CN101173314B - Reagent kit for detecting senility macular degeneration disease - Google Patents

Abstract

The invention provides a reagent kit for detecting age-related macular degeneration disease, which can be used for the early monitoring and the detection of AMD high-risk population to implement the early prevention and the treatment and decrease the eyesight damage. The reagent kit of the invention takes the 512G to A mutation of a HTRA1 gene and the relative locus / gene as goal, the degree of association is high with AMD, and in combination with the analysis of the CHF gene mutation, the detecting accuracy can be improved.

Description

Detect the test kit of agedness yellow spot degenerative disease

Technical field

The present invention relates to a kind of test kit that detects agedness yellow spot degenerative disease, specifically detect the test kit of relevant HTRA1 genovariation with agedness yellow spot degenerative disease.

Background technology

It is the different substantiality disease of feature with decline of carrying out property central vision and the retinal pigment epithelium sex change of macula lutea portion that macular degeneration disease is considered to a class, is important blinding illness in eye.Macular degeneration comprises age-related macular degeneration, macular dystrophy (stargart disease, best disease, sorsby fundus dystrophy etc.).Age-related macular degeneration (age-related macular degeneration, AMD) now become one of topmost blinding illness in eye of the elderly, whole world number of patients 1,000 ten thousand, China 60-69 year crowd AMD morbidity is can reach 15.33% more than 7.77%, 70 years old.Also do not have effective methods of treatment for AMD at present, its major cause is exactly that pathogenesis it be unclear that.

Recent study thinks that macular degeneration is one group of different substantiality disease, and inherited genetic factors plays an important role in its pathogenesis.Some have similar research clinical and the other types macular degeneration that histopathology shows to make important progress to age-related macular degeneration (AMD), such as: discover that in recent years recessive Stargardt macular degeneration has variation on the ABCR gene; Dominance Stargardt macular degeneration (STGD3) has variation on the ELOVL4 gene; Best macular dystrophy (BMD) has variation on the VMD2 gene; There is the some variation in Sorsby fundus dystrophy patient at the TIMP3 gene, also has macula lutea butterfly sample atrophy etc. relevant with Peripherin/RDS genovariation.

Senile macular degeneration SMD is the major cause that causes developing country's non-reversibility visual loss, studies show that much it has significant genetic predisposition.At the research of different crowd, just becoming one of international research focus at present with race's macular degeneration disease.The real as yet systematicness of domestic monogenic macular degeneration disease and disease crowd and the ethnic contrast crowd's that is complementary genetic analysis (multigenic disease analysis) work is carried out, and does not also have relevant gene profile analysis.

The early-stage Study of AMD confirms alternative pathway of complement member's encoding gene, comprises that complement factor H (CFH) and factor B/C2 are relevant with individual ill risk, and the CFH that especially is positioned on the karyomit(e) 1q31 is considered to the main risks and assumptions of AMD.Some other association analysis shows that also karyomit(e) 10q26 goes up the risk site that has another AMD.Yet its definite Disease-causing gene it be unclear that.

Age-related macular degeneration is a kind of retinopathy of the multifactor compound action relevant with age growth.Mostly occur more than 45 years old, the age is big more, and morbidity is high more.It is one of primary disease of whole world adult's blinding.Senile macular degeneration SMD develops very slow sometimes, makes the patient be difficult for discovering, and but makes much progress sometimes, deprive the patient rapidly and look the thing ability, thereby its early discovery and prevention seems most important.

The real etiology unknown of this disease is a multi-factor disease, and influence factor also has ethnic group, sex, metabolism etc. except that the age, and inherited genetic factors is most important.Be divided into atrophic type (dryness type) and exudative type (moist type) clinically.Dryness person, visible glassy membrane wart progressively causes the macular area attenuation on the eyeground, thereby influences function.Still there is not special effective means treatment at present.Moist person causes the serious hindrance of eyesight more, and its eyesight destructive reason mainly is that unusual new vessel is grown under retina, causes the destruction of retinal hemorrhage, oedema and retinal tissue, finally causes cicatrization, thereby causes the forfeiture of eyesight.Moist AMD is morbidity in a single day, and the course of disease is rapid, and the poor effect of conventional treatment is difficult to recover significantly the visual function that it has been lost.Therefore, early diagnosis is found, intervenes early, can better delay the course of disease, reduces patient's visual loss, improves the quality of living.

Present AMD diagnoses in dependence: 1, more than the elderly's morbidity more than 50 years old, the age is big more, and sickness rate is high more; 2, metamorphopsia, visual deterioration or obvious visual disorder successively take place in eyes; 3, there are significantly glassy membrane wart and amphitypy performance separately in the eyeground; 4, fluorescence fundus angiography.But, lack the monitoring, diagnosing index at patient's their early stage, can find AMD high risk population by early screening, and implement early detection and prevention, reduce its inpairment of vision.

Summary of the invention

The object of the present invention is to provide the test kit that detects agedness yellow spot degenerative disease, this test kit derives from whether there is HTRA1 gene 512G → A variation in the sample of individuality to be measured by detection, and the generation of monitoring or diagnosis agedness yellow spot degenerative disease in early days, and then provide reference for clinical diagnosis and treatment.

The susceptibility loci that the present invention is directed to karyomit(e) 10q26 has carried out the gene type assay of AMD and normal control, discovery be positioned at the G at 512bp place, 10q26 karyomit(e) HTRA1 genetic transcription starting point upstream → A variation be cause the main diseases of AMD because of, the change of allelotrope G → A herein belonged to mononucleotide sequence polymorphism (SNP rs11200638), found report during at genome research in 2003.Yet the meaning of this variation it be unclear that.The direct correlation of rs11200638 and AMD has been found in the whole world of the present invention first.Even with respect to the collator who comprises known AMD risk genes CHF variation, analyze the population attributable risk that obtains the rs11200638 variation and also reach 49.3%.With rs11200638 and the collaborative analysis of CHF genovariation, obtain population attributable risk and reach 71.4%.

In addition, the rs10490924G on the karyomit(e) 10q26 → T variation also with the AMD height correlation, and rs11200638 and rs10490924 are in a LD.

The result of study demonstration exists G → AMD patient's lymphocyte of A variation and the transcription and translation level of retinal pigment epithelium HTRA1 to raise, and immunoblotting also shows AMD patient's eye HTRA1 antibody response strong positive.This shows that the G of rs11200638 → A variation has caused the up-regulated of HTRA1, thereby occupy critical role in the AMD susceptibility.

HTRA1 genes encoding secretor type serine protease, its coded product all has expression at mouse retina and retinal pigment epithelium, discover that HTRA1 albumen participates in the degraded of regulating cell epimatrix proteoglycan, promoted the Degradation of stromatin degrading enzyme, for example collagenase and matrix metalloproteinase to substrate.Can infer, the overexpression meeting of HTRA1 destroys the integrity of Bruch ' s film, makes choroidal artery be easy to penetrate the intrusion extracellular matrix, forms moist AMD.In addition, TGF-β is bringing into play important effect in regulating extracellular matrix degradation and vasculogenesis, and HTRA1 can in conjunction with and suppress transforming growth factor TGF-β.Therefore, G → A of rs11200638 has raised the HTRA1 expression, and the change of HTRA1 albumen and downstream signal bioactive molecule thereof has influenced intraocular extracellular matrix metabolic degradation process and vasculogenesis then, has finally participated in the morbidity process of AMD.

According to an aspect of the present invention, be provided for detecting the test kit of agedness yellow spot degenerative disease, whether the detection principle of test kit exists for detecting with the closely-related HTRA1 rs11200638G of agedness yellow spot degenerative disease → A variant sites, can comprise in the test kit that optional being used to detects the reagent of HTRA1 gene 512G → A variation, for example detect increase HTRA1 gene or comprise the reagent of HTRA1 gene rs11200638 gene fragment of the reagent of HTRA1 gene 512G → A sudden change and optional being used to.At first increase to the sample extraction DNA of acquisition and at HTRA1 gene or its 512 bit base site, detection method can be with reference to the method in detection point mutation well known to those skilled in the art site, for example direct sequencing or use the restriction fragment length polymorphism method or single base primers extension method or Taqman method or chip hybridization etc. is judged the allelotype in rs11200638 site.

Preferably, can in test kit, further comprise amplification and detect the related reagent that CHF gene Y402H makes a variation, by detecting the synergy that two gene mutations of HTRA1 and CHF take place, because the chain degree of rs11200638 and rs10490924 is high, detects the effectiveness that rs10490924 also has diagnosis AMD.Can in test kit, add the amplification and the detection reagent in rs10490924 site, further improve accuracy.

According to a further aspect in the invention, the HTRA1 protein expression level that is in harmonious proportion on the HTRA1 transcription and translation level that causes AMD patient because HTRA1 gene 512G → A suddenlys change raises, therefore at the rna transcription level of HTRA1 gene and suitable equally the present invention of test kit of protein expression level design.Can adopt for example RT-PCR technology for the rna transcription level that detects the HTRA1 gene, extract mRNA, detect the HTRA1 gene transcription level in patient's lymphocyte and obtain detected result.For the detection of protein expression level, can derive from the total protein of retinal pigment epithelium by extraction, react with the HTRA1 protein antibodies, measure proteic expression level and obtain detected result.Further, can adopt immunohistochemistry and fluorescence in-situ hybridization method to carry out the detection of protein expression level in position by preparation proteic monoclonal antibody of HTRA1 or polyclonal antibody.

The invention provides the test kit that detects agedness yellow spot degenerative disease, can be used for early stage monitoring and detect AMD high risk population, implement early prevention and treatment, reduce its inpairment of vision.Test kit of the present invention is a target with HTRA1 gene 512G → A sudden change and related locus/gene thereof, and with the relational degree height of AMD, the analysis of collaborative CHF transgenation more can improve the accuracy of detection.

Description of drawings

Fig. 1 is the genotype figure that Snapshot detects SNP rs11200638;

Fig. 2 is the sequencing result figure that the HTRA1 gene contains the rs11200638 site;

Fig. 3 is immunohistochemical methods figure;

Fig. 4 is fluorescence immunoassay group figure;

Fig. 5 is HTRA1 mRNA abundance result in the quantitative RT-RCR detection of human lymphocyte;

Fig. 6 detects the expression of results of HTRA1 at human retina pigment epithelial cell (RPE) for Western;

Fig. 7 cuts the result for PCR product enzyme.

Embodiment

Agents useful for same in following examples unless stated otherwise, is commercially available purchase.

[embodiment 1] blood sample is collected

Choose 581 routine senile macular degeneration SMD (AMD) patients and 309 routine collators.The patient is the AMD of clinical definite, 392 routine moist AMD patients wherein, and 189 routine dryness AMD patients, contrast is the crowd who changes more than or equal to 60 years old, non-glass wart and retinochrome who meets AMD patient age section.

To all its medical history of participator's probe and family history, and it is carried out a medical examination and detailed ophthalmology training inspection.Everyone blood sample collection 5～10ml after the signature Informed Consent Form.

[embodiment 2] comprise the pcr amplification and the gene type assay of rs11200638 gene

1, extracts the poba gene group DNA of AMD patient and control group.

2, pcr amplification genomic DNA fragment (comprising rs11200638)

Primer sequence: forward: 5 '-ATGCCACCCACAACAACTTT-3 '

Oppositely: 5 '-CGCGTCCTTCAAACTAATCC-3 '

Reaction conditions: 95 ℃ of 3minutes, (94 ℃ of 30seconds, 52 ℃ of 30seconds, 72 ℃ of 45seconds) * 35cycles

3, gene type

Restriction enzyme is identified:

(1) the PCR product adopts the EagI enzyme to cut and identifies G allelotrope

The restriction enzyme EagI of employing NEB (5 ' ... C^G G C C G...3 '), reaction system is as follows: 3.9ul H ₂O+5ul PCR product+1ul buffer 3+0.1ul EagI; Reaction conditions is as follows: 37 ℃ of 12hour.

(2) 2% agarose gel electrophoresis enzymes are cut product, and (the AA homozygote is big or small DNA bands such as former PCR product according to electrophoretic band interpretation genotype; The GG homozygote comprises two products behind the complete degestion, and its big or small sum equals PCR product size; The AG heterozygote is three bands, wherein one bigger identical with former PCR product size, two other is identical with the complete degestion product).

The SnapShot gene type:

1. purifying PCR reaction product

Reaction system:

PCR product cumulative volume 9 μ l

Exol enzyme 0.1 μ l

SAPbuffer 0.1μl

ddH ₂O 0.8μl

SAP 2μl

Amount to 12 μ l

Annotate: PCR product cumulative volume 9 μ l decide on PCR reaction quantity, and N is to the PCR of primer, and then the product application of sample of every pair of primer acquisition is 9 μ l/N.

Reaction conditions: 37 ℃ of 1hour; 75 ℃ of 15min

2. single base primers extends

Reaction system:

Purified product 1 μ l

SNaPshot?Multiplex 1μl

SNaPshot?primer 0.2×N

DdH ₂O supplies 5 μ l

Amount to 5 μ l

Reaction conditions: 96 ℃ of 1min

3. purification reaction thing

Reaction system: 0.7 μ l SAP/well

Reaction conditions: 37 ℃ of 1hour; 75 ℃ of 15min

4. sex change

Reaction system:

Purified product 1 μ l

HD 9μl

Amount to 10 μ l

Reaction conditions: 95 ℃ of 5min; Ice bath cooling rapidly

5.ABI3100 go up the machine-readable genotype result that gets

The nucleosidase sequencing:

1, glue reclaims:

(1) will be placed with and add Binding Buffer30ulfangru in the pipe that glue reclaims and hatch 7min for 55 ℃.(treating to dissolve fully)

(2) after the peptizationization, take out column jecket, perform mark, liquid is changed in the column jecket, 10000rpm/1min is centrifugal, outwells supernatant liquor after finishing, and adds 700ulSpw again, leaves standstill 2～3min, and 10000rpm/1min is centrifugal, outwells supernatant liquor.

(3) repeat above-mentioned 2 operations once.

(4) outwell supernatant liquor after, column jecket 1000rpm/1min is dallied.

(5) add 15ulElution Buffer to post central authorities, leave standstill 2～3min, 10000rpm/1min is centrifugal.

(6) repeat aforesaid operations

(7) liquid light that elutes is gone in the clean pipe, perform mark, place refrigerator.

2, amplification before the order-checking

Reaction system:

DNA 1ul

5×Buffer 1ul

Primer(0.2pm) 0.5ul

Seq 0.5ul

ddH ₂O 2ul

Amount to 5ul

Reaction conditions:

3, the second step purifying:

NaAc 12ul

EDTA 12ul

4, go up the machine order-checking

1ul/well behind the mixing, the 25ul dehydrated alcohol/wel 15min that keeps in Dark Place, 4100rpm/30min is centrifugal.Blot supernatant liquor as far as possible, 70% ethanol 40ul/well, 4100rpm/10min is centrifugal..Blot supernatant liquor, the room temperature 15min that keeps in Dark Place.Add 9ul/well, lucifuge is placed 1hour.

Fig. 1 detects the genotype figure (green peak is A, and blueness is G) of SNP rs11200638 for Snapshot.This check result is the genotypic individuality of heterozygote, and it comprises a risk allelotrope A; Green unimodal if homozygote AA type just shows as, GG then is blue unimodal.Mix the ill risk of money AA type mutually and be significantly higher than the GG type, the ill risk of heterozygosis AG type is between between the two.

Fig. 2 contains the sequencing result figure in rs11200638 site for the HTRA1 gene.Arrow is depicted as this site signal, heterozygote A/G.The meaning of allelotype as previously shown.

[embodiment 3] comprise the pcr amplification and the gene type assay of rs10490924 gene

1, extracts the poba gene group DNA of AMD patient and contrast.

2, pcr amplification genomic DNA fragment (comprising rs10490924).

Primer sequence: forward: 5 '-TACCCAGGACCGATGGTAAC-3 ';

Oppositely: 5 '-GAGGAAGGCTGAATTGCCTA-3 '.

Reaction conditions: 95 ℃ of 3minutes, (94 ℃ of 30seconds, 52 ℃ of 30seconds, 72 ℃ of 45seconds) * 35cycles

3, gene type

Restriction enzyme is identified:

(1) the PCR product adopts the PVUII enzyme to cut and identifies G allelotrope.

The restriction enzyme PVUII of employing NEB (5 ' ... C A G^C T G...3 '), reaction system is as follows: 3.9ul H ₂O+5ul PCR product+1ul buffer 2+0.1ul PVUII; Reaction conditions is as follows: 37 ℃ of 12hour.

(2) 2% agarose gel electrophoresis enzymes are cut product, and (the TT homozygote is big or small DNA bands such as former PCR product according to electrophoretic band interpretation genotype; The GG homozygote comprises two products behind the complete degestion, and its big or small sum equals PCR product size; The TG heterozygote is three bands, wherein one bigger identical with former PCR product size, two other is identical with the complete degestion product).

The correlation analysis result of last 15 SNP of [embodiment 4] 10q26

Adopt the SNaPshot method, on the ABI3130 genetic analyzer, carry out.

The correlation analysis of 15 SNP of table 1

SNP

bp

chi_trend

trend_p

Orhet

Orhom

chi_dom

dom_p

Ordom

rs986960 rs1998345 rs2901307 rs4146894 rs2421016 rs1045216 rs10490924 rs3750847 rs3750846 rs2014307 rs11200638 rs1049331 rs4752700 rs2300431 rs714816

124082543 124114296 124118433 124145371 124157502 124179187 124204438 124205411 124205555 124207622 124210524 124211260 124227602 124232807 124246335

0.5141005 8.1847003 5.0544893 16.549416 11.094448 0 32.1381 27.1378 18.646568 24.366379 37.2931 0.8081998 1.4022982 1.6662006 6.812544

0.4733694 0.0042245 0.0245621 4.74E-05 0.0008661 1 8.14E-08 1.86E-07 1.57E-05 5.90E-07 1.02E-09 0.368653 0.236339 0.1967684 0.0090517

0.91(0.64，1.31) 1.37(0.99，1.90) 0.79(0.55，1.14) 1.76(1.22，2.53) 1.82(1.24，2.66) 0.88(0.64，1.23) 1.35(0.99，1.86) 1.44(1.04，1.98) 1.40(1.02，1.92) 0.61(0.44，0.85) 1.86(1.35，2.56) 0.71(0.49，1.03) 0.65(0.45，0.94) 0.74(0.51，1.08) 1.11(0.80，1.54)

0.85(0.55，1.32) 2.02(1.19，3.43) 0.59(0.38，0.93) 2.32(1.55，3.47) 2.21(1.38，3.54) 1.12(0.67，1.87) 6.09(3.27，11.34) 5.99(2.98，12.04) 4.86(2.32，10.19) 0.23(1.13，0.41) 6.56(3.23，13.31) 0.88(0.53，1.44) 0.85(0.51，1.41) 0.81(0.46，1.45) 2.26(1.33，3.84)

0.4099402 6.1691034 3.0248076 15.445303 11.989527 0.2458672 15.372106 14.608167 10.332306 14.867718 28.22876 2.4978763 4.176009 2.4436668 2.2571312

0.522 0.013 0.082 8.49E-05 0.0005352 0.62 8.80E-05 0.0001324 0.0013074 0.0001154 3.82E-07 0.114 0.041 0.118 0.133

0.89(0.64，1.26) 1.48(1.09，2.02) 0.73(0.52，1.04) 1.96(1.40，2.74) 1.91(1.32，2.77) 0.92(0.67，1.26) 1.81(1.35，2.45) 1.82(1.34，2.47) 1.65(1.22，2.24) 0.54(0.39，0.74) 2.21(1.62，3.01) 0.75(0.52，1.07) 0.69(0.49，0.99) 0.76(0.53，1.07) 1.27(0.93，1.73)

The correlation analysis result that genome 10q26 goes up 15SNP shows, the meaning of rs11200638 the most remarkable (P value), and the cognation of this single nucleotide polymorphism A risk allelotrope and AMD is the highest.

The ill risk of [embodiment 5] rs10490924 and rs11200638 relatively

At first 442 routine AMD patients (265 moist AMD and 177 dryness AMD) and 309 contrasts have been carried out gene type assay, compare the allelotrope frequency of occurrences, subsequently at 581 routine AMD patients (392 routine moist AMD and 189 routine dryness AMD), study rs10490924 and rs11200638 genotype, compared the ill risk of the two AMD.

The result shows that rs10490924 and AMD dependency are higher, with report is consistent before.Find that simultaneously rs11200638 is the highest (p=1 * 10, variation site of dependency in 15 SNP ^-9, OR _Het=1.86 (1.35,2.56), OR _Hom=6.56 (3.23,13.31), A allele:40.3% in cases versus 25.2% incontrols; ).And rs10490924 and rs11200638 are that the dependency of T-A type is higher than simple rs10490924 (T), but are lower than rs11200638 (A).

For further its dependency of research, appended 139 routine patients AMD, the difference of more above-mentioned two kinds of varients, result show that the dependency of the two and AMD all raises (rs10490924:p=1.2 * 10 ^-8, rs11200638:p=1.6 * 10 ^-11), and rs11200638 remains the highest single varient (OR of the degree of correlation _Het=1.90 (1.40,2.58), OR _Hom=7.51 (3.75,15.04)).

Rs11200638 and CFH gene rs1061170 varient (being positioned at the important ill risks and assumptions of known AMD of human chromosome 1q31) have been contrasted in addition.The result shows rs11200638 and AMD height correlation, even based on analysis (p=5.9 * 10 that comprise CFH equipotential risk genes (C) ^-8).All AMD patients' result of study shows that the AA homology allelotrope risk of rs11200638 is greater than AG allos allelotrope (OR _Hom=7.29 (3.18,16.74); OR _Het=1.83 (1.25,2.68)), confirmed the allelotrope dosage effect.Adopt the allelotrope dose modal, it is 49.3% that the AMD crowd who obtains rs11200638 retreats risk level.The binding pattern analysis of rs11200638 and CFH Y402H, it is 71.4% (being any one site variation) that the AMD crowd who obtains the two additive effect retreats risk level.

[embodiment 6] data analysis

10q26：

Adopt chi square test to analyze the allelotrope dependency, calculate odds ratio and 95% credibility interval simultaneously, estimate the ill risk of homozygote and heterozygote.

Last CFHY402H of 1q31 and 10q26 go up the rs11200638 two site assay:

Make up contingency table based on case-contrast and the combination of dibit point gene type, CFHY402H and the combination of rs11200638 dibit point gene type comprise: TT/GG, TT/AG, TT/AA, CT/GG, CT/AG, CT/AA, CC/GG, CC/AG and CC/AA.Dibit point 9 * 2 contingency tables all adopt 8 degree of freedom chi square tests to handle.With normal homozygote allelotrope combination TT/GG is contrast, calculates the odds ratio and 95% credibility interval of every kind of assortment of genes.Employing Levin formula calculating population attributable risk (populationattributable risks, PAR), this genotypic total ill risk among the reflection crowd.

The odds ratios result in table 2 white people HTRA1 rs11200638 and two sites of CFH rs1061170

SNP		HTRA1rs11200638
					CFHrs1061170 (Y402H)	TT CT CC	GG 1.00 1.07(0.59，1.94) 3.07(1.50，6.27)	AG 1.08(0.93，3.49) 2.31(1.28，4.17) 3.97(1.93，8.15)	AA 3.43(0.62，19.00) 7.31(2.68，19.93) 31.52(4.01，247.96)

The result shows that the individual ill risk that has HTRA1rs11200638 site AA risk allelotype and CFHrs1061170 site CC risk allelotype simultaneously is far longer than other individualities (oddsratios is 31.52)

Table 3 Chinese AMD association analysis result

The dependency of HTRA1 (rs11200638) and the same CNV of ARMS2 (rs10490924) (non-Drusen)

Phenotype

N (genotype counting)

The risk gene frequency

p-value

ORhom (95％CI)

OR?het(95％CI)

PAR

HTRA1(rs11200638)

CNV

90(AA：53；AG：34；GG：3)

77.78％

5.00E-12

32.98(8.80， 123.63)

5.04(1.43，17.79)

67.4

Drusen

74(AA：15；AG：43；GG：16)

49.33％

0.258

Control

106(AA：15；AG：63；GG：28)

43.87％

LOC387715(rs10490924)

CNV

90(TT：52；TG：35；GG：3)

77.22％

6.83E-12

33.51(8.95， 125.47)

5.46(1.55，19.22)

66.84

Drusen

74(TT：13；TG：45；GG：16)

47.97％

0.337

Control

106(TT：15；TG：62；GG：29)

43.40％

The result shows that HTRA1rs11200638 and Chinese AMD height correlation especially endanger serious moist AMD.

[embodiment 7] HTRA1 immunohistochemical methods

AMD patient's eyeball Paraformaldehyde 96 is fixed, and contains the methyl alcohol deactivation endogenous peroxidase activity of 0.3%H2O2.HTRA1 resists more is R﹠amp; D company product (HTRA1/PRSS11 MAb, Clone 275615,100UG), concentration 5 μ g/ml.Adopt VectorStain Elite ABC kit test kit, adopt Nikon Eclipse80i microscope acquired signal.Immunohistochemical methods result such as Fig. 3, fluorescence immunoassay chemical results such as Fig. 4.

HTRA1 mRNA abundance in [embodiment 8] quantitative RT-RCR detection of human lymphocyte:

Adopt the real-time quantitative PCR (Opticon of system; MJ Research, Watertown, MA) transcriptional level of reflection HTRA1 in AMD patient's lymphocyte.

1, adopt RNeasy test kit (Qiagen) to extract the total RNA of patient's peripheral blood lymphocyte.

2, use QuantiTect SYBR Green RT-PCR kit (Qiagen) RT-PCR

One step amplification HTRA1 mRNA,

Primer: forward: 5 '-AGCCAAAATCAAGGATGTGG-3 ';

Oppositely: 5 '-GATGGCGACCACGAACTC-3 '.

RNA consumption 100ng.Adopt the total RNA of 0-400ng, parallel amplification house-keeping gene GAPDH obtains typical curve.

Primer: forward: 5 '-CTGCACCACCAACTGCTTAG-3 ';

Oppositely: 5 '-GTCTTCTGGGTGGCAGTGAT-3 '.

3, calculate the AA genotype with respect to the genotypic HTRA1 RNA of GG abundance.The results are shown in Figure 5.

[embodiment 9] Western detects the expression of HTRA1 at human retina pigment epithelial cell (RPE):

Extract the total protein of the RPE of four genotypic moist AMD patients of AA and six genotypic normal controls of GG, get 25 μ g and carry out the SDS-PAGE electrophoresis, antibody concentration 1.5 μ g/ml carry out Westernblotting.The HTRA1 protein expression level is confidential reference items with β-actin.Adopt SPSS version 13.0 to carry out independent sample t check analysis data, calculate its statistical significance.Result such as Fig. 6, result show that the genotypic HTRA1 protein expression of AA is about 1.8 times of GG genotype protein expression.

[embodiment 10] test kit detects

This test kit is used for: 1, AMD patient's early diagnosis and somatotype reference; 2, the gene test of AMD Susceptible population and risk assessment.

[reagent]

The test kit inventory

Reagent can carry out 50 parts of mensuration. composition

PCR reaction mixture ml (1 bottle) 450ul

Eag-I restriction enzyme ul (1 bottle) 5ul

10 * enzyme is cut Buffer ul (1 bottle) 100ul

The genotypic contrast of Control DNA-1 ml (1 bottle) A/A DNA

The genotypic contrast of Control DNA-2 ml (1 bottle) G/G DNA

The genotypic contrast of Control DNA-3 ml (1 bottle) A/G DNA

[operation steps]

The pcr amplification of rs11200638 gene and gene type assay:

1 sampling: get patient's whole blood (EDTA anti-freezing) 1ml, extract AMD patient's poba gene group DNA.

2.PCR operation

Design of primers: pcr amplification genomic DNA fragment (comprising rs11200638)

Primer sequence: forward: 5 '-ATGCCACCCACAACAACTTT-3 '

Oppositely: 5 '-CGCGTCCTTCAAACTAATCC-3 '

3. application of sample system: (concentration is 50～100ng) to add reaction mixture 9ul to take out the 1ul genomic dna

4. reaction conditions:

1,95 ℃ of pre-sex change 5minutes

2,95 ℃ of sex change 30seconds

3,52 ℃ of annealing 30seconds

4,72 ℃ are extended 45seconds

5, step 2～4 totally 35 circulations

6,4 ℃ of preservations

5, the PCR product adopts the EagI enzyme to cut evaluation allelotrope

Reaction system: 3.9ulH2O+5ul PCR product+10 * Buffer 1ul+0.1ul Eag-I;

Reaction conditions: 37 ℃ of enzymes were cut 12 hours;

2% agarose gel electrophoresis, experimental result such as Fig. 7, swimming lane 1 is cut the result for the enzyme of AA homozygote sample DNA, and swimming lane 2 is cut the result for the enzyme of GG homozygote sample DNA, and swimming lane 6 is cut the result for the enzyme of AG heterozygote.

Claims (9)

1. the reagent that detects HTRA1 gene rs11200638 variation is used for predicting the application of the test kit of suffering from the agedness yellow spot degenerative disease risk in preparation, wherein said HTRA1 gene rs11200638 variation is the G at 512bp place, HTRA1 genetic transcription starting point upstream → A variation, and genotype A is ill risk genes.

2. the described application of claim 1, wherein said test kit comprise the reagent that detects HTRA1 gene rs11200638 variation; And optional be used to increase comprise the reagent of HTRA1 gene rs11200638 gene fragment.

3. the described application of claim 2, the reagent of wherein said detection HTRA1 gene rs11200638 variation is order-checking reagent.

4. the described application of claim 2, the reagent of wherein said detection HTRA1 gene rs11200638 variation is restriction fragment length polymorphism method reagent.

5. the described application of claim 2, the reagent of wherein said detection HTRA1 gene rs11200638 variation is Snapshot method reagent.

6. the described application of claim 2 it is characterized in that further comprising the reagent that detects CHF gene Y402H gene T → C variation, and/or optional being used to detects the reagent that 10q26 goes up rs10490924G → T variation.

7. the described application of claim 1, wherein said test kit also comprise the reagent that detects the HTRA1 protein expression, and described HTRA1 gene rs11200638G → A variation causes described HTRA1 protein content to raise; The reagent of described detection HTRA1 protein expression comprises the proteic antibody of HTRA1; And the reagent of optional detection HTRA1 expressing quantity.

8. the described application of claim 1, wherein said test kit also comprise the reagent that detects the HTRA1mRNA expression, and described HTRA1 gene rs11200638G → A variation causes described HTRA1mRNA to express rising.

9. the described application of claim 8 is characterized in that the reagent that described detection HTRA1mRNA expresses is quantitative RT-PCR reagent.

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