CN101586165A - Kit for detecting 2975-2978delAG mutation of OTOF gene - Google Patents

Abstract

The present invention relates to an OTOF mutant gene with 2975-2978delAG mutation, wherein the mutant gene is related with the auditory neuropathy. The invention provides a kit for detecting 2975-2978delAG mutation of OTOF gene, and a detecting method for detecting the cause and the type of the auditory neuropathy through detecting whether the mutant gene exists in the patient. The mutant gene and detecting method of the invention facilitate the clinically development of OTOF mutation screening operation of the auditory neuropathy patient, and provide criterion for diagnosing and treating the neuropath.

Description

Detect the test kit of OTOF gene 2975-2978delAG sudden change

Technical field

The present invention relates to be used to detect the test kit of OTOF gene 2975-2978delAG (X1000) mutator gene.

The invention still further relates to new OTOF mutator gene and in the application of diagnosis and/or treatment auditory nerve disease.

Background technology

(Auditory neuropathy is to prop up impaired and a kind of special sensorineural deafness that cause by listening of VIII cranial nerve AN) to the auditory nerve disease.Show as sound and can enter into inner ear normally, but voice signal can not synchronously be transferred to the auditory function abnormality disease of brain from inner ear by external ear, middle ear.The clinical audition inspection shows as that bringing out property otoacoustic emission is normal, one group of auditory function obstacle syndrome of auditory brain-stem response severely subnormal.The auditory nerve disease many from childhood or pubescence onset, sickness rate is about 0.23% in high-risk infant, the sickness rate in the unusual deaf sufferer youngster of the ABR due to a variety of causes is up to 11%, asexuality difference.Studies show that at present the morbidity of auditory nerve disease is main relevant with correlative factors such as heredity, immunity, infection, poisoning, metabolism.

Heredity auditory nerve disease can be divided into two classes according to whether merging the other system disease: a class is to be associated with the syndrome and type disease that other cranial nerve and/or peripheral neuropathy decrease, as Charot Marie Tooth syndrome: CMT1 type (demyelination type) and CMT2 type (aixs cylinder type), take Reed and rely and wish ataxia syndrome etc.; Another kind is the non-syndrome type auditory nerve disease (Non-syndromic auditoryneuropathy NSAN) that the auditory dysesthesia performance is only arranged.Non-syndrome type auditory nerve is sick to have good corresponding relationship because phenotype is single between genotype and the phenotype, becomes hot research in recent years gradually.Starr etc. are divided into TYPE I type (diseased region is at auditory nerve) and TYPE II type (diseased region is in inner hair cell and cynapse) according to the difference of site of pathological change with it.

Difference according to hereditary pattern can be divided into heredity auditory nerve disease autosomal dominant inheritance (AUNA1) [11], autosomal recessive inheritance (NSRAN) and x linked recessive heredity (AUNX1) and Mitochondrial DNA Mutation etc.2003, the method for utilization such as Varga linkage analysis was positioned at four familys in the 2p23 zone that comprises OTOF gene place, determined that OTOF is first discovery and the comparatively common gene sick relevant with non-syndrome type auditory nerve.OTOF gene DNA sequence total length 101496bp comprises 48 exons, is positioned 2p23.OTOF has 4 kinds of transcriptional variation bodies that length is different, and the mRNA length of its long hypotype varient is 7172bp, and coded protein otoferlin comprises 1997 amino acid, may participate in the fusion of synaptic vesicle.Otoferlin has 6 calcium ion calmodulin binding domain CaMs (C2 domain), and wherein back 4 contain 5 complete asparagicacid residues, can combine with calcium ion, and have 1 carboxyl end span diaphragm area (TM), and these structures all are high conservatives vertebrates.2006, the protein otoferlin that Petit professor research group observes mouse otof genes encoding was high expression level at cochlea inner hair cell and cynapse place of the formed banding pattern of afferent neuron.The mouse of isozygotying that knocks out the otof gene shows as utmost point severe deafness, it is normal that its inner hair cell banding pattern cynapse form keeps, the calcium ion circulation is also normal, but the exocytosis of synaptic vesicle is but abrogated fully, therefore reach a conclusion: the protein otoferlin of otof genes encoding is as a kind of calcium ion inductor block, trigger film in cynapse place of inner hair cell banding pattern and merge, thereby in the exocytosis process of inner hair cell synaptic vesicle, bringing into play important effect.Therefore the auditory nerve patient diseased region that is caused by this transgenation is many in cochlea inner hair cell or cynapse, and artificial cochlea's surgical result is better.And find for the examination of auditory nerve patient OTOF gene both at home and abroad, carry that the auditory nerve patient of this transgenation is clinical manyly to show as the master with the TypeII type, be suitable for the artificial cochlea and implant.And TYPE I type auditory nerve patient diseased region is at auditory nerve, and the position exists deeply, does not still have effective methods of treatment at present.Therefore, utilization is assisted somatotype to the detection of auditory nerve patient's OTOF gene, has important directive significance for clinical treatment.

Summary of the invention

The object of the present invention is to provide the test kit that is used to detect OTOF sudden change 2975-2978delAG (X1000) gene.

The present invention will be for carrying out tumor susceptibility gene examination certain method of providing convenience in the auditory nerve patient; Simultaneously can guiding clinical treatment, and provide solid basis for utilizing in the future this sudden change to carry out deaf gene therapy as target spot.

Whether the detection method of using test kit of the present invention for existing OTOF gene 2975-2978delAG sudden change in the sample to be tested that comes from the patient by detection, and judge sick occurrence cause of this patient's auditory nerve and type.Wherein, the 2975-2978delAG sequence change of OTOF gene causes that at 993 frameshit taking place in the amino acid cataloged procedure changes, cause encoding premature termination in the 1000th (being labeled as X1000), form the brachymemma peptide chain of Otoferlin, lose its function, jejune expression product is induced and is started regulation mechanism in the body, makes the mRNA degraded of variation.

The auditory nerve disease that the OTOF transgenation is relevant is transmitted in the mode of autosomal recessive inheritance.Examination is carried out in the method that the contriver uses the candidate gene examination and contrast that do not have family history normal to 76 routine non-syndrome type auditory nerve patients and 92 routine hearing, find 2975-2978delAG (X1000) sudden change of OTOF gene a routine temperature sensitive auditory nerve patient, in addition the patient also carries another sudden change that is positioned at exon region, mother of patient is the carrier of 2975-2978delAG sudden change, father is the carrier of another one sudden change, OTOF transgenation and auditory nerve disease phenotype be divided into from.The sudden change that report is sick relevant with auditory nerve in the world at present has 11 kinds, does not still have the report of 2975-2978delAG sudden change.

Fig. 1 provides the contrast figure of OTOF coding region amino acid and nucleic acid base, and mutational site (square frame mark) is shown, this sudden change is lost two adjacent A of the 2975-2978 position that is arranged in the OTOF gene coding region and G base, make the amino acid coding frameshit take place from the 993rd, amino acid code at the 1000th becomes terminator codon, and sudden change the back gene expression product takes place become 999 amino acid peptide chains by normal 1997 amino acid.The amino acid peptide chain that blocks starts regulation mechanism in the body, makes the mRNA degraded of variation.

Detect the method for any check point sudden change of this available this area that suddenlys change and carry out, for example PCR (polymerase chain reaction)-sequencing, adopt the OTOF gene DNA probe hybridization method of mark or with method of restriction fragment length polymorphism method or sequence specific primers or the like.

In one embodiment of the invention, adopt pcr amplification-direct sequencing to detect sample, specifically comprise the steps:

1) sample of collection individuality to be measured, for example blood, body fluid or tissue extract genomic dna;

2) be template with this DNA,, obtain pcr amplification product to carry out the PCR reaction near the PCR primer that designs OTOF gene or the OTOF coding region 2975-2978 bit base;

3) the PCR product that obtains is carried out direct sequencing analysis, the sequence of resulting sequence and OTOF normal gene is compared, determine whether to exist the OTOF mutational site.

4) judge according to above result whether individuality to be measured is the auditory nerve disease that OTOF transgenation 2975-2978delAG causes.

Further, this method can optionally comprise the steps:

5) translate to determine whether amino acid exists the coding from 1000 of the 993rd frameshit to the to stop changing by the normal reading frame.

In the experiment that the contriver carries out, collect various phonosensitive nerve deafness patients by deaf sick outpatient service and resource acquisition network, comprise the auditory nerve patient, set up resources bank.Under the voluntary prerequisite of patient, signature informed consent postscript is left and taken the 5-10ml blood sample, and is set up the patient medical history database, incidence in detail record conditions of patients, the family and contact method.Then, use the extractive method extraction of phenol chloroform genomic dna, quantitatively put in storage the back ,-20 ℃ of preservations, and every part of DNA sample is all in detail corresponding to the patient clinical data of registering.Then, use online primer-design software Primer3 design primer, comprise the whole coding region of OTOF, use pcr amplification.The PCR product directly checks order: sequencing primer is identical with the pcr amplification primer, and the ABI 3730DNA of company sequenator is used in forward and reverse order-checking.Sequence that obtains and the sequence among the Genbank (accession number: NC_000002) relatively determine the OTOF mutational site.Translate to determine the mutational site of OTOF by the normal reading frame.

Above-mentioned steps 2 resulting PCR reaction product can also detect with hybridization probe, and used hybridization probe can be and normal OTOF nucleotide sequence hybridization, or with the nucleotide sequence hybridization of sudden change, or with their probe of complementary sequence hybridization.These probes can be used radio isotope, chromonic material or fluorescent substance mark, especially can utilize the allele specific probe, and also whether the method examination of cutting of available constraints enzyme exists the sudden change that has been determined.

Therefore, the reagent of detection pcr amplification product is selected from order-checking detection reagent, digestion with restriction enzyme detection reagent, restricted length polymorphism detection reagent, sequence specific primers detection reagent and probe hybridization detection reagent.The PCR primer that uses in aforesaid method can be generally 15～30 bases according to known nucleotide sequence design, and GC content is about 45～50%, combines with terminal specificity under suitable temperature, and it can utilize special computer programming.In a specific embodiment of the present invention, to use online primer-design software primer 3.0 and designed one couple of PCR primers, its sequence is:

Upstream primer: OTOF-25F:5 '-TGCCTTCCCACCCGTCAG-3 ' (nt82540-nt82557)

Downstream primer: OTOF-25R:5 '-AGCCCGTAGCCTTTCCAG-3 ' (nt82962-nt82979)

The standard sequence of OTOF normal gene can reference example such as Genbank:NC_000002.

The test kit that is used for detecting OTOF gene 2975-2978delAG sudden change can comprise following reagent:

Be used near the PCR primer of amplified sample DNA OTOF gene or OTOF gene coding region 2975-2978 bit base; And following one or more combination of agents:

From testing sample, extract the reagent of DNA;

The PCR reaction reagent;

The reagent that pcr amplification product is directly checked order.

For example, a test kit that detects OTOF gene 2975-2978delAG sudden change is provided in one embodiment of the invention, be equipped with in the container in order to detect the composition of OTOF gene 2975-2978delAG sudden change, what provide simultaneously with it can be through manufacturing, use and the marketing information of the audit of medication management mechanism of government, relevant medicine or biological products.For example, after adopting pcr amplification, the direct test kit in OTOF gene 2975-2978delAG mutational site in the test sample can contain amplimer, dNTPs, be used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid or the required reagent of sequencing reaction etc.It is known to those skilled in the art that above component only is that schematically for example, described primer can adopt above-mentioned pair of O TOF-25F and OTOF-25R primer, the archaeal dna polymerase of the described PCR of being used for reaction is the enzyme that can be used in pcr amplification.

The using method of described test kit mainly comprises the steps:

(1) DNA of extraction blood sample to be measured utilizes above-mentioned pair of O TOF-25F and OTOF-25R primer, carries out the PCR reaction, obtains the PCR reaction product;

(2) directly order-checking behind the PCR reaction product purifying is with the sequence of gained and the existence in the relatively more definite mutational site of the standard sequence among the Genbank;

Described step also can further comprise:

(3) translate to determine whether to exist termination change by the normal reading frame from frameshit to the 1000 amino acids of the 993rd amino acids.

This test kit can detect the OTOF mutational site quickly and easily, thereby is applied in the detection and diagnosis or methods of treatment of the sick genes involved of auditory nerve.

The present invention also provides the OTOF mutator gene in diagnosis or treat application in the human auditory nerve disease.By detecting from whether existing OTOF gene 2975-2978delAG sudden change to judge this patient's sick occurrence cause of auditory nerve and type in patient's the sample to be tested, and then provide foundation for clinical diagnosis and treatment; In addition; aspect further clinical treatment,, normal gene can be imported cell and the expression therein that carry mutator gene detecting to after OTOF gene 2975-2978delAG sudden change has taken place; it can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.

Whether the present invention proposes by detecting among the patient exists the new sudden change of OTOF gene to diagnose the detection method of pathogenetic reason of auditory nerve and type, this will help being avoided the artificial cochlea to implant invalid and unnecessary financial loss that cause for dissimilar auditory nerve patients determine the individualized treatment scheme.

Below in conjunction with accompanying drawing,, describe in detail but do not limit the present invention by explanation to better embodiment of the present invention.

Brief description of drawings

Fig. 1 is OTOF gene coding region Nucleotide and amino acid whose contrast: sudden change is arranged in OTOF gene 2975-2978 two adjacent A in position and G base, enclosing what come with square frame is the base and the corresponding amino acid of sudden change, base sequence after the sudden change makes amino acid from 993 generation frameshit (amino acid of underscore part), and premature termination is in the 1000th amino acids (" * " mark);

Fig. 2 is a PCR reaction process synoptic diagram in the inventive method, shows temperature of reaction and time;

Fig. 3 is in the inventive method, and the PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis, there is shown the fragment position of quantitative Marker;

Fig. 4 is OTOF gene sequencing result in the inventive method, and the top is heterozygous mutant sequence (patient's sequence), and the centre is wild-type sequence (father's patient sequence), and following orientation heterozygous mutant sequence (mother's patient sequence) square frame shows the position, mutational site;

The embodiment of invention

The used test materials of the present invention if no special instructions, is commercially available purchase product.

Blood sample to be measured extracts the pcr amplification with the OTOF gene coding region

[embodiment 1]

One, the preparation of object blood sample DNA to be measured

1, research object

The hearing normal control that 76 examples is distributed non-syndrome type auditory nerve patient and 92 no family histories carries out the examination of OTOF gene according to following method.Among the 76 routine patients, 1 routine patient not only has the unusual auditory function feature of auditory nerve disease, show identification that speech when slight change of body temperature, occurs simultaneously, distinguish and the variation of hearing threshold value, often be diagnosed as temperature sensitivity auditory nerve disease after the auditory function inspection of clinical observation and system for a long time.The compound heterozygote of patient for 2975-2978delAG sudden change and another sudden change found in this routine temperature sensitivity auditory nerve patient's OTOF gene test.Mother of patient is the carrier of simple 2975-2978delAG sudden change, and father is the carrier of another kind of sudden change.Do not find 2975-2978delAG sudden change person in the examination to 92 normal persons of hearing.

To all its medical history of participator's probe and family history, and it is carried out a medical examination, the otology inspection comprises otoscopy, audiological evaluation.Everyone blood sample collection 5～10ml after the signature Informed Consent Form.

2, extracting genome DNA

Adopt the phenol chloroform extraction method.

First day

1) anticoagulation is done 1 times of dilution with PBS.

2) the lymph parting liquid (18 ℃～28 ℃) of 2 times of volumes of adding in centrifuge tube is spread the blood that 1 times of volume of one deck has diluted, room temperature, 1000 * g, centrifugal 20 minutes above.

3) supernatant liquor is abandoned in suction, and karyocyte layer in the middle of the careful sucking-off changes in the 5ml Ep pipe, and 5000 * g centrifugal 10 minutes, washes once with PBS then.5000 * g, centrifugal 10 minutes.

4) cell is suspended from 2ml TE damping fluid (10mM Tris.HCl, 1mM EDTA, pH8.0) in, add 10% SDS (dodecyl sulphate) to final concentration 0.5% (100 μ l), protein kinase k 100～200 μ g/ml (10mg/ml), 50 ℃ of water-baths 3～5 hours.

5) use the phenol chloroform extraction.With isopyknic saturated phenol, add mixing, 5000 * g, centrifugal 10 minutes.

6) suct clear liquid to new centrifuge tube, abandon lower sediment, add equal-volume phenol: chloroform mixture, mixing, 5000 * g, centrifugal 10 minutes.

7) suct clear liquid to new centrifuge tube, abandon lower sediment, add the equal-volume chloroform: iso pentane alcohol mixture, mixing, 5000 * g, centrifugal 10 minutes.

8) suct clear liquid to new centrifuge tube, abandon lower sediment, add the sodium acetate of 1/10 volume, mixing.

9) dehydrated alcohol of 2.5 times of volumes of adding.

10)-20 a ℃ deposit D NA spends the night.

Second day

11) high speed centrifugation, 10000 * g, 10 minutes, 4 ℃.

12) abandon supernatant liquor, add 75% ethanol 2ml, high speed centrifugation 10000 * g, 5 minutes

13) abandon supernatant liquor, dry up.

14) with TE damping fluid dissolving (200 μ l TE/5ml whole bloods, 400TE/10ml whole blood).

15) packing.1% agarose electrophoresis and spectrophotometer are quantitative.

Two, the pcr amplification of OTOF gene coding region

1, primer sequence

Upstream primer: OTOF-25F:5 '-TGCCTTCCCACCCGTCAG-3 ' (nt82540-nt82557)

Downstream primer: OTOF-25R:5 '-AGCCCGTAGCCTTTCCAG-3 ' (nt82962-nt82979)

Annotate: OTOF gene order searching number: NC_000002

2, the foundation (table 1) of PCR reaction system

The PCR reaction system of table 1OTOF gene

Wherein, damping fluid, general T aq enzyme are buied from precious Biogen, Inc., and dNTP buys from precious biotech firm, and primer is given birth to worker company by Shanghai and synthesized.

Reaction conditions: PCR is reflected on ABI company 9700 thermal cyclers and carries out, and reaction process (comprising temperature and time) as shown in Figure 2.

The purifying of OTOF gene coding region pcr amplification product and quantitative

[embodiment 2]

One, the purifying of PCR product---96 well plate method

1, in 96 orifice plates that the PCR product is housed, adds 50 μ l sterilized waters, mixing.

2, it is transferred in the Millipore purifying plate, be put on the vacuum pump suction filtration about 3 minutes, see in the purifying plate not having water to get final product.

3, the deionized water that adds 50 μ l in the purifying plate once more continues suction filtration, in the purifying plate, do not have water till.

4, the purifying plate is taken off from vacuum pump, in plate, add the deionized water of 20 μ l, static 15 minutes, shook again 15 minutes, be drawn onto then in new 96 orifice plates.

5, be kept in-20 ℃ of refrigerators.

Two, electrophoresis is quantitative

1, sample is prepared

Get one 96 hole point templates, every hole adds sample damping fluid 6 μ l earlier, and from the chamber plate that the PCR product is housed, PCR product (2 μ l) is shifted out with the volley of rifle fire in every hole, transfers on the point template, mixing, centrifugal (chamber plate hole number corresponding one by one with 96 hole point templates).

2, flow process

1) joins glue (0.8% agarose): take by weighing the 2.4g agarose, be suspended in (500ml Erlenmeyer flask) among 300ml 0.5 * TBE.

2) colloidal sol: high fire is heated to and boils in the microwave oven, and constantly boiling number minute is noted not boiling, and takes out mixing therebetween.

3) cool glue: treat that glue dissolves fully, from microwave oven, take out that cool to about 60 ℃, (about 10 μ l 10mg/ml), shake up to add 1 EB.

4) shop glue: obturage with adhesive plaster in dull and stereotyped two ends, the 250ml glue is all poured into flat board, inserts comb scale.

5) gluing: flat board is put in the electrophoresis chamber that fills electrophoresis liquid (0.5 * TBE, liquid level apart from glue face 1 to 2mm), pulled up comb scale.

6) application of sample:, add the DNA standard substance with single rifle at last with volley of rifle fire form application of sample in accordance with regulations.

7) walk glue: cover the electrophoresis chamber lid, check positive and negative level, open electrophoresis apparatus, regulate electrophoretic voltage.

8) quantitative: as to walk from well 1.5 to 2cm places when tetrabromophenol sulfonphthalein, close electrophoresis apparatus, carefully get glue, put into pickup camera and take a picture, carry out quantitatively.

Quantitatively marker is DL 2000, as shown in Figure 3, through behind the electrophoresis, 6 bands is arranged as seen, and fragment length is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp, the total concn of DL2000 is 300ng/5 μ l.Get 5 μ l DL2000 during electrophoresis, therefore the content of every band is 50ng.Get 3 μ l (PCR product)+5 μ l (sample-loading buffer) during PCR product electrophoresis and carry out electrophoresis.The content of relatively judging the PCR product according to the gray-scale value of gray-scale value behind the PCR product electrophoresis and DL2000.

The direct order-checking of the OTOF gene coding region pcr amplification product of purifying

[embodiment 3]

One, the purity of PCR product D NA template and consumption requirement

DNA purity: OD ₂₆₀/ OD ₂₈₀=1.6～2.0.

DNA concentration: PCR product 10ng/ μ l.

The DNA consumption:

The PCR product

100-200bp 1-3ng

200-500bp 3-10ng

500-1000bp 5-20ng

1000-2000bp 10-40ng

＞2000bp 40-100ng

Two, sequencing reaction

1, the required reagent of sequencing reaction should be fresh preparation, need can use after autoclaved reagent must be sterilized.The required equipment of sequencing reaction (first-class as 384 orifice plates, tip) should be cleaning sterile equally.

2, in order to guarantee the fresh of sample and reaction reagent that check order, should be during application of sample in operation on ice.

3, present reaction system is 5 μ l, and all ingredients add-on is as shown in table 2.

The sequencing reaction system of table 2OTOF gene PCR amplified production

* BigDye 3.1 is a kind of fluorescence dye that is used for sequencing reaction of u.s.a. applied biosystem company (ABI) production.

4, sample is put on the PCR instrument, and the process of the reaction of doing sees Table 3.

The sequencing reaction process of table 3OTOF gene PCR amplified production

Step	Effect
		1	96℃，2min.
2	Repeat 30 circulations of following process: ● 96 ℃, 10sec; ● 50 ℃, 5sec; ● 60 ℃, 4min.
		3	Remain on 4 ℃, up to purifying

5, the sample that has reacted will in time take off from the PCR instrument, and the sample that will carry out purifying in the short period of time is positioned in 4 ℃ of refrigerators, surpass more than one day could purifying sample to be positioned over-20 ℃ of refrigerators freezing.

Three, the purifying of sequencing reaction thing and order-checking

1, in every hole, adds 20 μ l, 80% ethanol, 4, the centrifugal 30min of 000rpm; Sample panel is placed on the paper handkerchief of rolling well, gets rid of in whizzer, speed can not surpass 1,000rpm when getting rid of;

2, in every hole, add 30 μ l, 70% ethanol, 4, the centrifugal 10min of 000rpm gets rid of;

3, repeat the operation in the 2nd step;

4, repeat the operation in the 2nd step;

5, sample panel is put in the clean drawer the dry 30min of lucifuge;

6, add 5 μ l methane amides, the envelope film is in the centrifugal being placed on-20 ℃ refrigerator;

7, go up the preceding 95 ℃ of sex change of machine 5 minutes, placed 2 minutes on ice, sample is gone up in centrifugal back.

Sequencer map as shown in Figure 4.

Detect deaf-related gene OTOF---2975-2978delAG mutational site test kit and application thereof

[embodiment 4]

1, the composition of test kit

(1) amplification primers:

Upstream primer: OTOF-25F:5 '-TGCCTTCCCACCCGTCAG-3 ' (nt82540-nt82557)

Downstream primer: OTOF-25R:5 '-AGCCCGTAGCCTTTCCAG-3 ' (nt82962-nt82979)

Annotate: OTOF gene order searching number: NC_000002

(2) pcr amplification Taq enzyme 5U/ μ l

(3) 10 * damping fluids (containing 15ml MgCl2)

(4)dNTP 2mM

(5)Big-Dye mix

2, using method

Mainly comprise the steps:

1) pcr amplification

With the coding region of 3 pairs of OTOF genes of software Primer design PCR primer, reaction conditions is 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 30 seconds, 61 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, 30 circulations, after reaction finishes again 72 ℃ extended 4 ℃ of preservations (Fig. 2) 7 minutes.

2) PCR product purification

The M μ ltiScreen-PCR plate that will contain the PCR product vacuumizes, and adds deionized water, leaves standstill, and M μ ltiScreen-PCR plate is placed on the mixing tank shake subsequently, is transferred in 96 orifice plates of another cleaning after the PCR product behind the purifying dissolves again.

3) sequencing reaction and checking

Carry out sequencing reaction with the PCR primer as sequencing primer, on the ABI9700 thermal cycler, carry out sequencing reaction.After reaction finished, extension products was splined on ABI PRISM 3730DNA sequenator.Resulting order-checking collection of illustrative plates is analyzed, with the standard sequence comparison of NC_000002 to determine whether the mutational site exists.

Concrete grammar is referring to embodiment 1,2,3,4.

Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.

Claims (6)

1, a kind of test kit that is used to detect the 2975-2978delAG that be positioned at OTOF gene (X1000) sudden change sick relevant with auditory nerve, described test kit comprise the increase reagent of OTOF gene of the reagent that is used to detect the 2975-2978delAG sudden change that is positioned at the OTOF gene and being used to of choosing wantonly.

2, test kit according to claim 1, described test kit comprises following combination of agents:

From testing sample, extract the reagent of DNA;

Be used for amplified sample DNA and comprise segmental PCR primer of OTOF gene 2975-2978 bit base and corresponding PCR reaction reagent;

Detect the reagent of pcr amplification product.

3, test kit as claimed in claim 2, wherein said PCR primer is:

OTOF-25F：5’-TGCCTTCCCACCCGTCAG-3’

OTOF-25R：5’-AGCCCGTAGCCTTTCCAG-3’。

4, test kit as claimed in claim 2, the reagent of wherein said detection pcr amplification product are selected from order-checking detection reagent, digestion with restriction enzyme detection reagent, restricted length polymorphism detection reagent, sequence specific primers detection reagent and probe hybridization detection reagent.

5, the step that test kit according to claim 1, described test kit are used to detect the 2975-2978delAG sudden change that is positioned at the OTOF gene comprises:

1) blood, body fluid or the tissue samples of collection individuality to be measured extract DNA;

2) be template with this DNA, carry out the PCR reaction, obtain the PCR reaction product with following PCR primer;

OTOF-25F：5’-TGCCTTCCCACCCGTCAG-3’

OTOF-25R：5’-AGCCCGTAGCCTTTCCAG-3’

3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and OTOF normal gene is compared, determine whether to exist the 2975-2978delAG mutational site of OTOF gene.

6, as test kit as described in the claim 5, described step also further comprises:

4) translate determining that the 2975-2978delAG sudden change makes aminoacid sequence from 993 frameshit take place and changes according to the normal reading framework, and make amino acid whose coding premature termination in the 1000th amino acid.

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