CN102925562B - Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene - Google Patents
Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene Download PDFInfo
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Abstract
The invention relates to the field of gene detection, and particularly relates to a kit and method for detecting an aminoglycoside drug-induced deafness-sensitive gene through a fluorescent PCR (polymerase chain reaction)-melting curve method. The technical scheme of the invention provides a kit for detecting an aminoglycoside drug-induced deafness-sensitive gene. The kit comprises a PCR solution, a negative quality control sample and a positive quality control sample, wherein the PCR solution comprises primers, a PCR Buffer, a dNTP (deoxyribonucleotide triphosphate) mixed solution, saturated fluorescent dyes, a Taq enzyme and a UNG enzyme. In the invention, the specific PCR primers are designed according to SNP (single nucleotide polymorphism) sites of the aminoglycoside drug-induced deafness-sensitive gene, two aminoglycoside drug-induced deafness-sensitive sites can be detected in one PCR, the operation can be finished in one tube, and the detection is quick, accurate and wide in range.
Description
Technical field
The present invention relates to gene test field, be specifically related to test kit and method that fluorescent PCR-melting curve method detects aminoglycoside medicaments induced deafness tumor susceptibility gene.
Background technology
According to China's the Second China National Sample Survey on Disability in 2006, show, hearing loss person 2,004 ten thousand people, account for the whole nation 8,296 ten thousand Disabled personss' 24.16%, and its Chinese traditional medicine causes the deaf 30%-40% that accounts for, and number reaches 8,000,000.Drug induced deafness refers to and uses some drugs to cure the disease or human body contacts the caused deafness of some chemicals.Found that at present ototoxic drug reaches kind more than 100, and ototoxicity microbiotic is the most common.Aminoglycoside antibiotics is modal ototoxicity microbiotic, comprises conventional clinically Streptomycin sulphate, gentamicin etc.Clear and definite nearly ten years several mitochondrial mutations relevant with aminoglycoside antibiotics, wherein the occurrence frequency in Chinese population such as A1555G, C1494T is up to 4%.Therefore, detect aminoglycoside medicaments induced deafness mitochondrial mutations site, to deaf diagnosis, early screening, and provide medication guide to have great significance.
The test kit kind detecting for deaf gene is in the market less, and common mitochondrial mutations detection method has PCR-RFLP method, micro-array chip method, ARMS-PCR method, fluorescence quantitative PCR method, direct sequencing etc.Domestic clinical conventional method is micro-array chip method and fluorescence quantitative PCR method etc., and their main technological deficiency is as follows:
1, micro-array chip method can be to the concrete somatotype of mutant gene locus, and it is relatively many to detect genotype, but it has several large shortcomings:
Detect and need to extract through whole blood DNA the steps such as (30min), pcr amplification (more than 2h), product hybridization analysis (more than 1h), detection time long (more than 4h), complex operation, can not meet clinical in causing the demand of a large amount of examinations of deaf gene; Because operating process is too much, easily cause sample cross contamination; Secondly, lower to the discrimination power of single base, be prone to false negative and false positive, the accuracy rate of result is affected; In addition, micro-array chip high cost, needs special chip scanning instrument, and testing cost is high, and as " brilliant core " series product of rich biological production difficult to understand, a person-portion detects to be needed more than thousand yuan, to be difficult to promote on a large scale clinically.
Therefore, be necessary to research and develop that detection time is short, cost is low and applicable medicine is caused to the product that deaf gene carries out examination.
2, the advantage of existing PCR method product is that detection time is short, operates relatively simply, and cost is low, but it has following shortcoming:
The amplification efficiency of common ARMS-PCR product is only the 1-10% of normal amplification, so amplification system deficient in stability; A reaction of four primer ARMS-PCR can only be examined a mutational site: the product that biological company limited is controlled in middle raw north can detect four deaf gene mutational sites, but each site needs a reaction tubes, and multiplex PCR is difficult to realize, and testing process is loaded down with trivial details.
The shortcoming that regular-PCR product is total: need to judge genotype by stripe size when result interpretation, lack intuitive.Detected through gel electrophoresis result easily causes PCR crossed contamination, detection sensitivity is low and length consuming time.
3, utilize Taqman probe to carry out gene type, highly sensitive, high specificity, but have following shortcoming:
Utilize Taqman probe to carry out SNP sudden change and detect, site of every detection needs 2 probes, and sensitivity has higher requirements to probe.The synthetic cost of probe is higher, and the port number of fluorescent PCR instrument is had to requirement, is unfavorable for promoting.
Each reaction of current detection kit only detects a site.As current Beijing Ke Lingjin instrument Bioisystech Co., Ltd, the fluoroscopic examination product of 301 Hospital of PLA exploitation is the single site of one-time detection only also.The product that there is no energy one-time detection multidigit point comes out.
4, the advantage of direct sequencing be can be definite the gene order of knowing institute's surveyed area, accuracy is high, but has a following shortcoming:
Testing process is loaded down with trivial details, excessive cycle: before order-checking, need object fragment to carry out pcr amplification, amplified production is carried out to purifying, then carry out capillary electrophoresis order-checking, whole process need strict quality control; Sequencing reaction cost is higher, needs expensive fluorescently-labeled nucleic acid and professional sequenator, is not suitable for clinical expansion.The interpretation more complicated of sequencing result, need to pass through special training.
In sum: existing common method, as fluorescence quantitative PCR method detects gene, single mutational site on detection line plastochondria only, most of fluorescence quantitative PCR method, without interior mark, can not carry out quality control; The microarray state chip product operating time is long, cost is high, accuracy is low and need specific apparatus.Extensive examination mitochondrial gene mutation, requirement is fast, low-cost, sensing range is wide, and above product cannot meet simultaneously.
For the problems referred to above, be badly in need of a kind of, sensing range wide, detection sensitivity high product more simple to operate than existing method, conscientiously meet the demand of aminoglycoside medicaments induced deafness tumor susceptibility gene examination.
Summary of the invention
The object of the invention is to utilize allele-specific PCR (Allele Specific PCR), associating melting curve analysis (Melting Curve Analysis), utilize the difference of product Tm value to carry out gene test to different samples, without probe, without primer is carried out to special modification, with low cost.Two susceptibility locis on quick, the accurate detection line plastochondria of energy.
Technical scheme of the present invention is for providing a kind of aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, and described test kit comprises PCR reaction solution, negative quality control product and positive quality control product; Described PCR reaction solution comprises primer, PCR Buffer, dNTP mixed solution, saturated fluorescence dyestuff, Taq enzyme and UNG enzyme; Described dNTP mixed solution is the mixed solution of dATP, dCTP, dGTP and dUTP;
Described primer comprises:
1494 locus specificity primer T, the sequence of described primer T is: SEQ ID NO:1;
1555 locus specificity primer G, the sequence of described primer G is: SEQ ID NO:2;
The downstream primer R that two sites are public, the sequence of described primer R is: SEQ ID NO:3;
A pair of internal reference beta-actin primer A1 and primer A2, the sequence of described primer A1 is SEQ ID NO:4, the sequence of described primer A2 is SEQ ID NO:5.
Wherein, described negative quality control product is the plasmid vector with normal people's mitochondria DNA fragment.
Described positive quality control product is divided into 1494 positive quality control product and 1555 positive quality control product, with the plasmid vector of human mitochondrial DNA fragments of 1494 sudden changes or 1555 sudden changes.
Preferably, above-mentioned aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, in described PCR reaction solution, primer final concentration is 50-500nM, the MgCl in PCR Buffer
2final concentration is 1.5-9mM, and each component final concentration of described dNTP mixed solution is 100-300nM, and Taq enzyme final concentration is 1-7.5IU/ reaction, and UNG enzyme final concentration is 0.05-0.3IU/ reaction.
Preferably, above-mentioned aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, described saturated fluorescence dyestuff is EvaGreen dyestuff, LC Green PLUS dyestuff, ResoLight dyestuff or SYTO 9 dyestuffs.
Preferably, above-mentioned aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, described each component concentration of PCR reaction solution is:
Deionized water: 16.14 μ L;
10×PCR?buffer：2.5μL；
The equal-volume mixed solution of 10mM dATP, dCTP, dGTP, dUTP: 2 μ L;
10mM?dUTP：0.5μL；
100 μ M primer A1:0.01 μ L;
100 μ M primer A2:0.01 μ L;
100 μ M primer G:0.04 μ L;
100 μ M primer T:0.03 μ L;
100 μ M primer R:0.02 μ L;
20×Evagreen：1.25μL；
The ultrapure Taq enzyme of 5U/ μ L: 0.2 μ L;
1U/μL?UNG：0.3μL。
Another technical scheme of the present invention, for a kind of aminoglycoside medicaments induced deafness tumor susceptibility gene detection method is provided, comprises the steps:
(1) adopt the test kit described in above-mentioned any one to carry out pcr amplification to the goal gene of the template DNA of sample;
(2) melting curve analysis is carried out in the mutational site of the goal gene of PCR product.
Above-mentioned aminoglycoside medicaments induced deafness tumor susceptibility gene detection method, the condition that described step (1) is carried out pcr amplification reaction is:
50℃、1-10min；
90-95℃、10min；
95 ℃, 5-30s, 50-65 ℃, 30-90s, 30-45 circulation.
Preferably, above-mentioned aminoglycoside medicaments induced deafness tumor susceptibility gene detection method, the condition that described step (1) is carried out pcr amplification reaction is specially:
50℃、5min；
90-95℃、10min；
95 ℃, 15s, 60 ℃, 30s, 72 ℃, 30s, 35 circulations.
Preferably, above-mentioned aminoglycoside medicaments induced deafness tumor susceptibility gene detection method, the program that described step (2) PCR product carries out melting curve analysis is: 70 ℃ of-90 ℃ of continuous detecting, 15 times/s.
Existing common method, as fluorescence quantitative PCR method detects gene, single mutational site on detection line plastochondria only, most of fluorescence quantitative PCR method, without interior mark, can not carry out quality control; The microarray state chip product operating time is long, cost is high, accuracy is low and need specific apparatus, extensive examination mitochondrial gene mutation, and requirement is fast, low-cost, sensing range is wide, and above product cannot meet simultaneously.The present invention is according to aminoglycoside medicaments induced deafness tumor susceptibility gene SNP site, design special PCR primer, the template that makes only to suddenly change can increase, when melting curve analysis, show Tm value tag peak, and the normal template internal control gene that can only increase, adopt multiplex PCR fluorescence detection and melting curve analysis method, can in same PCR reaction, detect 2 aminoglycoside medicaments induced deafness susceptibility locis, realize the somatotype of a pipe operation to mitochondrial gene mutation site.Gene test of the present invention is without probe, without primer is carried out to special modification, with low cost, and detects fast, accurately.Compare existing method simple to operate, sensing range is wider, and detection sensitivity is higher, conscientiously meets the demand of aminoglycoside medicaments induced deafness tumor susceptibility gene examination.
Accompanying drawing explanation
Fig. 1: the solubility curve of fluorescent PCR melting curve detection line plastochondria C1494T sudden change positive sample;
Fig. 2: the solubility curve of fluorescent PCR melting curve detection line plastochondria A1555G sudden change positive sample;
Fig. 3: fluorescent PCR melting curve detects the solubility curve of mitochondrial mutations negative sample.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, below in conjunction with embodiment and coordinate accompanying drawing to be explained in detail.
The present invention adopts allele-specific PCR, is to utilize Taq enzymatic defect 3 '-5 ' 5 prime excision enzyme activity, when 3 ' of primer, holds and can not match completely with template, and pcr amplification can not carry out.Therefore, under ideal state, primer 3 ' end is only designed to and sudden change template matches, and does not mate with normal template, this primer sudden change template that can only increase, and normal mode plate is not increased, thus reach the effect of somatotype.Meanwhile, at primer 3 ', hold other positions, as 2,3,4, introduce mutating alkali yl, can strengthen the specificity of primer, thoroughly suppress non-specific amplification.Therefore, for a certain site, can two contrary Auele Specific Primers of design direction, article one, sudden change template only increases, another normal template that only increases, the upstream and downstream primer that design matches simultaneously, makes the expanding fragment length of normal template variant with the expanding fragment length of sudden change template, the difference of clip size during by electrophoresis, genotype that can judgement sample.
From the side, allele-specific PCR is a kind of simple and easy to do gene tester, and primer does not need expensive special mark, easy and simple to handle, is applicable to detect the detection that aminoglycoside medicaments in the present invention causes deaf susceptibility loci.
Not homotactic DNA, Tm value and GC content, length is relevant with secondary structure.Utilize the chimeric dyestuff of DNA double chain can carry out melting curve analysis, in the process slowly heating up, DNA unwinds gradually and discharges dye molecule, and fluorescent value declines immediately, can calculate thus the Tm value of this DNA chain, according to the character of the difference judgement amplified fragments of Tm value.
Fluorescence melting curve method can be distinguished the product of different fragments length by the difference of product Tm value, higher compared with gel electrophoresis sensitivity; Can stop the PCR product Aerosol Pollution of uncapping and causing due to product simultaneously.Melting curve method and allele-specific PCR coupling, can manufacture low cost, simplicity and practical diagnostic kit, is very applicable to clinical diagnosis and extensive examination.
The preparation of embodiment 1 test kit of the present invention is used
Test kit of the present invention comprises PCR reaction solution and quality control product.In PCR reaction solution, comprise Taq enzyme, the chimeric dyestuff of DNA double chain, amplimer, amplification buffer and dN (U) TP etc., dN (U) TP is dNTP mixed solution.PCR response procedures moves and reads detected result on fluorescent PCR instrument.
1, aminoglycoside medicaments induced deafness tumor susceptibility gene detects design of primers
According to locus specificity design of primers principle, mutational site is made as to 3 ' end of primer, and introduces point mutation to improve specificity 3 ' the 2nd, 3,4 of holding.5 operable locus specificity primers finally from 20 primers, have been filtered out, wherein for two, 1555 site, for three, 1494 site.The position in mutational site is most important to the specificity of primer, and the amplification of the normal template of primer pair can not be checked in some mutational sites; Or due to excessively strong amplification restraining effect, cause sudden change template also can not increase.Guarantee the primer that obtains of the screening sudden change template that only can increase in certain amplification system, and normal mode plate is not increased.
Meanwhile, screen the primer in 10 1494 and 1555 common downstreams, make the Tm value of the PCR product of C1494T, A1555G sudden change template can be separated when melting curve analysis, the separation of Tm value can be by judging that Tm value reach the object of somatotype; Must avoid the generation of by product, by product can produce non-specific Tm peak value, and the interpretation of result is caused to interference simultaneously.
House-keeping gene beta-actin in Select gene group, as the internal control of amplification system checking.
Finally, selected 1555 locus specificity primers, 1494 locus specificity primers, a downstream primer that two sites are public and a pair of beta-actin primer.Primer sequence after screening is as shown in table 1.
Table 1
2, deaf locus specificity primer, internal control primer and other concentration of component of reaction system are determined:
Each primer final concentration is selected 50nM-500nM, and PCR buffer contains MgCl
2its final concentration is selected 1.5mM-9mM, dN (U) TP final concentration is selected 100nM-300nM, it comprises dATP, dCTP, dGTP and dUTP, Taq enzyme final concentration is selected 1-7.5IU/ reaction, and UNG selects 0.05-0.3IU/ reaction, utilizes orthogonal test method, by great many of experiments, contrast, finally determine that optimum PCR reaction system is as shown in table 2.
Table 2
In the PCR reaction solution of above-mentioned table 2, DNA application of sample amount is 2 μ L, and total reaction volume is 25 μ L, and wherein dUTP additionally adds, and has strengthened amplification efficiency.
Wherein: a pair of internal reference beta-actin primer A1 and primer A2, the sequence of described primer A1 is SEQ ID NO:4, the sequence of described primer A2 is SEQ ID NO:5;
1555 locus specificity primer G, the sequence of described primer G is: SEQ ID NO:2;
1494 locus specificity primer T, the sequence of described primer T is: SEQ ID NO:1;
The downstream primer R that two sites are public, the sequence of described primer R is: SEQ ID NO:3.
3, PCR reaction and melting curve method reaction conditions determines
This reagent adopts containing U (uridylic) system, and UNG can eliminate the pollution that product brings.Fluorescent PCR and melting curve analysis reaction conditions divide following steps optimization:
50 ℃: 1min-10min (UNG action time)
90-95 ℃: 10min (deactivation UNG)
Through great many of experiments, contrast is optimized, and final definite optimum reaction condition is:
50℃：5min
95℃：10min
Melting curve: 70 ℃ of-90 ℃ of continuous detecting, 15 times/s.
Annealing temperature and annealing time are larger on pcr amplification efficiency and specific amplification impact, above-mentioned condition optimizing result shows that annealing temperature meeting on the low side causes non-specific amplification signal, other melt peak on melting curve, to demonstrate product peak value other places, and to result, interpretation impacts.This experiment can accomplish that by controlling annealing temperature and annealing time, without other by products, specificity is good, and amplification efficiency is high, detects under DNA profiling and is limited to 2ng.
The result of use of embodiment 2 test kits of the present invention
1, the comparison of test kit of the present invention and existing similar technology
The present invention compared with the existing technology, has set up a kind of method that is applicable to the aminoglycoside antibiotics induced deafness site examination of rapid detection and a large amount of examinations.Only a tube reaction, can detect two mitochondrial mutations sites popular in Chinese population, has avoided troublesome operation, crossed contamination, the problem that accuracy is low.
2, use test kit of the present invention to detect C1494T, A1555G sudden change situation in clinical sample
Utilize this test kit to detect general population's sample 200 examples, detect C1494T, A1555G sudden change situation in clinical sample, all sample standard deviations adopt gold standard sequencing to carry out sequence verification, and test kit detected result of the present invention and sequencing result and contrast are in Table 3 and table 4.
Table 3
Table 4
Table 3, in the sequencing result of table 4, the positive refers to that in test kit sensing range of the present invention, genotype is positive, feminine gender refers to that other genotype samples outside test kit sensing range of the present invention are positive or negative.
Test kit of the present invention detects 200 routine clinical samples and the contrast of gold standard sequencing result, undetected 0 example, and accuracy is 100%; To other genotype positive and negative sample of non-test kit sensing range, this test kit detected result is all negative, and sensitivity is 100%, and specificity is 100%.
Test kit of the present invention detects 200 routine clinical sample results and gold standard sequencing result comparative analysis statistics, and in Table 5, table 5 is that in 200 routine clinical samples, each genotype sequencing result meets situation statistics with this test kit detected result.
Table 5
3, the performance index of test kit of the present invention:
3.1 minimum detectabilities: this product can be stablized under the required genomic dna of the mitochondrial mutations that detects and is limited to 2ng.
3.2 sensitivity: the sensitivity that this product detects mitochondrial mutations reaches more than 98% (100 routine positive sample, detected result sensitivity 100%);
3.3 specificitys: the specificity of this product detection line plastochondria A1555G, C1494T reaches more than 98% (30 routine normal genotype sample duplicate detection results are all negative);
3.4 repeatability: the repeatability of this product detection line plastochondria A1555G, C1494T be more than 98% (30 routine positive sample, in experiment, between experiment, in the daytime, respectively to repeat 3 results between personnel all consistent;
3.5 stability: this product is used before the deadline can meet above each index completely.
One embodiment of embodiment 3 test kits of the present invention
1, test kit chief component composition of the present invention is as shown in table 6
Table 6
2, applicable instrument
Comprise ABI Prism 7300/7500/7700/5700/7000/7900, Roche LightCycler 480 instrument such as augmentation detection such as fluorescent PCR such as grade.
3, condition of storage and validity period
Condition of storage: test kit lucifuge is stored in-20 ℃.
Validity period: 6 months.
4, sample requirement
(1) specimen types
Peripheral blood, buccal swab are processed in dried blood spot, anti-freezing.
(1) Sample preservation
-20 ℃ of preservations are no more than 1 year.
(2) sample transportation
Dried blood spot and buccal swab can be transported by normal temperature, blood sample sealing transportation on the rocks.
(3) sample size
Upper limit of detection is 100ng genomic dna, under be limited to 2ng genomic dna, a little less than can causing amplification extremely lower than 2ng.It is 20-30ng DNA that recommendation adds sample size, can reach best effect.
5, sample extraction
For anticoagulated whole blood, recommendation sky root poba gene group DNA extraction test kit, article No. DP319; For dried blood spot, buccal swab, recommendation sky root micro-example genome extracts test kit, article No. DP316.
6, the method for inspection
(1) amplifing reagent is prepared
From test kit, taking out PCR reaction solution is placed in room temperature and melts and vibrate and mix, the centrifugal 10sec of 2000rpm.
If needed PCR reaction tubes pipe number is n, negative quality control product+2 of pipe, n=sample number+1 to be checked pipe positive quality control product; PCR reaction solution is managed with 23 μ L/ pipe packing n.
(2) application of sample
In each PCR reaction tubes, add respectively sample DNA to be checked, negative quality control product DNA and positive quality control product DNA, each 2 μ L of each quantitative reference material DNA, cover tightly pipe lid.Of short duration centrifugal be placed in fluorescent PCR detector and record sample put order.Pcr amplification program is as shown in table 7.
Table 7
7, interpretation of result condition is set
Select fluorescent PCR instrument melting curve analysis pattern, instrument can detect product peak value automatically, or utilizes manual regulation, and choosing the corresponding transverse axis coordinate of longitudinal axis fluorescent value (dF/dT) position vertex is Tm value, then according to this Tm value, carries out result interpretation.
8, quality control standard is as shown in table 8
Table 8
9, the explanation of assay
The corresponding genotype of Tm value is as shown in table 9.
Table 9
As there is the peak value except feature Tm value, longitudinal axis fluorescent value (dF/dT) is worth lower than 5 (Roche LightCycler 480), is non-specific product, does not affect the interpretation to result.
Due to A1555G and C1494T sudden change, occurrence probability is minimum simultaneously, does not show corresponding result here.
Refer to Fig. 1, Fig. 2, Fig. 3, utilize embodiment 3 reaction conditionss, Fig. 1 is the solubility curve of fluorescent PCR melting curve detection line plastochondria C1494T sudden change positive sample; Fig. 2 is the solubility curve of fluorescent PCR melting curve detection line plastochondria A1555G sudden change positive sample; Fig. 3 is the solubility curve that fluorescent PCR melting curve detects mitochondrial mutations negative sample.Wherein the fluorescent value of Y coordinate axis is changed to: the negative first order derivative that fluorescent signal changes.
10, the limitation of the method for inspection
(1), this product is qualitative test kit, do not carry out quantitative assay.
(2), this product only judges 2 kinds of chondriogen types in sensing range, cannot other genotype outside sensing range be detected.
(3), the crossed contamination of sample may cause the generation of false positive results.
(4), extract in nucleic acid and contain PCR inhibition (as heparin), may cause increase unsuccessfully, or false negative result generation.
(5), nucleic acid concentration should not be lower than 2ng/uL, should not be higher than 100ng/uL.
11, beneficial effect of the present invention
(1) minimum detectability: can stablize under the required genomic dna of the mitochondrial mutations that detects and be limited to 2ng;
(2) sensitivity: the sensitivity that detects mitochondrial mutations reaches more than 98% (100 routine positive sample, detected result sensitivity 100%);
(3) specificity: the specificity of detection line plastochondria A1555G, C1494T reaches more than 98% (30 routine normal genotype sample duplicate detection results are all negative);
(4) repeatability: the repeatability of detection line plastochondria A1555G, C1494T be more than 98% (30 routine positive sample, in experiment, between experiment, in the daytime, respectively to repeat 3 results between personnel all consistent;
(5) stability: use and can meet above each index completely before the deadline.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes specification sheets of the present invention and accompanying drawing content to do; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. an aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, is characterized in that, described test kit comprises PCR reaction solution, negative quality control product and positive quality control product; Described PCR reaction solution comprises primer, PCR Buffer, dNTP mixed solution, saturated fluorescence dyestuff, Taq enzyme, UNG enzyme; Described dNTP mixed solution is the mixed solution of dATP, dCTP, dGTP and dUTP;
Described primer comprises:
1494 locus specificity primer T, the sequence of described primer T is: SEQ ID NO:1;
1555 locus specificity primer G, the sequence of described primer G is: SEQ ID NO:2;
The downstream primer R that two sites are public, the sequence of described primer R is: SEQ ID NO:3;
A pair of internal reference beta-actin primer A1 and primer A2, the sequence of described primer A1 is SEQ ID NO:4, the sequence of described primer A2 is SEQ ID NO:5.
2. aminoglycoside medicaments induced deafness kit for detecting susceptibility genes according to claim 1, is characterized in that, in described PCR reaction solution, primer final concentration is 50-500nM, the MgCl in PCR Buffer
2final concentration is 1.5-9mM, and each component final concentration of described dNTP mixed solution is 100-300nM, and Taq enzyme final concentration is 1-7.5IU/ reaction, and UNG enzyme final concentration is 0.05-0.3IU/ reaction.
3. aminoglycoside medicaments induced deafness kit for detecting susceptibility genes according to claim 1, is characterized in that, described saturated fluorescence dyestuff is EvaGreen dyestuff, LC Green PLUS dyestuff, ResoLight dyestuff or SYTO9 dyestuff.
4. aminoglycoside medicaments induced deafness kit for detecting susceptibility genes according to claim 1, is characterized in that, described each component concentration of PCR reaction solution is:
Deionized water: 16.14 μ L;
10×PCR?buffer：2.5μL；
The equal-volume mixed solution of 10mM dATP, dCTP, dGTP, dUTP: 2 μ L;
10mM?dUTP：0.5μL；
100 μ M primer A1:0.01 μ L;
100 μ M primer A2:0.01 μ L;
100 μ M primer G:0.04 μ L;
100 μ M primer T:0.03 μ L;
100 μ M primer R:0.02 μ L;
20×Evagreen：1.25μL；
The ultrapure Taq enzyme of 5U/ μ L: 0.2 μ L;
1U/μL?UNG：0.3μL。
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