Deafness-related gene mutation screening kit

Abstract

The invention relates to the field of kits, in particular to a deafness-related gene mutation screening kit. The kit comprises an amplification primer, a PCR (Polymerase Chain Reaction) amplification enzyme reaction reagent, enzyme-free water, a magnetic bead purification reagent, a sequencing primer, a sequencing reaction reagent and a sequencing product purification reagent, wherein the amplification primer is designed for mutation of 26 sites in GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes; the magnetic bead purification reagent is used for purifying amplification products. An amplification primer follows the design principle that the distance between the amplification primer and a mutation site is at least 50 bases or above, and high-frequency single nucleotide polymorphic sites are avoided, PCR amplification adopts a falling annealing temperature control program, an amplification product is purified through a magnetic bead purification system, sequencing reaction configures an optimized working enzyme system, a specific thermal cycle program is adopted, and the high-frequency single nucleotide polymorphisms are obtained. And finally carrying out first-generation sequencing analysis. The method can realize high-specificity, high-sensitivity and high-stability detection of deafness-related gene multi-site mutation, and has the advantages of simplicity and convenience in operation, accurate result and wide application range.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of kits, in particular to a deafness-related gene mutation screening kit. BACKGROUND

[0002] Deafness is one of the most common sensory system defects in humans, with an incidence of about 1‰ to 3‰ in newborns. Genetic deafness seriously affects population quality, increases the burden of social medical and rehabilitation, and has the characteristics of family aggregation and concealment, so early detection and intervention are of great significance.

[0003] Existing research shows that mutations in GJB2, SLC26A4, GJB3 and mitochondrial 12S rRNA genes are closely related to non-syndromic genetic deafness. Among them, GJB2 gene mutation is the most common in congenital deafness patients; SLC26A4 gene mutation is closely related to large vestibular aqueduct syndrome; GJB3 gene mutation is mostly seen in postnatal high-frequency sensorineural hearing loss; and 1555A>G and 1494C>T mutations in mitochondrial 12S rRNA gene are highly related to aminoglycoside-induced deafness. Detecting mutations in these four genes can cover the main genetic mutation types in the deaf population in China.

[0004] Existing deafness gene detection technologies mainly include microarray chips, fluorescence PCR melting curve method, PCR flow-through hybridization method, flight mass spectrometry, next-generation sequencing technology, etc. These technologies can achieve multi-site mutation screening, but there are still deficiencies in specificity, ease of operation, detection cost and clinical application popularity. At the same time, some detection methods still need to verify the positive results by Sanger sequencing, which increases the complexity of the detection process and the overall cost. Therefore, developing a new type of deafness gene screening kit with high specificity, simple operation process and direct application to Sanger sequencing confirmation has important clinical application value and popularization significance. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a deafness-related gene mutation screening kit.

[0006] The above-mentioned purpose of the present application is realized by the following technical solutions: A deafness-related gene mutation screening kit comprises: amplification primers designed for 26 site mutations in GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes; PCR amplification enzyme reagents; enzyme-free water; magnetic bead purification reagents for amplification product purification; sequencing primers; sequencing reaction reagent; sequencing product purification reagent.

[0007] Further, the amplification primer design rule is at least 50 bases or more from the mutation site, and the primer sequence does not contain a high-frequency single nucleotide polymorphism site.

[0008] Further, the amplification primer comprises: a primer pair for the GJB2 gene, the sequence being SEQ ID NO. 1, SEQ ID NO. 2; a primer pair for the GJB3 gene, the sequence being SEQ ID NO. 3, SEQ ID NO. 4; a primer pair for the SLC26A4 gene, the sequence being SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8; a primer pair for the mitochondrial 12S rRNA gene, the sequence being SEQ ID NO. 9, SEQ ID NO. 10.

[0009] The conventional deafness gene detection primer is mainly designed randomly near the site, lacks a unified standard, and is easy to mismatch with the template, produce non-specific amplification, primer dimers, cause subsequent sequencing difficulties, and affect the reading accuracy. The application designs the primer for the mutation site, strictly controls the primer distance from the mutation site to be greater than 50 bp, and avoids the high-frequency SNP region, significantly improves the specificity of the primer combined with the correct template, effectively reduces the background amplification, improves the quality of the subsequent sequencing template, and ensures the detection accuracy and reliability.

[0010] Further, the PCR amplification enzyme reaction reagent comprises a PCR amplification enzyme, and the PCR amplification enzyme is Taq DNA polymerase or a DNA polymerase with a hot start property.

[0011] Further, the PCR amplification program comprises: (1) 95℃ pre-denaturation for 3 minutes; (2) 96℃ denaturation for 30 seconds, annealing temperature from 68℃ gradually reduced to 62℃ annealing step for 20 seconds, 72℃ extension for 45 seconds, cycle 4 times; (3) 96℃ denaturation for 30 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 45 seconds, cycle 31 times; (4) 72℃ extension for 3 minutes to end.

[0012] The traditional PCR amplification procedure adopts a single fixed annealing temperature, cannot consider all different sequence templates, primers are prone to non-specific binding, causes mixed bands and mixed peaks of amplified fragments, affects sequencing clarity, and the problem is more prominent in a multi-site detection system. The application adopts a drop-style annealing temperature control procedure, the initial high-temperature annealing is gradually reduced to a stable temperature, high-specificity binding is preferentially generated, and the amplification efficiency is subsequently improved, specificities and yields are considered, and it is particularly suitable for parallel amplification of multiple genes and multiple sites, greatly improving the sensitivity and specificity of detection.

[0013] Further, the magnetic bead purification reagent comprises carboxyl modified magnetic beads, polyethylene glycol and sodium chloride.

[0014] Further, the concentration of the recovered product after purification of the magnetic bead purification reagent is 5 ng / μL.

[0015] Traditional PCR product purification often adopts gel electrophoresis gel cutting or column purification, which is tedious, large sample loss, high risk of pollution, and the purification efficiency is easily affected by human factors, which is difficult to standardize, and reduces the subsequent sequencing success rate. The application introduces a magnetic bead purification system, specifically adsorbs DNA, removes impurities by ethanol washing, and standardizes the process, realizes high recovery rate, high purity and low residue of PCR products, greatly improves the sample processing efficiency and sequencing success rate, and is particularly suitable for batch deafness gene screening applications.

[0016] Further, the sequencing reaction reagent comprises sequencing working enzymes and fluorescently labeled dideoxynucleotides.

[0017] Further, the sequencing reaction procedure comprises: (1) 96℃ denaturation for 1 minute; (2) 30 cycles, each cycle comprising 96℃ denaturation for 10 seconds, 50℃ annealing for 5 seconds, and 60℃ extension for 4 minutes; (3) 4℃ incubation.

[0018] The existing deafness gene detection sequencing reaction often uses a general procedure, lacks optimization for the characteristics of the amplification product, causes weak sequencing signal, overlapping peak shape, and high background noise, affects accurate identification of mutation sites, and is prone to missed detection or misjudgment. The application optimizes the sequencing reaction working system and the thermal cycle procedure, reasonably sets the three-step procedure of 96℃ denaturation, 50℃ annealing and 60℃ extension, makes the chain termination event uniformly distributed, the sequencing peak clearly separated, and the background extremely low, greatly improves the signal-to-noise ratio of sequencing and the accuracy of mutation detection, and meets the needs of clinical diagnosis.

[0019] Further, the purified sequencing product is washed with 85% ethanol three times, and single-stranded DNA is obtained by elution with deionized water for machine first-generation sequencing.

[0020] Compared with the prior art, the application can realize high specificity, high sensitivity, high stability and gene mutation detection by providing a set of screening kits for 26 sites of deafness-related GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes, which mainly embodies the following aspects: Specific amplification primers are designed for 26 sites of GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes, and the primer design principle of more than 50 bases away from the detection mutation site and avoiding common SNP interference is followed. The design significantly improves the matching specificity of the primer and the template, reduces the probability of non-specific binding of the primer and formation of primer dimers, thereby ensuring the singleness and high purity of the PCR amplification product, and providing an accurate and reliable template basis for the subsequent sequencing reaction.

[0021] The PCR amplification program of the drop-type annealing temperature control is adopted, the initial annealing temperature is set to 68℃, and each cycle is gradually reduced to 62℃, and then enters the fixed 60℃ annealing stage. The drop-type program effectively improves the binding selectivity of the primer and the correct template, inhibits mismatch binding and non-specific amplification, ensures the amplification efficiency, and further improves the specificity and consistency of the amplification, so that each target fragment in the multi-site amplification system can be stably and efficiently amplified.

[0022] The purification system based on carboxyl-modified magnetic beads is used to purify the PCR amplification product, and through DNA selective adsorption, ethanol washing and water elution, the residual primers, enzymes and small molecule reaction components are removed. The magnetic bead purification process is simple and efficient, the operation time is short, the recovery rate is high, the high-purity amplification fragment can be obtained, the background noise of the subsequent sequencing is significantly reduced, and the sensitivity and accuracy of the sequencing reaction are improved.

[0023] By optimizing the sequencing reaction enzyme configuration and the sequencing thermal cycle program, the three-step cycle of 96℃ denaturation, 50℃ annealing and 60℃ extension is set, and the cycle is 30 times, so as to ensure the efficient incorporation of fluorescently labeled double deoxyribonucleotides (ddNTP). The thermal cycle program makes the chain termination event of the sequencing reaction uniform and efficient, the generated sequencing chromatogram peak is clear and distinct, the background peak is less, the mutation site sequence can be accurately read, and the readability and reliability of the overall detection are improved. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 Sanger sequencing result graph for GJB2 c.416G>A heterozygous mutation sample.

[0025] Fig. 2 Sanger sequencing result graph for GJB3 c.421A>G heterozygous mutation sample.

[0026] Fig. 3Figure of Sanger sequencing result of SLC26A4 c.2162C>T heterozygous mutation sample. DETAILED DESCRIPTION

[0027] The application will be described in detail below with reference to examples.

[0028] Example 1 The present example discloses a deafness-related gene mutation screening kit, comprising: amplification primers designed for 26 site mutations in GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes; The primer pair for GJB2 gene has the sequence of SEQ ID NO. 1 and SEQ ID NO. 2. The primer pair for GJB3 gene has the sequence of SEQ ID NO. 3 and SEQ ID NO. 4. The primer pair for SLC26A4 gene has the sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. The primer pair for mitochondrial 12S rRNA gene has the sequence of SEQ ID NO. 9 and SEQ ID NO. 10.

[0029] PCR amplification enzyme reagent; Enzyme-free water; Magnetic bead purification reagent for amplification product purification; Sequencing primer; Sequencing reagent; Sequencing product purification reagent.

[0030] The primer sequences of SEQ ID NO. 1-NO. 10 are as follows: The amplification reaction and sequencing process are as follows: 1. DNA sample preparation Extract the DNA of the oral swab of the subject, obtain the genomic DNA using a commercial extraction kit, measure the DNA concentration using Nanodrop 2000, and adjust to 10-15 ng / μL for standby.

[0031] 2. Configuration of PCR amplification reaction system Add the following to a 25 μL system: (1) Sample DNA: 1 μL; (2) F-Primer and R-Primer: 1 μL each; F-Primer and R-Primer are primer pairs for GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA gene, respectively; (3) PCR amplification enzyme reaction solution: 12.5 μL (containing heat-stable Taq DNA polymerase, dNTPs and amplification buffer); (4) Enzyme-free water to 25 μL.

[0032] 3. PCR amplification procedure After the amplification reaction, the PCR product was detected by agarose electrophoresis, and the product had a bright single band.

[0033] 4. Purification and recovery of amplification product (1) Composition and ratio of magnetic bead purification reagent: Carboxyl modified magnetic beads: 10% (w / v); Polyethylene glycol (PEG 8000): 18% (w / v); Sodium chloride (NaCl): 1.25 M; Tris-HCl buffer (10 mM, pH 8.0); Deionized water to volume.

[0034] (2) Purification operation steps: (2.1) The PCR product after amplification was directly added to 24 μL of room temperature balanced magnetic bead purification reagent; (2.2) Mix well and centrifuge briefly to concentrate the liquid at the bottom of the tube; (2.3) Stand at room temperature for 8 minutes to allow DNA to bind to the surface of the magnetic beads; (2.4) Place the reaction tube on the magnetic stand and stand for 5 minutes. After the magnetic beads are adsorbed and fixed, carefully aspirate the supernatant; (2.5) Add 150 μL of 80% ethanol solution (pre-cooled), slowly add along the tube wall, and gently shake the magnetic stand; (2.6) After standing, aspirate the supernatant; (2.7) Repeat the 80% ethanol washing step once (total washing twice, 150 μL each time); (2.8) After aspirating all the supernatant, open the reaction tube and dry the magnetic beads at room temperature for 5-10 minutes until the surface of the magnetic beads loses its luster; (2.9) Add 40 μL of deionized water, mix well, and stand at room temperature for 5 minutes to elute the DNA from the magnetic beads; (2.10) Place the reaction tube on the magnetic rack, aspirate 38 μL of supernatant, and transfer it to a new PCR tube; (2.11) The product concentration was determined using Nanodrop and remained stable between 5-8 ng / μL.

[0035] 5. Sequencing reaction preparation Dilute the recovered PCR product with pure water to 5 ng / μL, then take 2 μL and add 1 μL of sequencing primers, 2 μL of sequencing working enzyme, and 5 μL of deionized water to form a 10 μL system.

[0036] The sequencing working enzyme is prepared by mixing component A and component B in a volume ratio of 1:3; Component A: DNA polymerase and reaction buffer; Component B: Contains fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP).

[0037] 6. Sequencing procedure 7. Sequencing product purification and detection A. The purification reagents for first-generation sequencing need to be equilibrated at room temperature for 30 minutes.

[0038] B. Add 4 μL of first-generation sequencing product and 30 μL of 85% ethanol to each reaction well, cap, shake for 15 seconds, briefly incubate for 2 seconds, and let stand at room temperature for 10 minutes.

[0039] C. Place the mixture sequentially onto the magnetic rack and let it stand for 5 minutes. Discard the cap, aspirate 40 μL of the reaction solution, and discard it. When aspirating the reaction solution, the pipette tip should be pointing in the opposite direction to the magnetic bead adsorption to avoid pulling out the magnetic beads. This applies to all subsequent operations on the magnetic rack.

[0040] D. Elution: Add 100 μL of 85% ethanol, and aspirate the supernatant with a pipette, being careful not to aspirate the magnetic beads. Repeat 3 times (add on the first setting, aspirate on the second setting).

[0041] E. Discard the original cap of the eight-tube strip, add a new cap, and momentarily remove it for 2 seconds, ensuring the magnetic bead side is facing outwards. Use a 10μL pipette tip to aspirate any remaining liquid, removing as much as possible.

[0042] F. Air dry for 10 minutes. After there are no water droplets, air dry for a few minutes, but do not exceed 15 minutes. Do not let the magnetic beads become too dry.

[0043] G. Add 30 μL of water to each reaction well, shake well to ensure no magnetic beads adhere to the wall, and incubate at room temperature for 5 min.

[0044] H. Put into magnetic stand for 5 min, and then 25 μL liquid was sucked out and added into 96-well plate. The empty wells of 96-well plate were supplemented with water, and there should be no empty wells. The magnetic beads should not be sucked in this step, and if the magnetic beads are sucked, they need to be re-sucked on the magnetic stand.

[0045] I. Seal the film, centrifuge, and check whether there are magnetic beads and air bubbles. 6. Sequencing Three samples were selected to obtain fluorescently labeled single-stranded DNA using the kit, and then Sanger sequencing was performed. The sequencing results are shown in the following table. Figs. 1-3 The sequencing results showed that sample 1 was a GJB2 c.416G>A heterozygous mutation, sample 2 was a GJB3 c.421A>G heterozygous mutation, and sample 3 was a SLC26A4 c.2162C>T heterozygous mutation.

[0046] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solutions falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. A kit for screening mutations in a deafness-related gene, characterized in that, It comprises: amplification primers designed for 26 mutation sites in GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA genes; PCR amplification enzyme reagents; enzyme-free water; magnetic bead purification reagents for amplification product purification; sequencing primers; sequencing reagents; sequencing product purification reagents.

2. The deafness-related gene mutation screening kit according to claim 1, characterized by, The design rules of the amplification primers are at least 50 bases away from the mutation sites, and the primer sequences do not contain high-frequency single nucleotide polymorphism sites.

3. The deafness-related gene mutation screening kit according to claim 1, characterized by, The amplification primers comprise: a pair of primers for GJB2 gene, the sequences of which are SEQ ID NO. 1 and SEQ ID NO. 2; a pair of primers for GJB3 gene, the sequences of which are SEQ ID NO. 3 and SEQ ID NO. 4; a pair of primers for SLC26A4 gene, the sequences of which are SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8; a pair of primers for mitochondrial 12S rRNA gene, the sequences of which are SEQ ID NO. 9 and SEQ ID NO.

10.

4. The deafness-related gene mutation screening kit according to claim 1, characterized by, The PCR amplification enzyme reagents comprise PCR amplification enzymes, which are Taq DNA polymerase or DNA polymerase with hot start characteristics.

5. The deafness-related gene mutation screening kit according to claim 1, characterized by, The PCR amplification program comprises: (1) 95℃ pre-denaturation for 3 minutes; (2) 96℃ denaturation for 30 seconds, annealing step of 20 seconds with annealing temperature gradually decreasing from 68℃ to 62℃, 72℃ extension for 45 seconds, 4 cycles; (3) 96℃ denaturation for 30 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 45 seconds, 31 cycles; (4) 72℃ extension for 3 minutes to end.

6. The deafness-related gene mutation screening kit according to claim 1, characterized by, The magnetic bead purification reagents comprise carboxyl-modified magnetic beads, polyethylene glycol and sodium chloride.

7. The deafness-related gene mutation screening kit according to claim 1, characterized by, The concentration of the recovered product after the magnetic bead purification reagents purification is 5 ng / μL.

8. The deafness-related gene mutation screening kit according to claim 1, characterized by, The sequencing reagents comprise sequencing working enzymes and fluorescently labeled dideoxynucleotides.

9. The deafness-related gene mutation screening kit according to claim 1, characterized by, The sequencing reaction program comprises: (1) 96℃ denaturation for 1 minute; (2) 30 cycles, each cycle comprising 96℃ denaturation for 10 seconds, 50℃ annealing for 5 seconds, and 60℃ extension for 4 minutes; (3) 4℃ incubation.

10. The deafness-related gene mutation screening kit according to claim 1, characterized by, The purified sequencing product is washed with 85% ethanol for three times, and single-stranded DNA is obtained by elution with deionized water for first-generation sequencing.

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