WO2013170744A1 - Kit de dépistage de dégénérescence maculaire sénile - Google Patents

Kit de dépistage de dégénérescence maculaire sénile Download PDF

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Publication number
WO2013170744A1
WO2013170744A1 PCT/CN2013/075618 CN2013075618W WO2013170744A1 WO 2013170744 A1 WO2013170744 A1 WO 2013170744A1 CN 2013075618 W CN2013075618 W CN 2013075618W WO 2013170744 A1 WO2013170744 A1 WO 2013170744A1
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WO
WIPO (PCT)
Prior art keywords
site
mutation
locus
gene
reagents
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PCT/CN2013/075618
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English (en)
Chinese (zh)
Inventor
杨正林
鲁芳
刘小琦
石毅
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四川省医学科学院（四川省人民医院）
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Application filed by 四川省医学科学院（四川省人民医院） filed Critical 四川省医学科学院（四川省人民医院）
Publication of WO2013170744A1 publication Critical patent/WO2013170744A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of SNPs, and in particular to SNPs associated with age-related macular degeneration.
  • Age-related macular degeneration which is considered to be a heterogeneous disease characterized by progressive central vision loss and macular retinal pigment epithelial degeneration, is one of the leading diseases in adult blindness worldwide.
  • the number of patients suffering from the world is 10 million.
  • the prevalence of AMD in China's 60-69 year old population is 7.77%, and that of over 70 years old is 15.33%.
  • Macular degeneration includes age-related macular degeneration, macular malnutrition (stargart disease, best disease, sorsby, dystrophy, etc.).
  • the present invention provides a screening test kit for a new age-related macular degeneration disease.
  • the present invention provides five variant bases, the variant base I is A, located at the SKIV2L locus rs429608; the variant base II is C, located at the SKIV2L locus rs2075702; the variant base III is C, located at the RDBP locus Rs760070; The variant base III is C, located at the RDBP locus rs3880457; the variant base III is A, located at the RDBP locus rs9501161.
  • the SNP is an abbreviation for Single Nucleotide Polymorphism, which translates into a single nucleotide polymorphism in Chinese and refers to the variation of a single nucleotide on the genome.
  • the aforementioned five variant bases are the result of a single nucleotide variation.
  • the invention also provides a reagent for detecting SKIV2L gene locus rs429608 mutation, locus rs2075702 mutation, RDBP locus rs760070 mutation, locus rs3880457 mutation or/and locus rs9501161 mutation in screening reagent for preparing age-related macular degeneration disease. the use of.
  • the reagent comprises detecting SKIV2L gene locus rs429608 G ⁇ A mutation, locus rs2075702 T ⁇ C mutation, RDBP locus rs760070 mutation T ⁇ C, locus rs3880457 T ⁇ C mutation or/and locus rs9501161 G ⁇ A variant of the reagent; further comprising an optional reagent for amplifying a gene fragment comprising the SKIV2L gene locus rs429608, the locus rs2075702, the RDBP locus rs760070, the locus rs3880457 or/and the locus rs9501161.
  • the reagent is Snapshot reagent.
  • the reagent is a reagent for sequencing.
  • the reagent-restriction fragment length polymorphism method uses a reagent or a reagent for single-strand conformation polymorphism analysis.
  • the invention also provides a screening kit for age-related macular degeneration diseases, which comprises optionally for detecting SKIV2L gene locus rs429608 mutation, locus rs2075702 mutation, RDBP gene locus rs760070 mutation, locus rs3880457 mutation or / And related reagents for the mutation of rs9501161.
  • locus rs429608 detects SKIV2L gene locus rs429608 G ⁇ A mutation, locus rs2075702 T ⁇ C mutation, RDBP locus rs760070 mutation T ⁇ C, locus rs3880457 T ⁇ C mutation or/and locus rs9501161 G ⁇ A variation Reagents; also include optional for amplification
  • the reagent for the A mutation is a Snapshot reagent.
  • the reagent for the A mutation is a reagent for sequencing.
  • the reagent for the A mutation is a reagent for restriction fragment length polymorphism method or a reagent for single strand conformation polymorphism analysis.
  • the present invention clarifies for the first time that the SKIV2L gene locus rs429608 mutation, locus rs2075702 mutation, RDBP locus rs760070 mutation, locus rs3880457 mutation or/and locus rs9501161 mutation are protective variants of age-related macular degeneration disease, and the aforementioned mutation can effectively reduce The prevalence of age-related macular degeneration.
  • the kit provided by the invention the resistance of the test population to age-related macular degeneration diseases can be effectively screened, and the possibility of suffering from age-related macular degeneration in the population to be examined is evaluated, and the application prospect is good.
  • the assay method of the present invention measures genomic DNA derived from humans, and the sample is not limited, such as body fluids (such as blood, ascites, and urine), tissue cells (such as liver tissue), and the like, and genomic DNA can be prepared by extracting and purifying these samples.
  • body fluids such as blood, ascites, and urine
  • tissue cells such as liver tissue
  • genomic DNA can be prepared by extracting and purifying these samples.
  • SNPs are mostly only two types of alleles in the population, so they are also called biallelic markers.
  • the detection is a kind of "+/ _" or "Full/None” detection method.
  • SNPs can be detected in a variety of ways, including sequencing, hybridization, dissolution, electrophoresis, chemical, enzymatic, physical, and combinations thereof. These methods are routine experiments for detecting nucleotide variations. means.
  • the invention discloses a base type of a variant of the SKIV2L gene locus rs429608, a locus rs2075702 variant, an RDBP locus rs760070 variant, a locus rs3880457 variant or/and a locus rs9501161 variant, and the sequences of the SKIV2L gene and the RDBP gene are known,
  • the polymorphisms of the above two sites can be detected by the existing methods for detecting nucleotide variation.
  • FIG. 1 Snapshot detection genotyping map of SNP rs429608 locus
  • dNTP deoxynucleoside triphosphate
  • the collected 5 ml of human peripheral blood was centrifuged at 3000 rpm for 30 minutes in the presence of an anticoagulant EDTA to remove serum. Then 0.2% NaCl solution was added to make the total volume 50 ml. The solution was gently shaken 5-6 times and placed on ice for 15 minutes. Thereafter, the mixture was centrifuged at 3000 rpm for 30 minutes, thereby collecting a precipitate. Washing was carried out in a manner similar to the previous one with a 0.2% NaCl solution. To the precipitate thus obtained, 10 mM Tris-HCl (pH 8.0) and 10 mM EDTA (4 ml) were added to suspend the precipitate.
  • the invention uses Snapshot genotyping technology to detect five sites of rs429608, rs2075702, rs760070, rs3880457 and rs9501161.
  • the gene amplification reagent and Snapshot primer A-E were used for typing according to the following methods.
  • PCR Denaturation at 95 °C for 5 minutes; 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 45 seconds, 35 cycles; 72 °C for 7 minutes, 4 °C for insulation.
  • Snapshot reaction Snapshot MULTIPLEX 1 ⁇ , ⁇ PCR mix 1 ⁇ , Snapshot primers each 0.2 l, DDH 2 O complement 5 ⁇ 1.
  • Snapshot Primer A (rs429608): Snapshot Primer B (rs2075702):
  • FIG. 5 ' - ACCACAGAATTTGTAAAATGGAGAATTCAGGAGTCGTCTA -3 '
  • Figure 1 to Figure 5 are genotype maps of Snapshot detection rs429608, rs2075702, rs760070, rs3880457 and rs9501161.
  • Example 3 Correlation between SNP locus and age-related macular degeneration
  • Primer pair B (rs2075702 locus) 5 ⁇ 400 ⁇ 1
  • Primer pair C (rs760070) 5 ⁇ 400 ⁇ 1
  • Primer pair D (rs3880457 site) 5 ⁇ 400 ⁇ 1
  • Primer pair E (rs9501161 locus) 5 ⁇ 400 ⁇ 1
  • the effect of the PCR reaction was examined by 2% agarose gel electrophoresis and the amount of the PCR reaction was added as a template in the subsequent reaction.
  • the first step purification of the PCR product (12 ⁇ 1 system):
  • Step 4 ABI Genetic Analyzer reads the results:
  • genotype results were read on the ABI PRISM 3130XL DNA Sequencer. This kit is used for: 1. Early diagnosis and classification reference for people with age-related macular degeneration disease; 2. Genetic detection and possibility assessment for people with age-related macular degeneration disease resistance.
  • the detection method of the present invention can be used to analyze the polymorphisms of five SNP loci of rs429608, rs2075702, rs760070, rs3880457 and rs9501161, and is used in the auxiliary diagnosis of age-related macular degeneration and the assessment of individual age-related macular degeneration disease resistance.
  • the nucleic acid sequence for detecting SNP locus polymorphism and the related gene/site of age-related macular degeneration established by the invention can be applied to the kit for genetic diagnosis of age-related macular degeneration with high sensitivity and specificity.
  • the polymorphisms of the five SNP loci of rs429608, rs2075702, rs760070, rs3880457, and rs9501161 were significantly associated with age-related macular degeneration. Therefore, according to the present invention, the polymorphism thereof can be determined for genetic diagnosis.
  • the present invention measures the variability of five SNP loci of rs429608, rs2075702, rs760070, rs3880457 and rs9501161, and evaluates the possibility of age-related macular degeneration of the sample to be tested. The sensitivity is high, and only a small amount of DNA sample is sufficient to determine the The variation of the locus achieves the purpose of early screening.

Abstract

La présente invention concerne les loci rs429608 et rs2075702 loci dans le gène SKIV2L et les mutations de bases des loci rs760070, rs3880457 et rs9501161 dans le gène RDBP. La présente invention concerne également l'utilisation d'un réactif pour détecter les mutations de bases susmentionnées en préparation du réactif de dépistage de la dégénérescence maculaire sénile et un kit de dépistage de la dégénérescence maculaire sénile. Le kit de la présente invention peut être utilisé pour un diagnostic auxiliaire de dégénérescence maculaire sénile et pour l'évaluation de la résistance de la dégénérescence maculaire sénile individuelle.
PCT/CN2013/075618 2012-05-14 2013-05-14 Kit de dépistage de dégénérescence maculaire sénile WO2013170744A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210148101 2012-05-14
CN201210148101.4 2012-05-14

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WO2013170744A1 true WO2013170744A1 (fr) 2013-11-21

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005082110A2 (fr) * 2004-02-26 2005-09-09 Illumina Inc. Marqueurs haplotypes pour le diagnostic de la susceptibilite aux conditions immunologiques
WO2011006161A2 (fr) * 2009-07-10 2011-01-13 The Regents Of The University Of Michigan Compositions et procédés permettant de diagnostiquer et de traiter la dégénérescence maculaire

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101857899A (zh) * 2009-04-03 2010-10-13 四川省医学科学院（四川省人民医院） 老年黄斑变性疾病的检测试剂盒

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005082110A2 (fr) * 2004-02-26 2005-09-09 Illumina Inc. Marqueurs haplotypes pour le diagnostic de la susceptibilite aux conditions immunologiques
WO2011006161A2 (fr) * 2009-07-10 2011-01-13 The Regents Of The University Of Michigan Compositions et procédés permettant de diagnostiquer et de traiter la dégénérescence maculaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BROOK ALLISON CATTELL LONGVILLE., GENETIC EPIDEMIOLOGY OF AGE-RELATED MACULAR DEGENERATION (AMD): THE ROLE OF THE COMPLEMENT COMPONENT 2 (C2) AND COMPLEMENT FACTOR B (CFB) GENES IN DETERMINING AMD SUBPHENOTYPES., 2010, MURDOCH UNIVERSITY LIBRARY RESEARCH REPOSITORY. *
LU, FANG ET AL.: "A Genetic Variant in the SKIV2L Gene Is Significantly Associated With Age-Related Macular Degeneration in a Han Chinese Population.", IOVS, vol. 54, no. 4, April 2013 (2013-04-01), pages 2911 - 2917 *
NAOSHI KONDO ET AL.: "Role of RDBP and SKIV2L Variants in the Major Histocompatibility Complex Class III Region in Polypoidal Choroidal Vasculopathy Etiology.", OPHTHALMOLOGY, vol. 116, no. 8, August 2009 (2009-08-01), pages 1502 - 1508 *

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CN104293953A (zh) 2015-01-21
CN103215265A (zh) 2013-07-24

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