CN111139293A - OTOF gene mutation detection kit related to auditory neuropathy spectrum system disorder - Google Patents

Abstract

The invention discloses an OTOF gene mutation detection kit related to auditory neuropathy spectrum system obstacle, which comprises a reagent for extracting DNA from a sample to be detected, a PCR reaction reagent for amplifying sample DNA and a reagent for sequencing PCR amplification products; the PCR reaction reagent for amplifying the sample DNA comprises PCR primers. The kit provided by the invention is used for detecting whether the OTOF gene c.4236delC deletion mutation exists in a patient, so that the cause of auditory neuropathy spectrum system disorder is diagnosed. The kit is favorable for clinically carrying out OTOF mutation screening work on the patients with the auditory neuropathy spectrum disorder, and provides a basis for diagnosis of the patients with the auditory neuropathy spectrum disorder.

Description

OTOF gene mutation detection kit related to auditory neuropathy spectrum system disorder

Technical Field

The invention relates to the field of gene detection, in particular to an OTOF gene mutation c.4236delC (p.E1414Sfs x 108) typing detection kit applied to diagnosis of auditory neuropathy/auditory neuropathy spectrum system disorders.

Background

Auditory Neuropathy (AN) is a hearing disorder caused by a malfunction of the cochlear inner hair cell, auditory nerve synapse or auditory nerve. It is an important disease causing language communication disorder of infants and teenagers, accounts for about 8% of newly-discovered children with hearing loss every year, and the onset of the disease is closely related to genetic factors.

Auditory neuropathy is divided into two types according to the disease site: type I auditory neuropathy is postsynaptic; type II auditory neuropathy is synaptic and presynaptic. Cochlear implant is the best treatment option for patients with severe sensorineural deafness in ears, but is only effective for type ii auditory neuropathy. The diagnosis of auditory neuropathy depends on audiological and imaging examinations, but cannot be classified according to audiological examination results, and the pathogenic genotype is the main basis for classification at present. The genes associated with auditory neuropathy type ii lineage disorders are primarily OTOF genes.

The otof (otoferlin) gene is the first cloned gene associated with non-syndromic acoustic neuropathy and is located within the DFNB9 locus of 2p 23. The total length of the OTOF gene DNA sequence is 101496bp, and there are 4 kinds of transcript variants with different lengths. The longest isoform of which contains 48 exons and encodes the protein Otoferlin of amino acid length 1997 aa. Yasunaga et al performed candidate gene studies on multiple autosomal recessive non-syndromic presbycusis families, and found that OTOF genes of 21 diseased members of the three families all have nonsense mutation in the 18 th exon, and generated stop codons in advance and formed truncated proteins, so that the encoded Otoferlin proteins are incomplete. In the inner ear, the Otoferlin protein is expressed only in inner hair cells in the mature cochlea, and is mostly expressed in the basolateral part of the inner hair cells. The protein is a calcium ion receptor, gene mutation can cause the exocytosis of vesicles in the moving area of inner hair cells to be inhibited, neurotransmitter release is reduced, and then nerve impulse is reduced; in addition, neurotransmitters may have a trophic effect on postsynaptic nerve fibers, such as a reduction in trophic effect may lead to degenerative changes in the postsynaptic membrane, perhaps explaining the progressive hearing loss in patients with Auditory Neuropathy Spectrum Disorders (ANSD).

OTOF gene mutations are reported in the literature to be widely present in the SNHL population worldwide and are also the major pathogenic mutations in ANSD patients. The OTOF gene is currently the most widely and extensively studied ANSD-related gene. According to literature statistics about 5.5% of sporadic cases in han ANSD patients can attribute the cause to mutations in the OTOF gene. The mutation spectrum of ANSD population in different regions and races is still imperfect, and there are over 100 types of OTOF gene mutation known at present, and most of the OTOF gene mutation types are nonsense mutation, truncation mutation or frame shift mutation.

Compared with the traditional hearing aid and drug therapy, the intervention effect of cochlear implant is more exact by combining the treatment experiences reported by domestic and foreign documents, but the difference is great, and the selection of an individual patient still needs to be careful. Previous studies have considered that post-implantation effects can be predicted with reference to several aspects: 1. preoperative MRI, if central system lesions are found, may indicate that the effect is not good after operation, especially when cochlear nerve loss or abnormal shape exists; 2. the relationship between the gene mutation type and the postoperative curative effect is uncertain, the existing research result can be used as a reference, the ANSD postoperative effect caused by presynaptic lesions caused by OTOF mutation is generally considered to be better, the direct electrical stimulation provided by the artificial cochlea is supposed to bypass the lesion part, and the ANSD postoperative effect caused by postsynaptic lesions and lesions related to the whole process of auditory nerve (such as hereditary ataxia and deafness-dystonia-optic neuron disease) caused by axonal injury is poorer. Therefore, the method has important significance for determining auditory neuropathy/auditory neuropathy spectrum system obstacle pathogenic genes, typing patients and evaluating the implantation effect of the artificial cochlea. Reports about OTOF gene mutation c.4236delC (p.E1414Sfs 108) are not seen at present.

Disclosure of Invention

The invention aims to provide an OTOF gene mutation detection kit related to auditory neuropathy pedigree disorder.

In order to achieve the purpose, the invention adopts the following technical scheme:

a kit for detecting the mutation of the OTOF gene c.4236delC (p.E1414Sfs. 108), which comprises PCR reaction reagents for amplifying a DNA fragment, wherein the PCR reaction reagents comprise a PCR primer, and a target fragment amplified by the PCR primer comprises a base corresponding to 4236 th position (NM-194248.3, chr2:26690093) of the CDS region of the human OTOF gene.

Preferably, the kit further comprises a reagent for extracting template DNA required for PCR amplification from an individual to be tested.

Preferably, the kit further comprises reagents for sequencing the PCR amplified target fragment.

Preferably, the PCR primers are selected from one of the following three pairs of primers P1, P2 and P3:

the sequence of primer pair P1 is:

OTOF-F-1:5’-AGACCCTTCTAGGGTGTGTGG-3’；

OTOF-R-1:5’-CTGCTGAGTCATGGGAGAGTC-3’；

the sequence of primer pair P2 is:

OTOF-F-2:5’-CTTCCTGCTCTGCTCATTTCT-3’；

OTOF-R-2:5’-GTGGCATTCAAAATAGCTAACA-3’；

the sequence of primer pair P3 is:

OTOF-F-3:5’-CTCTGCTCATTTCTGCTCCAC-3’；

OTOF-R-3:5’-GGGTCAGGGTGTAGGTTTGC-3’。

the method for detecting the mutation of the OTOF gene c.4236delC (p.E1414Sfs 108) by using the kit comprises the following steps:

1) collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

2) taking the DNA extracted in the step 1) as a template, and carrying out PCR reaction by using the PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and carrying out typing identification according to the condition of the base deletion of the target fragment corresponding to 4236 th site (NM-194248.3, chr2:26690093) of CDS region of the human OTOF gene.

Preferably, the typing identification adopts a method of directly sequencing the target fragment, and the genotype or the allelic gene type of the tested individual corresponding to 4236 th site (NM-194248.3, chr2:26690093) of the CDS region of the human OTOF gene is determined by comparing the sequencing result with a reference sequence (NC-000002.11 (26680071.. 26781566)).

Preferably, the genotype determined by the alignment includes wild homozygous W/W, mutant heterozygous W/D or mutant homozygous D/D, wherein W: a wild-type allele; d: is absent.

The kit is applied to analysis of etiology of auditory neuropathy spectrum system disorder. The kit is used for detecting whether c.4236delC (p.E1414Sfs x 108) deletion mutation exists in a sample (OTOF gene fragment of a patient) from a test sample corresponding to the 4236 th site (NM-194248.3, chr2:26690093) of a CDS region of a human OTOF gene, so as to judge the hereditary cause of auditory neuropathy system disorder of the patient. Among them, the OTOF gene is mutated c.4236delc (p.e1414Sfs x 108), so that the amino acid sequence is shifted from 649, and the coding of the amino acid is terminated at 660 th amino acid (p.e1414Sfs x 108) in advance, thereby forming a short peptide chain of Otoferlin, losing the function thereof, and immature expression products induce to start an in vivo regulation mechanism, so that the mutated mRNA is degraded.

The invention has the beneficial effects that:

the kit provided by the invention can be used for rapidly detecting the specific mutation sites of the OTOF gene, and can judge the occurrence reason of auditory neuropathy system disorders of a patient by detecting whether the OTOF gene c.4236delC (p.E1414Sfs x 108) mutation exists in a DNA sample from the patient, thereby providing a basis for clinical diagnosis.

The kit provided by the invention is used for diagnosing the auditory neuropathy spectrum system disorder: 1) the invention provides a convenient and reliable method for screening susceptibility genes in auditory neuropathy pedigree disorder patients; 2) through prenatal diagnosis and screening, whether the fetus carries c.4236delC (p.E1414Sfs x 108) mutation or not is determined, the birth rate of the deaf infant is reduced, and the burden is reduced for the society and families; 3) the method is beneficial to determining individualized treatment schemes for different types of auditory neuropathy pedigree disorder patients, and avoids unnecessary economic loss caused by invalid artificial cochlea implantation.

Drawings

FIG. 1 shows the nucleotide and amino acid alignment of coding regions of OTOF gene (NM-194248.3; NP-919224.1): the mutation site is located at the 4236 th base of OTOF gene, and due to frame shift mutation, a truncated protein is produced, and the first few mutated amino acids (inverted) are circled by a box.

FIG. 2 is a schematic diagram of the PCR reaction process: reaction temperature and time are shown, where ↓ represents a 0.5 ℃ decrease per cycle.

FIG. 3 is a diagram of agarose gel electrophoresis for PCR product electrophoresis quantification: the fragment positions of the quantitative Marker are shown, and the amplified target sequence is 381 bp.

FIG. 4A is a diagram showing the result of sequencing of OTOF gene: heterozygous mutant sequences (patient sequences) are shown, and arrows indicate the location of the deletion.

FIG. 4B is a diagram showing the result of sequencing of OTOF gene: the wild type sequence is shown and the arrow indicates the position of the deletion.

Detailed Description

The invention is described in detail below with reference to the drawings and examples, which are provided to illustrate the invention and not to limit the scope of the invention.

The invention searches pathogenic gene sites from a close family by a whole exon next generation sequencing method. In screening 200 patients with non-syndromic auditory neuropathy spectrum disorder and 100 controls with normal hearing and no family history, c.4236delC (p.E1414Sfs x 108) homozygous mutation was found in the gene of OTOF of one of the auditory neuropathy spectrum disorder patients, and the OTOF gene mutation was cosegregated with the auditory neuropathy spectrum disorder phenotype. OTOF gene mutation-associated auditory neuropathy/auditory neuropathy spectrum disorders are transmitted by autosomal recessive inheritance. 117 mutations related to auditory neuropathy/auditory neuropathy spectrum system disorder are reported at present, and the deletion mutation of c.4236delC is not reported.

Referring to FIG. 1, the above mutation (c.4236delC) deletes the base located at the 4236 th position (NM-194248.3, chr2:26690093) of the CDS region of OTOF gene, so that the amino acid code is shifted from the 1414 th position, the amino acid codon at the 1521 th position is changed into a stop codon, and the gene expression product is changed into 1521 amino acids from the normal peptide chain with the length of 1997 amino acids after the mutation. The truncated amino acid peptide chain initiates the regulatory mechanism in vivo, degrading the variant mRNA.

The detection of the above mutation (c.4236delC) can be carried out by various methods for detecting a mutation, for example, a PCR (polymerase chain reaction) -sequencing method, a DNA probe hybridization method using a labeled OTOF gene, a method using a restriction fragment length polymorphism or a method using a sequence-specific primer, and the like. Wherein, the method for detecting the sample by adopting a PCR amplification-direct sequencing method comprises the following steps:

1) collecting a sample of an individual to be tested, such as blood, and extracting genomic DNA;

2) carrying out PCR reaction by using the DNA as a template and a PCR primer designed aiming at 4236 th base (NM-194248.3, chr2:26690093) of CDS region of OTOF gene to obtain PCR amplification product;

3) performing target fragment sequencing analysis on the obtained PCR amplification product, comparing a sequence obtained by sequencing with an OTOF gene reference sequence (NC-000002.11 (26680071..26781566)) and determining whether c.4236delC mutation exists in the OTOF gene of an individual to be detected;

4) and judging whether the individual to be detected is the auditory neuropathy spectrum system disorder caused by the OTOF gene mutation c.4236delC according to the result.

The PCR primer used in the above step 2) can be designed based on the known primer nucleotide sequence: usually 15 to 30 bases, and GC content of about 45 to 50%, and specifically binds to the terminal at an appropriate temperature. Primers can be designed using existing computer programs.

If the PCR reaction product obtained in step 2) above is detected using a hybridization probe, the hybridization probe used may be a probe that hybridizes with a normal OTOF nucleotide sequence, or with a mutated nucleotide sequence, or with their complementary sequences. These probes may be labeled with a radioisotope, a chromogenic substance or a fluorescent substance, and particularly, allele-specific probes may be used.

According to different detection methods, the kit for detecting the c.4236delC mutation of the OTOF gene comprises a PCR reaction reagent and a reagent for detecting a PCR amplification product, wherein the reagent is specifically selected from a sequencing detection reagent, a restriction length polymorphism detection reagent, a sequence-specific primer detection reagent and a probe hybridization detection reagent.

The kit container is filled with a reagent component for detecting the c.4236delC mutation of the OTOF gene, and simultaneously provides manufacturing, using and selling information of related medicines or biological products approved by a government drug regulatory agency. The PCR reaction reagent may contain, for example, amplification primers, dNTPs, DNA polymerase used for PCR reaction, a buffer therefor, and the like.

Example 1

Various sound-sensing nerve deafness patients, including auditory neuropathy pedigree disorder patients, are collected through a deafness outpatient service and a resource collection network, and a resource library is established. On the premise of patient's voluntary, after signing the informed consent, blood samples are taken, and an outpatient medical record database is established to record the patient's condition, the incidence of disease in the family and the contact way in detail. Then, the genomic DNA is extracted by a protease degradation method, quantified and then stored at-20 ℃, and each DNA sample corresponds to the registered clinical data of the patient in detail. Then, primers were designed using Primer design software Primer5 (amplification target region is exon 33 of OTOF gene, reference sequence: NC-000002.11 (26680071..26781566), amplification target fragment size is 381bp), and PCR amplification was performed on a BIORAD My Cycle thermal cycler using genomic DNA as a template. The sequencing primer is the same as the PCR amplification primer, and is used for forward and reverse sequencing by using an ABI 3730DNA sequencer. The results of the sequence sequencing were compared to the sequence in GenBank (NC _000002.11(26680071..26781566)) to determine the OTOF c.4236delc mutation as follows:

firstly, extracting blood sample to be detected and PCR amplification of OTOF gene coding region

1. Preparation of DNA in blood sample of subject to be tested

1.1 study object

Screening of OTOF gene was performed for 200 patients with acoustic neuropathy deafness and 100 normal hearing controls without family history according to the following method.

The deaf subject suffering from sporadic auditory neuropathy is collected from deaf patients who are subjected to deafness gene screening in otolaryngology head and neck surgery outpatient service of Xijing hospital (xi' an city, Shaanxi province). The normal hearing control is a normal hearing subject without family history of deafness, and the medical history and family history of all participants are investigated in detail and subjected to physical examination, wherein the otological examination comprises otoscopy and audiological evaluation. 5-10 mL of blood samples are collected by each person after signing an informed consent, and the collection time is 10 months in 2009.

1.2 genomic DNA extraction

1.2.1 preparation before experiment and important precautions

(1) Protease K was dissolved by adding a predetermined amount of protease K Storage Buffer, and stored at-20 ℃. The prepared Proteinase K is not stored for a long time at room temperature, and repeated freeze thawing is avoided, so that the activity of the Proteinase K is not influenced.

(2) All centrifugation operations were done at room temperature.

(3) Storage of blood sample: the blood sample added with anticoagulant can be stored at 2-8 ℃ for 10 days at most, for some experiments such as Southern hybridization and the like, complete and full-length genome DNA is required, please store the blood sample at 2-8 ℃ for 3 days at most, and the degradation degree of the genome DNA is light.

1.2.2 working steps

1) The blood sample was centrifuged at low speed until it was stratified, and the supernatant serum was removed with a pipette, taking care not to aspirate or damage the middle buffy coat layer.

2) Transferring all blood cells into a 5mL centrifuge tube, adding erythrocyte lysate to the total volume of 4mL, and turning upside down and mixing uniformly for 20 times until the precipitate is fully dispersed.

3)6500g, centrifuged for 10min and the supernatant discarded.

4) 3mL of Buffer FG1 was added and vortexed for 15s to thoroughly disperse the precipitate.

5) And centrifuging at 6500g for 10min, discarding supernatant, reversely buckling the centrifugal tube on clean absorbent paper, and sucking water.

6) Preparing a mixed solution of the DNA extracting solution and the proteinase K according to the mixing ratio of the DNA extracting solution and the proteinase K being 100:1, mixing, fully and uniformly mixing by whirling for 15s, and preparing as required.

7) Adding 1mL of the prepared mixed solution of the DNA extracting solution and the proteinase K into the sample, immediately and fully whirling and oscillating for 1min until the solution has no lumps.

8) The sample was incubated in a 65 ℃ water bath for 15min, during which time the mixture was reversed and mixed 3 times until the color of the sample changed from red to light green, indicating complete digestion of the protein.

9) 2mL of isopropanol was added to the sample and mixed by inversion 10 times until a white flocculent precipitate was visible.

10) A clean sterile 1.5mL centrifuge tube was labeled and 500. mu.L of pre-cooled 75% ethanol was added.

11) Picking the white flocculent precipitate in the step 9) by using a clean and sterile 1mL pipette tip, transferring the white flocculent precipitate into 75% ethanol prepared in the step 10), reversing and mixing the white flocculent precipitate for 10 times, and slowly pouring off the supernatant, wherein the white flocculent precipitate is not poured off.

11) Add 500. mu.L of pre-cooled 75% ethanol again, mix well 10 times by inversion, pour off the supernatant slowly and suck to dryness.

12) The tube cap was opened and dried at room temperature for 15min until all liquid was completely evaporated.

13) 380. mu.L of a DNA solution was added, and the mixture was incubated in a water/metal bath at 65 ℃ for 2 hours while shaking to dissolve the DNA sufficiently.

14) And quantifying and detecting the purity by a spectrophotometer.

15) The DNA was stored at-20 ℃.

2. PCR amplification of coding region of OTOF Gene

2.1 primer sequences

Primer5 Primer design software was used, referenced to sequence gene NC _000002.11(26680071..26781566), sequence synthesized for this assay, with a design completion time of 2019, 5 months:

an upstream primer OTOF-F-1: 5'-AGACCCTTCTAGGGTGTGTGG-3';

the downstream primer OTOF-R-1: 5'-CTGCTGAGTCATGGGAGAGTC-3'.

The size of the fragment obtained by PCR amplification using this primer was 381 bp.

2.2 establishment of PCR reaction System (Table 1)

TABLE 1 PCR reaction System for OTOF Gene

In this case, the PCR Mix of Tiangen was used for PCR amplification.

Reaction conditions are as follows: the PCR reaction was performed on a BIORAD My Cycle thermal cycler, and the reaction sequence (including temperature and time) is shown in FIG. 2.

Electrophoresis process of PCR products:

1) gel (1% agarose): 0.4g of agarose was weighed and suspended in 40mL of XTAE (500mL Erlenmeyer flask).

2) Sol: heating with high fire in a microwave oven until boiling, continuously boiling for several minutes, taking out and mixing.

3) Cooling the glue: after the gel was completely dissolved, the gel was removed from the microwave oven, cooled to about 60 deg.C, added 1 drop of EB (about 10. mu.L, 10mg/mL), and shaken well.

4) Spreading glue: the two ends of the plate are sealed with adhesive tapes, 250mL of glue solution is poured into the plate, and a comb ruler is inserted.

5) Gluing: the plate was placed in an electrophoresis tank containing an electrophoresis solution (0.5 × TAE, liquid level 1 to 2mm from the surface of the gel), and the comb ruler was pulled out.

6) Sample adding: and (4) adding the sample according to a specified format by using a pipettor, and finally adding the MarkerDL 2000.

7) Glue spreading: covering the electrophoresis tank cover, checking the positive and negative levels, starting the electrophoresis apparatus, and adjusting the electrophoresis voltage.

8) Quantification: when the bromophenol blue leaves the sample adding hole by 1.5-2 cm, the electrophoresis apparatus is closed, the gel is carefully taken out, and the gel is placed into a camera for photographing. After electrophoresis, 6 bands can be seen, the lengths of the fragments of the MarkerDL2000(TaKaRa) are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively, and the total concentration of DL2000 is 300ng/5 mu L. The amount of each band was 50ng because 5. mu.L of DL2000 was used for electrophoresis. When the PCR product was electrophoresed, 5. mu.L (PCR product) was electrophoresed. The size and content of the PCR product were judged by comparing the gray value of the PCR product after electrophoresis with the gray value of DL2000 (see FIG. 3).

Purification and quantification of PCR amplification products from coding region of OTOF gene

Purification of PCR amplification products (96-well plate method):

1) after the electrophoresis of the PCR amplification product is finished, a target strip is cut off by a scalpel under a long-wave 365nm ultraviolet transilluminator, the mass of the cut gel block is less than 3g, and the gel block is placed in the plate hole number corresponding to the plate hole number.

2) Centrifuging at 4000rpm for 1min, adding 500ul sol solution, covering with sealing film, and water bathing at 65 deg.C for 15 min.

3) Checking whether the glue block in each hole is completely dissolved, if not, carrying out water bath at 65 ℃ for 3min again, uncovering the sealing film, adding 10 mu L of uniformly mixed magnetic beads in each hole by using a continuous liquid adding device, covering a silica gel pad, carrying out vortex oscillation for 30s, and transferring to a horizontal oscillator at 600-800 rpm for oscillation for 5 min.

4) And (3) clamping the 96-well plate into a magnetic frame, carrying out magnetic attraction for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

5) Discarding the waste liquid, slightly knocking on absorbent paper, transferring 500 mu L of lotion into each hole by using a 50-1200 mu L8-channel electric pipettor, covering a silica gel pad for vortex oscillation for 30s, clamping a 96-hole plate into a magnetic frame, magnetically attracting for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

6) Discarding the waste liquid, slightly knocking on absorbent paper, moving 500 mu L70% ethanol to each hole by using a 50-1200 mu L8-channel electric pipettor, covering a silica gel pad on each hole, carrying out vortex oscillation for 30s, clamping a 96-hole plate into a magnetic frame, carrying out magnetic attraction for 30s, slightly reversing the magnetic frame and the sample for 3 times, and standing for magnetic attraction for 1min again.

7) Abandoning the waste liquid, slightly knocking on absorbent paper, inverting and centrifuging to 600rpm, and horizontally shaking for 5 min.

8) Centrifuging to 1000rpm, clamping the 96-well plate into a magnetic frame, and magnetically attracting for 1 min.

9)2 mu L of sample +6 mu L of 1.4X bromophenol blue are mixed and dripped into 0.8% identification gel, the identification gel is transversely dripped according to the vertical sequence of A01-H01, 2 holes are left in the middle, 1 mu L of DL2000 and 2 mu L of DL2000 are respectively added, and 300V electrophoresis is carried out for 11 min.

10) And (3) putting the identification gel into a gel imager to collect images, wherein the images must ensure that the marker strips are clear.

11) And (3) contrasting gel images before and after purification, marking the concentration of the sequencing template obtained after the amplification product of each hole is purified on a PCR record table according to a PCR quantitative standard, diluting to the specified concentration, and carrying out electrophoretic re-identification on the recovered electrophoretic sample without bands according to 4 mu L sample +5 mu L1.4X bromophenol blue.

12) Centrifuging the diluted template for 2min to 4000rpm, marking the state of the Lims system template, checking the state of the template again before confirming submission, and storing the template in a refrigerator at 4 ℃ after confirming no error.

(III) direct sequencing of PCR amplification products of coding region of purified OTOF Gene

1. The purity and amount requirements of the PCR product DNA template are shown in Table 2.

DNA purity: OD260/OD280 is 1.6-2.0.

DNA concentration: PCR product 10 ng/. mu.L.

TABLE 2 DNA dosage

PCR product Length (bp)	Template addition for sequencing reactions (ng)
		100～200	1～3
200～500	3～10
		500～1000	5～20
1000～2000	10～40
		>2000	40～100

2. Sequencing reactions

1) The reagents required for the sequencing reaction should be freshly prepared, and the reagents that need to be autoclaved must be sterilized before use. The equipment required for the sequencing reaction (e.g., 96-well plates, tip heads, etc.) should also be clean and sterile.

2) In order to ensure the freshness of the sequencing sample and the reaction reagent, the sample should be loaded on ice.

3) The current reaction system is 5. mu.L, and the amounts of various reagents added are shown in Table 3.

TABLE 3 sequencing reaction System for PCR amplification products of OTOF Gene

Among them, BDT is a fluorescent dye produced by applied biosystems of America (ABI) for sequencing reactions. 5 XGCbuffer is the buffer for sequencing reactions produced by applied biosystems, Inc. (ABI) of USA.

4) The samples were placed on a PCR machine (thermal cycler) and the course of the reaction is shown in Table 4.

TABLE 4 sequencing reaction procedure for PCR amplification product of OTOF Gene

5) The reacted sample needs to be taken down from a PCR instrument (thermal cycler) in time, the sample to be purified in a short time is placed in a refrigerator at 4 ℃, and the sample which can be purified only after more than one day is placed in a refrigerator at-20 ℃ for freezing.

3. Purification and sequencing of sequencing reactions

1) Adding 20 μ L of 80% ethanol into each well, and centrifuging at 4000rpm for 30 min; putting the sample plate on a folded paper towel, and reversely throwing the sample plate in a centrifugal machine, wherein the speed rate cannot exceed 1000rpm when the sample plate is reversely thrown;

2) adding 30 μ L70% ethanol into each well, centrifuging at 4000rpm for 10min, and back-throwing;

3) repeating step 2) two more times;

4) placing the sample plate in a clean drawer, and drying for 30min in a dark place;

5) adding 5 mu L of formamide, sealing the membrane, centrifuging and placing in a refrigerator at the temperature of minus 20 ℃;

6) denaturalizing at 95 deg.C for 5min before loading, placing on ice for 2min, centrifuging, and loading on ABI 3730 sequencer.

The sequencing results are shown in FIGS. 4A and 4B. Out of 200 patients, 1 of auditory neuropathy pedigree disorder was detected as a compound heterozygous mutation consisting of c.4236delc and another well-defined disease-causing site c.907delAG in the OTOF gene test. No c.4236delC mutant was found in the screening of 100 normal hearing patients.

(IV) kit for detecting deafness related gene OTOF mutation site (c.4236delC) and application thereof

1. Composition of the kit

(1) Amplification primers:

an upstream primer:

OTOF-F-1:5’-AGACCCTTCTAGGGTGTGTGG-3’；

a downstream primer:

OTOF-R-1:5’-CTGCTGAGTCATGGGAGAGTC-3’；

(2) PCR Mix 2 for PCR amplification

(4)dNTP 2.5mM

(5) Big-Dye mix (manufactured by applied biosystems of America (ABI))

2. Application method

1) PCR amplification

Software Primer 5.0 was used to design PCR primers for the coding region of the OTOF gene, and the reaction conditions are shown in FIG. 2.

2) PCR product purification

And (3) performing electrophoresis on the PCR product, purifying the gel and performing electrophoresis quantification.

3) Sequencing reaction and validation

And (3) carrying out sequencing reaction by taking the PCR primer as a sequencing primer and carrying out sequencing reaction on a BIORAD My Cycle thermal cycler. After the reaction is finished, the extension product is loaded on an ABI 3730DNA sequencer. The resulting sequencing map was analyzed and compared to the normal sequence (NC _000002.11(26680071..26781566)) to determine whether a mutation was present.

Example 2

The amplification primers (design completion time: 2019, 5 months) were as follows, as in example 1 (amplification target region: exon 33 of OTOF gene, reference sequence: NC-000002.11 (26680071.. 26781566)):

an upstream primer OTOF-F-2: 5'-CTTCCTGCTCTGCTCATTTCT-3';

the downstream primer OTOF-R-2: 5'-GTGGCATTCAAAATAGCTAACA-3'.

Example 3

The amplification primers (design completion time: 2019, 5 months) were as follows, as in example 1 (amplification target region: exon 33 of OTOF gene, reference sequence: NC-000002.11 (26680071.. 26781566)):

OTOF-F-3:5’-CTCTGCTCATTTCTGCTCCAC-3’；

OTOF-R-3:5’-GGGTCAGGGTGTAGGTTTGC-3’。

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Claims (8)

1. A kit for detecting the c.4236delC mutation of an OTOF gene is characterized in that: the kit comprises PCR reaction reagents for amplifying DNA fragments, wherein the PCR reaction reagents comprise PCR primers, and a target fragment amplified by the PCR primers comprises a base corresponding to 4236 th site of CDS region of OTOF gene.

2. The kit for detecting the c.4236delC mutation of the OTOF gene according to claim 1, wherein: the kit also comprises a reagent for extracting template DNA required by PCR amplification from an individual to be detected.

3. The kit for detecting the c.4236delC mutation of the OTOF gene according to claim 1, wherein: the kit also includes reagents for sequencing the PCR amplified target fragment.

4. The kit for detecting the c.4236delC mutation of the OTOF gene according to claim 1, wherein: the PCR primer is selected from one of the following three pairs of primers P1, P2 and P3:

the sequence of primer pair P1 is:

OTOF-F-1:5’-AGACCCTTCTAGGGTGTGTGG-3’；

OTOF-R-1:5’-CTGCTGAGTCATGGGAGAGTC-3’；

the sequence of primer pair P2 is:

OTOF-F-2:5’-CTTCCTGCTCTGCTCATTTCT-3’；

OTOF-R-2:5’-GTGGCATTCAAAATAGCTAACA-3’；

the sequence of primer pair P3 is:

OTOF-F-3:5’-CTCTGCTCATTTCTGCTCCAC-3’；

OTOF-R-3:5’-GGGTCAGGGTGTAGGTTTGC-3’。

5. a method for detecting the c.4236delC mutation of an OTOF gene is characterized in that: the method comprises the following steps:

1) collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

2) taking the DNA extracted in the step 1) as a template, and carrying out PCR reaction by using a PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and carrying out typing identification according to the base corresponding to 4236 th site of CDS region of OTOF gene in the target fragment.

6. The method for detecting the c.4236delC mutation of the OTOF gene according to claim 5, wherein: the typing identification adopts a method of directly sequencing the target fragment, and the genotype or allele type of the individual to be detected corresponding to 4236 th site of CDS region of OTOF gene is determined by comparing the sequencing result with the reference sequence.

7. The method for detecting the c.4236delC mutation of the OTOF gene according to claim 5, wherein: the genotype determined by the alignment includes wild homozygous W/W, mutant heterozygous W/D or mutant homozygous D/D, where D represents a deletion.

8. Use of the kit of claim 1 for analysis of the etiology of disorders of the auditory neuropathy spectrum.

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