

[0091] Table 4

[0092]

[0093] Table 5

[0094]

[0095] It can be seen from the test results that the kit of the present invention can accurately detect the genotypes of the 27 deafness gene mutation sites.

[0096] The same samples were subjected to Sanger sequencing, and the sequencing results and mass spectrometry results were exactly the same. The detection spectra of the three representative variant sites of samples 85 and 86 were compared. Figures 1 to 12 shown.

[0097] Figure 1 This is the mass spectrometry result of the c.109G>A site of sample 85, and the result is boxed; Figure 2 This is the Sanger sequencing result of the c.109G>A site of sample 85, and the result is boxed; Figure 3This is the mass spectrometry result of the c.109G>A site of sample 86, and the result is boxed; Figure 4 This is the Sanger sequencing result of the c.109G>A site of sample 86, and the result is boxed; Figure 5 This is the mass spectrometry result of the c.919-2A>G site of sample 85, and the result is boxed; Figure 6 This is the Sanger sequencing result of the c.919-2A>G site of sample 85, and the result is boxed; Figure 7 This is the mass spectrometry result of the c.919-2A>G site of sample 86, the result is boxed; Figure 8 This is the Sanger sequencing result of the c.919-2A>G site of sample 86, and the result is boxed; Fig. 9 This is the mass spectrometry result of the m.1555A>G site of sample 85, the result is boxed; Fig.10 This is the Sanger sequencing result of the m.1555A>G site of sample 86, and the result is boxed; Fig.11 This is the mass spectrometry result of the m.1555A>G site of sample 85, the result is boxed; Fig.12 This is the Sanger sequencing result of the m.1555A>G site of sample 86, and the result has been boxed.

[0098] Figures 1 to 12 The results show that the sequencing method of the present invention is completely consistent with the results of Sanger sequencing.

Claims

1. A primer set for detecting polymorphism of hereditary deafness gene loci, characterized in that The primer set includes multiple PCR amplification primers, the sequences of which are shown in SEQ ID NOs: 1 to 54, and are used to amplify 27 hereditary deafness-related gene loci:

2. The primer set according to claim 1, characterized in that The primer set also includes a UEP primer for single base extension reaction, the sequences of which are shown in SEQ ID NO:55 to SEQ ID NO:81:

3. The use of the primer set according to any one of claims 1 to 2, characterized in that: The application is at least one of the following (1)-(2): (1) detecting the polymorphism of the genetic deafness gene locus on the sample; (2) preparing a kit for detecting the polymorphism of the genetic deafness gene locus on the sample.

4. A kit for detecting polymorphism of hereditary deafness gene loci, characterized in that The kit comprises the primer set according to claim 2.

5. The kit as claimed in claim 4, characterized in that The kit comprises a multiplex PCR amplification reaction system containing the multiplex PCR amplification primers, a SAP purification reaction system, and a single base extension reaction system containing the UEP primers.

6. The kit as claimed in claim 5, characterized in that The multiplex PCR amplification reaction solution includes multiplex PCR amplification primers with base sequences as shown in SEQ ID NOs: 1 to 54, and the concentration of each primer is 0.1 to 1 μM; The single base extension reaction system includes UEP primers with base sequences as shown in SEQ ID NOs: 55 to 81, and the concentration of each primer is 0.1 to 1 μM.

7. The kit according to claim 6, characterized in that The multiplex PCR amplification reaction solution includes multiplex PCR amplification primers, PCR buffer, dNTP, DNA polymerase, and MgCl2; The SAP purification reaction system comprises SAP enzyme and SAP buffer; The single base extension reaction system includes UEP primer, iPLEX Buffer Plus, iPLEX Termination mix and iPLEX Pro Enzyme.

8. Use of the kit as described in any one of claims 4 to 7 in detecting polymorphisms of hereditary deafness gene loci.

9. The use according to claim 8, characterized in that The hereditary deafness genes include one or more of GJB2, GJB3, GJB6, SLC26A4, KCNQ4, COCH, POU3F4, TMIE, and MT-RNR1; Genetic polymorphisms of hereditary deafness include one or more of the following gene mutations: GJB2: c.109G>A, c.167del, c.176_191de, c.235del, c.299_300del, c.35del; GJB3: c.538C>T, c.547G>A; GJB6: c.14C>T; KCNQ4: c.827G>C; COCH: c.151C>T; POU3F4: c.652del, c.896del; TMIE: c.274C>T; SLC26A4: c.1174A>T, c.1226G>A, c.1229C>T, c.1707+5G>A, c.1975G>C, c.2027T>A, c.2162C>T, c.2168A>G, c.281C>T, c.589G>A, c.919-2A>G; MT-RNR1: m.1494C>T, m.1555A>G.

10. A method for detecting hereditary deafness gene polymorphism in vitro for non-diagnostic purposes using the kit according to any one of claims 4 to 7, characterized in that The method comprises the following steps: (1) Multiplex PCR amplification reaction Extracting template DNA from samples, preparing a multiplex PCR amplification reaction system, performing multiplex PCR amplification reactions, and obtaining multiplex PCR amplification products; (2) SAP purification reaction: Purifying the multiplex PCR amplification products with SAP enzyme to obtain purified products; (3) Single base extension reaction The purified product of step (2) is subjected to a single base extension reaction to obtain an extension product; (4) Nucleic acid mass spectrometry analysis The extension products were detected by nucleic acid mass spectrometer to obtain the polymorphism information of the related sites of hereditary deafness gene.

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