CN113136426A - Kit for detecting MYO15A gene variation and detection method and application thereof - Google Patents

Abstract

The invention provides a kit for detecting MYO15A gene c.8582T > C variation, which comprises a PCR reaction reagent for amplifying DNA fragments, wherein the PCR reaction reagent comprises a PCR primer, a target fragment amplified by the PCR primer comprises a base corresponding to the 8582 th position of a MYO15A gene coding region, and further provides a method and application of the kit for detecting MYO15A gene c.8582T > C variation, and the kit is used for etiological analysis of sensorineural deafness. The kit can be used for rapidly detecting the specific mutation site of the MYO15A gene, and can judge the occurrence reason of the sensorineural deafness of a patient by detecting whether the mutation of the C.8582T > C of the MYO15A gene exists in a DNA sample from the patient, thereby providing a basis for clinical diagnosis.

Description

Kit for detecting MYO15A gene variation and detection method and application thereof

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a kit for detecting mutation of MYO15A gene c.8582T > C, and a detection method and application thereof.

Background

The MYO15A gene is one of common deafness causing genes and can cause autosomal recessive inheritance non-syndrome deafness DFNB3(OMIM 600316). The finding of DFNB3 was that Friedman, equal to 1995, found a family of autosomal recessive inherited non-syndromic deafness in Indonesia, first localized to the autosomal 17p11.2 by linkage analysis in a 3 centimorgan range, designated the DFNB3 locus. In 1998, it was demonstrated in 3 unrelated DFNB3 families that homozygous variation in MYO15A was responsible for the disease in these 3 families, and studies on the mutation in the MYO15A gene began.

The MYO15A gene is located at 17p 11.2. A total of 66 exons, encoding myosin 15, which comprises 3530 amino acids; the protein structure is divided into three large areas of head, neck and tail, wherein the head comprises an N-terminal structural domain and a movement structural domain responsible for ATP activity; the neck comprises a calmodulin light chain binding-associated IQ motif; the tail region comprises two domains of My TH4 (myosin tail homolog 4), two FERM domains, an SH3 domain, and binding motifs for C-terminal subtypes i and PDZ. The function of myosin 15 is to bind the cytoskeletal actin filaments within the cell and to provide energy for the development of motor tension, and plays a very crucial role in the differentiation and elongation of hair cell cilia, and dysfunction of this protein can interrupt the mechanical transport mechanism between the cilia on the surface of the hair cell. Thus, myosin 15 is essential for normal hearing maintenance, and myosin 15 changes due to the mutation in the MYO15A gene are responsible for the occurrence of DFNB3 deafness in humans.

The screening of MYO15A gene mutation of a patient with sensorineural deafness has been widely carried out at home and abroad, and nearly 200 reported pathogenic mutation sites comprise missense mutation, frame shift mutation, nonsense mutation and splice site mutation and are mainly distributed in a coding region. Because of the characteristics of close marriage and more common population in the middle east, south Asia and other countries in the population, research reports of the sensorineural deafness caused by the MYO15A variation in the region are more common, and compared with other countries and regions, the proportion of the MYO15A gene variation in the sensorineural deafness in the regions is higher. The number of MYO15A gene variation carriers is higher in China.

The knowledge by scholars both at home and abroad of the genotype-hearing phenotype association of the MYO15A variation is a process of increasing progress. Studies have shown that the genotype of the MYO15A variation appears to be more complex to correlate with hearing phenotype than previously envisioned. There are more non-congenital hearing phenotypes of severe sensorineural deafness in ears reported in the literature, with more diverse phenotypic characteristics: in addition to residual hearing in the low frequency region, congenital moderate-severity sensorineural deafness with descending hearing curve, all-frequency moderate-severity sensorineural deafness, progressive high-frequency descending severe sensorineural deafness and the like, delayed-onset and progressive moderate-severity sensorineural deafness (the onset age is up to 14 years old at the latest) are included. It is important to determine the severity of the genotype and phenotype.

The variant of MYO15A gene is diversified, each site has different effects on the function of protein after variant, and the pathogenicity and phenotype severity of the novel variant site are determined. This is the basis of genetic counseling and is the basis for prenatal diagnosis for preventing the birth of deaf children. No report about mutation of MYO15A gene c.8582T > C (p.F2861S) is found at present.

Disclosure of Invention

The invention aims to solve the technical problems that the prior art is not enough, and provides a kit for detecting MYO15A gene c.8582T > C variation, a detection method and application thereof, wherein the kit can be used for quickly detecting a specific variation site of the MYO15A gene, and can judge the occurrence reason of sensorineural deafness of a patient by detecting whether MYO15A gene c.8582T > C variation exists in a DNA sample from the patient, so that a basis is provided for clinical diagnosis.

In order to solve the technical problems, the invention adopts the technical scheme that: a kit for detecting mutation of a MYO15A gene c.8582T > C, the kit comprises PCR reaction reagents for amplifying DNA fragments, the PCR reaction reagents comprise PCR primers, and a target fragment amplified by the PCR primers comprises a base corresponding to the 8582 th position of a MYO15A gene coding region.

Preferably, the kit further comprises reagents 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 primer pair P1, primer pair P1 includes

MYO15A-F-1 and MYO15A-R-1, wherein the MYO15A-F-1 has a nucleotide sequence of a sequence table SEQ ID.No.1; the MYO15A-R-1 has a nucleotide sequence of a sequence table SEQ ID No. 2.

Preferably, the PCR primers are selected from a primer pair P2, the primer pair P2 comprises MYO15A-F-2 and MYO15A-R-2, and the MYO15A-F-2 has a nucleotide sequence of a sequence table SEQ ID No. 3; the MYO15A-R-2 has a nucleotide sequence of a sequence table SEQ ID No. 4.

The invention also provides a method for detecting MYO15A gene c.8582T > C variation by using the kit for detecting MYO15A gene c.8582T > C variation, which comprises the following steps:

s1, collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

s2, carrying out PCR reaction by taking the DNA extracted from S1 as a template and a PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and typing and identifying the base contained in the target fragment and corresponding to 8582 th of the MYO15A gene coding region.

Preferably, the typing identification adopts a method of directly sequencing the target fragment, and the genotype or the allelic gene type of the individual to be tested, which corresponds to 8582 of the MYO15A gene coding region, is determined by comparing the sequencing result with a reference sequence (MYO15A gene reference sequence (NM-016239)).

Preferably, the genotype or allele type determined from the alignment comprises wild homozygous T/T, variant heterozygous T/C or variant homozygous C/C.

The invention also provides application of the kit for detecting the C.8582T > C variation of the MYO15A gene, wherein the kit is used for etiological analysis of sensorineural deafness, and whether the T > C variation exists at the 8582 th position corresponding to the coding region of the human MYO15A gene reference sequence in a sample (the MYO15A gene fragment of a patient) to be detected is detected by using the kit, so that the hereditary cause of the occurrence of the sensorineural deafness of the patient is judged. Wherein c.8582T > C mutation of the MYO15A gene is missense mutation, so that the 2861 th amino acid coded by the MYO15A gene is changed from phenylalanine to serine (p.F2861S), the hydrophobicity of the amino acid is changed, and the tertiary structure of the protein is influenced.

Compared with the prior art, the invention has the following advantages:

the kit provided by the invention can be used for rapidly detecting the specific mutation site of the MYO15A gene, and can judge the occurrence reason of sensorineural deafness of a patient by detecting whether the mutation of the C.8582T > C of the MYO15A gene exists in a DNA sample from the patient, thereby providing a basis for clinical diagnosis.

When the kit provided by the invention is used for diagnosing sensorineural deafness: 1) the invention provides a convenient and reliable method for screening susceptibility genes in patients with sensorineural deafness; 2) by prenatal diagnosis and screening, whether the fetus carries c.8582T > C homozygous mutation or the compound heterozygous mutation of the site and other definite recessive pathogenic sites is determined, the birth rate of the deaf infant is reduced, and the burden is relieved for the society and families.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Drawings

FIG. 1 is a conservative analysis of the amino acids in the coding region of the MYO15A gene of the invention: the mutation is located at the T base 8582 of the coding region of the MYO15A gene, and the boxed circle is the pre-mutation amino acid.

FIG. 2 is a diagram showing the sequencing result of the MYO15A gene of an individual to be tested in example 1 (A is a variation heterozygote sequence, and an arrow indicates a variation site position; B is a homozygote sequence, and an arrow indicates a variation site position).

FIG. 3 is an electropherogram of the amplification product of example 1 of the present invention.

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 screens 100 patients with non-syndrome type phonosensitive nerve deafness and 100 controls with normal hearing and no family history by using a candidate gene screening method, and finds c.8582T > C homozygous variation of MYO15A gene in a non-syndrome type phonosensitive nerve deafness family. The audiological test results of the proband the sisters are extremely severe hearing loss, CT and MRI results do not show abnormality, the sisters and the parents of the proband have normal hearing, and the parents of the proband have normal hearing. The genotype of the predecessor and the sister is c.8582T > C homozygous variation, the parents carry c.8582T > C heterozygous variation, and the sensorineural deafness related to MYO15A gene variation is transmitted in an autosomal recessive inheritance mode. The MYO15A gene variation was co-segregated with the sensorineural deafness phenotype in this patient. MYO15A gene c.8582T > C is a pathogenic variant. At present, MYO15A gene reports more than 200 variation related to sensorineural deafness, and no c.8582T > C variation is reported.

The above variation (c.8582t > C) switches the base 8582 in the coding region of the MYO15A gene, resulting in a change in the hydrophobicity of the encoded amino acid 2861 from phenylalanine to serine (reference for the standard sequence of the wild-type MYO15A gene can be made, for example, NM — 016239). The sequence is highly conserved across species (FIG. 1).

The detection of the above-mentioned mutation (c.8582T > C) can be carried out by various methods for detecting a mutation in a nucleic acid sequence, for example, a PCR (polymerase chain reaction) -sequencing method, a hybridization method using a labeled MYO15A gene DNA probe, a method using a Restriction Fragment Length Polymorphism (RFLP) method or a sequence-specific primer method. Wherein, the method for detecting the sample by adopting a PCR amplification-direct sequencing method comprises the following steps:

s1, collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

s2, carrying out PCR reaction by using the DNA extracted from S1 as a template and a PCR primer designed near the 8582 th base of the MYO15A gene coding region to obtain a PCR amplification product;

s3, sequencing and analyzing the obtained PCR amplification product, comparing the sequence obtained by sequencing with a MYO15A gene reference sequence (NM-016239), and determining whether the MYO15A gene of the individual to be detected has c.8582T > C variation;

s4, judging whether the individual to be detected is a sensorineural deafness gene carrier caused by MYO15A gene mutation c.8582T > C according to the results, and whether genetic counseling is needed before childbirth.

The PCR primers used in the above step S2 can be designed based on the known primer nucleotide sequences: 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 amplification product obtained in step S2 is detected by using a hybridization probe, the hybridization probe can be a probe that hybridizes with the normal MYO15A nucleotide sequence, or with the variant nucleotide sequence, or with the complementary sequence thereof. 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 mutation of the MYO15A gene c.8582T > C 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 mutation of MYO15A gene c.8582T > C, and provides manufacturing, using and selling information of related medicines or biological products approved by a government medicine 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 sensorineural deafness 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, genomic DNA was extracted by protease degradation, quantified and stored at-20 ℃ with each DNA sample corresponding in detail to the clinical data of the registered patients. Then, on-line primer design software Primer5.0 was used to design primers (amplification target region is MYO15A coding region, reference sequence NM-016239, amplification target fragment size is 692bp), and PCR amplification was performed on a BIORAD My Cycle thermocycler using genomic DNA as a template. Direct sequencing of PCR amplification products: 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 sequenced sequence was compared to the sequence in Genbank (NM — 016239) to determine if a mutation in the MYO15A gene c.8582t > C was present. The method comprises the following specific steps:

(I) extraction of blood sample to be tested and PCR amplification of MYO15A gene coding region

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

1.1 study object

Screening for the MYO15A gene was performed in the following manner for 100 patients with non-syndromic sensorineural deafness and 100 healthy hearing controls without family history.

The sporadic non-syndrome deafness subjects are collected from deafness 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 2 months in 2019.

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 the anticoagulant can be stored at 2-8 ℃ for 10 days at most, for some experiments such as Southern hybridization and the like, complete full-length genome DNA is required to be obtained, and the blood sample is stored at 2-8 ℃ for not more than 3 days, wherein 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 37 ℃ water/metal bath 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 the coding region of the MYO15A gene

2.1 primer sequences

Primer5 Primer design software was used, reference sequence gene NM — 016239, for this assay after sequence synthesis, with design completion time of 2019, 3 months:

an upstream primer MYO15A-F-1 has a nucleotide sequence of a sequence table SEQ ID No.1,

the downstream primer MYO15A-R-1 has a nucleotide sequence of a sequence table SEQ ID No. 2.

The size of the fragment obtained by PCR amplification using this primer was 692 bp.

2.2 establishment of PCR reaction System (Table 1)

TABLE 1 PCR reaction System for MYO15A Gene

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

Reaction conditions are as follows: the PCR reaction is carried out on a BIORAD My Cycle thermal cycler, and the reaction process is as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 61 ℃ (each cycle is reduced by 0.5 ℃ in turn) for 30s, 40s at 72 ℃ and 12 cycles; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature constant at 4 ℃.

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: adding sample according to a specified format by using a pipettor, and finally adding

Plus DNA Marker。

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, 8 bands are visible,

the lengths of the Plus DNA Marker (TaKaRa) fragments are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively. During electrophoresis, 5. mu.L of Marker was used, wherein the band concentration of 750bp was 100 ng/5. mu.l, which indicated bright bands, and the band concentrations of the rest were 50 ng/5. mu.l. When the PCR product was electrophoresed, 5. mu.L (PCR product) was electrophoresed. The size and content of the PCR product can be judged by comparing the gray value of the PCR product after electrophoresis with the gray value of Marker, see FIG. 3, lane 1 is Marker, and lane 2 is amplified target sequence (692 bp).

Purification and quantification of PCR amplification products from the coding region of the MYO15A 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 500 μ L 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 muL of 70% ethanol to each hole by using a 50-1200 muL 8-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) And mixing 2 mu L of sample and 6 mu L of 1.4X bromophenol blue, then dropping the mixture into 0.8% identification gel, horizontally dropping the mixture according to the vertical sequence of A01-H01, leaving 2 holes in the middle, respectively adding 1 mu L and 2 mu L of Marker, and performing 300V electrophoresis 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 a specified concentration, and carrying out electrophoretic re-identification on the recovered electrophoretic sample without bands according to 4 mu L of sample +5 mu L of 1.4X 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 the coding region of the purified MYO15A 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 product of MYO15A gene

Among them, BDT is a fluorescent dye produced by applied biosystems of America (ABI) for sequencing reactions. 5 XGC buffer is a 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 products of MYO15A gene

Step (ii) of	Function of
		1	96℃,2min
2	96 ℃ for 10 s; 50 ℃ for 5 s; 60 ℃ for 4 min; repeat step 2, 30 cycles
		3	Kept at 4 ℃ until purification

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 result is shown in FIG. 2, wherein, A in FIG. 2 is a variant heterozygote sequence, and the arrow points to the position of a variant site; FIG. 2, panel B, is a homozygous sequence with the position of the mutation site indicated by the arrow.

(IV) kit for detecting mutation site (c.8582T > C) of deafness related gene MYO15A and application thereof

1. Composition of the kit

(1) Amplification primers:

an upstream primer MYO15A-F-1 has a nucleotide sequence of a sequence table SEQ.ID.No.1;

a downstream primer MYO15A-R-1 which has a nucleotide sequence of a sequence table SEQ.ID.No.2;

(2) 2 XPCR Mix for PCR amplification

(4)dNTP 2.5mM

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

2. Application method

The method mainly comprises the following steps:

1) PCR amplification

Software Primer5.0 is used for designing PCR primers (an upstream Primer MYO15A-F-1 and a downstream Primer MYO15A-R-1) for a coding region of a MYO15A gene, and the reaction conditions are as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 61 ℃ (each cycle is reduced by 0.5 ℃ in turn) for 30s, 40s at 72 ℃ and 12 cycles; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature constant at 4 ℃.

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 (NM — 016239) to determine if a variation was present.

The MYO15A gene detection of 1 of 100 patients with sensorineural deafness shows c.8582T > C homozygous variation. No c.8582T > C variant was found in the screening of 100 normal hearing subjects.

Example 2

The amplification primers (design completion time 2019, 3 months) are as follows, and the other same as example 1 (the amplification target region is a 714bp fragment of MYO15A gene including c.8582 site, and the reference sequence NM-016239):

an upstream primer MYO15A-F-2 has a nucleotide sequence of a sequence table SEQ ID No.3,

the downstream primer MYO15A-R-2 has a nucleotide sequence of a sequence table SEQ ID No. 4.

The other reagents and reaction conditions were the same as in example 1.

The MYO15A gene detection of 1 of 100 patients with sensorineural deafness shows c.8582T > C homozygous variation. No c.8582T > C variant was found in the screening of 100 normal hearing subjects.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.

Sequence listing

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Claims (9)

1. A kit for detecting mutation of a MYO15A gene c.8582T > C, which is characterized by comprising 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 the 8582 th position of a coding region of a MYO15A gene.

2. The kit for detecting mutation of MYO15A gene c.8582T > C according to claim 1, wherein the kit further comprises a reagent for extracting template DNA required by PCR amplification from an individual to be detected.

3. The kit for detecting mutation of MYO15A gene c.8582T > C according to claim 1, wherein the kit further comprises reagents for sequencing PCR-amplified target fragments.

4. The kit for detecting mutation of C.8582T > C of MYO15A gene according to claim 1, wherein the PCR primer is selected from primer pair P1, and primer pair P1 comprises

MYO15A-F-1 and MYO15A-R-1, wherein the MYO15A-F-1 has a nucleotide sequence of a sequence table SEQ ID.No.1; the MYO15A-R-1 has a nucleotide sequence of a sequence table SEQ ID No. 2.

5. The kit for detecting mutation of MYO15A gene c.8582T > C according to claim 1, wherein the PCR primer is selected from a primer pair P2, the primer pair P2 comprises MYO15A-F-2 and MYO15A-R-2, and the MYO15A-F-2 has a nucleotide sequence of a sequence table SEQ ID No. 3; the MYO15A-R-2 has a nucleotide sequence of a sequence table SEQ ID No. 4.

6. A method for detecting MYO15A gene c.8582T > C variation by using the kit for detecting MYO15A gene c.8582T > C variation, which is disclosed by any one of claims 1-5, and comprises the following steps:

s1, collecting blood, body fluid or tissue of an individual to be detected, and then extracting DNA;

s2, carrying out PCR reaction by taking the DNA extracted from S1 as a template and a PCR primer to obtain a PCR reaction product; separating the target fragment amplified by the PCR reaction from the PCR reaction product, and typing and identifying the base contained in the target fragment and corresponding to 8582 th of the MYO15A gene coding region.

7. The method as claimed in claim 6, wherein the typing identification is carried out by directly sequencing the target fragment, and determining the genotype or allele type of the tested individual corresponding to 8582 of the MYO15A gene coding region by comparing the sequencing result with a reference sequence.

8. The method of claim 6, wherein the genotype or allelic type determined from the alignment includes wild-homozygous T/T, variant-heterozygous T/C, or variant-homozygous C/C.

9. Use of a kit according to any one of claims 1-5 for detecting a variation in the MYO15A gene c.8582t > C for etiology of sensorineural deafness.

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