CN101892306A - Screening test kit for detecting pathologic moyopia - Google Patents
Screening test kit for detecting pathologic moyopia Download PDFInfo
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Abstract
The invention discloses a test kit for detecting pathologic moyopia, comprising optionally related regents for screening relating to pathologic moyopia screening for and detecting rs2073135 variation of BAI3 genes, rs4697489 variation of CCDC149 genes, rs248014 variation, rs1105191 variation, rs1943049 variation or/and rs7035322 variation of TNC genes; and six SNP loci of a tester, including rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and rs7035322 are measured to judge whether variation exists, so that the susceptibility of the tester to the pathologic moyopia are predicted. The invention has the advantages of firstly, clarifying the correlation of the six SNP loci and the pathologic moyopia, having simple method for predicting the susceptibility of the pathologic moyopia, being capable of being applied to prevention, assistant diagnosis and cure for the pathologic moyopia and being applied to research and development of new medicines.
Description
Technical field
The present invention relates to a kind of method and reagent of predicting the pathologic myopia susceptibility, more specifically say so by measuring mononucleotide polymorphism site rs2073135 (BAI3 gene, G → A), rs4697489 (CCDC149 gene, C → T), rs248014 (A → G), rs1105191 (T → C), rs1943049 (G → A) and rs7035322 (TNC gene, the prediction of C → A) experimenter is for the susceptibility of pathologic myopia, this method can be used for auxiliary diagnosis, treatment and the new drug development of disease, belongs to biological technical field.
Background technology
Pathologic myopia (pathological myopia, PM) be meant patient's dioptry-more than the 6.0D, usually axiallength is greater than 26 millimeters, the myopia number of degrees continue to deepen, and the distortion of the back utmost point portion of often occurring together changes, comprise the growth of sclera attenuation, choroidal atrophy attenuation and axis oculi, can be with the illness in eye of multiple complications such as amblyopia, glaucoma, cataract, vitreous opacity, retinal detachment, being a kind of important diseases that has a strong impact on vision health and even life quality, also is the one of the main reasons of blinding.
Along with the process that civilises of modern society, the sickness rate of myopia sharply rises, and has become at present the highest a kind of illness in eye of sickness rate in the world, and WHO has listed it in one of blinding illness in eye that needs to be resolved hurrily.Pathologic myopia is 1.7%-2.1% at overall crowd's sickness rate, accounts for near-sighted crowd's 27%～33%.There is racial difference in PM, yellow's morbidity height, and white people's morbidity is lower.At present, the near-sighted sickness rate of China is 33%, is 1.5 times of world average level 22%, and near-sighted total number of persons is nearly 400,000,000, and wherein PM surpasses 2,000,000 people.Investigation in 2006 shows that China's myopia of student sickness rate is 50-60%, is only second to Singapore and occupies the 2nd in the world, and the myopia of student number occupies first place in the world, and visual deterioration directly influences entering a higher school, obtaining employment of child, and its physiology, psychology are caused in various degree obstruction.
Pathologic myopia has a strong impact on the human life quality, and is unfortunately up to the present also very limited for the treatment of PM.Optical correction is as wearing glasses or the refractive defects of contact lense correction of myopia, though help temporarily to recover normal or near normal eyesight, can not control the myopia progress effectively; Operation on vitreous and the laser therapy adopted at the PM maculopathy, and delay the time of ocular fundus pathology appearance and alleviate its lesion degree by operation control axis oculi, it is the interference method of normal employing clinically, these treatment meanss can make Postoperative visual acuity improve or keep stable, stable or the minimizing of dioptry, the axis oculi growth is tending towards slow, functional change there is slight improvement effect, but these treatments can not be effective to all patients, and there is certain risk in operation, postoperative complication mainly contains postoperative retinal detachment and venae vorticosae, the arteria ciliares damage, its long-term effect yet waits textual criticism.The pathological change of report by medicine control axis oculi growth and PM arranged in addition, but still at the experimental stage, be still waiting further research.Owing to lack effective PM preventive means and medicine and method, therefore, PM is studied, inquire into its pathophysiological mechanism, seem of far-reaching significance in the hope of from the source, it being prevented and treated.
The morbidity of pathologic myopia mainly is subjected to inherited genetic factors and Effect of Environmental, and environmental factors comprises closely operation, reading habit, habitual light, often uses a computer, nutrition and disease etc., but environmental factors effect degree report differs.Different nationalities PM sickness rate difference, PM has obvious family to assemble tendency.Gwiazda etc. discover that the myopic danger of father and mother myopia children is bigger than the non-near-sighted children of father and mother.Guggenheim etc. show by calculating near-sighted heredity grade: ill risk level is 20 between the PM compatriot, and the risk level of simple myopia only is 1.5.The morbidity that shows PM has very obvious genetic factor effect.Even there is the scholar to think that PM is exactly simple heredopathia.
Along with the genetics research of Chinese scholars to myopia further gos deep into, use full genome scanning and candidate gene examination, technology such as linkage analysis are carried out the assignment of genes gene mapping of myopia and clone's screening of candidate gene, have found the genetic linkage site of a plurality of myopia, be positioned xq28 (MYP1, OMIM 310460), 18p11.31 (MYP2, OMIM 160700), 12q21-23 (MYP3, OMIM 603221), 7q36 (MYP4, OMIM 608367), 17q21-q23 (MYP5, OMIM 608474), 22q12 (MYP6, OMIM 608908), 11p13 (MYP7, OMIM 609256), 3q26 (MYP8, OMIM 609257), 4q12 (MYP9, OMIM 609258), 8p23 (MYP10, OMIM 609259), 4q22-q27 (MYP11, OMIM 609994), 2q37.1 (MYP12, OMIM 609994), xq23-25 (MYP13, OMIM300613), 1p36 (MYP14, OMIM 0320), 7p15.3,10q21.1,15q12-13.Sure PM gene locus has 6 at present, comprises MYP1, MYP2, MYP3, MYP4, MYP5 and MYP11; Clear and definite chromosomal localization, what wait gene locus name has 7p15.3,10q21.1, a 15q12-13.Dependency checking by to the PM disease gene comprises that mainly about 16 PM candidate genes such as PAX6, TGIF, DSPG3, COL2A1, FBN1 have carried out examination.But, still do not find clear and definite PM Disease-causing gene up to now.And China's pathologic myopia gene locus is different with external report, has genetic heterogeneity, and its hereditary pattern may not be single single-gene autosomal dominant inheritance.Therefore, be more prone at present think that pathologic myopia may be a multigenic disease.
The development trend of following pathologic myopia treatment should be to seek effective, the persistent treatment of curative effect, in the hope of preserving the useful visual function of patient more muchly.Should in PM high risk population, carry out early stage screening, pathological development is found, correctly diagnoses, in time treats, prevented to its ocular fundus pathology as early as possible.Press at present and find some more effectively genetic marker and detection methods, can find PM high risk population by early screening, and implement early detection and prevention, reduce its inpairment of vision.
Summary of the invention
The present invention at first provides BAI3 gene rs2073135 variation, CCDC149 gene rs4697489 variation, rs248014 variation, rs1105191 variation, rs1943049 variation or/and TNC gene rs7035322 variation is used for the application of pathologic myopia screening agent in preparation.
Whether the present invention is by existing BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191T → C variation, rs1943049 G → A variation or/and TNC gene rs7035322 C → A variation in the individuality that detects sample to be tested, and the generation of examination pathologic myopia in early days, and then provide reference for clinical diagnosis and treatment.
The contriver finds under study for action, in Chinese, as long as morph in one of above-mentioned six sites, has all increased the ill risk of pathologic myopia to some extent, proves that these six site variations are directly related with pathologic myopia.
Thereby screening agent of the present invention comprises detection BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A makes a variation or/and the reagent of TNC gene rs7035322 C → A variation; And optional be used to increase comprise BAI3 gene rs2073135, CCDC149 gene rs4697489, rs248014, rs1105191, rs1943049 or/and the reagent of TNC gene rs7035322 gene segment.
Described detection BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A variation or/and the reagent of TNC gene rs7035322 C → A variation for order-checking with reagent, restriction fragment length polymorphism method with reagent or Snapshot reagent.
According to a further aspect in the invention, a kind of test kit that is used to detect pathologic myopia that comprises above-mentioned screening agent also is provided, comprises that optional being used to detects BAI3 gene rs2073135 variation, CCDC149 gene rs4697489 variation, rs248014 variation, rs1105191 variation, rs1943049 variation or/and TNC gene rs7035322 variation is used for the related reagent of pathologic myopia examination.
Described test kit comprises detection BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A makes a variation or/and the reagent of TNC gene rs7035322 C → A variation; And optional be used to increase comprise BAI3 gene rs2073135, CCDC149 gene rs4697489, rs248014, rs1105191, rs1943049 or/and the reagent of TNC gene rs7035322 gene segment.
Further, described detection BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A variation are or/and the reagent of TNC gene rs7035322 C → A variation is order-checking reagent.
Perhaps, the described detection of mentioned reagent box BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A variation are or/and the reagent of TNC gene rs7035322 C → A variation is restriction fragment length polymorphism method reagent.
Perhaps, the described detection of mentioned reagent box BAI3 gene rs2073135 G → A variation, CCDC149 gene rs4697489 C → T variation, rs248014 A → G variation, rs1105191 T → C variation, rs1943049 G → A variation are or/and the reagent of TNC gene rs7035322 C → A variation is Snapshot reagent.
By the mentioned reagent box, can predict the ill risk of pathologic myopia.
In the present invention, by extracting the genomic dna of host cell, measure experimenter's rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and rs7035322 loci gene type, predict the susceptibility of experimenter: 1.463 times of the ill risks of the A allelotrope increase pathologic myopia in rs2073135 site pathologic myopia; 1.822 times of the ill risks of the T allelotrope increase pathologic myopia in rs4697489 site; 1.364 times of the ill risks of the A allelotrope increase pathologic myopia in rs248014 site; 1.745 times of the ill risks of the T allelotrope increase pathologic myopia in rs1105191 site; 1.795 times of the ill risks of the A allelotrope increase pathologic myopia in rs1943049 site; 1.420 times of the ill risks of the A allelotrope increase pathologic myopia in rs7035322 site.
It is right to the invention provides allele specific PCR primer, has primer to the base sequence shown in the A-F, and length is 18-20bp, and can amplify the amplified production shown in the sequence 1-6 specifically.
The present invention also provides allele specific Sanpshot primer, has the base sequence shown in the Snapshot primer A-F, and length is 20-60bp, and can detect the different genotype in six SNP sites specifically.
Contain in the test kit of the present invention the segmental PCR primer of specific amplification purpose of the present invention to the conventional assembly, reagent, damping fluid etc. that are used for pcr amplification and carry out the test kit that the Sanpshot somatotype detects, those skilled in the art know these conventional assembly and detection methods.
Measuring method of the present invention is measured the genomic dna that derives from the people, and sample as body fluid (as blood, ascites and urine), histocyte (as hepatic tissue) etc., all can prepare genomic dna by extraction and these samples of purifying without limits.
The present invention has illustrated the dependency of above-mentioned pleomorphism site and pathologic myopia first, and a kind of method of predicting the pathologic myopia susceptibility is provided, and this method can be used for early stage screening, auxiliary diagnosis and the treatment of pathologic myopia, can also be used for new drug development.
Description of drawings:
Fig. 1 is the gene type collection of illustrative plates that Snapshot detects SNP rs2073135 site;
Fig. 2 is the gene type collection of illustrative plates that Snapshot detects SNP rs4697489 site;
Fig. 3 is the gene type collection of illustrative plates that Snapshot detects SNP rs248014 site;
Fig. 4 is the gene type collection of illustrative plates that Snapshot detects SNP rs1105191 site;
Fig. 5 is the gene type collection of illustrative plates that Snapshot detects SNP rs1943049 site;
Fig. 6 is the gene type collection of illustrative plates that Snapshot detects SNP rs7035322 site;
The invention will be further described below in conjunction with embodiment, so that the public has more deep understanding to summary of the invention, is not limitation of the present invention.
Embodiment
Be used for the following example expression reagent and be commercially available product, its english abbreviation is as follows.
When needing, with pressure kettle (120 ℃, 20 minutes) sterilization
EDTA: disodium ethylene diamine tetraacetate
SDS: sodium lauryl sulphate
TE：10mM?Tris-HCl(pH7.5)、1mM?EDTA(pH8.0)
DNTP: deoxynucleoside triphosphate
The extraction of embodiment 1 sample collection and genomic dna
Sample is the case of being collected by the state natural sciences fund problem that disease gene research Sichuan Province key lab of People's Hospital, Sichuan Prov. of Sichuan Academy of Medical Sciences bears.Collect pathologic myopia patient 289 examples altogether, 35.7 ± 9.7 years old mean age, wherein the male sex accounts for 54.3%, contrast 543 examples (normal control, inclusion criteria is: more than 40 years old, be engaged near work for a long time, dioptry＞-1.0D.), 46.3 ± 10.5 years old mean age, wherein the male sex accounts for 55.6%.All persons under inspection are Han nationality, and the signature Informed Consent Form, and this research has also obtained Ethics Committee's approval.
According to following method, prepare genomic dna with human peripheral.In the presence of antithrombotics EDTA, at 2500rpm, centrifugation removed serum deprivation in 30 minutes with the 5ml human peripheral collected.Then add 0.2%NaCl solution, making cumulative volume is 50ml.Vibrate gently solution 5-6 time, and it was positioned over 15 minutes on ice.After this, at 2500rpm centrifugation 30 minutes, collecting precipitation thing whereby.NaCl solution with 0.2% washs in the mode that is similar to the front again.In the throw out that so obtains, add 10mMTris-HCl (pH8.0) and 10mM EDTA (4m1), with this throw out that suspends.With 10%SDS, the Proteinase K of 25mg/ml and the RNaseA of 10mg/ml add in the suspension, and its add-on is respectively 4ml, 16 μ l and 20 μ l, and the suspension that then turns upside down mixes gently.Then, at 37 ℃ of incubation suspensions that spend the night.After spending the night, add 4ml phenol/Tris solution, the mixture of the mixture mixing gained that turns upside down.Carry out centrifugation with 3000rpm and removed water layer in 10 minutes.Water layer and 4ml phenol/chloroformic solution are mixed, then against mixing and with 3000rpm centrifugation 10 minutes.Remove water layer.At last, with chloroform extraction twice, obtaining water, toward wherein adding 1/10,3M NaAC (pH5.2), the cold dehydrated alcohol of doubling dose makes the DNA precipitation.Washing with alcohol with 70% is to obtain genomic dna.Make the DNA of such acquisition, genomic dna is dissolved among the TE, and the quantitative assay mixture is in the specific absorption of 260nm then.DNA working fluid concentration correction is put-20 ℃ of refrigerators and is preserved to 50ng/ μ l.
Embodiment 2 comprises the pcr amplification and the gene type assay of rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and rs7035322 single nucleotide polymorphism dna fragmentation
The present invention adopts the Snapshot genotyping technique that rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and six sites of rs7035322 are detected.Adopt above-described specific PCR and Snapshot primer A-F to carry out somatotype according to above-mentioned corresponding method respectively.
Experimental procedure is as follows:
1, PCR:95 ℃ of sex change is 5 minutes; 95 ℃ 30 seconds, 54 ℃ annealing were extended 35 circulations 45 seconds for 30 seconds, 72 ℃; 72 ℃ were extended 4 ℃ of insulations 7 minutes.
2, purifying 1:PCR product is respectively got 1-3 μ l (decide according to need blended PCR number, total amount is 9 μ l, adds mixed solution 3 μ l (SAP 2 μ l, 1: 10Exol enzyme 1 μ l).1: the preparation of 10Exol enzyme: 1 volume Exol enzyme, 1 volume SAP 10Xbuffer, 8 volume ddH
2O.Program: 37 ℃ of 1h, 75 ℃ of 15min.
3, Snapshot reaction: Snapshot MULTIPLEX 1 μ l, purified PCR mix 1 μ l, each is 0.2 μ l for the Snapshot primer, DDH
2O supplies 5 μ l.Program: 96 ℃ 1 minute, 96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 30 seconds, cycle to step 2 for 25 more times, 12 ℃ of insulations.
4, purifying 2: each reaction adds SAP 0.5-1.0 μ l, program: 37 ℃ 1 hour, 75 ℃ 15 minutes, 4 ℃ of insulations.
5, the above-mentioned product of 1 μ l adds 9 μ l HD.Program: 95 ℃, 5 minutes, place rapidly on ice, machine testing is gone up in the cooling back.
The PCR primer is to A (rs2073135):
F：5’-AGCCTCATTTATTCATCTGT-3’
R：5’-GATTCTGGTGGTGAGTGAT-3’
The PCR primer is to B (rs4697489):
F：5’-GGTTTGTAGCCCAGAAGTA-3’
R：5’-GTGTTCCCAACCATTAGTC-3’
The PCR primer is to C (rs248014):
F：5’-TAAATGGGGAAATAGATG-3’
R：5’-ATAATGCTACAGAGATGAAC-3’
The PCR primer is to D (rs1105191):
F：5’-TACTGGTGATCCCTGTGAC-3’
R：5’-TGTAAATCGGCTGTGACTC-3’
The PCR primer is to E (rs1943049):
F：5’-GGCATCCTTGTCTTGTTC-3’
R：5’-GGGTTTTGTTCCAACTTCT-3’
The PCR primer is to F (rs7035322):
F：5’-TGTTCCGTGATTCTTAGAC-3’
R：5’-GTATCTGGGTTCCTTTGAC-3’
Resulting extension increasing sequence is as follows:
Sequence 1 (rs2073135) 211bp:
AGCCTCATTTATTCATCTGTgggagacaaattctcctcacaaatttgctg
ttaagattagaggtaatgaatattaatgtttaacacatttttggtacata
tgaagagctcaataaacagctatttccacaaaagtttct
atatcaccga
ctccactatcaccactatcatcccatcgtcatggccattaccATCACTCA
CCACCAGAATC
Sequence 2 (rs4697489) 300bp:
GGTTTGTAGCCCAGAAGTAacaagccataccatgtagcctcgggatgcag
cggactataccatgtaggtttgtgtcagtacactctagtacagtcgcaca
atgatgaaattgcataacaaagcatttctcaggacatatccctgtcatta
agcaatg
atgggtatagctaatagatggtaataagtagctgattatcat
tcactaatagtggctaactacagtacactaaacactttaaaagagacaca
aaactcccagaaaagaaagaaaagcagtttaGACTAATGGTTGGGAACAC
Sequence 3 (rs248014) 334bp:
TAAATGGGGAAATAGATGttgggtatatttttaaaaagatgttcattata
tgaaataaggaagatttggaaaaagagaatggagaaaagtggttatgtag
aatttact
tatgttttacctacatttgttgacaatttcttatttaatga
caaccccaagagataaatgcttatgttggataaagctgagaaaagggaaa
tgatctctaatatttacttcagagaaaatgtgcccacaggactaaattat
aatctaatctttcaagctttaggatgtttatattcataattatacactga
gaaaaatatgtatgGTTCATCTCTGTAGCATTAT
Sequence 4 (rs1105191) 258bp:
TACTGGTGATCCCTGTGACcccagcccttgtgtacgatctgtcctggaaa
gtattatttaatttctgatgcaaatgtggcaatgcccttccatcagcaga
ggcaggcaaagctcccgagagtgaggaggagggatggtgttaccatggta
acgagatgaccagctgagtgtagatggagctaagcatctttctactgaag
aacaggagtagctat
cttagcctctacttgtacctgagGAGTCACAGCC
GATTTACA
Sequence 5 (rs1943049) 330bp:
GGCATCCTTGTCTTGTTCctaatcttagaggtccagtacttttaatggtg
acttggcaggtatgccaatgaattaaaaaagatttttgtggcagcctact
gaacactgccagagtaatgctatcac
tattaactttgtttgctgattct
ctaaagaagataaagtagttgacccaattatatcagcttctattttttct
cttaggtcttaggataaaataaccccttgaaaaatctttcctacccttct
ttctggcatgttatccttagaggagatgcgccaaggagaagctggttctg
ctcaggtggacAGAAGTTGGAACAAAACCC
Sequence 6 (rs7035322) 217bp:
TGTTCCGTGATTCTTAGACaagaagcccattaacatgtactactatttgg
aaatctaatttgtaatttaatatattgaatttttgaagcatttcagtacc
attgctttgtgggtctcctcactcccaatccag
caagtgctgtgcacaa
acatttcaagactctgaggcattctaccgtcataatttggagcaagttGT
CAAAGGAACCCAGATAC
Snapshot primer A (rs2073135):
5’-CTATTTCCACAAAAGTTTCT-3’
Snapshot primer B (rs4697489):
5’-CAGGACATATCCCTGTCATTAAGCAATG-3’
Snapshot primer C (rs248014):
5’-AAGAGAATGGAGAAAAGTGGTTATGTAGAATTTACT-3’
Snapshot primer D (rs1105191):
5’-AGATGGAGCTAAGCATCTTTCTACTGAAGAACAGGAGTAGCTAT-3’
Snapshot primer E (rs1943049):
5’-
AAAAAAGATTTTTGTGGCAGCCTACTGAACACTGCCAGAGTAATGCTAT
CAC-3’
Snapshot primers F (rs7035322):
5’-
ATTGAATTTTTGAAGCATTTCAGTACCATTGCTTTGTGGGTCTCCTCACT
CCCAATCCAG-3’
Fig. 1～Fig. 6 detects the genotype figure of rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and rs7035322 for Snapshot.
The dependency of embodiment 3SNP site and pathologic myopia
Statistical method: utilize SPSS13.0 software to analyze, check Hardy-Weinberg balance detection, quantitative variation adopts the t check, and quality variable adopts chi square test.Statistical significant difference is thought in bilateral P＜0.05.
The result:
1) the basic clinical data of case and contrast
Annotate:
*, P＜0.01.
2) SNP loci polymorphism and pathologic myopia are closely related
rs2073135：
Under the situation of not considering pathologic myopia tradition risk factor, A allelotrope can make pathologic myopia dangerously significantly increases (OR=1.463,95%CI=1.192-1.797), promptly the ill risk of pathologic myopia increases by 1.463 times; Wherein homozygote carrier (AA) can make ill risk increase by 1.607 times, and heterozygote (AG) increases by 1.241 times of ill risks.See Fig. 1.
rs4697489：
Under the situation of not considering pathologic myopia tradition risk factor, T allelotrope can make pathologic myopia dangerously significantly increases (OR=1.822,95%CI=1.474-2.252), promptly the ill risk of pathologic myopia increases by 1.822 times; Wherein homozygote carrier (TT) can make ill risk increase by 3.307 times, and heterozygote (CT) increases by 1.139 times of ill risks.See Fig. 2.
rs248014：
Under the situation of not considering pathologic myopia tradition risk factor, A allelotrope can make pathologic myopia dangerously significantly increases (OR=1.364,95%CI=1.074-1.732), promptly the ill risk of pathologic myopia increases by 1.364 times; Wherein homozygote carrier (AA) can make ill risk increase by 1.452 times, and heterozygote (AG) can not increase ill risk.See Fig. 3.
rs1105191：
Under the situation of not considering pathologic myopia tradition risk factor, T allelotrope can make pathologic myopia dangerously significantly increases (OR=1.745,95%CI=1.415-2.152), promptly the ill risk of pathologic myopia increases by 1.745 times; Wherein homozygote carrier (TT) can make ill risk increase by 1.877 times, and heterozygote (CT) can not increase ill risk.See Fig. 4.
rs1943049：
Under the situation of not considering pathologic myopia tradition risk factor, A allelotrope can make pathologic myopia dangerously significantly increases (OR=1.795,95%CI=1.446-2.228), promptly the ill risk of pathologic myopia increases by 1.795 times; Wherein homozygote carrier (AA) can make ill risk increase by 2.258 times, and heterozygote (AG) increases by 1.425 times of ill risks.See Fig. 5.
rs7035322：
Under the situation of not considering pathologic myopia tradition risk factor, A allelotrope can make pathologic myopia dangerously significantly increases (OR=1.420,95%CI=1.156-1.744), promptly the ill risk of pathologic myopia increases by 1.420 times; Wherein homozygote carrier (AA) can make ill risk increase by 1.714 times, and heterozygote (AC) can not increase ill risk.See Fig. 6.
Embodiment 4 detection kit
Whole components, content and using method in the test kit of the present invention are as follows:
Pcr amplification reagent (50 person-portion):
Sanpshot typing detection reagent (50 person-portion)
The standard DNA sample:
Using method:
1) DNA extraction
Get patient's whole blood (EDTA anti-freezing) 2ml, extract its genomic dna.
2) contain the dna fragmentation in the SNP site of detection to some extent by pcr amplification
Pcr amplification (20 μ l system):
Reaction conditions:
The PCR product detects:
Agarose gel electrophoresis with 2% detects the PCR product, observes the effect of PCR reaction, and its amount that adds in subsequent reactions as template of determining.
3) the Sanpshot somatotype detects
The first step: PCR product purification (12 μ l system):
Reaction conditions:
1,37 ℃ of enzymes were cut 1 hour
2,75 ℃ of deactivations are 15 minutes
3,4 ℃ of preservations
Second step: single base primers extension (5 μ l):
Reaction conditions:
The 3rd step: purification reaction thing:
Reaction system: 0.7 μ l serum alkaline phosphatase/hole
Reaction conditions: 37 ℃ 1 hour, 75 ℃ 15 minutes, 4 ℃ of preservations.
The 4th step: the ABI genetic analyzer reads the result:
Reaction system: purified product 1 μ l+HD 9 μ l
Reaction conditions: 95 ℃ 5 minutes, rapidly ice bath cooling
The machine-readable genotype result that gets on the ABI PRISM 3100DNA Sequencer.
This test kit is used for: 1, pathologic myopia patient's early diagnosis and somatotype reference; 2, the gene test of pathologic myopia Susceptible population and risk assessment.
1) detection method of the present invention can be used for analyzing the polymorphism in rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and six SNP sites of rs7035322, whether have great pathologic myopia ill risk assess, be beneficial to carry out the early intervention and the treatment of pathologic myopia to the complementary diagnosis of pathologic myopia with to individuality if being applied in.
2) utilize the polymorphism in pathologic myopia tumor susceptibility gene/site that the present invention sets forth, as one of biomarker, the screening of the molecular target of useful as drug design has the bioactive molecule of regulating these genetic expressions to help to seek, and promotes new drug development.
3) nucleotide sequence and the pathologic myopia genes involved/site of the detection SNP loci polymorphism set up of the present invention, but highly sensitive, be applied to the test kit that the pathologic myopia gene diagnosis is used specifically.
As mentioned above, reach a conclusion the polymorphism in rs2073135, rs4697489, rs248014, rs1105191, rs1943049 and six SNP sites of rs7035322 and pathologic myopia tool significant correlation.Therefore, according to the present invention, measure its polymorphism and can be used for carrying out gene diagnosis.
The invention discloses the relevant new tumor susceptibility gene/site of pathologic myopia, and the test kit that provides these sites to be used for the application of pathologic myopia screening agent and to comprise these reagent in preparation, test kit of the present invention is highly sensitive, only a small amount of DNA sample of needs just is enough to measure the variation in described site, reaches the purpose of early screening.
PM detection kit .ST25.txt
SEQUENCE?LISTING
＜110〉Medical Scientific Academy in Sichuan(Sichuan People's Hospital)
＜120〉a kind of kit for screening that detects pathologic myopia
<130>CD537-10P108122
<150>200910058834.7
<151>2009-04-03
<160>24
<170>PatentIn?version?3.2
<210>1
<211>20
<212>DNA
＜213〉the PCR primer is to A (rs2073135) F
<400>1
agcctcattt?attcatctgt 20
<210>2
<211>19
<212>DNA
＜213〉the PCR primer is to A (rs2073135) R
<400>2
gattctggtg?gtgagtgat 19
<210>3
<211>19
<212>DNA
＜213〉the PCR primer is to B (rs4697489) F
<400>3
ggtttgtagc?ccagaagta 19
<210>4
<211>19
<212>DNA
＜213〉the PCR primer is to B (rs4697489) R
<400>4
gtgttcccaa?ccattagtc 19
<210>5
<211>19
<212>DNA
＜213〉the PCR primer is to C (rs248014) F
<400>5
tactggtgat?ccctgtgac 19
<210>6
<211>20
<212>DNA
＜213〉the PCR primer is to C (rs248014) R
<400>6
ataatgctac?agagatgaac 20
<210>7
<211>19
<212>DNA
＜213〉the PCR primer is to D (rs1105191) F
<400>7
tactggtgat?ccctgtgac 19
<210>8
<211>19
<212>DNA
＜213〉the PCR primer is to D (rs1105191) R
<400>8
tgtaaatcgg?ctgtgactc 19
<210>9
<211>18
<212>DNA
＜213〉the PCR primer is to E (rs1943049) F
<400>9
ggcatccttg?tcttgttc 18
<210>10
<211>19
<212>DNA
＜213〉the PCR primer is to E (rs1943049) R
<400>10
gggttttgtt?ccaacttct 19
<210>11
<211>19
<212>DNA
＜213〉the PCR primer is to F (rs7035322) F
<400>11
tgttccgtga?ttcttagac 19
<210>12
<211>19
<212>DNA
＜213〉the PCR primer is to F (rs7035322) R
<400>12
gtatctgggt?tcctttgac 19
<210>13
<211>211
<212>DNA
＜213〉Kuo Zeng sequence 1 (variation)
<400>13
agcctcattt?attcatctgt?gggagacaaa?ttctcctcac?aaatttgctg?ttaagattag 60
aggtaatgaa?tattaatgtt?taacacattt?ttggtacata?tgaagagctc?aataaacagc 120
tatttccaca?aaagtttcta?atatcaccga?ctccactatc?accactatca?tcccatcgtc 180
atggccatta?ccatcactca?ccaccagaat?c 211
<210>14
<211>300
<212>DNA
＜213〉Kuo Zeng sequence 2 (variation)
<400>14
ggtttgtagc?ccagaagtaa?caagccatac?catgtagcct?cgggatgcag?cggactatac 60
catgtaggtt?tgtgtcagta?cactctagta?cagtcgcaca?atgatgaaat?tgcataacaa 120
agcatttctc?aggacatatc?cctgtcatta?agcaatgtat?gggtatagct?aatagatggt 180
aataagtagc?tgattatcat?tcactaatag?tggctaacta?cagtacacta?aacactttaa 240
aagagacaca?aaactcccag?aaaagaaaga?aaagcagttt?agactaatgg?ttgggaacac 300
<210>15
<211>334
<212>DNA
＜213〉Kuo Zeng sequence 3 (variation)
<400>15
taaatgggga?aatagatgtt?gggtatattt?ttaaaaagat?gttcattata?tgaaataagg 60
aagatttgga?aaaagagaat?ggagaaaagt?ggttatgtag?aatttactgt?atgttttacc 120
tacatttgtt?gacaatttct?tatttaatga?caaccccaag?agataaatgc?ttatgttgga 180
taaagctgag?aaaagggaaa?tgatctctaa?tatttacttc?agagaaaatg?tgcccacagg 240
actaaattat?aatctaatct?ttcaagcttt?aggatgttta?tattcataat?tatacactga 300
gaaaaatatg?tatggttcat?ctctgtagca?ttat 334
<210>16
<211>258
<212>DNA
＜213〉Kuo Zeng sequence 4 (variation)
<400>16
tactggtgat?ccctgtgacc?ccagcccttg?tgtacgatct?gtcctggaaa?gtattattta 60
atttctgatg?caaatgtggc?aatgcccttc?catcagcaga?ggcaggcaaa?gctcccgaga 120
gtgaggagga?gggatggtgt?taccatggta?acgagatgac?cagctgagtg?tagatggagc 180
taagcatctttctactgaag?aacaggagta?gctatcctta?gcctctactt?gtacctgagg 240
agtcacagcc?gatttaca 258
<210>17
<211>330
<212>DNA
＜213〉Kuo Zeng sequence 5 (variation)
<400>17
ggcatccttg?tcttgttcct?aatcttagag?gtccagtact?tttaatggtg?acttggcagg 60
tatgccaatg?aattaaaaaa?gatttttgtg?gcagcctact?gaacactgcc?agagtaatgc 120
tatcacatat?taactttgtt?tgctgattct?ctaaagaaga?taaagtagtt?gacccaatta 180
tatcagcttc?tattttttct?cttaggtctt?aggataaaat?aaccccttga?aaaatctttc 240
ctacccttct?ttctggcatg?ttatccttag?aggagatgcg?ccaaggagaa?gctggttctg 300
ctcaggtgga?cagaagttgg?aacaaaaccc 330
<210>18
<211>217
<212>DNA
＜213〉Kuo Zeng sequence 6 (variation)
<400>18
tgttccgtga?ttcttagaca?agaagcccat?taacatgtac?tactatttgg?aaatctaatt 60
tgtaatttaa?tatattgaat?ttttgaagca?tttcagtacc?attgctttgt?gggtctcctc 120
actcccaatc?cagacaagtg?ctgtgcacaa?acatttcaag?actctgaggc?attctaccgt 180
cataatttgg?agcaagttgt?caaaggaacc?cagatac 217
<210>19
<211>20
<212>DNA
＜213〉Snapshot primer A (rs2073135)
<400>19
ctatttccac?aaaagtttct 20
<210>20
<211>28
<212>DNA
＜213〉Snapshot primer B (rs4697489)
<400>20
caggacatat?ccctgtcatt?aagcaatg 28
<210>21
<211>36
<212>DNA
＜213〉Snapshot primer C (rs248014)
<400>21
aagagaatgg?agaaaagtgg?ttatgtagaa?tttact 36
<210>22
<211>44
<212>DNA
＜213〉Snapshot primer D (rs1105191)
<400>22
agatggagct?aagcatcttt?ctactgaaga?acaggagtag?ctat 44
<210>23
<211>52
<212>DNA
＜213〉Snapshot primer E (rs1943049)
<400>23
aaaaaagatt?tttgtggcag?cctactgaac?actgccagag?taatgctatc?ac 52
<210>24
<211>60
<212>DNA
＜213〉Snapshot primers F (rs7035322)
<400>24
attgaattt?tgaagcattt?cagtaccatt?gctttgtggg?tctcctcact?cccaatccag 60
Claims (10)
1. a test kit that detects pathologic myopia comprises that optional being used to detects BAI3 gene rs2073135 variation, CCDC149 gene rs4697489 variation, rs248014 variation, rs1105191 variation, rs1943049 variation or/and TNC gene rs7035322 variation is used for the related reagent of pathologic myopia examination.
2. the described test kit of claim 1, it is characterized in that described test kit comprises detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A makes a variation or/and the reagent of TNC gene rs7035322C → A variation; And optional be used to increase comprise BAI3 gene rs2073135, CCDC149 gene rs4697489, rs248014, rs1105191, rs1943049 or/and the reagent of TNC gene rs7035322 gene segment.
3. test kit according to claim 2 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is order-checking reagent.
4. the described test kit of claim 2 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is restriction fragment length polymorphism method reagent.
5. the described test kit of claim 2 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is Snapshot reagent.
6.BAI3 gene rs2073135 variation, CCDC149 gene rs4697489 variation, rs248014 variation, rs1105191 variation, rs1943049 variation are or/and TNC gene rs7035322 variation is used for the application of pathologic myopia screening agent in preparation.
7. application according to claim 6 is characterized in that described screening agent comprises detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A makes a variation or/and the reagent of TNC gene rs7035322C → A variation; And optional be used to increase comprise BAI3 gene rs2073135, CCDC149 gene rs4697489, rs248014, rs1105191, rs1943049 or/and the reagent of TNC gene rs7035322 gene segment.
8. application according to claim 7 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is order-checking reagent.
9. application according to claim 7 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is restriction fragment length polymorphism method reagent.
10. application according to claim 7 is characterized in that described detection BAI3 gene rs2073135G → A variation, CCDC149 gene rs4697489C → T variation, rs248014A → G variation, rs1105191T → C variation, rs1943049G → A variation are or/and the reagent of TNC gene rs7035322C → A variation is Snapshot reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010162356 CN101892306B (en) | 2009-04-03 | 2010-04-02 | Screening test kit for detecting pathologic moyopia |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910058834.7 | 2009-04-03 | ||
| CN200910058834 | 2009-04-03 | ||
| CN 201010162356 CN101892306B (en) | 2009-04-03 | 2010-04-02 | Screening test kit for detecting pathologic moyopia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101892306A true CN101892306A (en) | 2010-11-24 |
| CN101892306B CN101892306B (en) | 2013-06-12 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010162356 Active CN101892306B (en) | 2009-04-03 | 2010-04-02 | Screening test kit for detecting pathologic moyopia |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732607A (en) * | 2011-03-07 | 2012-10-17 | 四川省医学科学院（四川省人民医院） | Kit for detecting high myopia |
| CN105734149A (en) * | 2016-04-01 | 2016-07-06 | 天津脉络生物科技有限公司 | Kit for predicting myopia risk of child and application thereof |
| CN109609618A (en) * | 2019-01-08 | 2019-04-12 | 青岛大学 | A kind of kit detecting pathological myopia and its application method and purposes |
| CN114395620A (en) * | 2021-12-20 | 2022-04-26 | 温州谱希医学检验实验室有限公司 | Biomarker combination for detecting high-myopia susceptible population |
| CN116287199A (en) * | 2023-02-09 | 2023-06-23 | 山东中医药大学附属眼科医院(山东施尔明眼科医院) | Primer combination and kit for detecting high myopia risk and application of primer combination and kit |
| CN117187375A (en) * | 2023-09-12 | 2023-12-08 | 上海谱希和光基因科技有限公司 | Application of KDELR3 biomarker in diagnosis of high myopia |
-
2010
- 2010-04-02 CN CN 201010162356 patent/CN101892306B/en active Active
Non-Patent Citations (8)
| Title |
|---|
| 《NCBI网站，dbSNP数据库》 20000630 登录号 rs248014 , 2 * |
| 《NCBI网站，dbSNP数据库》 20000810 登录号 rs2073135 , 2 * |
| 《NCBI网站，dbSNP数据库》 20000906 登录号 rs1105191 , 2 * |
| 《NCBI网站，dbSNP数据库》 20010103 登录号 rs1943049 , 2 * |
| 《NCBI网站，dbSNP数据库》 20030629 登录号 rs7035322 , 2 * |
| 《NCBI网站，dbSNP数据库》 20040320 登录号 rs4697489 , 2 * |
| 《眼科新进展》 20010430 李寿玲等 病理性近视与HLA-DQB1基因关联的家系研究 全文 1-10 第21卷, 第2期 2 * |
| 《眼科研究》 20060831 于志强等 病理性近视的基因位点筛查 全文 1-10 第24卷, 第4期 2 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732607A (en) * | 2011-03-07 | 2012-10-17 | 四川省医学科学院（四川省人民医院） | Kit for detecting high myopia |
| CN102732607B (en) * | 2011-03-07 | 2014-02-19 | 四川省医学科学院（四川省人民医院） | Kit for detecting high myopia |
| CN105734149A (en) * | 2016-04-01 | 2016-07-06 | 天津脉络生物科技有限公司 | Kit for predicting myopia risk of child and application thereof |
| CN109609618A (en) * | 2019-01-08 | 2019-04-12 | 青岛大学 | A kind of kit detecting pathological myopia and its application method and purposes |
| CN109609618B (en) * | 2019-01-08 | 2021-11-26 | 青岛大学 | Kit for detecting pathological myopia and use method and application thereof |
| CN114395620A (en) * | 2021-12-20 | 2022-04-26 | 温州谱希医学检验实验室有限公司 | Biomarker combination for detecting high-myopia susceptible population |
| CN116287199A (en) * | 2023-02-09 | 2023-06-23 | 山东中医药大学附属眼科医院(山东施尔明眼科医院) | Primer combination and kit for detecting high myopia risk and application of primer combination and kit |
| CN117187375A (en) * | 2023-09-12 | 2023-12-08 | 上海谱希和光基因科技有限公司 | Application of KDELR3 biomarker in diagnosis of high myopia |
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|---|---|
| CN101892306B (en) | 2013-06-12 |
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