CN108179187A - A kind of pre-computed altitude risks of myopia gene detecting kit and detection method - Google Patents
A kind of pre-computed altitude risks of myopia gene detecting kit and detection method Download PDFInfo
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- CN108179187A CN108179187A CN201810247823.2A CN201810247823A CN108179187A CN 108179187 A CN108179187 A CN 108179187A CN 201810247823 A CN201810247823 A CN 201810247823A CN 108179187 A CN108179187 A CN 108179187A
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Abstract
The present invention provides a kind of pre-computed altitude risks of myopia gene detecting kit and detection methods.Wherein, the kit includes：It is one or more in the first reagent for MYP11 gene mutation sites rs10034228 detections, the second reagent for GJD2 gene mutation sites rs524952 detections and the third reagent for RSPO1 gene mutation sites rs4074961 detections.The present invention carries out the prediction to high myopia risk using MYP11 gene mutation site rs10034228, GJD2 gene mutation site rs524952 and RSPO1 gene mutation sites rs4074961, substantially increase the accuracy of prediction, it ensure that the high accurancy and precision of detection, effectively assess the influence of inherent cause and other factors collective effect to high myopia risk.
Description
Technical field
The invention belongs to technical field of gene detection more particularly to a kind of pre-computed altitude risks of myopia gene detection reagents
Box and detection method.
Background technology
High myopia rate has been gradually increasing in recent years, and most important problem is exactly the age more Come of generation myopia younger
Change, once myopia generates, the number of degrees are typically to be growing on and on, and the study found that suffer from the myopic age smaller, number of degrees intensification is cured
Soon, the chance for becoming high myopia later is just higher, the complication such as detached retina, glaucoma easily occurs, and may cause
Blindness.
Therefore, high myopia class disease to early be found, early to intervene, the visual loss for reducing patient is that this year is next national
The precisely important goal of medical treatment development.Current method is subsequent control such as wearing spectacles are adjusted and performed the operation to eye
It treats, therapeutic effect is poor.
Existing mechanism is detected high myopia neurological susceptibility using gene tester at present, for example, based on ZNF644
Only have wherein through studying confirmation to predict neurological susceptibility of the sample to be checked to high myopia in one or more of gene site
Partial gene mutation is related with heredity high myopia morbidity.Although it is carried out in short, having at present by the detection of a certain gene
Prediction to high myopia risk, validity also needs to further verify, forecasting accuracy is low, it is difficult to effectively assessment heredity
Factor and other factors collective effect are to the risk of high myopia.
Invention content
To solve the above problems, the present invention provides a kind of pre-computed altitude risks of myopia gene detecting kit, including：
For MYP11 gene mutation sites rs10034228 detection the first reagent,
For the second reagent of GJD2 gene mutation sites rs524952 detections and for RSPO1 gene mutation sites
It is one or more in the third reagent of rs4074961 detections.
Preferably,
First reagent includes the reagent for MYP11 gene magnifications and the reagent for MYP11 genes digestion detection；
Second reagent includes the reagent for GJD2 gene magnifications and the reagent for GJD2 genes digestion detection；
The third reagent includes the reagent for RSPO1 gene magnifications and the reagent for RSPO1 genes digestion detection.
Preferably,
The reagent for MYP11 gene magnifications includes the first primer and the second primer；
The sequence of the first primer is SEQ ID NO.1；
The sequence of second primer is SEQ ID NO.2.
Preferably,
The reagent for GJD2 gene magnifications includes third primer and the 4th primer；
The sequence of the third primer is SEQ ID NO.3；
The sequence of 4th primer is SEQ ID NO.4.
Preferably,
The reagent for RSPO1 gene magnifications includes the 5th primer and the 6th primer；
The sequence of 5th primer is SEQ ID NO.5；
The sequence of 6th primer is SEQ ID NO.6.
Preferably,
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
Preferably,
4th reagent includes the reagent for BCMO1 gene magnifications and the reagent for BCMO1 genes digestion detection；
5th reagent includes the reagent for TTR gene magnifications and the reagent for TTR genes digestion detection.
Preferably,
The reagent for BCMO1 gene magnifications includes the 7th primer and the 8th primer；
The reagent for TTR gene magnifications includes the 9th primer and the tenth primer；
The sequence of 7th primer is SEQ ID NO.7；
The sequence of 8th primer is SEQ ID NO.8；
The sequence of 9th primer is SEQ ID NO.9；
The sequence of tenth primer is SEQ ID NO.10.
In addition, to solve the above problems, the present invention also provides a kind of vitamin A deficiency risk gene detecting kit,
Including：
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
In addition, to solve the above problems, the present invention also provides a kind of pre-computed altitude risks of myopia gene tester,
It is characterized in that, including：
Extract the genomic DNA in biochemical sample；
Synthetic primer, and using genomic DNA described in the primer amplification, obtain segment；
Pass through the primer extend base；
The site in the segment is detected to know the base information in the site；
Analyze the high myopia risk of the sample.
The present invention provides a kind of pre-computed altitude risks of myopia gene detecting kit and detection method, the kit packet
Include the first reagent detected for MYP11 gene mutation sites rs10034228, for GJD2 gene mutation sites rs524952
It is one or more in second reagent of detection and the third reagent detected for RSPO1 gene mutation sites rs4074961.This
Invention utilizes MYP11 gene mutation site rs10034228, GJD2 gene mutation site rs524952 and RSPO1 gene mutations position
Point rs4074961 carries out the prediction to high myopia risk, substantially increases the accuracy of prediction, ensure that the high-precision of detection
Accuracy effectively assesses the influence of inherent cause and other factors collective effect to high myopia risk.
Description of the drawings
Fig. 1 is PCR product through agarose electrophoresis testing result.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
Technical scheme of the present invention is described in further detail with reference to the mode of specific embodiment, but not structure
Into any limitation of the invention, the modification of anyone limited number of time made within the scope of the invention as claimed, still in this hair
Within bright right.
The present invention provides a kind of pre-computed altitude risks of myopia gene detecting kit, including：
For MYP11 gene mutation sites rs10034228 detection the first reagent,
For the second reagent of GJD2 gene mutation sites rs524952 detections and for RSPO1 gene mutation sites
It is one or more in the third reagent of rs4074961 detections.
It is above-mentioned, it is to be understood that research points out that MYP11 genes are related with the high myopia of Autosome dominant inheritance,
Its variation can cause the risk of high myopia to increase.MYP11 genes and high myopia are closely related.In one of SNP site
Genotype when becoming T from C, do not mutate low 1.2 times of the Hazard ratio for suffering from high myopia.If the otherwise genotype in the site
During for C, the risk for suffering from high myopia then increases by 1.2 times.
It is above-mentioned, it is to be understood that GJD2 genes are inserted by connexin, belong to a kind of neuron specificity albumen.
GJD2 shows intraocular photoreceptor, without aixs cylinder nerve cell (amacrine) and Beale's ganglion cells (bipolarcell), can
To regulate and control the nerve information transmittance process on retina.Research points out that GJD2 is related with development with the growth of eyeball crystalline, and
SNP variations near GJD2 genes are also high with the correlation of myopia.
It is above-mentioned, it is to be understood that the protein product of RSPO1 genes is secreted protein, belongs to R-spondin eggs
White family member can regulate and control Wnt/ β-catenin metabolic pathways, and in tissue differentiation, orga- nogenesis and the disease of organism
It plays a significant role in sick generating process.Research points out that the SNP variations on RSPO1 genes can influence the length of axis oculi, if gene
Type is then longer compared with axis oculi of the genotype with C with T, has the tendency that myopia.
The present invention chooses MYP11rs10034228, GJD2rs524952, RSPO1rs4074961 as high myopia risk
Site in pre-computed altitude risks of myopia gene detecting kit, is detected one or more combinations in above-mentioned site,
Realize the assessment for high myopia risk.
The present invention utilizes MYP11 gene mutation site rs10034228, GJD2 gene mutation sites rs524952 and RSPO1
Gene mutation site rs4074961 carries out the prediction to high myopia risk, substantially increases the accuracy of prediction, ensure that
The high accurancy and precision of detection effectively assesses the influence of inherent cause and other factors collective effect to high myopia risk.
Preferably,
First reagent includes the reagent for MYP11 gene magnifications and the reagent for MYP11 genes digestion detection；
Second reagent includes the reagent for GJD2 gene magnifications and the reagent for GJD2 genes digestion detection；
The third reagent includes the reagent for RSPO1 gene magnifications and the reagent for RSPO1 genes digestion detection.
Preferably,
The reagent for MYP11 gene magnifications includes the first primer and the second primer；
The sequence of the first primer is SEQ ID NO.1；
The sequence of second primer is SEQ ID NO.2.
It is above-mentioned, it is to be understood that primer (primer) also known as introduction.It is a bit of single stranded DNA or RNA, as DNA
The starting point of duplication in nucleic acid synthetic reaction, works more as the starting point that each polynucleotide chain is extended
Nucleotide chain, on 3 '-OH of primer, nucleotide is synthesized in the form of diester chain, therefore 3 '-OH of primer, it is necessary to be trip
From.Why need primer be because DNA synthesis in archaeal dna polymerase new nucleotide can be only added to it is existing
In DNA chain.
Above-mentioned, the form of presentation of sequence is from left to right form of presentation well known in the art, and as 5 ' ends are held to 3 '.
In the present embodiment, it is as follows for detecting MYP11 gene primers sequence difference：
For the primer sequence of myp11：
The sequence of the first primer is SEQ ID NO.1, gatgcccgttgaaagagt；
The sequence of second primer is SEQ ID NO.2, gggagataaagccctgtg；
Fragment length is 370bp；
In the present embodiment, it in order to detect some site, needs to need the fragment amplification containing the site in each position
2 primers of point design, to expand generation homologous segment.The segment is spare as the template that follow-up base extends；Extension needs to use
To the primer of extension base, the base to extend out needs sequenator, such as 3730 sequenators are detected and could read specifically
It is certain base.
It is atgactgccacactaacaaa, length 20bp by the base that primer is extended；
By the first primer and the second primer, i.e. the MYP11 pieces of sequence SEQ ID NO.1 and sequence SEQ ID NO.2 amplifications
Duan Xulie is：SEQ ID NO.11.
Preferably,
The reagent for GJD2 gene magnifications includes third primer and the 4th primer；
The sequence of the third primer is SEQ ID NO.3；
The sequence of 4th primer is SEQ ID NO.4.
In the present embodiment, it is as follows for detecting GJD2 gene primers sequence difference：
For the primer sequence of GJD2：
The sequence of the third primer is SEQ ID NO.3, aggctattatacaccaggaa；
The sequence of 4th primer is SEQ ID NO.4, tgaaccagtttggctctt；
Fragment length is 465bp；
It is cccccctaattttcagatgtagttt, length 25bp by the base that primer is extended；
By third primer and the 4th primer, i.e. the GJD2 segments of sequence SEQ ID NO.3 and sequence SEQ ID NO.4 amplifications
Sequence is：SEQ ID NO.12.
Preferably,
The reagent for RSPO1 gene magnifications includes the 5th primer and the 6th primer；
The sequence of 5th primer is SEQ ID NO.5；
The sequence of 6th primer is SEQ ID NO.6.
In the present embodiment, it is as follows for detecting RSPO1 gene primers sequence difference：
For the primer sequence of RSPO1：
The sequence of 5th primer is SEQ ID NO.5, tactcagtgcgagggatg；
The sequence of 6th primer is SEQ ID NO.6, atgagcctcagggttgtt；
Fragment length is 362bp；
It is cccccccccgctccatgcaggcagaccatc a, length 31bp by the base that primer is extended；
By the 5th primer and the 6th primer, i.e. the RSPO1 pieces of sequence SEQ ID NO.5 and sequence SEQ ID NO.6 amplifications
Duan Xulie is：SEQ ID NO.13.
Preferably,
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
Preferably,
4th reagent includes the reagent for BCMO1 gene magnifications and the reagent for BCMO1 genes digestion detection；
It is above-mentioned, it is to be understood that BCMO1 genes are the key enzymes that carotenoid is converted into vitamin A, in human body
Digestion, metabolism, growth and development and immunological regulation play the part of important role.The SNP variations on BCMO1 genes are pointed out in research,
The outer secretory volume of BCMO1 enzymes can be reduced, and then influences the conversion of vitamin A, if genotype carries the variation of A, is tieed up in blood
The concentration of raw element A is low compared with common people.
It is above-mentioned, it is to be understood that TTR genes are responsible for generating the instruction for being known as transthyretin.This protein exists
Vitamin A (retinol) and a kind of hormone for being known as thyroxine are transported in vivo.In order to transport vitamin A, turn thyroxine egg
In vain the tetramer necessarily is formed to be combined with RBP ELISA.
It is above-mentioned, it should be noted that vitamin A (vitaminA) is also known as retinol (its aldehyde derivatives retinene) or anti-dry
The eye disease factor is a unsaturated monohydric alcohol with alicyclic ring, and two kinds of the retinol1 including animal food source, A2 are
One kind has the substance of retinol bioactivity.Retinol1 is stored in the liver of mammal and saltwater fish more, dehydroretinol
Often it is stored in the liver of fresh-water fishes.Since the expression activitiy of dehydroretinol is low, so usually said vitamin A refers to vitamin
A1。
The photoreceptor of eye is rhabdocyte and cone cell in retina.All there are photosensitive colors for both cells
Element feels the rhodopsin of dim light and feels the visual violet of strong light.Rhodopsin and visual violet are all with regarding Huang by opsin
What aldehyde was formed.After light irradiates, 11-cis retinal isomery is detached with opsin and eclipsed rhodopsin into trans-retinene,
This process claims " to bleach ".If into dark place, because of the rhodopsin disappearance to dim light sensitivity, therefore object cannot be seen.
Retinene after separation is reduced to all-trans-retinal, is further transformed to trans- retinyl ester and (or tautomerizes to suitable
Formula) and be stored in pigment epithelium.By retinyl ester hydrolase in retina, retinyl ester is changed into trans- retinol, it is oxidized
And isomerization, form 11-cis retinal.It is recombined with albumen again as rhodopsin, restores the sensibility to dim light, so as to
See object in the dark place of certain illumination, this process claims dark (DarkAdaptation).By the retinol of liver release and retinol knot
Hop protein (RBP) combines, and is combined again with prealbumin in blood plasma, is transported to retina, participates in the photochemical reaction of retina,
If vitamin A is sufficient, the regeneration of rhodopsin is fast and complete, therefore dark adaptation recovery time is short；If hypovitaminosis A, depending on
Rhodopsin regeneration is slow and incomplete, therefore dark adaptation recovery time extends, and yctalopia (NightBlindness) can be generated when serious.
So one kind in carrying out using MYP11 rs10034228, GJD2rs524952 and RSPO1 rs4074961
Or a variety of sites can will embody the BCMO1 rs6564851 of vitamin A situation when high myopia risk is detected and is assessed
And/or TTR rs1667255 are detected jointly as one of evaluation index, it is accurate that high myopia is predicted with increase
Property.
5th reagent includes the reagent for TTR gene magnifications and the reagent for TTR genes digestion detection.
Preferably,
The reagent for BCMO1 gene magnifications includes the 7th primer and the 8th primer；
The reagent for TTR gene magnifications includes the 9th primer and the tenth primer；
The sequence of 7th primer is SEQ ID NO.7；
The sequence of 8th primer is SEQ ID NO.8；
The sequence of 9th primer is SEQ ID NO.9；
The sequence of tenth primer is SEQ ID NO.10.
In the present embodiment, it is as follows for detecting BCMO1 gene primers sequence difference：
For the primer sequence of BCMO1：
The sequence of 7th primer is SEQ ID NO.7, aatcctccacagcatcac；
The sequence of 8th primer is SEQ ID NO.8, tcagttcttccgcttaca；
Fragment length is 463bp；
It is cccccccccctgagtgcctactgtattttaagcactg, length 37bp by the base that primer is extended；
By the 7th primer and the 8th primer, i.e. the BCMO1 pieces of sequence SEQ ID NO.7 and sequence SEQ ID NO.8 amplifications
Duan Xulie is：SEQ ID NO.14.
In the present embodiment, it is as follows for detecting TTR gene primers sequence difference：
For the primer sequence of TTR：
The sequence of 9th primer is SEQ ID NO.9, ttttcccattaaccatcc；
The sequence of tenth primer is SEQ ID NO.10, cttaggcagcaccactcc；
Fragment length is 301p；
It is cccccccccccccccgcaatgccagagatgggactatttcttctt by the base that primer is extended,
Length 45bp；
By the 9th primer and the tenth primer, i.e. the TTR segments of sequence SEQ ID NO.9 and sequence SEQ ID NO.10 amplifications
Sequence is：SEQ ID NO.14.
In addition, the present invention also provides a kind of vitamin A deficiency risk gene detecting kit, including：
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
In addition, the present invention also provides a kind of pre-computed altitude risks of myopia gene testers, which is characterized in that including：
Extract the genomic DNA in biochemical sample；
Synthetic primer, and using genomic DNA described in the primer amplification, obtain segment；
Pass through the primer extend base；
The site in the segment is detected to know the base information in the site；
Analyze the high myopia risk of the sample.
It is above-mentioned, in the present embodiment, pre-computed altitude risks of myopia genetic test is carried out based on Snapshot technologies,
Snapshot technologies are that the U.S. is developed using biotech firm (ABI), are a kind of points based on fluorescent marker Single base extension principle
Type technology, also referred to as small sequencing, mainly for the SNP parting projects of moderate fluxes.
It is above-mentioned, in the present embodiment, a kind of pre-computed altitude risks of myopia gene tester is provided, testing process can
To include but not limited to following steps：
Testing process：
(1) genomic DNAs such as mouth desquamated cells, vein blood leukocytes, sperm, skin are extracted；
(2) it synthetic primer and is diluted；
(3) template amplification is carried out in same reaction tube with primer pair progress, respectively obtained containing MYP11
One or more sites and BCMO1 in rs10034228, GJD2 rs524952 and RSPO1 rs4074961
The segment in the sites such as rs6564851 and/or TTR rs1667255；
(4) pass through primer extend base；
(5) it is detected with 3730 detectors and knows MYP11 rs10034228, GJD2 rs524952 and RSPO1
The sites such as one or more sites and BCMO1 rs6564851 and/or TTR rs1667255 in rs4074961 are corresponding
Base；
(6) risk of sample height myopia and the scheme of follow-up replenishing vitamins A are learnt after analyzing base.
Above-mentioned detection method can include but is not limited to the gene tester provided in the present embodiment, in addition, also may be used
The methods of be sequenced using quantitative fluorescent PCR (realtime PCR), mass spectrum, generation sequencing, two generations, is realized.
For the ease of understanding the present invention, the technical solution further illustrated the present invention with reference to embodiment.Applicant
Statement, the present invention illustrate the detailed process equipment of the present invention and technological process, but not office of the invention by above-described embodiment
It is limited to above-mentioned detailed process equipment and technological process, that is, does not mean that the present invention has to rely on above-mentioned detailed process equipment and technique
Flow could be implemented.Person of ordinary skill in the field is each to product of the present invention it will be clearly understood that any improvement in the present invention
The equivalence replacement of raw material and the addition of auxiliary element, the selection of concrete mode etc. all fall within protection scope of the present invention and openly
Within the scope of.
Embodiment：
First, the extraction of sample DNA：
Sampling：It is wiped in cheek 10 times using cotton swab.It please don't feed or drink water in 30 minutes before sampling.
Processing：
1st, swab is transferred in 2mL centrifuge tubes, cotton swab part is cut from its bar with scissors, adds in 400 μ L solution As.
2nd, the RNaseA (10mg/mL) of 20 μ L, 55 DEG C of placement 15min are added in into solution.
3rd, the Proteinase K (10mg/mL) of 20 μ L is added in, fully reverse mixing, 55 DEG C of water-baths digest 60min, during digestion
It is recommended that reverse centrifuge tube mixing was primary every ten minutes.
4th, 400 μ L bulk solution B are added in, fully reverse mixing white precipitate such as occurs, can be positioned over 75 DEG C of 15-
30min, precipitation can disappear, and not influence subsequent experimental.
The 5th, solution is transferred to new centrifuge tube, add in 400 μ L absolute ethyl alcohols, abundant mixing, at this time it is possible that wadding
Shape precipitates, and does not influence the extraction of DNA, can all add in adsorption column solution and flocculent deposit and (be transferred in two times after mixing same
Adsorption column).
6th, 12000rpm centrifuges 1min, abandons waste liquid, and adsorption column is put into collecting pipe to (being repeated twice will be obtained by previous step
Solution is all transferred to).
7th, 600 μ L rinsing liquids (please first checked whether before use and added in absolute ethyl alcohol) are added in into adsorption column,
12000rpm centrifuges 1min, abandons waste liquid, adsorption column is put into collecting pipe.
8th, 600 μ L rinsing liquids are added in into adsorption column, 12000rpm centrifugation 1min abandon waste liquid, adsorption column is put into collection
Guan Zhong.
9th, 12000rpm centrifuges 2min, and adsorption column opening is placed in room temperature or 50 DEG C of incubators are placed several minutes, it is therefore an objective to will
Remaining rinsing liquid removes in adsorption column, and otherwise the ethyl alcohol in rinsing liquid can influence subsequent experiment such as digestion, PCR.
10th, adsorption column is put into a clean centrifuge tube, to the hanging 50-200 μ L that are added dropwise in adsorbed film center through 65 DEG C
The eluent of water-bath preheating is placed at room temperature for 5min, 12000rpm centrifugations 2min.
11st, centrifugation gained eluent can be added in adsorption column, 12000rpm centrifugations 2min, you can obtain high quality
Genomic DNA.
2nd, it synthetic primer and is diluted
1st, above designed primer 1-10 and 5 extension base primers are synthesized.
2nd, primer dilutes, and the primer of 2OD is diluted to 100mM.The amount of the TE of addition is 660000/MW.
3rd, one group of primer is added into ddH before doing experiment2O is diluted to the working concentration of 10mM.
3rd, fragment amplification
1st, PCR reaction systems：It is shown in Table 1；
Each reagent dosage in PCR reaction systems in table 1, embodiment
| Reagent | Dosage |
| 10×Buffer(15Mm Mg2+) | 1μL |
| Primer(10Mm) | 0.4μL |
| dNTP(10Mm) | 0.3μL |
| HotStar enzymes (5u/ μ L) | 0.1μL |
| Genomic DNA | 1μL |
| Mg2+(25Mm) | 0.52μL |
| ddH2O | 6.68μL |
| Total Volume | 10μL |
2nd, multi-PRC reaction uses the PCR response procedures of Touch-down：
95℃10min；
94℃40s；
63 DEG C of 40s each recycle 0.5 DEG C of decline；
72 DEG C of 1min, 15 cycles；
94℃40s；
56℃30s；
72 DEG C of 1min, 25 cycles；
72℃5min；After 4 DEG C preservation；
PCR product has the Success in Experiment that shows of segment, the result is shown in Figure 1 through agarose electrophoresis detection.
3rd, the purifying of PCR product
Polychrome SNP experiments are with the PCR product (0.01-0.4pmol) of step 2 for template.But PCR product must first perform pure
Change, using SAP enzymes by remaining dNTPs dephosphorylations and ExoI degradation dissociate single-stranded primer.
(1) enzyme is purified：SAP and Exo I；
(2) action principle：
SAP removes extra dNTP and primer；
Exo I remove single-stranded primer and other single-stranded DNA products；
(3) reaction system：
6 μ L+2 μ L enzyme mixations (SAP containing 2U and 2U ExoI) of PCR product vibrate mixing.
(4) reaction condition：
37 DEG C are incubated heat preservation 1hr, then keep the temperature 15min to inactivate SAP and Exo I enzymes for 75 DEG C.Purified template can be with
R for 24 hours or -20 DEG C long-term is preserved at 4 DEG C to preserve.
4.Snapshot PCR
Hybrid template：If using PCR product as the template of Snapshot PCR, after purifying is good, each respectively takes 2 μ L,
Mixing.
Mix the PCR primer of Snapshot：The final concentration of each primer in the reaction system is respectively 0.2 μM.It is (each
SNP primers add 1-2 μ L to 500 μ LddH2In O)
Snapshot PCR systems：It is shown in Table 2；
Each reagent dosage in Snapshot PCR reaction systems in table 2, embodiment
PCR cycle condition is：
96℃10sec→【96℃10sec→50℃(53℃)5sec→60℃30sec】* 25 → 60 DEG C of 30sec of cycle →
4℃forever
5th, the purifying of Snapshot PCR products
0.5USAP 1U CIP are added in the above-mentioned Snapshot PCR products of 5 μ L, shake mixing, 37 DEG C of heat preservations
With inactivator, 4 DEG C preserve r for 24 hours or -20 DEG C long-term and preserve by 1hr, 75 DEG C of heat preservation 15min.
6th, prepared by 310,3100,3730 electrophoresis Samples
Each reagent dosage in electrophoretic system is sequenced in Snapshot in table 3, embodiment
| Reagent | Dosage (μ L) |
| Hi-Di Formamide | 9.25 |
| GS-120LIZ | 0.1 |
| Snapshot products | 1 |
| Total volume | 10.35 |
95 DEG C of denaturation 5min → freeze rapidly 4min.
Each electrophoresis sample adds LIZ-120 internal standards, is accurately positioned the length scale of extension segment.
4th, results and discussion：
In the present embodiment, data analysis is carried out using GeneMapper4.0 softwares；Output data result after software analysis
It is as follows：It is shown in Table 4 and table 5；
The testing result in each 5 sites of 2 samples in table 4, embodiment
| Sample Name | Marker | Allele 1 | Allele 2 |
| Sample 1 | MYP11 | A | G |
| Sample 1 | GJD2 | A | A |
| Sample 1 | RSPO1 | T | T |
| Sample 1 | BCMO1 | T | G |
| Sample 1 | TTR | C | C |
| Sample 2 | MYP11 | A | A |
| Sample 2 | GJD2 | A | A |
| Sample 2 | RSPO1 | T | C |
| Sample 2 | BCMO1 | G | G |
| Sample 3 | TTR | A | A |
Sample system comprehensive analysis risks of myopia and hypovitaminosis Risk Results in table 5, embodiment
| Sample 1 | Sample 2 | |
| MYP11 testing results | Risk | Low-risk |
| GJD2 testing results | High risk | High risk |
| RSPO1 testing results | High risk | Risk |
| Risks of myopia result | High risk | High risk |
| BCMO1 testing results | Risk | Low-risk |
| TTR testing results | Low-risk | High risk |
| Vitamin A deficiency Risk Results | Low-risk | High risk |
In the present embodiment, the testing result carried out is directed to the risks of myopia and vitamin A of two children's individuals
Lacking risk, the childhood risks of myopia in sample 1 is high, but the shortage risk of vitamin A is low, therefore, according to the testing result,
In subsequent parent looks after, in addition to eye hygiene to be paid special attention to and with other than eye custom, it is also necessary to the youngster recommended according to country
Virgin vitamin A magnitude of recruitment supplements appropriate vitamin A.
Its risks of myopia of the children that 2 kinds of sample belongs to higher, and the shortage risk of vitamin A is higher.Corresponding health control
In, parent is when children are looked after it is noted that with eye custom and eye hygiene, in addition, it is also necessary to recommend supplement according in country
Increase the supplement of vitamin A on the basis of amount, specific used in amounts will seek advice from related doctor.
In the present embodiment, two genes are detected and have considered hereditary caused risks of myopia and the day after tomorrow for a long time
Read the influence that this important child growth external cause may be brought.In sample 1, MYP11 is detected as low-risk, still
APLP2 is detected as high risk, and parent can not still lose vigilance in the case of this kind, is still to strengthen the consciousness of protection child's eyesight, note
Meaning not allow child continuously to read for a long time, form child and good be accustomed to eye.
SEQUENCE LISTING
<110>Guangzhou Hexie Health Management Co., Ltd.
<120>A kind of pre-computed altitude risks of myopia gene detecting kit and detection method
<130> 2018
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
gatgcccgtt gaaagagt 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gggagataaa gccctgtg 18
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
aggctattat acaccaggaa 20
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
tgaaccagtt tggctctt 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> 5
tactcagtgc gagggatg 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> 6
atgagcctca gggttgtt 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence
<400> 7
aatcctccac agcatcac 18
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> 8
tcagttcttc cgcttaca 18
<210> 9
<211> 18
<212> DNA
<213>Artificial sequence
<400> 9
ttttcccatt aaccatcc 18
<210> 10
<211> 18
<212> DNA
<213>Artificial sequence
<400> 10
cttaggcagc accactcc 18
<210> 11
<211> 900
<212> DNA
<213>Genus Homo
<400> 11
gtatcttaaa gttaaagaat ttctttcagt tgacaataca gtagtctttt tggtaaactt 60
tgttgtcaat gttaatgttg tttataagta accagtttta cagggtttca tcctcatcct 120
tctaactgcc tgccccaagg gaagatgcag gtctgatatc tgtgataaaa gagagagaag 180
gagaaatctc agactggcca ggccaatcag gagtccttaa gccaaagatg cccgttgaaa 240
gagtcctgaa tttcaccgga aagtccactt ttgtgcctcc actattctta gtcatttgct 300
agaagtagcc tgtaggaagt gtggcctcca tggaaacaca gcgggtggat ccaggggcag 360
acacagcagt ggaggctctc catccactgc gttccacact gcaggagatc tgagtggtgc 420
attttcatga ctgccacact aacaaaatac acatctctga cttcttcccc tcataaccac 480
agtcaaatta tttcggctga gtgcggcaga aggtggctaa ggcttgggaa tgattgcaaa 540
acttagtgct atgccaaaat ctggaaggtg agggtatgca cagggcttta tctccctgta 600
agctgcacgt ggtgacttga ggtagcacaa aggcttgaga gagcaccaga acactgatga 660
ttttaagcag agcattgcct atgtgatctt tgaaatgctt gtggaaggct tgtccctgta 720
ctcatggaag gtcctgctag gacctgggag acactactgc ctatgaatga agagtagaga 780
atatgcacta attagctgat attcttctta tccagggact gggagggggc taattgtctt 840
cagttttttt ttttttcttc cttcttggtg ttttagcata aaactgatgg catgcaaatc 900
<210> 12
<211> 900
<212> DNA
<213>Genus Homo
<400> 12
gtaaaatggg tgtaagctga cataagccta gaaaaggaag ttggaaattt attggaaaga 60
ctcttaagtg ccaggctctg gaaactggat gtgagttagt agtcaatggg atcactgaag 120
gttctctaat aggggagaat gtgatcaaaa cagtgcactg gggaggttaa cacaggaggt 180
ggccaggctg ccttttgaga agacagagac tagaggcagg gaggcagatg tggaagctcc 240
tcttaacatc taggactaag ttgacagaag ctagagctca gtgatgcttg aaggaatgga 300
aaaaggaagg gaggctatta tacaccagga aaagggcttc aatccaaggc acttacttat 360
gagcccaaag gagagaaaag acaaatgtaa tttcaatata caatgtttca taacagtcat 420
gagactaatt ttcagatgta gtttttagaa ttaccctctt cattatacag gcacatataa 480
aaaaaggcta taattccatt gttgatgggc cattatctgt gaaggcacgc acaggagtgg 540
aagagccaaa ctggttcaca aatgtcatga gcacatcaga gcccagagga gaaagtgttc 600
ctaactctca ggtctcaatg gaatgtcctc attaggtcac agcacaaggt tttccaagct 660
gggggaaggt cactttcccc tcaaatgatt gaagtgttct aaatgctgaa ccatgcaatg 720
ccctgagcac atgtagcaga tggagcagag ggggcaagag gggcttttgg aaaatccact 780
tttatttcag tagcttgata ataattgttc ctttgaagca tttacaagct agacttttca 840
cccatcccaa cacagatgca ggcaaccagt gctcaccttg ctggaaagag gcacagactt 900
<210> 13
<211> 900
<212> DNA
<213>Genus Homo
<400> 13
gtgagaagag gcacaaaggg ggctcctggc ttctggagaa gtcagggctc aatgaaggaa 60
tgatggcttc ctgggaggca ggactgagag agggggccca agaatcagag aggagaaggg 120
gctcaggaag gagaattact cagtgcgagg gatgagtggg aatgagggct caggaaggtg 180
cagggacccc agatcagggg tgagggctag caagaagaca aggggcttag tgaacaggag 240
ttcagggagg ggcagggtcc tcaatgagtg gaggagggga acttcaaggg gacttcacca 300
ctcacagcta aagagaagtt ctttgctcaa cttttctcgc tccatgcagg cagaccatca 360
ctcagactcc gaaatcagac atggcttcaa aactagtgta taatagctca gaatgagatt 420
tagattgagt ggcttctggt ggccctccta ccctgcagcc gaggactgtc agccagagtc 480
aacaaccctg aggctcatac acagcacacc tacccctgcc ccgaggtcca cccagtgccc 540
ttccacgctt gtcttatcga gaggccaggc acacacagct gtcttggtct tccttcttcc 600
cttccctact cccccagaat cagtaaggca aaatggtcta cacattgttt atataacaat 660
gatttgcacc cagagtctac acagaacttg atgttttaca aaggtgttaa aaatatatat 720
ctattgaggt gggtggatca cctgaggtca agagttcaag accagcctgg ccaacatgac 780
aaaactttgt ctctactaaa aatacaaaaa ttagccaggc atggtgacac atgcctgtaa 840
tcccagctac ttgggaggct gaggcaggag aatcacttga acccgggagg cggaggttgc 900
<210> 14
<211> 900
<212> DNA
<213>Genus Homo
<400> 14
gcatatataa agctctcaat aaatggccaa tagtcctcct gcctggagag ttcaccccct 60
cctccttccc catccaaata cttcccactc tcaagggctc agctgtgcac cccctcctcc 120
aggaagcctt ctttcctaat cctccacagc atcaccaagc tttccttcct cagaacccct 180
atgatagttc ctatctgtgc ctcttgtaga gcagctattc taggcaacta attattactt 240
actgtgtatg tatatggctt cacagccagg ctaaaagaga cttaaatccc aacgttcctg 300
tgtattggtg tctatttttc aactgcatgc aagtaactca aaattagtat gtttgattat 360
tatattggct ggccgtttca gtttttctgt ctttcccaag aaaagaaagg gggaaagaat 420
gctctgagtg cctactgtat tttaagcact gtgacataca cagttttaca ctgtttaatt 480
taaactttgt agccagtcaa tgaggcagat gttataattt ccatttcaga gatcaggaaa 540
ctgagggtct gagacttaag tacgtggttc agacatttag tttgtaagcg gaagaactga 600
aacacaaatg caaatgagtc tgtcggggta gctagtttcc aaagatggcc ccaaagggac 660
catatagccc agaccctata gtcacaacct tgaatggtcc ctcctcacgg aatttttgct 720
ggccctgtga ttggttctaa ccaatagaag gcagtggaag ctaaactatc tcaattctgg 780
tgtagcctta aaaggtattg gcaacttctg cttcctctct ctcgtgtcac tcacctttgg 840
gagacatttg atgctatgta ttataagaag tttggctact gtgctggtaa gatctcacag 900
<210> 15
<211> 900
<212> DNA
<213>Genus Homo
<400> 15
tttaaacaag agtgtcattt aaaagaaggg tattcattct cataaaatgt ccctgccctt 60
tttaataaga tgaaaaagct acatatgatg tctatgtaag gcacactcaa ataaacataa 120
tatttgaatg ggacattaat atgtgagagg ctatttcaca attacctctt ctaaaggctc 180
tcctttgcca catattctat cttgagggaa ccaggaatcc ccgttgaaat cagtggatgc 240
cctgagacta tgaacctaga ctttacaggt aaaaattact ggtctggtca catatagaag 300
ttgattagtg attttttccc attaaccatc ctcatatttc aagatgctag attgctaaag 360
cctggaaaat catttctgat ataagaacag atattcagca gttttggaga tggaagcaat 420
gccagagatg ggactatttc ttcttattgt tttagatgta aacattaaaa aaaaaaaaac 480
aggatgcaca cttagtccct acctctaagt gtatttgtct ctctctctag tcatcttctc 540
ctctcccttc ccagagccca gggctgtgtt ttgggagctg agttagaagc agctgcggag 600
tggtgctgcc taaggacaaa agagaaatta ttggagacga tataataaat gaatggagtc 660
atgtgaaata ggggaggcat ggaaatgagc acgtgtggtc agcactgctg gagaacccag 720
gtcagagagc agagggcagc ttgatggatg tggatagctc atgcaccttg ccccttgtat 780
gtcaggccag gcactttgtt agtgaaagca acatagagga gttgtttctc ccacatcaat 840
cgcaggtttt taagcagaga tgcgtcatgt ttagattagt attccaaatc agtgtttctt 900
Claims (10)
1. a kind of pre-computed altitude risks of myopia gene detecting kit, which is characterized in that including：
For MYP11 gene mutation sites rs10034228 detection the first reagent, for GJD2 gene mutation sites
One kind in second reagent of rs524952 detections and the third reagent detected for RSPO1 gene mutation sites rs4074961
It is or a variety of.
2. pre-computed altitude risks of myopia gene detecting kit as described in claim 1, which is characterized in that
First reagent includes the reagent for MYP11 gene magnifications and the reagent for MYP11 genes digestion detection；
Second reagent includes the reagent for GJD2 gene magnifications and the reagent for GJD2 genes digestion detection；
The third reagent includes the reagent for RSPO1 gene magnifications and the reagent for RSPO1 genes digestion detection.
3. pre-computed altitude risks of myopia gene detecting kit as claimed in claim 2, which is characterized in that
The reagent for MYP11 gene magnifications includes the first primer and the second primer；
The sequence of the first primer is SEQ ID NO.1；
The sequence of second primer is SEQ ID NO.2.
4. pre-computed altitude risks of myopia gene detecting kit as claimed in claim 2, which is characterized in that
The reagent for GJD2 gene magnifications includes third primer and the 4th primer；
The sequence of the third primer is SEQ ID NO.3；
The sequence of 4th primer is SEQ ID NO.4.
5. pre-computed altitude risks of myopia gene detecting kit as claimed in claim 2, which is characterized in that
The reagent for RSPO1 gene magnifications includes the 5th primer and the 6th primer；
The sequence of 5th primer is SEQ ID NO.5；
The sequence of 6th primer is SEQ ID NO.6.
6. pre-computed altitude risks of myopia gene detecting kit as described in claim 1, which is characterized in that further include：
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
7. pre-computed altitude risks of myopia gene detecting kit as claimed in claim 6, which is characterized in that
4th reagent includes the reagent for BCMO1 gene magnifications and the reagent for BCMO1 genes digestion detection；
5th reagent includes the reagent for TTR gene magnifications and the reagent for TTR genes digestion detection.
8. pre-computed altitude risks of myopia gene detecting kit as claimed in claim 7, which is characterized in that
The reagent for BCMO1 gene magnifications includes the 7th primer and the 8th primer；
The reagent for TTR gene magnifications includes the 9th primer and the tenth primer；
The sequence of 7th primer is SEQ ID NO.7；
The sequence of 8th primer is SEQ ID NO.8；
The sequence of 9th primer is SEQ ID NO.9；
The sequence of tenth primer is SEQ ID NO.10.
9. a kind of vitamin A deficiency risk gene detecting kit, which is characterized in that including：
For the 4th reagent of BCMO1 gene mutation sites rs6564851 detections and for TTR gene mutation sites
One or both of the 5th reagent of rs1667255 detections.
10. a kind of pre-computed altitude risks of myopia gene tester, which is characterized in that including：
Extract the genomic DNA in biochemical sample；
Synthetic primer, and using genomic DNA described in the primer amplification, obtain segment；
Pass through the primer extend base；
The site in the segment is detected to know the base information in the site；
Analyze the high myopia risk of the sample.
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Application publication date: 20180619 |