CN102925562A - 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
- Publication number
- CN102925562A CN102925562A CN2012103938664A CN201210393866A CN102925562A CN 102925562 A CN102925562 A CN 102925562A CN 2012103938664 A CN2012103938664 A CN 2012103938664A CN 201210393866 A CN201210393866 A CN 201210393866A CN 102925562 A CN102925562 A CN 102925562A
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- induced deafness
- kit
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 the 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
Show according to China's the Second China National Sample Survey on Disability in 2006, 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 the ototoxicity microbiotic is the most common.Aminoglycoside antibiotics is modal ototoxicity microbiotic, comprises Streptomycin sulphate commonly used clinically, gentamicin etc.Clear and definite several mitochondrial mutations relevant with aminoglycoside antibiotics over past ten years, 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 diagnosis, the early screening of deafness, and provide medication guide to have great significance.
Less for the test kit kind of deaf gene detection in the market, 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 method commonly used is micro-array chip method and fluorescence quantitative PCR method etc., and their main technological deficiency is as follows:
1, the 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:
Detecting needs to extract the steps such as (30min), pcr amplification (more than the 2h), product hybridization analysis (more than the 1h) through whole blood DNA, and detection time long (more than the 4h), complex operation can not satisfy clinical in causing the demand of a large amount of examinations of deaf gene; Because operating process is too much, easily causes sample cross contamination; Secondly, lower to the discrimination power of single base, be prone to false negative and false positive, result's accuracy rate is affected; In addition, the micro-array chip high cost needs special chip scanning instrument, and testing cost is high, and such as " brilliant core " series product of rich biological production difficult to understand, a person-portion detects to be needed to be difficult to promote on a large scale clinically more than thousand yuan.
Therefore, be necessary to research and develop that detection time is short, cost is low and suitable medicine is caused 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 only is the 1-10% of normal amplification, so the amplification system deficient in stability; The reaction of four primer ARMS-PCR can only be examined a mutational site: middle product of giving birth to the north biological company limited of control can detect four deaf gene mutational sites, but each site needs a reaction tubes, and multiplex PCR is difficult to realization, and testing process is loaded down with trivial details.
The shortcoming that the regular-PCR product is total: when as a result interpretation, need to judge genotype by stripe size, lack intuitive.Detected through gel electrophoresis result easily causes the PCR crossed contamination, detection sensitivity is low and length consuming time.
3, utilize the Taqman probe to carry out gene type, highly sensitive, high specificity, but following shortcoming is arranged:
Utilize the Taqman probe to carry out the 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 requirement, is unfavorable for promoting.
Each reaction of current detection kit only detects a site.Listen Jin Yi Bioisystech Co., Ltd such as present Beijing section, 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 following shortcoming arranged:
Testing process is loaded down with trivial details, excessive cycle: need the purpose fragment is carried out pcr amplification before the order-checking, amplified production is carried out purifying, carry out the capillary electrophoresis order-checking, whole process need strict quality control again; The sequencing reaction cost is higher, needs the sequenator of expensive fluorescently-labeled nucleic acid and specialty, is not suitable for clinical expansion.The interpretation more complicated of sequencing result needs through special training.
In sum: existing common method, detect gene such as fluorescence quantitative PCR method, single mutational site on the detection line plastochondria only, most of fluorescence quantitative PCR method can not carry out quality control without interior mark; The microarray attitude chip product operating time is long, cost is high, accuracy is low and need specific apparatus.Extensive examination mitochondrial gene mutation, requirement are fast, low-cost, sensing range is wide, and above product can't satisfy 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 satisfy the demand of aminoglycoside medicaments induced deafness tumor susceptibility gene examination.
Summary of the invention
The objective 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 that different samples are carried out gene test, need not probe, need not primer is carried out 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 confidential reference items 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, namely 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, the primer final concentration is 50-500nM in the described PCR reaction solution, the MgCl among the 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 the 1-7.5IU/ reaction, and UNG enzyme final concentration is the 0.05-0.3IU/ reaction.
Preferably, above-mentioned aminoglycoside medicaments induced deafness kit for detecting susceptibility genes, described saturated fluorescence dyestuff are 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 comprises the steps: for a kind of aminoglycoside medicaments induced deafness tumor susceptibility gene detection method is provided
(1) adopt above-mentioned each described test kit that the goal gene of the template DNA of sample is carried out pcr amplification;
(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 detects gene such as fluorescence quantitative PCR method, single mutational site on the detection line plastochondria only, and most of fluorescence quantitative PCR method can not carry out quality control without interior mark; The microarray attitude 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 can't satisfy simultaneously.The present invention is according to aminoglycoside medicaments induced deafness tumor susceptibility gene SNP site, design special PCR primer, template can increase so that only suddenly change, 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 that a pipe operation is to the somatotype in mitochondrial gene mutation site.Gene test of the present invention need not probe, need not primer is carried out special modification, and is with low cost, and detects fast, accurately.Compare existing method simple to operate, sensing range is wider, and detection sensitivity is higher, conscientiously satisfies the demand of aminoglycoside medicaments induced deafness tumor susceptibility gene examination.
Description of drawings
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: the 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 purpose and effect, below in conjunction with embodiment and cooperate accompanying drawing to give in detail explanation.
The present invention adopts allele-specific PCR, is to utilize Taq enzymatic defect 3 '-5 ' 5 prime excision enzyme activity, holds and can not match fully with template when 3 ' of primer, and then pcr amplification can not carry out.Therefore, under the ideal state, primer 3 ' end only is designed to and the sudden change template matches, and does not mate with normal template, this primer sudden change template that namely can only increase, and the normal mode plate is not increased, thus reach the effect of somatotype.Simultaneously, hold other positions at primer 3 ', such 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 opposite Auele Specific Primers of design direction, article one, the sudden change template only increases, another normal template that only increases, the upstream and downstream primer that simultaneously design matches is so that the expanding fragment length of normal template is 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, and is easy and simple to handle, is applicable to detect the detection that aminoglycoside medicaments among 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 that slowly heats up, DNA unwinds gradually and discharges dye molecule, and fluorescent value descends immediately, can calculate thus the Tm value of this DNA chain, judge the character of amplified fragments according to the difference of Tm value.
Fluorescence melting curve method can be distinguished by the difference of product Tm value the product of different fragments length, and is higher than gel electrophoresis sensitivity; Can stop simultaneously because the PCR product Aerosol Pollution that product is uncapped and caused.Melting curve method and allele-specific PCR coupling can be made low cost, simplicity and practical diagnostic kit, very are fit 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.Comprise Taq enzyme, the chimeric dyestuff of dna double chain, amplimer, amplification buffer and dN (U) TP etc. in the PCR reaction solution, dN (U) TP is the dNTP mixed solution.The PCR response procedures is in the operation of fluorescent PCR instrument and read detected result.
1, aminoglycoside medicaments induced deafness tumor susceptibility gene detects design of primers
According to locus specificity design of primers principle, the mutational site is made as 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 in 1555 sites, for three in 1494 sites.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; Perhaps because excessively strong amplification restraining effect causes the sudden change template also can not increase.Guarantee to screen the primer that the obtains sudden change template that in certain amplification system, only can increase, and the normal mode plate is not increased.
Simultaneously, screen the primer in 10 1494 and 1555 common downstreams, so that 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 reach by judgement Tm value the purpose of somatotype; Must avoid simultaneously the generation of by product, by product can produce non-specific Tm peak value, and result's interpretation is caused interference.
House-keeping gene beta-actin on the Select gene group is 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.As shown in table 1 through the primer sequence after the screening.
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 the 1-7.5IU/ reaction, and UNG selects the 0.05-0.3IU/ reaction, utilizes orthogonal test method, by the great many of experiments contrast, final definite optimum PCR reaction system is as shown in table 2.
Table 2
DNA application of sample amount is 2 μ L in the PCR reaction solution of above-mentioned table 2, and total reaction volume is 25 μ L, and wherein dUTP additionally adds, and has strengthened amplification efficiency.
Wherein: a pair of confidential reference items 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 and contains the U(uridylic) system, 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)
Contrast is optimized through great many of experiments, and the final optimum reaction condition of determining 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 efficient and specific amplification impact, above-mentioned condition optimizing result shows that annealing temperature meeting on the low side causes the non-specific amplification signal, other melt the peak to demonstrate product peak value other places at melting curve, and as a result interpretation is impacted.This experiment can accomplish that by control annealing temperature and annealing time without other by products, specificity is good, and amplification efficiency is high, detects under the 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 compares with prior art, 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 the clinical sample
Utilize this test kit to detect general population's sample 200 examples, detect C1494T, A1555G sudden change situation in the 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 see Table 3 and table 4.
Table 3
Table 4
Table 3, the positive refers to that genotype is positive in the test kit sensing range of the present invention in the sequencing result of table 4, feminine gender refers to that other genotype samples outside the 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, and undetected 0 example, accuracy are 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, sees Table 5, and table 5 is that each genotype sequencing result meets the situation statistics with this test kit detected result in the 200 routine clinical samples.
Table 5
3, the performance index of test kit of the present invention:
3.1 minimum detectability: 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 (100 routine positive sample, detected result sensitivity 100%) more than 98%;
3.3 specificity: the specificity of this product detection line plastochondria A1555G, C1494T reaches (30 routine normal genotype sample duplicate detection results are all negative) more than 98%;
3.4 repeatability: the repeatability of this product detection line plastochondria A1555G, C1494T be 98% or more (30 routine positive sample, in the experiment, test between, in the daytime, respectively to repeat 3 results between personnel all consistent;
3.5 stability: this product is used before the deadline can satisfy above each index fully.
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: the 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
But dried blood spot and the transportation of buccal swab normal temperature, blood sample sealing transportation on the rocks.
(3) sample size
Upper limit of detection is the 100ng genomic dna, is limited to the 2ng genomic dna down, and being lower than 2ng can cause a little less than the amplification extremely.Recommending to add sample size is 20-30ng DNA, 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
Taking out the PCR reaction solution from test kit places room temperature to melt and the vibration mixing the centrifugal 10sec of 2000rpm.
If needed PCR reaction tubes pipe number is n, the n=sample number to be checked+negative quality control product of 1 pipe+2 pipe positive quality control product; The 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 the pipe lid.Of short duration centrifugal be placed in the fluorescent PCR detector and record sample put the order.The 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 the product peak value automatically, or utilizes manual regulation, and (dF/dT) vertex corresponding transverse axis coordinate in position is the Tm value, then carries out as a result interpretation according to this Tm value to choose longitudinal axis fluorescent value.
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 the peak value except feature Tm value occurring, longitudinal axis fluorescent value (dF/dT) value is lower than 5(Roche LightCycler 480) then is non-specific product, does not affect the interpretation to the result.
Because occurrence probability is minimum simultaneously for A1555G and C1494T sudden change, does not show corresponding result here.
See also 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 the fluorescent PCR melting curve detects the 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 the sensing range, can't detect other outer genotype of sensing range.
(3), the crossed contamination of sample may cause the generation of false positive results.
(4), extract in the nucleic acid and contain PCR inhibition (such as heparin), may cause increase unsuccessfully, or the 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 (100 routine positive sample, detected result sensitivity 100%) more than 98%;
(3) specificity: the specificity of detection line plastochondria A1555G, C1494T reaches (30 routine normal genotype sample duplicate detection results are all negative) more than 98%;
(4) repeatability: the repeatability of detection line plastochondria A1555G, C1494T be more than 98% (30 routine positive sample, in the experiment, between experiment, in the daytime, respectively to repeat 3 results between personnel all consistent;
(5) stability: use before the deadline and can satisfy above each index fully.
The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Sequence table
SEQUENCE LISTING
＜110〉Yaneng Biotechnology (Shenzhen) Co., Ltd.
＜120〉aminoglycoside medicaments induced deafness kit for detecting susceptibility genes and method
<160>5
<170>PatentIn version 3.3
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<400>1
tacacaccgc ccgtgact 18
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<400>2
cctacgcatt tatatagagg tcg 23
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<400>3
gtccaagtgc actttccagt a 21
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<400>4
gtggacatcc gcaaagac 18
<210>5
<211>19
<212>DNA
＜213〉artificial sequence
<400>5
gaaagggtgt aacgcaact 19
Claims (8)
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 confidential reference items 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, it is characterized in that, the primer final concentration is 50-500nM in the described PCR reaction solution, MgCl2 final concentration among the PCR Buffer is 1.5-9mM, each component final concentration of described dNTP mixed solution is 100-300nM, Taq enzyme final concentration is the 1-7.5IU/ reaction, and UNG enzyme final concentration is the 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 SYTO 9 dyestuffs.
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。
5. an aminoglycoside medicaments induced deafness tumor susceptibility gene detection method is characterized in that, comprises the steps:
(1) adopt each described aminoglycoside medicaments induced deafness kit for detecting susceptibility genes of claim 1 to 4 that the goal gene of the template DNA of sample is carried out pcr amplification;
(2) melting curve analysis is carried out in the mutational site of the goal gene of PCR product.
6. according to claims 5 described aminoglycoside medicaments induced deafness tumor susceptibility gene detection methods, it is characterized in that, 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.
7. according to claims 6 described aminoglycoside medicaments induced deafness tumor susceptibility gene detection methods, it is characterized in that, 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.
8. according to claims 5 described aminoglycoside medicaments induced deafness tumor susceptibility gene detection methods, it is characterized in that, the program that described step (2) PCR product carries out melting curve analysis is: 70 ℃ of-90 ℃ of continuous detecting, 15 times/s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210393866.4A CN102925562B (en) | 2012-10-17 | 2012-10-17 | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210393866.4A CN102925562B (en) | 2012-10-17 | 2012-10-17 | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102925562A true CN102925562A (en) | 2013-02-13 |
| CN102925562B CN102925562B (en) | 2014-02-12 |
Family
ID=47640485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210393866.4A Active CN102925562B (en) | 2012-10-17 | 2012-10-17 | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102925562B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232750A (en) * | 2014-05-30 | 2014-12-24 | 嘉兴雅康博医学检验所有限公司 | NRAS gene mutation detection kit |
| CN104404164A (en) * | 2014-12-18 | 2015-03-11 | 亚能生物技术（深圳）有限公司 | Hereditary deafness gene mutation detection kit |
| CN104711366A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Drug-induced deafness gene multichannel fluorescence PCR (polymerase chain reaction) detection kit |
| CN106282335A (en) * | 2016-08-10 | 2017-01-04 | 四川金域医学检验中心有限公司 | A kind of detect the primer of juvenile's deaf inheritance risk, test kit, detection method |
| CN106957902A (en) * | 2016-11-01 | 2017-07-18 | 复旦大学 | A kind of non-marked probe for being used to detect drug-induced deafness related locus |
| CN108588213A (en) * | 2018-05-02 | 2018-09-28 | 宁波美丽人生医药生物科技发展有限公司 | Aminoglycoside medicaments correlation 12s rRNA gene mutation site detection kits |
| CN111172250A (en) * | 2020-03-03 | 2020-05-19 | 肇庆医学高等专科学校 | Probe composition for detecting drug-induced deafness gene and application thereof |
| CN112430649A (en) * | 2020-12-10 | 2021-03-02 | 杭州方略生物科技有限公司 | Fluorescent PCR (polymerase chain reaction) detection kit and detection method for mitochondrial DNA A1555G and C1494T mutations |
-
2012
- 2012-10-17 CN CN201210393866.4A patent/CN102925562B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 郑璐等: "碱基淬灭探针技术单管检测线粒体DNA 12S rRNA A1555G及C1494T", 《中华耳鼻咽喉头颈外科杂志》 * |
| 郑静等: "多重等位基因PCR检测线粒体12S rRNA基因突变", 《中华检验医学杂志》 * |
| 黄荷凤: "《高危妊娠》", 31 May 2003 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232750A (en) * | 2014-05-30 | 2014-12-24 | 嘉兴雅康博医学检验所有限公司 | NRAS gene mutation detection kit |
| CN104404164A (en) * | 2014-12-18 | 2015-03-11 | 亚能生物技术（深圳）有限公司 | Hereditary deafness gene mutation detection kit |
| CN104711366A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Drug-induced deafness gene multichannel fluorescence PCR (polymerase chain reaction) detection kit |
| CN106282335A (en) * | 2016-08-10 | 2017-01-04 | 四川金域医学检验中心有限公司 | A kind of detect the primer of juvenile's deaf inheritance risk, test kit, detection method |
| CN106957902A (en) * | 2016-11-01 | 2017-07-18 | 复旦大学 | A kind of non-marked probe for being used to detect drug-induced deafness related locus |
| CN106957902B (en) * | 2016-11-01 | 2020-12-04 | 复旦大学 | Non-labeled probe for detecting drug-induced deafness related sites |
| CN108588213A (en) * | 2018-05-02 | 2018-09-28 | 宁波美丽人生医药生物科技发展有限公司 | Aminoglycoside medicaments correlation 12s rRNA gene mutation site detection kits |
| CN111172250A (en) * | 2020-03-03 | 2020-05-19 | 肇庆医学高等专科学校 | Probe composition for detecting drug-induced deafness gene and application thereof |
| CN112430649A (en) * | 2020-12-10 | 2021-03-02 | 杭州方略生物科技有限公司 | Fluorescent PCR (polymerase chain reaction) detection kit and detection method for mitochondrial DNA A1555G and C1494T mutations |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102925562B (en) | 2014-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102925562B (en) | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene | |
| CN105506118B (en) | Primer pair, fluorescence probe, kit and method for detecting CYP2C19 Genotypings | |
| CN102337338B (en) | Method for simultaneously and quickly detecting number of five types of chromosomes, and kit and application thereof | |
| CN104120080A (en) | Alpha-globin gene mutation detection kit as well as preparation method and use thereof | |
| CN103757091B (en) | Sudden cardiac death rapid gene detection kit and detection method | |
| CN102586473B (en) | Hepatitis B virus genotyping PCR (polymerase chain reaction) test kit | |
| CN104263848A (en) | Deafness susceptibility gene mutation detection kit as well as preparation method and application thereof | |
| CN107227361A (en) | Primer, probe and detection kit for detecting CYP2C19 gene pleiomorphisms | |
| CN102154503A (en) | Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe | |
| CN105274190A (en) | HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T | |
| CN111118138A (en) | Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR | |
| CN108642156A (en) | A kind of the digital pcr detection kit and its detection method of T790M gene mutations | |
| CN103045717B (en) | Pyrosequencing techniques detects the method for mycobacterium tuberculosis detection of rifampin resistant | |
| CN103103259A (en) | Method, kit and primers for determining whether two predetermined loci of nucleic acid sample mutate or not | |
| CN109182493A (en) | The primer and kit and its detection method of people's 16p11.2 microdeletion syndrome detection | |
| CN105624315A (en) | Primers and reagent kit for detecting polymorphism of ALDH2 gene c.1510 locus | |
| CN104087672A (en) | Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique | |
| CN106319079A (en) | Method for detecting 22q11.2 copy number deletion | |
| CN105950766A (en) | Primer group and kit for detecting HLA-B*5801 allelic genes | |
| CN116254371A (en) | Primer molecular beacon combination for wild type and mutant molecular typing of monkey pox virus and application thereof | |
| CN103045716A (en) | Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology | |
| CN108531579A (en) | Primer composition and kit for detecting sulfonylureas related gene | |
| CN108277273A (en) | With detecting non-deletion type α probe, primer and the kit of poor gene mutation | |
| CN102912038A (en) | RT-HDA kit and primer for detecting avian influenza virus | |
| CN103215356A (en) | Assay kit for detecting human leukocyte antigen-B (HLA-B)*57:01 and HLA complex P5 (HCP5) alleles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |