CN104419757A - Novel deafness related gene mutation detection system and kit - Google Patents
Novel deafness related gene mutation detection system and kit Download PDFInfo
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Abstract
The invention discloses a novel deafness related gene mutation detection system, for detecting whether deafness gene mutation sites have mutation or not by using multiple PCR primers and a detection probe. The deafness gene mutation sites comprise a newly found CEACAM16 gene (NM_001039213.2)c.418A>C mutation site. The 32 sites selected by the detection kit are screened according to the latest human genetic deafness related gene mutation information and firsthand information accumulated by the inventor in clinical detection, and the selected sites are sites with relatively high mutation frequency in people of China and other countries and sites which are newly found in clinical detection by the inventor. Compared with deafness related gene mutation detection products available in the market, the kit covers relatively more detection sites so that the mutation detection rate is increased.
Description
Technical field
The present invention relates to a kind of detection in Gene Mutation system in hereditary hearing impairment Molecular Detection field, in particular to a kind of new deaf-related gene mutation detection system and test kit.
Background technology
Deafness is a kind of common clinical disease, main and inherited genetic factors and environmental factors closely related, namely may be caused by associated gene mutation, also may be caused by environmental exposure, wound, medicine etc.It is reported that for neonatal congenital deafness 50% be that inherited genetic factors causes, in the congenital case of relevant report, having an appointment 70%, to belong to non-syndrome deaf.This type of case can be divided into again deaf four classes of autosomal dominant, autosomal recessive, sex linkage and mitochondrial inheritance, the deaf related locus had been reported more than 114.
At present for hereditary hearing impairment checkout and diagnosis method, more generally using is detect the relevant gene of patient's deafness whether existing defects, relates to the correlation technique application of DNA detection.Conventional detection technique comprises: the technology such as restriction endonucleases fingerprinting single strand conformation polymorphism analysis, restriction fragment length polymorphism analysis, dhplc analysis, gene chip, flight time mass spectrum, exon trapping technology, direct Sequencing, above various detection technique respectively deposits relative merits.
Restriction endonucleases fingerprinting single strand conformation polymorphism analysis is the pcr amplification product with restriction endonuclease cutting target gene, and agarose gel electrophoresis detects plum and cuts product, carries out the judgement of target gene with or without sudden change according to abnormal conformational band.The topmost problem of the method all sudden changes to be detected, and the sudden change recall rate reported by each laboratory punching 99% to 35% is not etc.The method requires repeatedly to grope condition simultaneously, and as electrophoresis temperature, in glue, glycerol concentration and glue connection degree etc. all can affect the sensitivity of detection.In addition, the method can not determine the exact position of sudden change.
Restriction fragment length polymorphism is the pcr amplification product with specific Rastriction enzyme target gene, then the electrophoretogram feature of enzyme analysis hydrolysis products, judge whether measuring samples exists certain transgenation according to the comparison result with normal control, its weakness complex operation, recall rate is low, because also the transgenation of not all is positioned at the cog region of certain restriction endonuclease all just.
This technology of dhplc analysis is a new heteroduplex mutation detection techniques grown up in single-strand conformation polymorphism analysis and denaturing gradient gel electrophoresis basis.It can carry out examination to pcr amplification product in enormous quantities, and it demonstrates the susceptibility of height in the different sequences detecting a large amount of Disease-causing gene, is applicable to doing gene screening fast.But it also has some parts not fully up to expectations: (1) it merely provide information qualitatively, and concrete mutation type and mutational site cannot be drawn.Order-checking of still needing waits subsequent processes to confirm; (2) its result judges normally to be undertaken by operator, easily produces and observes difference, is unfavorable for that remolding sensitivity between each laboratory comparatively; (3) many fragments have multiple main melting temperature(Tm), need the temperature of examination more, the workload of increase.This technology is mainly used to the DNA fragmentation of detection 200 ~ 300bp size at present, and there is not been reported in the detection of long DNA fragmentation.
Gene chip has microminiaturization, intensive, stdn and high-throughout feature because of it, there is unique advantage in the clinical diagnosis of the diseases such as infectious diseases, heredopathia, Severe Communicable Disease and malignant tumour, can the oligonucleotide probe synthetic point or point that correspond to mutantional hotspot be added in DNA chip, by once having hybridized the examination to testing sample various mutations possibility, realize the efficient quick diagnosis to disease, but chip detection platform price is high, unfavorable clinical expansion; Existing clinical deaf gene detection chip only detects 9 mutational sites, and sudden change recall rate is lower.
It is first carry out pcr amplification object segmented regions that flight time mass spectrum method detects gene, then add multipair Auele Specific Primer, carry out single-basic extension, then by mass spectroscopy after single base extension product purifying, different according to the mass-to-charge ratio of different primers amplified production, determine whether base mutation.At present, deaf gene detects by flight time mass spectrum and exon trapping two kinds of detection methods in BGI-Shenzhen.Exon trapping technology can detect 51 genes and 2 mutational sites of non-syndromic cleft lip and palate at present, and other more common syndromic deafness, such as Alport syndrome, Waardenburg syndrome etc.Flight time mass spectrum detection technique mainly detects 20 mutational sites occurred frequently in Chinese population at present, and accuracy is high but testing cost is higher, and the time used is longer.
Direct Sequencing is by after polymerase chain reaction (PCR) amplification product purification, sex change, and sequenator checks order, for finding the gold standard of sudden change.But its plant and instrument is expensive, and complicated operation, consuming time longer.In addition, the problem such as existence of heterozygous mutant, glue laminated contracting, GC enrichment region makes to be difficult to obtain accurate data by once sequencing.
Summary of the invention
The object of the invention is to the above problem overcoming prior art existence, a kind of new deaf-related gene mutation detection system and test kit are provided, filter out 32 deaf-related gene detection site, comprise do not report in the world new and send out site, and synthesize efficiently and accurately detection system fast in conjunction with technology groups such as PCR, realization to rapid detection while 32 deaf gene mutational sites, can be applicable to biological study and hereditary hearing impairment Molecular Detection field in this system.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of deaf-related gene mutation detection system, by using multiple PCR primer and detection probes to detect in deaf gene mutational site whether there is sudden change, described deaf gene mutational site comprises CEACAM16 gene (NM_001039213.2) c.418A>C mutational site.
Further, described deaf gene mutational site comprises
Totally 32 deaf gene mutational sites.
Further, following reactions steps is comprised:
1) detection sample is carried out multi-PRC reaction; 2) multiple PCR products purifying; 3) somatotype ligation; 4) fluoroscopic examination.
Further, described multi-PRC reaction comprises DNA sample to be measured, bi-distilled water, 2xGC I damping fluid, divalent magnesium ion solution, deoxyribonucleoside triphosphate, primer mixed solution, the archaeal dna polymerase of thermostability; To go forward side by side performing PCR cycling program.
Further, described primer mixed solution is 18 pairs of multiple PCR primers, and the sequence of described primer is as shown in SEQ ID NO.:1-SEQ ID NO.:36.
Further, described multiple PCR products purifying is in PCR primer, add the SAP enzyme of 1 unit of enzyme and the Exonuclease I enzyme of 1 unit of enzyme, deactivation after temperature bath.
Further, described somatotype ligation comprises multiple PCR products, 10x ligase enzyme damping fluid, 4xGC solution, Taq ligase enzyme, mark mixed solution, PiMLDR mixed solution, bi-distilled water; And carry out circulating reaction.
Further, described PiMLDR mixed solution is 32 groups of detection probes, and often organize detection probes and comprise 2 discriminating probes and 1 general probe, the sequence of described detection probes is as shown in SEQ ID NO.:37 to SEQ IDNO.:132.
A kind of deaf-related gene mutation detection kit, comprising: bi-distilled water; 2xGC I damping fluid; Divalent magnesium ion solution; Deoxyribonucleoside triphosphate; Primer mixed solution; The archaeal dna polymerase of thermostability; SAP enzyme; Exonuclease I enzyme; 10x ligase enzyme damping fluid; 4xGC solution; Taq ligase enzyme; Mark mixed solution; PiMLDR mixed solution.
The invention has the beneficial effects as follows:
1,32 sites that this detection kit is chosen are that the firsthand information utilizing human inheritance's property deaf-related gene mutation information of latest report and contriver to accumulate in clinical detection screens, the site chosen is site and the contriver newfound site in clinical detection that domestic and international crowd's mutation frequency is higher, except the deaf site of common non-syndrome, also add the syndromic abrupt climatic change site of syndromic deafness Waardenburg common clinically, eliminate the site that the frequency of occurrences is low.Compare the existing deaf abrupt climatic change product in current market, this test kit covers more detection site, improves sudden change recall rate.
2,1 mankind's hereditary hearing impairment associated gene mutation novel site filtered out, did not also report in international literature, had enriched the correlative studys such as hereditary hearing impairment and the site range of choice in detecting.
3, by designing detection probes sequence, reaction conditions, reaction system and testing process and optimizing, make this test kit can more accurately, more efficient 32 gene mutation sites to be detected, the DNA sample of 20-30 nanogram is only needed to detect, be applicable to comprising blood collecting card and organizing the DNA sample in the multiple sources such as extracting to detect, 32 sites can be carried out in a reaction system, complete in 24 hours, improve the working efficiency of testing staff.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.The specific embodiment of the present invention is provided in detail by following examples and accompanying drawing thereof.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 overhaul flow chart;
The structural representation of Fig. 2 detection probes;
Fig. 3 detected peaks type figure.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
This technology screening goes out 32 more common deaf gene mutational site composition deaf gene abrupt climatic change systems.The new mutant do not reported that the screening in 32 mutational sites finds in clinical diagnosis according to common mutations site and the contriver for international SCI bibliographical information.(database with reference to cut-off in June, 2013 before NCBIhttp: //www.ncbi.nlm.nih.gov/pubmed database and international man's genoid mutation database http://www.hgmd.org/).Detect mutational site information in table 1, wherein on No. 17 karyomit(e)s CEACAM16 gene c.505G>A sport newfound sudden change:
The essential information in table 1 test kit detection in Gene Mutation site
Design the sequence information of 18 pairs of multiple PCR primers in table 2:
Table 2 multiple PCR primer sequence information
And the detection probes sequence information in mutational site is in table 3:
Table 3 detection probes sequence information
Embodiment
1, DNA sample prepares
Within the hospital blood (also can be the blood collecting card, biological tissue etc. containing individual of sample DNA) is gathered to 1 deafness patient chosen and a normal control individuals; Obtain DNA sample by DNA extraction kit extracting and respectively get 1 microlitre 1%agarose electrophoresis quality inspection and concentration sealing are carried out to its sample, then according to the concentration estimated by Sample Dilution to working concentration 10-20 nanogram/microlitre.
2, PCR reaction:
Multi-PRC reaction system forms: system cumulative volume 10 microlitre, wherein bi-distilled water (ddH2O) 1.13 microlitre, 2xGC I damping fluid (2*GC I buffer) 5 microlitres, divalent magnesium ion (Mg
2+) 0.2 microlitre, deoxyribonucleoside triphosphate (dNTP) 1.6 microlitre, primer mixed solution (Primer mix) 1 microlitre, archaeal dna polymerase (Taq enzyme) 0.07 microlitre of thermostability, detected sample DNA 1 microlitre.
PCR cycling program: 95 DEG C of sex change 2 minutes; Then 11 circulations are set, in each circulation reaction conditions be 94 DEG C 20 seconds, 65 DEG C 40 seconds (each circulation reduction by 0.5 DEG C), 72 DEG C 1.5 minutes; 24 circulations are then set after 11 loop ends, in each circulation reaction conditions be 94 DEG C 20 seconds, 59 DEG C 30 seconds, 72 DEG C 1.5 minutes; Arrange 72 DEG C after 24 loop ends to keep 2 minutes; Reaction product is preserved at last 4 DEG C.
3, multiple PCR products purifying
The SAP enzyme of 1 unit of enzyme and the Exonuclease I enzyme of 1 unit of enzyme is added, 37 DEG C of temperature baths 1 hour, then 75 DEG C of deactivations 15 minutes in 10 microlitre PCR primer.
4, somatotype ligation test.
Packing after mixing, each reaction system 7 microlitre, then add 3 microlitre multiple PCR products templates, supply 10 microlitres
Ligation system (10 μ l):
Response procedures:
38 circulations are set, each circulation 94 DEG C 1 minute, 58 DEG C 4 minutes, then preserve reaction product at 4 DEG C.
5, loading
Get 1 microlitre and connect product, add containing after 8.9 microlitre HiDi reagent and 0.1 microlitre Liz500 reagent, vibration mixing, 95 DEG C of sex change in 5 minutes, upper ABI3130XL sequenator runs STR program.
6, analyze
As shown in Figure 1, in schema, particle represents each fluorophor, in order to distinguish different PCR primer in capillary electrophoresis peak figure; Small arrow represents multiple PCR primer, and the direction of arrow is primer extension direction.After the reaction of the first step multiplexed PCR amplification, add detection probes and carry out ligation.Detect 1 site and need 2 detection probes, be respectively 5 ' end detection probes and 3 ' end detection probes.3 ' end detection probes is common PCR primer.
As shown in Figure 2, need to add mark ligation masterplate, fluorescent mark universal primer, detection probes and the component needed for other ligations in ligation system simultaneously.What mark ligation masterplate can design with us simultaneously 5 ' holds detection probes and fluorescent mark universal primer to be combined, and under ligase enzyme effect, make fluorescent dye primer be connected with 5 ' end detection probes, simultaneously, when the base of detection site is detection probes correspondence base, 5 ' end detection probes and 3 ' holds detection probes to be also connected under ligase enzyme effect, these 3 parts connect above is an overall connection product, the special dual link reaction of the multiple height namely in Fig. 1.
Because primer is detected with fluorescence in mutational site used, the am-plified fragments of different colours fluorescence and different size is detected by capillary electrophoresis, detect the peak figure (as shown in Figure 3) obtained and show whether each detection site undergos mutation with the information comparison of table 4, table 5 to analyze, and judge that this sports homozygous mutation or heterozygous mutant (as shown in table 4, table 5).Shown in contrast display Fig. 3 upper part, peak, each site type is normal peak type, and illustrating in this sample that detecting 32 mutational sites does not all undergo mutation, is normal control sample; C.235delC, the peak type correspondence of lower part arrow mark is suddenlyd change, and this site also exists normal peak type simultaneously, illustrates that this sample there occurs c.235delC heterozygous mutant.
Table 4 peak type comparative information table 1
Table 5 peak type comparative information table 2
Note: PET, VIC, NED, FAM tetra-kinds of fluorescence dyes (Applied Biosystems, USA company).
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a deaf-related gene mutation detection system, it is characterized in that: by using multiple PCR primer and detection probes to detect in deaf gene mutational site whether there is sudden change, described deaf gene mutational site comprises CEACAM16 gene (NM_001039213.2) c.418A>C mutational site.
2. deaf-related gene mutation detection system according to claim 1, is characterized in that: described deaf gene mutational site comprises
Totally 32 deaf gene mutational sites.
3. deaf-related gene mutation detection system according to claim 2, is characterized in that, comprises following reactions steps:
1) detection sample is carried out multi-PRC reaction;
2) multiple PCR products purifying;
3) somatotype ligation;
4) fluoroscopic examination.
4. deaf-related gene mutation detection system according to claim 3, is characterized in that: described multi-PRC reaction comprises
DNA sample to be measured,
Bi-distilled water,
2xGC I damping fluid,
Divalent magnesium ion solution,
Deoxyribonucleoside triphosphate,
Primer mixed solution,
The archaeal dna polymerase of thermostability;
To go forward side by side performing PCR cycling program.
5. deaf-related gene mutation detection system according to claim 4, is characterized in that: described primer mixed solution is 18 pairs of multiple PCR primers, and the sequence of described primer pair is as shown in SEQ ID NO.:1 to SEQID NO.:36.
6. deaf-related gene mutation detection system according to claim 3, is characterized in that: described multiple PCR products purifying is in PCR primer, add the SAP enzyme of 1 unit of enzyme and the Exonuclease I enzyme of 1 unit of enzyme, deactivation after temperature bath.
7. deaf-related gene mutation detection system according to claim 3, is characterized in that: described somatotype ligation comprises
Multiple PCR products,
10x ligase enzyme damping fluid,
4xGC solution,
Taq ligase enzyme,
Mark mixed solution,
PiMLDR mixed solution,
Bi-distilled water;
And carry out circulating reaction.
8. deaf-related gene mutation detection system according to claim 7, it is characterized in that: described PiMLDR mixed solution is 32 groups of detection probes, often organize detection probes and comprise 2 discriminating probes and 1 general probe, the sequence of described detection probes is as shown in SEQ ID NO.:37 to SEQ ID NO.:132.
9. a deaf-related gene mutation detection kit, is characterized in that, comprising:
Bi-distilled water;
2xGC I damping fluid;
Divalent magnesium ion solution;
Deoxyribonucleoside triphosphate;
Primer mixed solution;
The archaeal dna polymerase of thermostability;
SAP enzyme;
Exonuclease I enzyme;
10x ligase enzyme damping fluid;
4xGC solution;
Taq ligase enzyme;
Mark mixed solution;
PiMLDR mixed solution.
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Cited By (5)
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| CN105543370A (en) * | 2016-01-14 | 2016-05-04 | 中南大学湘雅医院 | New comprehensive-deafness related-gene mutation detection system and kit |
| CN106811533A (en) * | 2017-03-06 | 2017-06-09 | 亚能生物技术（深圳）有限公司 | A kind of hereditary hearing impairment gene detecting kit |
| CN106987637A (en) * | 2017-04-25 | 2017-07-28 | 郑州大学第附属医院 | A kind of amplimer, kit and application for detecting the mutation of familial retinal pigment degeneration Disease-causing gene |
| CN108753954A (en) * | 2018-06-26 | 2018-11-06 | 中南大学湘雅医院 | Capture probe group, kit, library constructing method and the purposes of dull-witted related gene |
| CN111893177A (en) * | 2020-08-19 | 2020-11-06 | 南通大学 | Mutation screening method and mutation detection kit for hereditary hearing loss inducing gene |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543370A (en) * | 2016-01-14 | 2016-05-04 | 中南大学湘雅医院 | New comprehensive-deafness related-gene mutation detection system and kit |
| CN105543370B (en) * | 2016-01-14 | 2019-06-04 | 中南大学湘雅医院 | New comprehensive deaf-related gene mutation detection architecture and kit |
| CN106811533A (en) * | 2017-03-06 | 2017-06-09 | 亚能生物技术（深圳）有限公司 | A kind of hereditary hearing impairment gene detecting kit |
| CN106811533B (en) * | 2017-03-06 | 2020-10-16 | 亚能生物技术（深圳）有限公司 | Genetic deafness gene detection kit |
| CN106987637A (en) * | 2017-04-25 | 2017-07-28 | 郑州大学第附属医院 | A kind of amplimer, kit and application for detecting the mutation of familial retinal pigment degeneration Disease-causing gene |
| CN106987637B (en) * | 2017-04-25 | 2020-06-02 | 郑州大学第一附属医院 | Amplification primer and kit for detecting familial retinitis pigmentosa disease-causing gene mutation and application |
| CN108753954A (en) * | 2018-06-26 | 2018-11-06 | 中南大学湘雅医院 | Capture probe group, kit, library constructing method and the purposes of dull-witted related gene |
| CN111893177A (en) * | 2020-08-19 | 2020-11-06 | 南通大学 | Mutation screening method and mutation detection kit for hereditary hearing loss inducing gene |
| CN111893177B (en) * | 2020-08-19 | 2022-10-04 | 南通大学 | Mutation screening method and mutation detection kit for hereditary hearing loss inducing gene |
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Application publication date: 20150318 |