CN101768637B - Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof - Google Patents

Abstract

The invention provides a kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T related to maternally inherited drug-induced deafness and a using method thereof. The kit comprises a reagent for extracting sample genomic DNA, a PCR amplification reactive reagent, a primer mixed liquor, a positive control template and a negative control template. The using method of the kit mainly comprises the following steps: using blood, hair with follicle, oral mucosa doctor blade, saliva, and the like, as a sample; adopting a proteinase K digestion pyrolysis method to extract genomic DNA; and then simultaneously detecting mutations in A1555G and C1494T by multiple allele specific PCR. The kit is used for detecting the mutations in mitochondria DNA A1555G and C1494T related to maternally inherited drug-induced deafness and is more rapid, economical and simpler than the single detection for mutation in A1555G or C1494T, and the kit has low requirements for equipment and environment and is conducive to promotion and application.

Description

The test kit and the method for use thereof that are used for while detection line mitochondrial DNA A1555G and C1494T sudden change

Technical field

The present invention relates to a kind of test kit and method of use thereof that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change with the matrilinear inheritance drug induced deafness.

Background technology

Mitochondrial DNA (Mitochondria DNA, mtDNA) sudden change is one of major reason that causes phonosensitive nerve deafness, these sudden changes mainly are positioned on plastosome 12S rRNA and the tRNA gene.Be positioned at homogeneity A1555G and the C1494T sudden change and aminoglycoside antibiotics (Aminoglycoside Antibiotics of 12SrRNA gene; AmAn) deaf relevant (the Prezant TR etal. of the non-syndrome type that causes; Nat Genet, 1993,4:259-294; Guan MX, Ann N Y Acad Sci, 2004,1011:259-271; Fischel Ghodsian, Pharmacogenomics, 2005,6:27-36).Although mtDNAA1555G or C1494T sudden change itself can cause deafness, most A1555G or C1494T carriers of mutation the hearing irreversible loss occurs after having used AmAn (like Streptomycin sulphate, qingfengmeisu qiong, kantlex, tobramycin, micronomicin etc.).Though there was the microbiotic of many novel powerfuls constantly to come out in recent years; But characteristics such as AmAn is cheap with it, has a broad antifungal spectrum, germicidal action are fast; Still be widely used clinically (especially for the treatment gram positive bacterial infection), its consequence is in deaf crowd, to occupy higher ratio by the deaf case that AmAn causes.The individuality that carries these two sudden changes is extremely sensitive to AmAn, causes common clinically " pin causes deaf " phenomenon, is acquired deaf major reason (Fischel-Ghodsian N, Hum Mutat, 1999, the 13:261-270 that the takes place day after tomorrow; Guan MX, Ann N Y AcadSci, 2004,1011:259-271; Van Camp G et al., C lin Genet, 2000,57:409-414.), especially at China remote countryside area, this situation ubiquity.At present; Only the matrilineal inherited deafness family of the mtDNAA1555G of Zhejiang area discovery or C1494T sudden change is above 80; 1 carriers of mutation of average discovery; Just can carry out early intervention, avoid the use of aminoglycoside antibiotics medicine in this crowd, can effectively reduce the generation of drug induced deafness about 17 maternal members that do not fall ill in its family.Therefore, in high risk population and Susceptible population, carry out the examination of mtDNA12S rRNA transgenation, for the important role that has of prevention and control matrilinear inheritance drug induced deafness.

Since nineteen ninety-three; Both at home and abroad to the detection of mtDNA A1555G sudden change except PCR directly checks order; Widespread usage PCR-restriction fragment length polymorphism (PCR-RFLP) technology identifies that used restriction enzyme is Alw26 I (BsmA I) (Fischel Ghodsian et al., Am J Otolaryngol; 1993,14:399-403; Fischel Ghodsian et al., Am J Otolaryngol, 1997b, 18:173-178).Since the relation of finding mtDNA C1494T sudden change and drug induced deafness, the detection method in this mutational site mainly still sequencing (Guan MX, Ann N Y Acad Sci, 2004,1011:259-271).Direct sequencing is a golden standard of identifying sudden change, but this complex operation, expensive has limited its widespread use.Though it is convenient and swift that enzyme is cut evaluation, reliable results, insufficient Alw26 I (BsmA I) enzyme price is more expensive, compares with Restriction Enzyme commonly used, and price variance reaches 50～100 times.

In recent years; Domestic improvement PCR-RFLP technology and the fluorescent quantitative PCR technique reported in succession; To satisfy the needs that mtDNAA1555G and C1494T are suddenlyd change and carry out extensive examination; " detection method and the test kit thereof of 1555 A of matrilineal inherited deafness chondriogen → G sudden change " (publication number: CN1490415) like people such as Dai Piao research and development; " being used to probe that detects matrilinear inheritance chondriosome deafness gene A 1555 G and uses thereof " (publication number: CN1987462), " being used to Taqman MGB probe that detects matrilinear inheritance chondriosome deafness gene C 1494 T mutation and uses thereof " (publication number: CN1987463).Though these detection methods have reduced cost to a certain extent, operation steps is easy not enough, can not detect mtDNAA1555G and C1494T sudden change simultaneously; To some degree, testing cost still is higher, and this is still a no small spending concerning the people of many families Ji difficulty.Crystalline substance

the hereditary hearing impairment gene diagnosis chip test kit of rich biological autonomous innovation research and development difficult to understand be for quick, the known hereditary hearing impairment gene mutation site of high-throughput examination is custom-designed; Adopted the principle of gene chip; Detect 4 deaf-related gene (GJB2 simultaneously; GJB3, SLC26A4 and 12S rRNA) 9 mutantional hotspots.But the gene chip kit detection relates to specific apparatus and the bulk data Treatment Analysis is had relatively high expectations to laboratory equipment, environment and operator, and expensive, is difficult to promote the use of in general hospital or laboratory.Traditionally, the method that obtains the deafness patient genomic dna is from blood, to extract, and everyone approximately needs 3～5ml.This blood-sampling method has brought some painful and injuries to a lot of examinees especially infant, and when trans-regional transfer samples, has certain risk and trouble.Therefore; Pressing for does not a kind ofly have the method for obtaining the deafness patient genomic dna and a kind of quick, easy, accurate, economic drug induced deafness associated gene mutation detection method of injury, no pain to human body, to satisfy the needs that mtDNA A1555G and C1494T are suddenlyd change and carry out extensive examination.

Summary of the invention

To above-mentioned shortcoming, the present invention provides a kind of test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant with the matrilinear inheritance drug induced deafness and C1494T sudden change, and this test kit comprises:

(1) reagent of extraction sample genomic dna;

(2) pcr amplification reaction reagent comprises dNTP, 10 * PCR damping fluid, MgCl ₂, Taq enzyme and tri-distilled water;

(3) two pipe primers, wherein a pipe is the mixed solution of primer C1, N1, N2 and C2, another pipe is the mixed solution of primer C1, M1, M2 and C2;

Said two pipe primers are according to the human mtdna sequences Design shown in the SEQ ID NO:7; Article one, primer sequence N1 is an antisense strand; Being the downstream universal primer, is the Nucleotide 1494-1516 of Mitochondrial DNA, and its sequence is the SEQ ID NO:1 in the sequence table; Second primer sequence M1 is an antisense strand, is the downstream universal primer, is the Nucleotide 1494-1516 of Mitochondrial DNA, and wherein the G in 1494 sites is replaced into A, and its sequence is the SEQ ID NO:2 in the sequence table; Article three, primer sequence C1 is a sense strand, is upper reaches universal primer, is the Nucleotide 1030-1052 of Mitochondrial DNA, and its sequence is the SEQID NO:3 in the sequence table; Article four, primer sequence N2 is a sense strand, is upper reaches universal primer, is the Nucleotide 1533-1555 of Mitochondrial DNA, and its sequence is the SEQ ID NO:4 in the sequence table; Article five, primer sequence M2 is a sense strand, is upper reaches universal primer, is the Nucleotide 1533-1555 of Mitochondrial DNA, and wherein the A in 1555 sites is replaced into G, and its sequence is the SEQ ID NO:5 in the sequence table; Article six, primer sequence C2 is an antisense strand, is the downstream universal primer, is the Nucleotide 2214-2235 of Mitochondrial DNA, and its sequence is the SEQ ID NO:6 in the sequence table;

(4) carry A1555G sudden change the positive control template, carry the positive control template and the negative control template of C1494T sudden change.

In above-mentioned primer, 3 ' end of primer N1 and M1 is positioned at 1494 sites of Mitochondrial DNA, corresponds respectively to 1494 site wild-type G and mutant A; 3 ' end of primer N2 and M2 is positioned at 1555 sites of Mitochondrial DNA, corresponds respectively to 1555 site wild-type A and mutant G (with reference to Fig. 1).

In above-mentioned primer; Introducing sulfuration phosphoric acid between first of every primer 3 ' end and second bit base modifies; And 3 of primer N1, M1, N2 and M2 ' end the 4th bit base is introduced mispairing; Promptly be replaced into A corresponding to mitochondrial 1497 G, 1552 G is replaced into A, to improve the specificity of extension.

In above-mentioned primer, N1 and C1 pairing is used to increase and contains the mitochondria DNA fragment in the normal site of C1494C, and N2 and C2 match to be used to increase and contain the mitochondria DNA fragment in the normal site of A1555A; M1 and C1 pairing is used to increase and contains the mitochondria DNA fragment in C1494T mutational site, and M2 and C2 match to be used to increase and contain the mitochondria DNA fragment in A1555G mutational site; Primer C1 and C2 pairing are used to generate a constant amplified band, as the molecule internal control index of PCR reaction.

In the reagent of said extracted sample genomic dna, comprise cell pyrolysis liquid and solution I, the staple of said solution I is a Proteinase K.In above-mentioned pcr amplification reaction reagent, add DMSO 99.8MIN., to improve the extension specificity.

The present invention also provides the method for use of mentioned reagent box, and this method of use may further comprise the steps:

(1) extraction of genomic dna: use the reagent of above-mentioned extraction sample genomic dna,, from tested sample, extract genomic dna through the protease K digesting cracking process;

(2) multiple allele-specific pcr amplification (Multiplex Allele-specific PCR, MASPCR): use above-mentioned pcr amplification reaction reagent and two pipe primers, the genomic dna of sample is carried out the MASPCR amplification; The present invention combines allele-specific round pcr and multiple PCR technique; Every part of dna sample is that primer C1, N1 and N2, C2 and primer C1, M1 and M2, C2 carry out the MASPCR amplification in two PCR reaction tubess with two pairs of allele-specific primerses respectively, just can in 2～3 hours, detect C1494T sudden change and A1555G sudden change simultaneously through a PCR.

(3) sudden change detects: agarose gel electrophoresis detects, the dna fragmentation quantity and the dna fragmentation size that directly produce according to pcr amplification, and whether the detection line mitochondrial DNA A1555G sudden change and C1494T sudden change take place simultaneously.

In the extraction step of above-mentioned genomic dna, can be from 20～200 μ l blood samples, 1cm ²Hair, oral mucosa scraping blade or the saliva sample of blood filter paper, band hair follicle in the rapid extraction genomic dna, different according to sampling mode or tested sample form, different samples are carried out corresponding pre-treatment.

(1) gets 20～200 μ l blood sample or 1cm ²The blood filter paper put into 1.5ml EP pipe (Eppendorf pipe), add 900 μ l erythrocyte splitting damping fluid (20mmol/L Tris-HCl, pH 7.6) room temperatures and leave standstill 10min, abundant splitting erythrocyte, the centrifugal 1min of 12000rpm collects white corpuscle and precipitates;

(2) wash the hair 1 time of band hair follicle with 70% ethanol, after use the distilled water flushing hair again 2 times.In 1.5ml EP pipe, put into 2～4 hairs respectively, hair follicle places EP pipe bottom, cuts off the part that hair is higher than the EP pipe with clean scissors;

(3) before the collection saliva, must let the person of being collected gargle.To remove materials such as aged cells too much in the oral cavity, swill, toothpaste, coffee, cigarette.Do not drink water in half a hour, feed, smoking, begin to collect oral mucosa scraping blade or saliva then, visual condition selects following method to collect saliva sample: 1. tongue is scraped around the oral cavity and is got mucomembranous cell, will about 0.1ml saliva directly tells in the EP pipe; 2. saline water is gargled, and tells in the EP pipe, draws PBS solution to the EP pipe that the saliva sample of collecting through above-mentioned any method is housed, and after the piping and druming, the centrifugal 5min of 12000rpm removes supernatant, repeats this process once repeatedly; 3. scrape the cheek inwall repeatedly 6 times with cotton swab, air drying carefully tears its outside surface off with the tweezers of sterilizing then, puts into the EP pipe; 4. saliva is told on filter paper, form salivary stain behind the air drying, be cut into the about 1cm of size with clean scissors again ²A scrap of paper places the EP pipe.

With cell pyrolysis liquid and Proteinase K above-mentioned sample is digested cracking then, salting-out process is removed impurity such as albumen again, and isopropanol precipitating DNA is subsequent use in-20 ℃ of preservations with autoclaving water or TE damping fluid dissolving back at last.

With 4 pairs of primers that the present invention designed; DNA with respective sample is a template respectively, the constant amplified band through obtaining the about 1206bp of size limpid in sight behind the pcr amplification respectively, corresponding to the 703bp electrophoretic band in 1555 sites and corresponding to the 487bp electrophoretic band in 1494 sites.Proof primer 3 ' end is introduced base mismatch and is carried out the efficient that thio-modification does not influence PCR.

When normal sample promptly do not carry plastosome A1555G and C1494T the sudden change genomic dna after two couples of primer C1, N1 and N2, C2 carry out MASPCR; Except producing a constant amplified band of 1206bp, also produce 2 and correspond respectively to the 703bp in 1555 sites, 1494 sites and the normal amplified band of 487bp; And behind two couples of C1, M1 and M2, C2 primer amplification, only produce the constant amplified band of a 1206bp, prove that this sample does not contain mtDNA A1555G sudden change and C1494T sudden change.When the genomic dna that carries mtDNAA1555G sudden change deafness patient after two couples of primer C1, N1 and N2, C2 carry out MASPCR, normal amplified band and constant amplified band of 1 487bp appearred; And after two couples of primer C1, M1 and M2, C2 amplification, sudden change amplified band and constant amplified band of 1 703bp appearred, proved that this sample carries mtDNA A1555G sudden change.When the genomic dna that carries mtDNA C1494T sudden change deafness patient after two couples of primer C1, N1 and N2, C2 carry out MASPCR, normal amplified band and constant amplified band of 1 703bp appearred; And after two couples of primer C1, M1 and M2, C2 amplification, sudden change amplified band and constant amplified band of 1 487bp appearred, proved that this sample carries mtDNA C1494T sudden change (with reference to Fig. 2).

In theory; Sample genomic dna is through a PCR reaction; Be only to add in the system just can detect to mutant site (1494T/1555G) primer C1, M1 and M2, C2; But we also use primer C1, N1 and N2, C2 to detect wild-type site (C1494/A1555), authenticate reverse experimental result simultaneously.That is to say that same sample genomic dna is realized bi-directional verification, thereby made detected result more accurate, credible through twice PCR.

Test kit of the present invention and method of use thereof have following characteristics:

1. the method that obtains the deafness patient genomic dna traditionally is from blood, to extract, and everyone approximately needs 3～5ml.This blood-sampling method has brought some painful and injuries to a lot of examinees especially infant, and when trans-regional transfer samples, has certain risk and trouble.Use test kit of the present invention, can from one bleed the liquid sample, the band hair follicle hair, oral mucosa scraping blade or saliva equal samples the rapid extraction genomic dna.This method has not only solved the problem of from deafness patient blood, extracting DNA traditionally, alleviates examinee's misery, but also has solved the problem of trans-regional transfer samples.Blood filter paper, hair and oral mucosa scraping blade (cotton swab) or salivary stain filter paper etc. can be placed in the envelope easily, and can be through the trans-regional transmission of mode of mailing.

2. primer used in the test kit of the present invention is all through careful design.At first, normally with mutation allele between different bases be designed in 3 of each bar primer ' end, thereby improved the specificity that increases greatly; The product that is obtained by primer C1 and C2 pairing amplification in addition can be used as the molecule internal control index of whole PCR reaction, thereby avoids the appearance of false negative result, improves the stability of detected result.Concentration through adjusting different primers and ratio and the PCR reaction conditions is optimized are with specificity and the stability of guaranteeing the result.

3. several kinds of matrilinear inheritance induced deafness gene testers are arranged at present both at home and abroad; Cut identification method, fluorescent quantitative PCR detection method, gene chip detection method etc. like direct sequencing, enzyme; Because himself defective, as waste time and energy, complex operation and testing cost be higher and be difficult for promoting clinical.Compare with these methods, test kit provided by the invention relates to multiple allele-specific round pcr, in conjunction with allele-specific round pcr and both advantages of multiple PCR technique.Every part of dna sample is that primer C1, N1 and N2, C2 and primer C1, M1 and M2, C2 carry out the MASPCR amplification in two PCR reaction tubess with two pairs of allele-specific primerses respectively, just can in 2～3 hours, detect C1494T sudden change and A1555G sudden change simultaneously through a PCR.

4. use test kit provided by the invention and carry out mtDNAA1555G and C1494T sudden change detection, cost is lower than other detection methods; Testing process is easy, quick, and interpretation is directly perceived as a result; Experimental installation and operator there is not particular requirement; Be adapted at general hospital, carry out in the molecular biosciences laboratory; Be convenient to the particularly extensive examination and the preventive inspection of low developed area implementation matrilinear inheritance drug induced deafness mtDNA A1555G and C1494T sudden change in China, effectively reduce the chance that drug induced deafness takes place the responsive crowd of aminoglycoside antibiotics.

Description of drawings

Fig. 1 is a multiple allele-specific PCR primer synoptic diagram of the present invention;

Fig. 2 is a multiple allele-specific pcr amplification scheme synoptic diagram of the present invention;

Fig. 3 is the matrilinear inheritance drug induced deafness family family tree that carries A1555G sudden change (WZD34 family) and C1494T sudden change (WZD102 family);

Fig. 4 is the 2% agarose gel electrophoresis figure that multiple allele-specific pcr amplification is identified mtDNA A1555G and C1494T sudden change.

Nomenclature

C1 is a upper reaches universal primer; N1 is the normal primer sequence, and 3 ' end is positioned at 1494, corresponding to 1494 site wild-type G, uses with the C1 pairing; M1 is the mutant primer sequence, and 3 ' end is positioned at 1494, corresponding to 1494 site mutation type A, uses with the C1 pairing; C2 is the downstream universal primer; N2 is the normal primer sequence, and 3 ' end is positioned at 1555, corresponding to 1555 site wild-type A, uses with the C2 pairing; M2 is the mutant primer sequence, and 3 ' end is positioned at 1555, corresponding to 1555 site mutation type G, uses with the C2 pairing; 1494C representes that 1494 sites of Mitochondrial DNA are wild-type C; 1494T representes that 1494 sites of Mitochondrial DNA are mutant T; 1555A representes that 1555 sites of Mitochondrial DNA are wild-type A; 1555G representes that 1555 sites of Mitochondrial DNA are mutant G; is that normal male is individual among the figure; Zero is normal female individual; ■ is the morbidity male individual; ● be the morbidity female individual;

is the propositus; * be aminoglycoside medicaments induced deafness patient; / be dead individuality; I is the first-generation member of this family; II is the s-generation member of this family; III is the third generation member of this family; A representes the electrophorogram of PC R amplification (C1, N1, N2, C2) product of wild-type site detection; B representes the electrophorogram of PCR (C1, M1, M2, the C2) amplified production that the mutant site is detected; Y1 is the propositus (WZD102-III-1) who carries the C1494T sudden change; Y2 is father propositus (WZD102-II-1) who carries the C1494T sudden change; Y3 is mother propositus (WZD102-II-2) who carries the C1494T sudden change; Y4 is the propositus (WZD34-III-2) who carries the A1555G sudden change; Y5 is father propositus (WZD34-II-1) who carries the A1555G sudden change; Y6 is mother propositus (WZD34-II-2) who carries the A1555G sudden change; The negative contrast template of Y7; Y8 is the positive control template of carrying the C1494T sudden change; Y9 is the positive control template of carrying the A1555G sudden change; Y10 is no template blank; YM is DL2000 DNA Marker.

Embodiment

Below in conjunction with nucleotide sequence table, accompanying drawing and specific embodiment the present invention is described further so that those skilled in the art can better understand the present invention and implementing, but the embodiment that lifts not conduct to qualification of the present invention.

Embodiment 1

1. design of primers

Use Primer 5.0 softwares and Oligo7.0 software assistance design improved primer; According to disclosed human mtdna Cambridge reference sequences (Human Mitochondrial DNA Revised CambridgeReference Sequence; The GenBank number of landing: NC_012920.1) or the SEQ ID NO:7 in the sequence table design, the primer design scheme is following:

(1) to mtDNA 1494 site; We design two length identical, 3 ' ends and 1494 site wild-type G and corresponding downstream primer N1 of 1494 site mutation type A and M1; Introduce mispairing at two allele-specific primerses 3 ' end the 4th bit base simultaneously; And 3 ' introduce sulfuration phosphoric acid between first of end and second bit base to modify, to strengthen specificity.The reverse primer N1 of the 1494 site wild-types of designing thus is mtDNA nt1494-nt1516, promptly 5 '-CCT TTG AAG TAT ACT TGA GAA G ^aG-3 ' (put for sulfuration phosphoric acid modifier bit at a place), wherein 1497 G is replaced into A, and this sequence is set at SEQ IDNO:1; The reverse primer M1 of 1494 site mutation types is mtDNA nt1494-nt1516, promptly 5 '-CCT TTGAAG TAT ACT TGA GAA G ^aA-3 ' (put for sulfuration phosphoric acid modifier bit at a place), wherein 1497 G is replaced into A, and the G in 1494 sites is replaced into A, and this sequence is set at SEQ ID NO:2.Forward primer C1 is a upper reaches universal primer, with N1 or M1 pairing use, is mtDNA nt1030-nt1052, i.e. GGCTTT AAC ATA TCT GAA CAC A ^aC (put for sulfuration phosphoric acid modifier bit at a place), this sequence is set at SEQ ID NO:3.

(2) to mtDNA 1555 sites; We design two length identical, 3 ' ends and 1555 site wild-type A and corresponding upstream primer N2 of 1555 site mutation type G and M2; Introduce mispairing at two allele-specific primerses 3 ' end the 4th bit base simultaneously; And 3 ' introduce sulfuration phosphoric acid between first of end and second bit base to modify, to strengthen specificity.The forward primer N2 of the 1555 site wild-types of designing thus is mtDNA nt1533-nt1555, promptly 5 '-CCT ACG CAT TTA TAT AGA GAA G ^aA-3 ' (put for sulfuration phosphoric acid modifier bit at a place), wherein 1552 G is replaced into A, and this sequence is set at SEQ ID NO:4; The forward primer M2 of 1555 site mutation types is mtDNA nt1533-nt1555, promptly 5 '-CCT ACG CAT TTA TAT AGA GAA G ^aG-3 ' (put for sulfuration phosphoric acid modifier bit at a place), wherein 1552 G is replaced into A, and the A in 1555 sites is replaced into G, and this sequence is set at SEQ ID NO:5.Reverse primer C2 is the downstream universal primer, with N2 or M2 pairing use, is mtDNA nt2214-nt2235, i.e. GGA TTT TTT AGG TAG TGG GTG ^aT (put for sulfuration phosphoric acid modifier bit at a place), this sequence is set at SEQ ID NO:6.

(3) introducing a size that is produced by C1 and C2 pairing is the constant amplified band of 1206bp, as the molecule internal control index of pcr amplification.

2. the test kit (100 person-portion) that is used for detecting simultaneously matrilinear inheritance drug induced deafness Mitochondrial DNA A1555G and C1494T sudden change comprises following component:

(1) extract the reagent of sample genomic dna: comprise cell pyrolysis liquid 50ml * 2 bottle and solution I 2ml, the staple of cell pyrolysis liquid is the Tris-Cl (pH8.0) of 10mmol/L, the EDTA (pH8.0) of 1mmol/L and the SDS of 0.1% (m/V); The staple of solution I is the Proteinase K of 20mg/ml;

(2) pcr amplification reaction reagent: comprise 2.5mM dNTP mixed solution 800 μ l, 10 * PCR damping fluid 1ml, 25mM MgCl ₂1ml, 5U/ μ l Taq enzyme 50 μ l, tri-distilled water 1ml * 4 pipes;

(3) primer 2 pipe: concentration is 10 μ M, every pipe 150 μ l; Wherein a pipe is primer C1, N1, N2 and C2 mixed solution, and another pipe is primer C1, M1, M2 and C2 mixed solution, and the primer volume ratio is C1: N1: N2: C2 is 1: 2: 2: 1, and C1: M1: M2: C2 is 1: 2: 2: 1;

(4) carry A1555G sudden change the positive control template, carry the positive control template and the negative control template of C1494T sudden change, the concentration of positive control template and negative control template is 20ng/ μ l, every pipe 300 μ l.The positive control template is the genomic dna that carries A1555G sudden change or C1494T sudden change behind the extraction purifying, and the negative control template is not carry the genomic dna of said mutation.

2. detected object

Select 1 matrilinear inheritance drug induced deafness family (WZD34 family) and 1 matrilinear inheritance drug induced deafness family (WZD102 family) of carrying the C1494T sudden change of carrying the A1555G sudden change.2 family comprehensive conditions are seen table 2, and its pedigree chart is seen Fig. 3.These two familys are matrilinear inheritance drug induced deafness family; All there is the patient to use aminoglycoside antibiotics in each family; Characteristics are that all fell ill early, deaf degree is heavy by aminoglycoside antibiotics if use for maternal member; Then the normal or deaf morbidity of hearing was late if do not use aminoglycoside antibiotics, and degree is light.These two family total numbers of persons are 50 people, and wherein deafness patient has 15 people.In the WZD34 family, it is 2 people that deafness patient receives the inspection number, and it is 1 people that the normal person of hearing receives the inspection number; In the WZD102 family, it is 2 people that deafness patient receives the inspection number, and it is 1 people (referring to table 2) that the normal person of hearing receives the inspection number.

3. the extraction of genomic dna

Obtain these 6 person under inspections' blood filter paper (bleeds) respectively, the blood filter paper is cut into the about 1cm of size with clean scissors ²A scrap of paper put into 1.5ml EP pipe (Eppendorf pipe), add 900 μ l erythrocyte splitting damping fluid (20mmol/L Tris-HCl, pH 7.6) room temperatures and leave standstill 10min, abundant splitting erythrocyte, the centrifugal 1min of 12000rpm collects white corpuscle and precipitates; , the sedimentary 1.5mlEP pipe of white corpuscle adds the cell pyrolysis liquid of 600 μ l and the solution I of 3 μ l, abundant mixing, 55 ℃ of constant temperature digestion cracking 2～3h in being housed; Treat that Digestive system reduces to room temperature, add 200 μ l precooling 5mol/L liquor kalii aceticis, abundant mixing, this moment visible significantly albumen precipitation, 12000rpm-4 ℃ of centrifugal 15min; Get about 700 μ l supernatants and add 600 μ l Virahols in a new 1.5mlEP manages, abundant mixing, behind-20 ℃ of ice bath 30min, the centrifugal 10min of 12000rpm collects the DNA deposition; Add 600 μ l washing with alcohol DNA deposition, the centrifugal 5min of 12000rpm abandons supernatant, treats behind the ethanol volatile dry subsequent use with autoclaving water or TE damping fluid dissolving-20 ℃ of preservations afterwards.

4. multiple allele-specific pcr amplification

6 primer sequences according to above-mentioned design; Obtain 4 pairs of primers through synthetic (entrusting biotech firm); Seized sample genomic dna with said extracted is a template, and the agarose gel electrophoresis that the positive control template that in test kit, provides and negative control template are carried out multiple allele-specific pcr amplification and 2% is identified.Every part of dna sample is that primer C1, N1 and N2, C2 and primer C1, M1 and M2, C2 carry out the MASPCR amplification in two PCR reaction tubess with two pairs of allele-specific primerses respectively; Wherein primer C1, N1, N2 and C2 pairing detect the wild-type site; Primer C1, M1, M2 and C2 pairing detect the mutant site; C1 and C2 can match and produce a constant amplified band, as the molecule internal control index of PCR reaction.

The multiple allele-specific pcr amplification reaction of table 1 system

Annotate: before the use, all ingredients is diluted to the concentration shown in the table.

The MASPCR loop parameter is 94 ℃ of preparatory sex change 5min, follows 94 ℃ of sex change 30s, 63 ℃ of annealing 30s; 72 ℃ are extended 30s, circulate 8 times, and 1 annealing temperature of every circulation reduces by 1 ℃; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; After the recirculation 30～35 times, extend 5min again in 72 ℃ at last.PCR product 5～10 μ l electrophoresis in 2% sepharose is observed and the photographic recording result on ultraviolet detector after EB dyeing.

5. experimental result

Can know according to Fig. 4; When the negative control template promptly do not carry plastosome A1555G and C1494T the sudden change genomic dna after two couples of primer C1, N1 and N2, C2 carry out MASPCR; Except producing a constant amplified band of 1206bp, also produce 2 and correspond respectively to the 703bp in 1555 sites, 1494 sites and the normal amplified band of 487bp (referring to Fig. 4 A swimming lane Y7); And after two couples of primer C1, M1 and M2, C2 amplification, only produce the constant amplified band (referring to Fig. 4 B swimming lane Y7) of a 1206bp, prove that this sample does not contain mtDNAA1555G sudden change and C1494T sudden change.

When the genomic dna of the positive control template of carrying plastosome C1494T sudden change after two couples of primer C1, N1 and N2, C2 carry out MASPCR; Except producing a constant amplified band of 1206bp, the normal amplified band (referring to Fig. 4 A swimming lane Y8) of 1 703bp has also appearred; And after two couples of primer C1, M1 and M2, C2 amplification, except producing a constant amplified band of 1206bp, the normal amplified band (referring to Fig. 4 B swimming lane Y8) of 1 487bp has appearred also.

When the genomic dna of the positive control template of carrying plastosome A1555G sudden change after two couples of primer C1, N1 and N2, C2 carry out MASPCR; Except producing a constant amplified band of 1206bp, the normal amplified band (referring to Fig. 4 A swimming lane Y9) of 1 487bp has also appearred; And after two couples of primer C1, M1 and M2, C2 amplification, except producing a constant amplified band of 1206bp, the normal amplified band (referring to Fig. 4 B swimming lane Y9) of 1 703bp has appearred also.

The propositus's of family WZD102 (WZD102-III-1) genomic dna a normal amplified band and the constant amplified band (referring to Fig. 4 A swimming lane Y1) of 1 703bp occurred after two couples of primer C1, N1 and N2, C2 carry out MASPCR; And after two couples of primer C1, M1 and M2, C2 amplification, a sudden change amplified band and the constant amplified band (referring to Fig. 4 B swimming lane Y1) of 1 487bp appearred, proved that this sample carries mtDNA C1494T sudden change.

The propositus of family WZD34 (WZD34-III-2) genomic dna a normal amplified band and the constant amplified band (referring to Fig. 4 A swimming lane Y4) of 1 487bp occurred after two couples of primer C1, N1 and N2, C2 carry out MASPCR; And after two couples of primer C1, M1 and M2, C2 amplification, a sudden change amplified band and the constant amplified band (referring to Fig. 4 B swimming lane Y4) of 1 703bp appearred, proved that this sample carries the mtDNAA1555G sudden change.

In like manner can get, A1555G sudden change and C1494T sudden change (referring to Fig. 4 A swimming lane Y2, Fig. 4 B swimming lane Y2) do not take place in propositus's (WZD102-III-1) father's (WZD102-II-1) mtDNA; Its mother (WZD102-II-2) then carries mtDNA C1494T sudden change (referring to Fig. 4 A swimming lane Y3, Fig. 4 B swimming lane Y3).A1555G sudden change and C1494T sudden change (referring to Fig. 4 A swimming lane Y5, Fig. 4 B swimming lane Y5) do not take place in propositus's (WZD34-III-2) father's (WZD34-II-1) mtDNA; Its mother (WZD34-II-2) then carries mtDNAA1555G sudden change (referring to Fig. 4 A swimming lane Y6, Fig. 4 B swimming lane Y6).

6. certificate authenticity

The individual mtDNA 12S rRNA gene of all examinees all passes through direct sequencing analysis; Sequencing result and multiple allele-specific pcr amplification qualification result match, and the reliability and stability according to test kit of the present invention and method of use detection line mitochondrial DNA A1555G and C1494T sudden change are described.

Table 22 a matrilinear inheritance drug induced deafness family situation and plastosome 1555 sites and 1494 site detected results

AmAn ¹: aminoglycoside antibiotics; MASPCR2: multiple allele-specific pcr amplification.

Embodiment 2

1. identical among the component that comprises of the test kit (100 person-portion) of vitro detection matrilinear inheritance drug induced deafness Mitochondrial DNA A1555G and C1494T sudden change and the embodiment 1.

2. detected object

Select 15 not have the related sporadic deafness patient sample of heredity, obtain the hair of these 15 person under inspections' band hair follicle respectively.

3. detection method

Before extracting the hair follicle cell genomic dna, need carry out corresponding pre-treatment to hair: send out (band hair follicle) 1 time with 70% ethanol scouring of wool, after use the distilled water flushing hair again 2 times; In 1.5ml EP pipe, put into 2～4 hairs respectively, hair follicle places EP pipe bottom, cuts off the part that hair is higher than test tube with clean scissors., the 1.5mlEP pipe of hair (band hair follicle) adds the cell pyrolysis liquid of 600 μ l and the solution I of 3 μ l in being housed, abundant mixing, 55 ℃ of constant temperature digestion cracking 2～3h; Treat that Digestive system reduces to room temperature, add 200 μ l precooling 5mol/L liquor kalii aceticis, abundant mixing, this moment visible significantly albumen precipitation, 12000rpm-4 ℃ of centrifugal 15min; Get about 700 μ l supernatants and add 600 μ l Virahols in a new 1.5mlEP manages, abundant mixing, behind-20 ℃ of ice bath 30min, the centrifugal 10min of 12000rpm collects the DNA deposition; Add 600 μ l washing with alcohol DNA deposition, the centrifugal 5min of 12000rpm abandons supernatant, treats behind the ethanol volatile dry subsequent use with autoclaving water or TE damping fluid dissolving-20 ℃ of preservations afterwards.

The DNA of said extracted that gets about 50ng is as the template of pcr amplification; And the positive control template that in test kit, provides and negative control template are carried out multiple allele-specific pcr amplification; Every part of dna sample is managed primers in two PCR reaction tubess with two respectively; One pipe detects the wild-type site for the mixed solution of primer C1, N1, N2 and C2, and another pipe detects the mutant site for primer C1, M1, M2 and C2 mixed solution.PCR reaction system and loop parameter are seen embodiment 1.PCR product 5～10 μ l electrophoresis in 2% sepharose is observed and the photographic recording result on ultraviolet detector after EB dyeing.

4. interpretation of result and checking

Behind multiple allele-specific pcr amplification, we detect 1 A1555G sudden change positive individuals in 15 deafness patients, do not detect C1494T sudden change positive individuals.Further the pcr amplification product with these samples carries out sequencing analysis, and sequencing result and multiple allele-specific pcr amplification result match, and the reliability and stability of using the inventive method detection line mitochondrial DNA A1555G and C1494T sudden change are described.

Multiple allele-specific round pcr and the enzyme that existing patent relates to are cut authentication method (PCR-RFLP technology), fluorescent quantitative PCR technique and biochip technology and are compared and have following advantage (seeing table 3).

Table 3 this patent and the contrast of other related patent U.S. Patent No. technical indicators

The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited thereto.Being equal to that the technician in present technique field is done on basis of the present invention substitutes or conversion, all within protection scope of the present invention.

Sequence table

SEQUENCE?LISTING

< 110>Wenzhou Medical College

< 120>be used for test kit and the method for use thereof that while detection line mitochondrial DNA A1555G and C1494T suddenly change

<160>7

<170>PatentIn?version?3.5

<210>1

<211>23

<212>DNA

< 213>artificial sequence

<220>

<221>mutation

<222>(20)..(20)

<220>

<221>modified-base

<222>(23)..(23)

<223>3-terminal?phosphorothioate-modified?nucleotide

<400>1

cctttgaagt?atacttgaga?agg 23

<210>2

<211>23

<212>DNA

< 213>artificial sequence

<220>

<221>mutation

<222>(20)..(20)

<220>

<221>modified-base

<222>(23)..(23)

<223>3-terminal?phosphorothioate-modified?nucleotide

<220>

<221>mutation

<222>(23)..(23)

<400>2

cctttgaagt?atacttgaga?aga 23

<210>3

<211>23

<212>DNA

< 213>artificial sequence

<220>

<221>modified-base

<222>(23)..(23)

<223>3-terminal?phosphorothioate-modified?nucleotide

<400>3

ggctttaaca?tatctgaaca?cac 23

<210>4

<211>23

<212>DNA

< 213>artificial sequence

<220>

<221>mutation

<222>(20)..(20)

<220>

<221>modified-base

<222>(23)..(23)

<223>3-terminal?phosphorothioate-modified?nucleotide

<400>4

cctacgcatt?tatatagaga?aga 23

<210>5

<211>23

<212>DNA

< 213>artificial sequence

<220>

<221>mutation

<222>(20)..(20)

<220>

<221>modified_base

<222>(23)..(23)

<223>3-terminal?phosphorothioate-modified?nucleotide

<220>

<221>mutation

<222>(23)..(23)

<400>5

cctacgcatt?tatatagaga?agg 23

<210>6

<211>22

<212>DNA

< 213>artificial sequence

<220>

<221>modified_base

<222>(22)..(22)

<223>3-terminal?phosphorothioate-modified?nucleotide

<400>6

ggatttttta?ggtagtgggt?gt 22

<210>7

<211>16569

<212>DNA

<213>Homo?sapiens

<220>

<221>misc_feature

<222>(3107)..(3107)

<223>n?is?a，c，g，or?t

<400>7

gatcacaggt?ctatcaccct?attaaccact?cacgggagct?ctccatgcat?ttggtatttt 60

cgtctggggg?gtatgcacgc?gatagcattg?cgagacgctg?gagccggagc?accctatgtc 120

gcagtatctg?tctttgattc?ctgcctcatc?ctattattta?tcgcacctac?gttcaatatt 180

acaggcgaac?atacttacta?aagtgtgtta?attaattaat?gcttgtagga?cataataata 240

acaattgaat?gtctgcacag?ccactttcca?cacagacatc?ataacaaaaa?atttccacca 300

aaccccccct?cccccgcttc?tggccacagc?acttaaacac?atctctgcca?aaccccaaaa 360

acaaagaacc?ctaacaccag?cctaaccaga?tttcaaattt?tatcttttgg?cggtatgcac 420

ttttaacagt?caccccccaa?ctaacacatt?attttcccct?cccactccca?tactactaat 480

ctcatcaata?caacccccgc?ccatcctacc?cagcacacac?acaccgctgc?taaccccata 540

ccccgaacca?accaaacccc?aaagacaccc?cccacagttt?atgtagctta?cctcctcaaa 600

gcaatacact?gaaaatgttt?agacgggctc?acatcacccc?ataaacaaat?aggtttggtc 660

ctagcctttc?tattagctct?tagtaagatt?acacatgcaa?gcatccccgt?tccagtgagt 720

tcaccctcta?aatcaccacg?atcaaaagga?acaagcatca?agcacgcagc?aatgcagctc 780

aaaacgctta?gcctagccac?acccccacgg?gaaacagcag?tgattaacct?ttagcaataa 840

acgaaagttt?aactaagcta?tactaacccc?agggttggtc?aatttcgtgc?cagccaccgc 900

ggtcacacga?ttaacccaag?tcaatagaag?ccggcgtaaa?gagtgtttta?gatcaccccc 960

tccccaataa?agctaaaact?cacctgagtt?gtaaaaaact?ccagttgaca?caaaatagac 1020

tacgaaagtg?gctttaacat?atctgaacac?acaatagcta?agacccaaac?tgggattaga 1080

taccccacta?tgcttagccc?taaacctcaa?cagttaaatc?aacaaaactg?ctcgccagaa 1140

cactacgagc?cacagcttaa?aactcaaagg?acctggcggt?gcttcatatc?cctctagagg 1200

agcctgttct?gtaatcgata?aaccccgatc?aacctcacca?cctcttgctc?agcctatata 1260

ccgccatctt?cagcaaaccc?tgatgaaggc?tacaaagtaa?gcgcaagtac?ccacgtaaag 1320

acgttaggtc?aaggtgtagc?ccatgaggtg?gcaagaaatg?ggctacattt?tctaccccag 1380

aaaactacga?tagcccttat?gaaacttaag?ggtcgaaggt?ggatttagca?gtaaactaag 1440

agtagagtgc?ttagttgaac?agggccctga?agcgcgtaca?caccgcccgt?caccctcctc 1500

aagtatactt?caaaggacat?ttaactaaaa?cccctacgca?tttatataga?ggagacaagt 1560

cgtaacatgg?taagtgtact?ggaaagtgca?cttggacgaa?ccagagtgta?gcttaacaca 1620

aagcacccaa?cttacactta?ggagatttca?acttaacttg?accgctctga?gctaaaccta 1680

gccccaaacc?cactccacct?tactaccaga?caaccttagc?caaaccattt?acccaaataa 1740

agtataggcg?atagaaattg?aaacctggcg?caatagatat?agtaccgcaa?gggaaagatg 1800

aaaaattata?accaagcata?atatagcaag?gactaacccc?tataccttct?gcataatgaa 1860

ttaactagaa?ataactttgc?aaggagagcc?aaagctaaga?cccccgaaac?cagacgagct 1920

acctaagaac?agctaaaaga?gcacacccgt?ctatgtagca?aaatagtggg?aagatttata 1980

ggtagaggcg?acaaacctac?cgagcctggt?gatagctggt?tgtccaagat?agaatcttag 2040

ttcaacttta?aatttgccca?cagaaccctc?taaatcccct?tgtaaattta?actgttagtc 2100

caaagaggaa?cagctctttg?gacactagga?aaaaaccttg?tagagagagt?aaaaaattta 2160

acacccatag?taggcctaaa?agcagccacc?aattaagaaa?gcgttcaagc?tcaacaccca 2220

ctacctaaaa?aatcccaaac?atataactga?actcctcaca?cccaattgga?ccaatctatc 2280

accctataga?agaactaatg?ttagtataag?taacatgaaa?acattctcct?ccgcataagc 2340

ctgcgtcaga?ttaaaacact?gaactgacaa?ttaacagccc?aatatctaca?atcaaccaac 2400

aagtcattat?taccctcact?gtcaacccaa?cacaggcatg?ctcataagga?aaggttaaaa 2460

aaagtaaaag?gaactcggca?aatcttaccc?cgcctgttta?ccaaaaacat?cacctctagc 2520

atcaccagta?ttagaggcac?cgcctgccca?gtgacacatg?tttaacggcc?gcggtaccct 2580

aaccgtgcaa?aggtagcata?atcacttgtt?ccttaaatag?ggacctgtat?gaatggctcc 2640

acgagggttc?agctgtctct?tacttttaac?cagtgaaatt?gacctgcccg?tgaagaggcg 2700

ggcataacac?agcaagacga?gaagacccta?tggagcttta?atttattaat?gcaaacagta 2760

cctaacaaac?ccacaggtcc?taaactacca?aacctgcatt?aaaaatttcg?gttggggcga 2820

cctcggagca?gaacccaacc?tccgagcagt?acatgctaag?acttcaccag?tcaaagcgaa 2880

ctactatact?caattgatcc?aataacttga?ccaacggaac?aagttaccct?agggataaca 2940

gcgcaatcct?attctagagt?ccatatcaac?aatagggttt?acgacctcga?tgttggatca 3000

ggacatcccg?atggtgcagc?cgctattaaa?ggttcgtttg?ttcaacgatt?aaagtcctac 3060

gtgatctgag?ttcagaccgg?agtaatccag?gtcggtttct?atctacnttc?aaattcctcc 3120

ctgtacgaaa?ggacaagaga?aataaggcct?acttcacaaa?gcgccttccc?ccgtaaatga 3180

tatcatctca?acttagtatt?atacccacac?ccacccaaga?acagggtttg?ttaagatggc 3240

agagcccggt?aatcgcataa?aacttaaaac?tttacagtca?gaggttcaat?tcctcttctt 3300

aacaacatac?ccatggccaa?cctcctactc?ctcattgtac?ccattctaat?cgcaatggca 3360

ttcctaatgc?ttaccgaacg?aaaaattcta?ggctatatac?aactacgcaa?aggccccaac 3420

gttgtaggcc?cctacgggct?actacaaccc?ttcgctgacg?ccataaaact?cttcaccaaa 3480

gagcccctaa?aacccgccac?atctaccatc?accctctaca?tcaccgcccc?gaccttagct 3540

ctcaccatcg?ctcttctact?atgaaccccc?ctccccatac?ccaaccccct?ggtcaacctc 3600

aacctaggcc?tcctatttat?tctagccacc?tctagcctag?ccgtttactc?aatcctctga 3660

tcagggtgag?catcaaactc?aaactacgcc?ctgatcggcg?cactgcgagc?agtagcccaa 3720

acaatctcat?atgaagtcac?cctagccatc?attctactat?caacattact?aataagtggc 3780

tcctttaacc?tctccaccct?tatcacaaca?caagaacacc?tctgattact?cctgccatca 3840

tgacccttgg?ccataatatg?atttatctcc?acactagcag?agaccaaccg?aacccccttc 3900

gaccttgccg?aaggggagtc?cgaactagtc?tcaggcttca?acatcgaata?cgccgcaggc 3960

cccttcgccc?tattcttcat?agccgaatac?acaaacatta?ttataataaa?caccctcacc 4020

actacaatct?tcctaggaac?aacatatgac?gcactctccc?ctgaactcta?cacaacatat 4080

tttgtcacca?agaccctact?tctaacctcc?ctgttcttat?gaattcgaac?agcatacccc 4140

cgattccgct?acgaccaact?catacacctc?ctatgaaaaa?acttcctacc?actcacccta 4200

gcattactta?tatgatatgt?ctccataccc?attacaatct?ccagcattcc?ccctcaaacc 4260

taagaaatat?gtctgataaa?agagttactt?tgatagagta?aataatagga?gcttaaaccc 4320

ccttatttct?aggactatga?gaatcgaacc?catccctgag?aatccaaaat?tctccgtgcc 4380

acctatcaca?ccccatccta?aagtaaggtc?agctaaataa?gctatcgggc?ccataccccg 4440

aaaatgttgg?ttataccctt?cccgtactaa?ttaatcccct?ggcccaaccc?gtcatctact 4500

ctaccatctt?tgcaggcaca?ctcatcacag?cgctaagctc?gcactgattt?tttacctgag 4560

taggcctaga?aataaacatg?ctagctttta?ttccagttct?aaccaaaaaa?ataaaccctc 4620

gttccacaga?agctgccatc?aagtatttcc?tcacgcaagc?aaccgcatcc?ataatccttc 4680

taatagctat?cctcttcaac?aatatactct?ccggacaatg?aaccataacc?aatactacca 4740

atcaatactc?atcattaata?atcataatag?ctatagcaat?aaaactagga?atagccccct 4800

ttcacttctg?agtcccagag?gttacccaag?gcacccctct?gacatccggc?ctgcttcttc 4860

tcacatgaca?aaaactagcc?cccatctcaa?tcatatacca?aatctctccc?tcactaaacg 4920

taagccttct?cctcactctc?tcaatcttat?ccatcatagc?aggcagttga?ggtggattaa 4980

accaaaccca?gctacgcaaa?atcttagcat?actcctcaat?tacccacata?ggatgaataa 5040

tagcagttct?accgtacaac?cctaacataa?ccattcttaa?tttaactatt?tatattatcc 5100

taactactac?cgcattccta?ctactcaact?taaactccag?caccacgacc?ctactactat 5160

ctcgcacctg?aaacaagcta?acatgactaa?cacccttaat?tccatccacc?ctcctctccc 5220

taggaggcct?gcccccgcta?accggctttt?tgcccaaatg?ggccattatc?gaagaattca 5280

caaaaaacaa?tagcctcatc?atccccacca?tcatagccac?catcaccctc?cttaacctct 5340

acttctacct?acgcctaatc?tactccacct?caatcacact?actccccata?tctaacaacg 5400

taaaaataaa?atgacagttt?gaacatacaa?aacccacccc?attcctcccc?acactcatcg 5460

cccttaccac?gctactccta?cctatctccc?cttttatact?aataatctta?tagaaattta 5520

ggttaaatac?agaccaagag?ccttcaaagc?cctcagtaag?ttgcaatact?taatttctgt 5580

aacagctaag?gactgcaaaa?ccccactctg?catcaactga?acgcaaatca?gccactttaa 5640

ttaagctaag?cccttactag?accaatggga?cttaaaccca?caaacactta?gttaacagct 5700

aagcacccta?atcaactggc?ttcaatctac?ttctcccgcc?gccgggaaaa?aaggcgggag 5760

aagccccggc?aggtttgaag?ctgcttcttc?gaatttgcaa?ttcaatatga?aaatcacctc 5820

ggagctggta?aaaagaggcc?taacccctgt?ctttagattt?acagtccaat?gcttcactca 5880

gccattttac?ctcaccccca?ctgatgttcg?ccgaccgttg?actattctct?acaaaccaca 5940

aagacattgg?aacactatac?ctattattcg?gcgcatgagc?tggagtccta?ggcacagctc 6000

taagcctcct?tattcgagcc?gagctgggcc?agccaggcaa?ccttctaggt?aacgaccaca 6060

tctacaacgt?tatcgtcaca?gcccatgcat?ttgtaataat?cttcttcata?gtaataccca 6120

tcataatcgg?aggctttggc?aactgactag?ttcccctaat?aatcggtgcc?cccgatatgg 6180

cgtttccccg?cataaacaac?ataagcttct?gactcttacc?tccctctctc?ctactcctgc 6240

tcgcatctgc?tatagtggag?gccggagcag?gaacaggttg?aacagtctac?cctcccttag 6300

cagggaacta?ctcccaccct?ggagcctccg?tagacctaac?catcttctcc?ttacacctag 6360

caggtgtctc?ctctatctta?ggggccatca?atttcatcac?aacaattatc?aatataaaac 6420

cccctgccat?aacccaatac?caaacgcccc?tcttcgtctg?atccgtccta?atcacagcag 6480

tcctacttct?cctatctctc?ccagtcctag?ctgctggcat?cactatacta?ctaacagacc 6540

gcaacctcaa?caccaccttc?ttcgaccccg?ccggaggagg?agaccccatt?ctataccaac 6600

acctattctg?atttttcggt?caccctgaag?tttatattct?tatcctacca?ggcttcggaa 6660

taatctccca?tattgtaact?tactactccg?gaaaaaaaga?accatttgga?tacataggta 6720

tggtctgagc?tatgatatca?attggcttcc?tagggtttat?cgtgtgagca?caccatatat 6780

ttacagtagg?aatagacgta?gacacacgag?catatttcac?ctccgctacc?ataatcatcg 6840

ctatccccac?cggcgtcaaa?gtatttagct?gactcgccac?actccacgga?agcaatatga 6900

aatgatctgc?tgcagtgctc?tgagccctag?gattcatctt?tcttttcacc?gtaggtggcc 6960

tgactggcat?gtattagca aactcatcac?tagacatcgt?actacacgac?acgtactacg 7020

ttgtagccca?cttccactat?gtcctatcaa?taggagctgt?atttgccatc?ataggaggct 7080

tcattcactg?atttccccta?ttctcaggct?acaccctaga?ccaaacctac?gccaaaatcc 7140

atttcactat?catattcatc?ggcgtaaatc?taactttctt?cccacaacac?tttctcggcc 7200

tatccggaat?gccccgacgt?tactcggact?accccgatgc?atacaccaca?tgaaacatcc 7260

tatcatctgt?aggctcattc?atttctctaa?cagcagtaat?attaataatt?ttcatgattt 7320

gagaagcctt?cgcttcgaag?cgaaaagtcc?taatagtaga?agaaccctcc?ataaacctgg 7380

agtgactata?tggatgcccc?ccaccctacc?acacattcga?agaacccgta?tacataaaat 7440

ctagacaaaa?aaggaaggaa?tcgaaccccc?caaagctggt?ttcaagccaa?ccccatggcc 7500

tccatgactt?tttcaaaaag?gtattagaaa?aaccatttca?taactttgtc?aaagttaaat 7560

tataggctaa?atcctatata?tcttaatggc?acatgcagcg?caagtaggtc?tacaagacgc 7620

tacttcccct?atcatagaag?agcttatcac?ctttcatgat?cacgccctca?taatcatttt 7680

ccttatctgc?ttcctagtcc?tgtatgccct?tttcctaaca?ctcacaacaa?aactaactaa 7740

tactaacatc?tcagacgctc?aggaaataga?aaccgtctga?actatcctgc?ccgccatcat 7800

cctagtcctc?atcgccctcc?catccctacg?catcctttac?ataacagacg?aggtcaacga 7860

tccctccctt?accatcaaat?caattggcca?ccaatggtac?tgaacctacg?agtacaccga 7920

ctacggcgga?ctaatcttca?actcctacat?acttccccca?ttattcctag?aaccaggcga 7980

cctgcgactc?cttgacgttg?acaatcgagt?agtactcccg?attgaagccc?ccattcgtat 8040

aataattaca?tcacaagacg?tcttgcactc?atgagctgtc?cccacattag?gcttaaaaac 8100

agatgcaatt?cccggacgtc?taaaccaaac?cactttcacc?gctacacgac?cgggggtata 8160

ctacggtcaa?tgctctgaaa?tctgtggagc?aaaccacagt?ttcatgccca?tcgtcctaga 8220

attaattccc?ctaaaaatct?ttgaaatagg?gcccgtattt?accctatagc?accccctcta 8280

ccccctctag?agcccactgt?aaagctaact?tagcattaac?cttttaagtt?aaagattaag 8340

agaaccaaca?cctctttaca?gtgaaatgcc?ccaactaaat?actaccgtat?ggcccaccat 8400

aattaccccc?atactcctta?cactattcct?catcacccaa?ctaaaaatat?taaacacaaa 8460

ctaccaccta?cctccctcac?caaagcccat?aaaaataaaa?aattataaca?aaccctgaga 8520

accaaaatga?acgaaaatct?gttcgcttca?ttcattgccc?ccacaatcct?aggcctaccc 8580

gccgcagtac?tgatcattct?atttccccct?ctattgatcc?ccacctccaa?atatctcatc 8640

aacaaccgac?taatcaccac?ccaacaatga?ctaatcaaac?taacctcaaa?acaaatgata 8700

accatacaca?acactaaagg?acgaacctga?tctcttatac?tagtatcctt?aatcattttt 8760

attgccacaa?ctaacctcct?cggactcctg?cctcactcat?ttacaccaac?cacccaacta 8820

tctataaacc?tagccatggc?catcccctta?tgagcgggca?cagtgattat?aggctttcgc 8880

tctaagatta?aaaatgccct?agcccacttc?ttaccacaag?gcacacctac?accccttatc 8940

cccatactag?ttattatcga?aaccatcagc?ctactcattc?aaccaatagc?cctggccgta 9000

cgcctaaccg?ctaacattac?tgcaggccac?ctactcatgc?acctaattgg?aagcgccacc 9060

ctagcaatat?caaccattaa?ccttccctct?acacttatca?tcttcacaat?tctaattcta 9120

ctgactatcc?tagaaatcgc?tgtcgcctta?atccaagcct?acgttttcac?acttctagta 9180

agcctctacc?tgcacgacaa?cacataatga?cccaccaatc?acatgcctat?catatagtaa 9240

aacccagccc?atgaccccta?acaggggccc?tctcagccct?cctaatgacc?tccggcctag 9300

ccatgtgatt?tcacttccac?tccataacgc?tcctcatact?aggcctacta?accaacacac 9360

taaccatata?ccaatgatgg?cgcgatgtaa?cacgagaaag?cacataccaa?ggccaccaca 9420

caccacctgt?ccaaaaaggc?cttcgatacg?ggataatcct?atttattacc?tcagaagttt 9480

ttttcttcgc?aggatttttc?tgagcctttt?accactccag?cctagcccct?accccccaat 9540

taggagggca?ctggccccca?acaggcatca?ccccgctaaa?tcccctagaa?gtcccactcc 9600

taaacacatc?cgtattactc?gcatcaggag?tatcaatcac?ctgagctcac?catagtctaa 9660

tagaaaacaa?ccgaaaccaa?ataattcaag?cactgcttat?tacaatttta?ctgggtctct 9720

attttaccct?cctacaagcc?tcagagtact?tcgagtctcc?cttcaccatt?tccgacggca 9780

tctacggctc?aacatttttt?gtagccacag?gcttccacgg?acttcacgtc?attattggct 9840

caactttcct?cactatctgc?ttcatccgcc?aactaatatt?tcactttaca?tccaaacatc 9900

actttggctt?cgaagccgcc?gcctgatact?ggcattttgt?agatgtggtt?tgactatttc 9960

tgtatgtctc?catctattga?tgagggtctt?actcttttag?tataaatagt?accgttaact 10020

tccaattaac?tagttttgac?aacattcaaa?aaagagtaat?aaacttcgcc?ttaattttaa 10080

taatcaacac?cctcctagcc?ttactactaa?taattattac?attttgacta?ccacaactca 10140

acggctacat?agaaaaatcc?accccttacg?agtgcggctt?cgaccctata?tcccccgccc 10200

gcgtcccttt?ctccataaaa?ttcttcttag?tagctattac?cttcttatta?tttgatctag 10260

aaattgccct?ccttttaccc?ctaccatgag?ccctacaaac?aactaacctg?ccactaatag 10320

ttatgtcatc?cctcttatta?atcatcatcc?tagccctaag?tctggcctat?gagtgactac 10380

aaaaaggatt?agactgaacc?gaattggtat?atagtttaaa?caaaacgaat?gatttcgact 10440

cattaaatta?tgataatcat?atttaccaaa?tgcccctcat?ttacataaat?attatactag 10500

catttaccat?ctcacttcta?ggaatactag?tatatcgctc?acacctcata?tcctccctac 10560

tatgcctaga?aggaataata?ctatcgctgt?tcattatagc?tactctcata?accctcaaca 10620

cccactccct?cttagccaat?attgtgccta?ttgccatact?agtctttgcc?gcctgcgaag 10680

cagcggtggg?cctagcccta?ctagtctcaa?tctccaacac?atatggccta?gactacgtac 10740

ataacctaaa?cctactccaa?tgctaaaact?aatcgtccca?acaattatat?tactaccact 10800

gacatgactt?tccaaaaaac?acataatttg?aatcaacaca?accacccaca?gcctaattat 10860

tagcatcatc?cctctactat?tttttaacca?aatcaacaac?aacctattta?gctgttcccc 10920

aaccttttcc?tccgaccccc?taacaacccc?cctcctaata?ctaactacct?gactcctacc 10980

cctcacaatc?atggcaagcc?aacgccactt?atccagtgaa?ccactatcac?gaaaaaaact 11040

ctacctctct?atactaatct?ccctacaaat?ctccttaatt?ataacattca?cagccacaga 11100

actaatcata?ttttatatct?tcttcgaaac?cacacttatc?cccaccttgg?ctatcatcac 11160

ccgatgaggc?aaccagccag?aacgcctgaa?cgcaggcaca?tacttcctat?tctacaccct 11220

agtaggctcc?cttcccctac?tcatcgcact?aatttacact?cacaacaccc?taggctcact 11280

aaacattcta?ctactcactc?tcactgccca?agaactatca?aactcctgag?ccaacaactt 11340

aatatgacta?gcttacacaa?tagcttttat?agtaaagata?cctctttacg?gactccactt 11400

atgactccct?aaagcccatg?tcgaagcccc?catcgctggg?tcaatagtac?ttgccgcagt 11460

actcttaaaa?ctaggcggct?atggtataat?acgcctcaca?ctcattctca?accccctgac 11520

aaaacacata?gcctacccct?tccttgtact?atccctatga?ggcataatta?taacaagctc 11580

catctgccta?cgacaaacag?acctaaaatc?gctcattgca?tactcttcaa?tcagccacat 11640

agccctcgta?gtaacagcca?ttctcatcca?aaccccctga?agcttcaccg?gcgcagtcat 11700

tctcataatc?gcccacgggc?ttacatcctc?attactattc?tgcctagcaa?actcaaacta 11760

cgaacgcact?cacagtcgca?tcataatcct?ctctcaagga?cttcaaactc?tactcccact 11820

aatagctttt?tgatgacttc?tagcaagcct?cgctaacctc?gccttacccc?ccactattaa 11880

cctactggga?gaactctctg?tgctagtaac?cacgttctcc?tgatcaaata?tcactctcct 11940

acttacagga?ctcaacatac?tagtcacagc?cctatactcc?ctctacatat?ttaccacaac 12000

acaatggggc?tcactcaccc?accacattaa?caacataaaa?ccctcattca?cacgagaaaa 12060

caccctcatg?ttcatacacc?tatcccccat?tctcctccta?tccctcaacc?ccgacatcat 12120

taccgggttt?tcctcttgta?aatatagttt?aaccaaaaca?tcagattgtg?aatctgacaa 12180

cagaggctta?cgacccctta?tttaccgaga?aagctcacaa?gaactgctaa?ctcatgcccc 12240

catgtctaac?aacatggctt?tctcaacttt?taaaggataa?cagctatcca?ttggtcttag 12300

gccccaaaaa?ttttggtgca?actccaaata?aaagtaataa?ccatgcacac?tactataacc 12360

accctaaccc?tgacttccct?aattcccccc?atccttacca?ccctcgttaa?ccctaacaaa 12420

aaaaactcat?acccccatta?tgtaaaatcc?attgtcgcat?ccacctttat?tatcagtctc 12480

ttccccacaa?caatattcat?gtgcctagac?caagaagtta?ttatctcgaa?ctgacactga 12540

gccacaaccc?aaacaaccca?gctctcccta?agcttcaaac?tagactactt?ctccataata 12600

ttcatccctg?tagcattgtt?cgttacatgg?tccatcatag?aattctcact?gtgatatata 12660

aactcagacc?caaacattaa?tcagttcttc?aaatatctac?tcatcttcct?aattaccata 12720

ctaatcttag?ttaccgctaa?caacctattc?caactgttca?tcggctgaga?gggcgtagga 12780

attatatcct?tcttgctcat?cagttgatga?tacgcccgag?cagatgccaa?cacagcagcc 12840

attcaagcaa?tcctatacaa?ccgtatcggc?gatatcggtt?tcatcctcgc?cttagcatga 12900

tttatcctac?actccaactc?atgagaccca?caacaaatag?cccttctaaa?cgctaatcca 12960

agcctcaccc?cactactagg?cctcctccta?gcagcagcag?gcaaatcagc?ccaattaggt 13020

ctccacccct?gactcccctc?agccatagaa?ggccccaccc?cagtctcagc?cctactccac 13080

tcaagcacta?tagttgtagc?aggaatcttc?ttactcatcc?gcttccaccc?cctagcagaa 13140

aatagcccac?taatccaaac?tctaacacta?tgcttaggcg?ctatcaccac?tctgttcgca 13200

gcagtctgcg?cccttacaca?aaatgacatc?aaaaaaatcg?tagccttctc?cacttcaagt 13260

caactaggac?tcataatagt?tacaatcggc?atcaaccaac?cacacctagc?attcctgcac 13320

atctgtaccc?acgccttctt?caaagccata?ctatttatgt?gctccgggtc?catcatccac 13380

aaccttaaca?atgaacaaga?tattcgaaaa?ataggaggac?tactcaaaac?catacctctc 13440

acttcaacct?ccctcaccat?tggcagccta?gcattagcag?gaataccttt?cctcacaggt 13500

ttctactcca?aagaccacat?catcgaaacc?gcaaacatat?catacacaaa?cgcctgagcc 13560

ctatctatta?ctctcatcgc?tacctccctg?acaagcgcct?atagcactcg?aataattctt 13620

ctcaccctaa?caggtcaacc?tcgcttcccc?acccttacta?acattaacga?aaataacccc 13680

accctactaa?accccattaa?acgcctggca?gccggaagcc?tattcgcagg?atttctcatt 13740

actaacaaca?tttcccccgc?atcccccttc?caaacaacaa?tccccctcta?cctaaaactc 13800

acagccctcg?ctgtcacttt?cctaggactt?ctaacagccc?tagacctcaa?ctacctaacc 13860

aacaaactta?aaataaaatc?cccactatgc?acattttatt?tctccaacat?actcggattc 13920

taccctagca?tcacacaccg?cacaatcccc?tatctaggcc?ttcttacgag?ccaaaacctg 13980

cccctactcc?tcctagacct?aacctgacta?gaaaagctat?tacctaaaac?aatttcacag 14040

caccaaatct?ccacctccat?catcacctca?acccaaaaag?gcataattaa?actttacttc 14100

ctctctttct?tcttcccact?catcctaacc?ctactcctaa?tcacataacc?tattcccccg 14160

agcaatctca?attacaatat?atacaccaac?aaacaatgtt?caaccagtaa?ctactactaa 14220

tcaacgccca?taatcataca?aagcccccgc?accaatagga?tcctcccgaa?tcaaccctga 14280

cccctctcct?tcataaatta?ttcagcttcc?tacactatta?aagtttacca?caaccaccac 14340

cccatcatac?tctttcaccc?acagcaccaa?tcctacctcc?atcgctaacc?ccactaaaac 14400

actcaccaag?acctcaaccc?ctgaccccca?tgcctcagga?tactcctcaa?tagccatcgc 14460

tgtagtatat?ccaaagacaa?ccatcattcc?ccctaaataa?attaaaaaaa?ctattaaacc 14520

catataacct?cccccaaaat?tcagaataat?aacacacccg?accacaccgc?taacaatcaa 14580

tactaaaccc?ccataaatag?gagaaggctt?agaagaaaac?cccacaaacc?ccattactaa 14640

acccacactc?aacagaaaca?aagcatacat?cattattctc?gcacggacta?caaccacgac 14700

caatgatatg?aaaaaccatc?gttgtatttc?aactacaaga?acaccaatga?ccccaatacg 14760

caaaactaac?cccctaataa?aattaattaa?ccactcattc?atcgacctcc?ccaccccatc 14820

caacatctcc?gcatgatgaa?acttcggctc?actccttggc?gcctgcctga?tcctccaaat 14880

caccacagga?ctattcctag?ccatgcacta?ctcaccagac?gcctcaaccg?ccttttcatc 14940

aatcgcccac?atcactcgag?acgtaaatta?tggctgaatc?atccgctacc?ttcacgccaa 15000

tggcgcctca?atattcttta?tctgcctctt?cctacacatc?gggcgaggcc?tatattacgg 15060

atcatttctc?tactcagaaa?cctgaaacat?cggcattatc?ctcctgcttg?caactatagc 15120

aacagccttc?ataggctatg?tcctcccgtg?aggccaaata?tcattctgag?gggccacagt 15180

aattacaaac?ttactatccg?ccatcccata?cattgggaca?gacctagttc?aatgaatctg 15240

aggaggctac?tcagtagaca?gtcccaccct?cacacgattc?tttacctttc?acttcatctt 15300

gcccttcatt?attgcagccc?tagcaacact?ccacctccta?ttcttgcacg?aaacgggatc 15360

aaacaacccc?ctaggaatca?cctcccattc?cgataaaatc?accttccacc?cttactacac 15420

aatcaaagac?gccctcggct?tacttctctt?ccttctctcc?ttaatgacat?taacactatt 15480

ctcaccagac?ctcctaggcg?acccagacaa?ttatacccta?gccaacccct?taaacacccc 15540

tccccacatc?aagcccgaat?gatatttcct?attcgcctac?acaattctcc?gatccgtccc 15600

taacaaacta?ggaggcgtcc?ttgccctatt?actatccatc?ctcatcctag?caataatccc 15660

catcctccat?atatccaaac?aacaaagcat?aatatttcgc?ccactaagcc?aatcacttta 15720

ttgactccta?gccgcagacc?tcctcattct?aacctgaatc?ggaggacaac?cagtaagcta 15780

cccttttacc?atcattggac?aagtagcatc?cgtactatac?ttcacaacaa?tcctaatcct 15840

aataccaact?atctccctaa?ttgaaaacaa?aatactcaaa?tgggcctgtc?cttgtagtat 15900

aaactaatac?accagtcttg?taaaccggag?atgaaaacct?ttttccaagg?acaaatcaga 15960

gaaaaagtct?ttaactccac?cattagcacc?caaagctaag?attctaattt?aaactattct 16020

ctgttctttc?atggggaagc?agatttgggt?accacccaag?tattgactca?cccatcaaca 16080

accgctatgt?atttcgtaca?ttactgccag?ccaccatgaa?tattgtacgg?taccataaat 16140

acttgaccac?ctgtagtaca?taaaaaccca?atccacatca?aaaccccctc?cccatgctta 16200

caagcaagta?cagcaatcaa?ccctcaacta?tcacacatca?actgcaactc?caaagccacc 16260

cctcacccac?taggatacca?acaaacctac?ccacccttaa?cagtacatag?tacataaagc 16320

catttaccgt?acatagcaca?ttacagtcaa?atcccttctc?gtccccatgg?atgacccccc 16380

tcagataggg?gtcccttgac?caccatcctc?cgtgaaatca?atatcccgca?caagagtgct 16440

actctcctcg?ctccgggccc?ataacacttg?ggggtagcta?aagtgaactg?tatccgacat 16500

ctggttccta?cttcagggtc?ataaagccta?aatagcccac?acgttcccct?taaataagac 16560

atcacgatg 16569

Claims (6)

1. be used for detecting simultaneously the test kit of Mitochondrial DNA A1555G relevant and C1494T sudden change, it is characterized in that, comprising with the matrilinear inheritance drug induced deafness:

(1) reagent of extraction sample genomic dna;

(2) pcr amplification reaction reagent comprises dNTP, 10 * PCR damping fluid, MgCl ₂, Taq enzyme and tri-distilled water;

(3) two pipe primers, wherein a pipe is the mixed solution of primer C1, N1, N2 and C2, another pipe is the mixed solution of primer C1, M1, M2 and C2;

Said two pipe primers are according to the human mtdna sequences Design shown in the SEQ ID NO:7; Article one, primer sequence N1 is an antisense strand; Being the downstream universal primer, is the Nucleotide 1494-1516 of Mitochondrial DNA, and its sequence is the SEQ ID NO:1 in the sequence table; Second primer sequence M1 is an antisense strand, is the downstream universal primer, is the Nucleotide 1494-1516 of Mitochondrial DNA, and wherein the G in 1494 sites is replaced into A, and its sequence is the SEQ ID NO:2 in the sequence table; Article three, primer sequence C1 is a sense strand, is upper reaches universal primer, is the Nucleotide 1030-1052 of Mitochondrial DNA, and its sequence is the SEQ ID NO:3 in the sequence table; Article four, primer sequence N2 is a sense strand, is upper reaches universal primer, is the Nucleotide 1533-1555 of Mitochondrial DNA, and its sequence is the SEQ ID NO:4 in the sequence table; Article five, primer sequence M2 is a sense strand, is upper reaches universal primer, is the Nucleotide 1533-1555 of Mitochondrial DNA, and wherein the A in 1555 sites is replaced into G, and its sequence is the SEQ ID NO:5 in the sequence table; Article six, primer sequence C2 is an antisense strand, is the downstream universal primer, is the Nucleotide 2214-2235 of Mitochondrial DNA, and its sequence is the SEQ ID NO:6 in the sequence table;

(4) carry A1555G sudden change the positive control template, carry the positive control template and the negative control template of C1494T sudden change.

2. the test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change according to claim 1 with the matrilinear inheritance drug induced deafness; It is characterized in that; 3 ' end of primer N1 and M1 is positioned at 1494 sites of Mitochondrial DNA, corresponds respectively to 1494 site wild-type G and mutant A; 3 ' end of primer N2 and M2 is positioned at 1555 sites of Mitochondrial DNA, corresponds respectively to 1555 site wild-type A and mutant G.

3. the test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change according to claim 1 with the matrilinear inheritance drug induced deafness; It is characterized in that; Introduce sulfuration phosphoric acid between first of every primer 3 ' end and second bit base and modify, and 3 ' end the 4th bit base of primer N1, M1, N2, M2 introduces mispairing, promptly be replaced into A corresponding to mitochondrial 1497 G; 1552 G is replaced into A, to improve the specificity of extension.

4. according to each described test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change of claim 1～3 with the matrilinear inheritance drug induced deafness; It is characterized in that; In said primer; N1 and C1 pairing is used to increase and contains the mitochondria DNA fragment in the normal site of C1494C; N2 and C2 pairing is used to increase and contains the mitochondria DNA fragment in the normal site of A1555A, and pairing is used to increase and contains the mitochondria DNA fragment in C1494T mutational site M1 with C1, and M2 and C2 match to be used to increase and contain the mitochondria DNA fragment in A1555G mutational site; Primer C1 and C2 pairing are used to generate a constant amplified band, as the molecule internal control index of PCR reaction.

5. the test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change according to claim 1 with the matrilinear inheritance drug induced deafness; It is characterized in that; In the reagent of said extraction sample genomic dna; Comprise cell pyrolysis liquid and solution I, the staple of said solution I is a Proteinase K.

6. the test kit that is used for detecting simultaneously Mitochondrial DNA A1555G relevant and C1494T sudden change according to claim 1 with the matrilinear inheritance drug induced deafness; It is characterized in that interpolation is used to improve the specific DMSO 99.8MIN. of extension in said pcr amplification reaction reagent.

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