CN114317721A - Kit for in vitro detection of autosomal recessive inheritance non-syndromic deafness gene MYO15A - Google Patents

Abstract

The invention relates to the field of medicine, in particular to a kit for in vitro detection of an autosomal recessive inheritance non-syndromic deafness gene MYO 15A. Aiming at the research situation of the prior art, the inventor of the application firstly discovers three pathogenic gene mutation sites of MYO15A, namely a G-to-A mutation site of c.3952, a G-to-T mutation site of c.6177+1 and a G-to-A mutation site of c.4252. The kit for in-vitro detection of the autosomal recessive inheritance non-syndromic deafness gene MYO15A provided by the invention aims at the three mutation sites, mainly comprises a specific primer, a PCR (polymerase chain reaction) mixed solution, an enzyme digestion mixed solution, a restriction enzyme, a positive control specimen and a negative control specimen, and can be used for simultaneously identifying the pathogenic gene mutation of the three MYO 15A. In the identification process, the reagents and the gene sequences cannot influence each other, the identification process is simple and efficient, and the result is safe and reliable.

Description

Kit for in vitro detection of autosomal recessive inheritance non-syndromic deafness gene MYO15A

Technical Field

The invention relates to the field of medicine, in particular to a kit for in vitro detection of an autosomal recessive inheritance non-syndromic deafness gene MYO 15A.

Background

Commonly known as deafness, also called as hearing impairment, refers to the decrease of hearing caused by organic or functional abnormalities of nerve center generators at all levels of the comprehensive analysis of the transmission, perception and sound in the auditory system. Current research indicates that there are many factors that affect hearing loss, including age, noise, ototoxic drugs and genes. Deafness is divided into hereditary deafness and non-hereditary deafness, more than half of which are hereditary deafness. Non-syndromic deafness accounts for about 70% of hereditary deafness, autosomal recessive hereditary non-syndromic deafness accounts for about 75% -85% of non-syndromic deafness, and clinical manifestations are non-progressive severe-extremely severe sensorineural deafness before binaural learning. At present, 77 genes are clinically found to be related to autosomal recessive inheritance non-syndromic deafness, but the pathogenesis of the genes still needs to be further researched, and meanwhile, a large number of genes related to hereditary deafness are not found. The MYO15A mutation was originally found in mystery, a mysterious village-Beng-kala village in which Bali island of Indonesia was communicated in a unique sign language, found by Friedman et al in 1995, and 47 (2.2%) of 2185 people in this village had very severe deafness, and these deafness patients conformed to the characteristic of autosomal recessive inheritance non-syndrome type deafness, and the linkage interval found on chromosome 17p11.2 was named as DFNB3 locus, which is the third autosomal recessive inheritance non-syndrome type deafness locus reported after DFNB1 (mutations in GJB2 and GJB6 genes) and DFNB2 (mutation in MYO7A gene). The MYO15A gene includes 66 exons, encoding three distinct isoforms, one of which is myosin 15, which plays an important role in the development and maintenance of hair cell cilia for sensory delivery. Currently 625 recessive mutations of MYO15A are related to hearing loss, wherein 225 mutations are classified into suspected pathogenic mutations, 60 mutations are suspected pathogenic mutations, 340 mutations are of unknown significance, and the MYO15A gene mutation is clinically manifested by non-progressive severe-extremely severe full-frequency hearing loss before study and no forecourt symptoms; the shaker-2 mice had shortened static cilia and broken actin axes.

The MYO15A gene is one of the common deafness genes, and it is counted that 225 pathogenic mutation points have been identified so far. So far, methods for detecting mutation sites aiming at the existing known sites include a direct sequencing method, a fluorescent quantitative PCR method, a gene chip method and the like, wherein the direct sequencing method is a gold standard for identifying mutations, but the operation cost is high, and the steps are complex, so that the method cannot be widely applied.

Disclosure of Invention

Aiming at the research situation of the prior art, the inventor of the application firstly discovers three pathogenic gene mutation sites of MYO15A, namely a G-to-A mutation site of c.3952, a G-to-T mutation site of c.6177+1 and a G-to-A mutation site of c.4252. The invention provides a kit for detecting an autosomal recessive inheritance non-syndromic deafness gene MYO15A in vitro aiming at the three mutation sites. The kit mainly comprises a specific primer, a PCR mixed solution, an enzyme digestion mixed solution, a restriction enzyme, a positive control specimen and a negative control specimen, and can be used for simultaneously identifying the three pathogenic gene mutations of MYO 15A. In the identification process, reagents and gene sequences cannot influence each other, the identification process is simple and efficient, the result is safe and reliable, a foundation is provided for large-scale screening and preventive inspection of deafness related gene mutation in a large range, the detection cost is low, the operation is simple, and the accuracy is high.

Specifically, the kit of the invention comprises a specific primer, a PCR mixed solution, an enzyme digestion mixed solution, a restriction enzyme, a positive control specimen and a negative control specimen, wherein:

the specific primers comprise three pairs, which are respectively as follows:

primers 1-F: 5'-CCTTGGAAGATTGCCTGGTA-3' (shown in SEQ ID NO: 1);

primers 1-R: 5'-GACAGGCAGGAACTCTCACC-3' (shown in SEQ ID NO: 2);

primer 2-F: 5'-CATTCCCTCCTCTTTCCACA-3' (shown in SEQ ID NO: 3);

primers 2-R: 5'-GGCTCCACCAGTTGTTCAAT-3' (shown in SEQ ID NO: 4);

primer 3-F: 5'-AAATCCCACCAGAACCCTCT-3' (shown in SEQ ID NO: 5);

primer 3-R: 5'-AAACTCACCCTCCCCAAATC-3' (shown in SEQ ID NO: 6);

the restriction enzymes are BsrBI, MseI and HpaII;

the positive control sample 1 is as shown in SEQ ID NO: 8, the DNA fragment contains a DNA fragment which cannot be subjected to enzyme digestion by the G-to-A heterozygous mutation of the MYO15A gene c.3952; the corresponding negative control specimen 1 is as shown in SEQ ID NO: 7, does not contain the DNA segment of which the mutation site can be subjected to enzyme digestion;

positive control specimen 2 is as set forth in SEQ ID NO: 10, containing a DNA fragment which can be subjected to enzyme digestion by the hybrid mutation from G to T of the MYO15A gene c.6177+ 1; the corresponding negative control sample 2 is as shown in SEQ ID NO: 9, and a DNA fragment which does not contain the mutation site and cannot be digested.

Positive control specimen 3 is as set forth in SEQ ID NO: 12, and the DNA fragment contains a DNA fragment which cannot be subjected to enzyme digestion by the hybrid mutation from G to A of the MYO15A gene c.4252; the corresponding negative control specimen 3 is as shown in SEQ ID NO: 11, and does not contain a DNA fragment in which the mutation site can be cut.

In the technical scheme, the primer 1 and the restriction endonuclease BsrBI aim at a G-to-A mutation site of c.3952, and the BsrBI can recognize a 5 'CCG/CTC 3' sequence; primer 2 and restriction enzyme MseI can recognize 5 'T/TAA 3' sequence aiming at the G-to-T mutation site of c.6177+ 1; primer 3 and restriction enzyme HpaII are directed against the G to A mutation site of c.4252, HpaII can recognize the 5 'C/CGG 3' sequence.

The PCR mixed solution comprises 10 times of PCR buffer and Taq enzyme.

The inventor also provides the following specific steps for detection by using the kit:

(1) extraction of genomic DNA: the genomic DNA is extracted from a sample to be detected by utilizing the prior art, and the detection of G to A mutation sites of three mutation points c.3952 of MYO15A, G to T mutation sites of c.6177+1 and G to A mutation sites of c.4252 can be realized by using the kit;

(2) amplification of the fragment of interest: PCR amplification is carried out by utilizing specific primers in the kit to obtain corresponding DNA fragments containing three mutation points of MYO15A (namely, a G to A mutation site of c.3952, a G to T mutation site of c.6177+1 and a G to A mutation site of c.4252), and PCR products are respectively: 418bp, 408bp and 474 bp;

(3) respectively carrying out enzyme digestion on the 418bp, 408bp and 474bp PCR products by using corresponding restriction enzymes BsrBI, MseI and HpaII;

(4) and (3) identifying an enzyme digestion product: and (3) carrying out electrophoresis by using 2% agarose gel, and judging whether mutation exists in the mutation site according to the size of the enzyme digestion product.

The kit provided by the invention is adopted to detect the mutation of three mutation points of deaf related gene MYO15A c.3952 from G to A mutation sites of c.3952, from G to T mutation sites of c.6177+1 and from G to A mutation sites of c.4252, and by combining a specific PCR technology and an enzyme digestion technology, each DNA sample is respectively subjected to common PCR amplification in a PCR reaction tube by using a specific primer, then the enzyme digestion at the mutation points is carried out, and the three mutation points of the deaf related gene MYO15A c.3952 can be detected within a few hours by one-time PCR: c.3952G to A mutation site, c.6177+1G to T mutation site, c.4252G to A mutation site, and heterozygous mutation or homozygous mutation can be also be clear at a glance.

For the G to A mutation site of c.3952, when no mutation occurs, the kit of the invention is used for enzyme digestion, two bands of 138bp and 280bp are generated; when mutation occurs, only one band of 418bp is generated when the kit is used for enzyme digestion, and only one mutated gene is generated for a recessive mutation carrier from G site to A site of MYO15A c.3952, so that three bands of 418bp, 138bp and 280bp are generated when the kit is used for enzyme digestion.

For the G to T mutation site of c.6177+1, the main judgment standard is that when no mutation occurs, only one 408bp band is generated when the enzyme digestion is carried out by using the kit; when mutation occurs, two bands of 153bp and 255bp are generated when the kit is used for enzyme digestion, and only one mutant allele is generated for a G-to-T site recessive mutation carrier of MYO15Ac.6177+1, so that three bands of 408bp, 153bp and 255bp are generated when the kit is used for enzyme digestion.

For the G to A mutation site of c.4252, when no mutation occurs, the kit provided by the invention is used for enzyme digestion, two bands of 200bp and 274bp are generated; when mutation occurs, only one strip of 474bp is generated when the kit is used for enzyme digestion, and only one mutated gene is generated for a carrier of recessive mutation from G to A sites of MYO15A c.4252, so that three strips of 474bp, 200bp and 274bp are generated when the kit is used for enzyme digestion; this makes a clear decision. Meanwhile, the negative control and the positive control enable the detection result to be more accurate and credible.

The kit developed by the invention has the following advantages:

1. the sample to be detected has no strict requirements: the genomic DNA extracted from blood, tissue, hair and the like by using a self-made reagent or a kit can be detected by using the kit, so that the detection is easier;

2. PCR-restriction enzyme fragment polymorphism analysis (PCR-RFLP) in the kit combines the advantages of a specific PCR technology and a restriction enzyme digestion technology, each DNA sample is subjected to specific PCR amplification in a PCR reaction tube by using a specific primer, namely a primer F/R, and three mutation sites of the gene MYO15A can be simultaneously detected within several hours by one-time PCR-RFLP: c.3952G to A mutation site, c.6177+1G to T mutation site and c.4252G to A mutation site, can detect recessive mutation carrier condition. Particularly, the primers used in the method are carefully designed, firstly, normal and recessive mutation carriers have different sites formed on specific restriction endonuclease in the enzyme digestion process, so that the enzyme digestion specificity is greatly improved; secondly, products of the normal control and the blank control can be used as molecular internal control indexes of the whole PCR reaction, so that the occurrence of false negative results is avoided, and the stability of detection results is improved. Dimer or hairpin structure can not be formed between the primers, and the primers can not initiate DNA polymerization reaction at the non-target sites of the template, thereby ensuring the specificity of the detection result.

3. The kit provided by the invention is applied to the three mutation sites of the deafness related gene MYO 15A: c.3952, c.6177+1 and c.4252, compared with other detection methods, the method has the advantages of low cost, simple operation, visual result interpretation, high accuracy, no special requirements on experimental equipment and operators, simple method and wide adaptability, and particularly carries out mutation detection of three mutation sites of the deafness related gene MYO 15A: large scale screening and preventive examination of the G to A mutation site of c.3952, the G to T mutation site of c.6177+1, and the G to A mutation site of c.4252.

Drawings

FIG. 1 is a gel electrophoresis diagram of enzyme digestion identification of mutation situation of C.3952 site of MYO15A gene;

FIG. 2 is a gel electrophoresis diagram of enzyme digestion identification of mutation condition of C.6177+1 site of MYO15A gene;

FIG. 3 is a gel electrophoresis diagram of enzyme digestion identification of mutation situation of C.4252 site of MYO15A gene.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention. Except as otherwise noted, the following examples were carried out using conventional techniques.

Example 1 extraction of genomic DNA

The extraction of genomic DNA was carried out using a conventional DNA extraction kit (in this example, Axygen manufactured by Corning Corp., product number AP-MD-FBL-GDNA-20), and the specific steps were as follows:

1) adding 2ml of blood sample into 5ml of Buffer AP1, and violently mixing uniformly;

2) adding 1ml Buffer AP2, mixing immediately, centrifuging 4630g for 15 min;

3) transferring the supernatant into a medium preparation tube, starting negative pressure, maintaining the negative pressure, and adding 7ml of Buffer W1, 8ml of Buffer W2 and 4ml of Buffer W2;

4) discharging 12000g of the tube head of the middle burette, and centrifuging for 2 min;

5) adding 0.4ml of Elution buffer, standing at room temperature for 5min, centrifuging at 12000g for 2min, and storing at-20 ℃ for later use.

Example 2 kit for in vitro detection of autosomal recessive inheritance non-syndromic deafness gene MYO15A

The kit comprises a specific primer, a PCR mixed solution, an enzyme digestion mixed solution, a restriction enzyme, a positive control specimen and a negative control specimen, wherein:

the specific primers comprise three pairs, which are respectively as follows:

primers 1-F: 5'-CCTTGGAAGATTGCCTGGTA-3' (shown in SEQ ID NO: 1);

primers 1-R: 5'-GACAGGCAGGAACTCTCACC-3' (shown in SEQ ID NO: 2);

primer 2-F: 5'-CATTCCCTCCTCTTTCCACA-3' (shown in SEQ ID NO: 3);

primers 2-R: 5'-GGCTCCACCAGTTGTTCAAT-3' (shown in SEQ ID NO: 4);

primer 3-F: 5'-AAATCCCACCAGAACCCTCT-3' (shown in SEQ ID NO: 5);

primer 3-R: 5'-AAACTCACCCTCCCCAAATC-3' (shown in SEQ ID NO: 6);

the restriction enzymes are BsrBI, MseI and HpaII;

the positive control sample 1 is as shown in SEQ ID NO: 8, or a DNA fragment thereof; the corresponding negative control specimen 1 is as shown in SEQ ID NO: 7.

Positive control specimen 2 is as set forth in SEQ ID NO: 10; the corresponding negative control sample 2 is as shown in SEQ ID NO: 9.

Positive control specimen 3 is as set forth in SEQ ID NO: 12; the corresponding negative control specimen 3 is as shown in SEQ ID NO: 11, or a fragment of DNA as set forth in fig. 11.

The PCR mixed solution comprises 10 times of PCR buffer and Taq enzyme.

Example 3PCR amplification of fragments of interest

The target fragment is amplified by PCR using specific primers and the like in the kit described in example 2:

1) primers specific to three mutation points within the kit were used: the lengths of the target products of amplification are respectively: 418 bp;

408bp；474bp。

2) PCR amplification was performed according to the following system and procedure:

and (3) PCR reaction system:

PCR reaction procedure:

3) the correct size of the PCR product was checked on a 1% agarose gel, and the procedure was as follows:

(1) preparing gel (1% agarose), weighing 0.3g agarose and dissolving in 30ml 1XTAE solution;

(2) sol: heating the mixture in a microwave oven to boil and then taking out the mixture;

(3) cooling the glue: taking out, cooling to about 40 ℃, and adding 1.5 mu l G-Red;

(4) spreading glue: pouring 30ml of glue solution into the flat plate, and inserting a comb ruler;

(5) gluing: putting the flat plate into an electrophoresis tank containing 1xTAE liquid, wherein the distance between the liquid level and the glue surface is 1-2 mm, and pulling out a comb ruler;

(6) sample adding: add PCR product 3. mu.l to the gel well;

(7) glue spreading: covering an electrophoresis tank cover, checking the anode and the cathode, starting an electrophoresis apparatus, and adjusting the voltage to 100V to run glue;

(8) quantification: when the electrophoresis is carried out for 20min, the electrophoresis apparatus is closed, the gel is carefully taken out, the gel is placed into an imager for photographing, and 418bp is obtained; 408 bp; 474bp of three target sequences.

Wherein the 418bp base sequence without G to A mutation of c.3952 is shown as SEQ ID NO: 7, the base sequence of the G to A mutation of c.3952 is shown in SEQ ID NO: 8 is shown in the specification; the base sequence of the 408bp sequence without C.6177+1G to T mutation is shown as SEQ ID NO: 9, the base sequence of G to T mutation of c.6177+1 is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of 474bp without C.4252G to A mutation is shown in SEQ ID NO: 11 is shown in the figure; base sequence diagram of the G to A mutation at c.4252 SEQ ID NO: shown at 12.

Example 4 enzyme cleavage identification

The restriction enzyme in the kit is used for enzyme digestion and gel electrophoresis identification:

1) the PCR products obtained in example 2 were digested with restriction enzymes BsrBI, MseI and HpaII in the kit, respectively:

the G to A mutation site system of c.3952 is as follows:

c.6177+1G to T mutation site system as follows:

the G to A mutation site system of 4252 is as follows:

respectively carrying out water bath at 37 ℃ for 45min2) taking a positive control specimen 1, a negative control specimen 1 and BsrBI enzyme digestion products in the steps as a group;

in the kit, a positive control sample 2, a negative control sample 2 and the MseI enzyme digestion product in the step are combined into a group;

in the kit, a positive control sample 3, a negative control sample 3 and the enzyme digestion product HpaII in the step are taken as a group;

mu.L of each of the three groups of samples was spotted, and the electrophoresis was performed for 25 minutes using 2% agarose gel at 110V in TAE buffer, and the results of the electrophoresis were observed in a gel imaging system.

The method of the above example 1-3 was used to test three samples, I-2, II-1, and I-2, and in addition, to further verify the effect of the present invention, a corresponding target sequence, i.e., not digested with II-1, was added to each of the three groups of samples as a control. The results are shown in fig. 1, 2 and 3:

as shown in FIG. 1, the Positive control specimen 1(Positive control) and the samples II-1 and II-2 have three bands of 418bp, 138bp and 280bp after enzyme digestion, and the sample II-1 without enzyme digestion (undigested II-1) has only one band of 418 bp; negative control sample 1(Negative control) and sample I-2 have two bands of 139bp and 279bp, which indicates that hybrid mutation occurs at C.3952G > A of MYO15A gene; the fact that the mutation does not occur at the C.3952 position of the MYO15A gene is shown.

As shown in FIG. 2, the Positive control sample 2(Positive control) and the samples II-1 and II-2 have three bands of 408bp, 153bp and 255bp after enzyme digestion; the Negative control sample 2(Negative control), the sample I-2 and the sample II-1 (undigested II-1) which are not enzyme-digested have only one 408bp band, which indicates that the MYO15A gene c.6177+1G > A of the samples II-1 and II-2 has heterozygous mutation, and the MYO15A gene c.6177+1 of the sample I-1 has no mutation.

As shown in FIG. 3, the Positive control specimen 3(Positive control) and the samples II-1 and II-2 have three bands of 474bp, 200bp and 274bp after enzyme digestion; the Negative control sample 3(Negative control) and the sample I-2 have two bands of 200bp and 274bp, and the sample II-1 which is not added with enzyme for enzyme digestion (undigested II-1) only has one band of 474 bp; it is indicated that the MYO15A gene c.4252G > A of samples II-1 and II-2 has heterozygous mutation, and the MYO15A gene c.4252 of sample II-1 has no mutation.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (2)

1. A kit for in vitro detection of an autosomal recessive inheritance non-syndromic deafness gene MYO15A comprises a specific primer, a PCR mixed solution, an enzyme digestion mixed solution, a restriction enzyme, a positive control specimen and a negative control specimen, and is characterized in that:

the specific primers comprise three pairs, which are respectively as follows:

primers 1-F: 5'-CCTTGGAAGATTGCCTGGTA-3', as shown in SEQ ID NO: 1 is shown in the specification;

primers 1-R: 5'-GACAGGCAGGAACTCTCACC-3', as shown in SEQ ID NO: 2 is shown in the specification;

primer 2-F: 5'-CATTCCCTCCTCTTTCCACA-3', as shown in SEQ ID NO: 3 is shown in the specification;

primers 2-R: 5'-GGCTCCACCAGTTGTTCAAT-3', as shown in SEQ ID NO: 4 is shown in the specification;

primer 3-F: 5'-AAATCCCACCAGAACCCTCT-3', as shown in SEQ ID NO: 5 is shown in the specification;

primer 3-R: 5'-AAACTCACCCTCCCCAAATC-3', as shown in SEQ ID NO: 6 is shown in the specification;

the restriction enzymes are BsrBI, MseI and HpaII;

the positive control sample 1 is as shown in SEQ ID NO: 8, or a DNA fragment thereof; the corresponding negative control specimen 1 is as shown in SEQ ID NO: 7;

positive control specimen 2 is as set forth in SEQ ID NO: 10; the corresponding negative control sample 2 is as shown in SEQ ID NO: 9;

positive control specimen 3 is as set forth in SEQ ID NO: 12; the corresponding negative control specimen 3 is as shown in SEQ ID NO: 11, or a fragment of DNA as set forth in fig. 11.

2. The kit of claim 1, wherein the PCR mixture comprises 10 XPCR buffer and Taq enzyme.

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