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WO2018040320A1 - Procédé et kit de détection du gène humain de la fructose bisphosphate aldolase b - Google Patents

Procédé et kit de détection du gène humain de la fructose bisphosphate aldolase b Download PDF

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Publication number
WO2018040320A1
WO2018040320A1 PCT/CN2016/107577 CN2016107577W WO2018040320A1 WO 2018040320 A1 WO2018040320 A1 WO 2018040320A1 CN 2016107577 W CN2016107577 W CN 2016107577W WO 2018040320 A1 WO2018040320 A1 WO 2018040320A1
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WIPO (PCT)
Prior art keywords
gene
primer
fructose diphosphate
human fructose
human
Prior art date
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PCT/CN2016/107577
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English (en)
Chinese (zh)
Inventor
杜蔚安
黄源坚
郑文彦
高静
王邦超
彭百华
周玉
吴智锋
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Guangdong Homy Genetech Inc
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Guangdong Homy Genetech Inc
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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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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 invention relates to the technical field of genetic engineering, in particular to a kit for detecting human fructose diphosphate aldolase B gene, and a detection method thereof.
  • HFI Hereditary Fructose Intolerance
  • the methods for detecting human fructose diphosphate aldolase B gene polymorphism are mainly direct sequencing and allele-specific oligonucleotide analysis (ASO).
  • Direct sequencing the accuracy is high, but it takes a long time and costs.
  • the allele-specific oligonucleotide assay uses PCR to amplify the product, denatures it on a nylon membrane, fixes it, then hybridizes with labeled primers, and finally autoradiographs.
  • the disadvantage of this method is that the operation is complicated, the technical requirements of the operator are high, the radiation development is required, the resolution is not high, and the time is long.
  • the present invention aims to provide a high-accuracy, low-cost, high-throughput, rapid detection kit for detecting the presence or absence of a mutation in the human fructose bisphosphate aldolase B gene.
  • a human fructose diphosphate aldolase B gene detecting kit comprising a primer pair for specifically amplifying three polymorphic sites on a human fructose diphosphate aldolase B gene, three The polymorphic sites are A150P, A175D, and N335K, respectively.
  • the primer pair includes a common upstream primer and a downstream primer, and the downstream primer includes a wild-type specific primer and a mutant-specific primer, corresponding to three polymorphisms.
  • the public upstream primers, wild-type specific primers, and mutant specific primers at the loci are shown in the following table:
  • F represents a common upstream primer
  • W represents a wild-type specific primer
  • M represents a mutant-specific primer
  • each common upstream primer is labeled with a fluorescent dye.
  • the fluorescent dye is one or more of FAM, HEX, TAMRA, ROX, Cy3, Cy5, JOE, VIC, PET.
  • the human fructose diphosphate aldolase B gene detecting kit of the present invention further comprises a control gene sequence comprising a normal sequence and a mutant sequence.
  • the human fructose diphosphate aldolase B gene detection kit further comprises a PCR buffer, a hot-start Taq enzyme, ultrapure water, a fluorescent molecular weight internal standard and an allelic genotyping standard.
  • the present invention also provides a method for using the above human fructose diphosphate aldolase B gene detection kit, which comprises using a polymerase for amplifying three polymorphic sites of human fructose diphosphate aldolase B gene in a sample.
  • the amplification products are detected by high performance liquid chromatography, multiple fluorescent capillary electrophoresis or polyacrylamide gel electrophoresis.
  • the amplification procedure of the polymerase chain reaction is: 95 ° C for 2 minutes; 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, a total of 10 cycles; 94 ° C for 30 seconds, 59 ° C for 30 seconds, 72 ° C 30 seconds for a total of 18 cycles; 72 ° C for 10 minutes.
  • the common upstream primers can detect up to 4-96 samples in a single electrophoresis with different fluorophores, which is highly efficient and economical;
  • the DNA extraction step can be omitted, and the saliva card, FTA card and blood filter paper can be directly expanded, the operation steps are reduced, and the operation process is simplified;
  • the present invention can accurately judge the genotyping of normal and mutant samples.
  • Figure 1 is a three-site detection map of a normal sequence control wild-type specific primer in the kit
  • Figure 2 is a three-site detection map of the mutant-specific mutant-specific primers in the kit. Spectrum
  • Figure 3 is a detection map of Example 1
  • Figure 4 is a detection map of Example 2.
  • Figure 5 is a detection map of Example 3.
  • Figure 6 is a detection map of Example 4.
  • Figure 7 is a detection map of Example 5.
  • Figure 8 is a detection map of Example 6.
  • W represents a wild type sequence and M represents a mutant sequence.
  • a human fructose diphosphate aldolase B gene detecting kit comprising a primer pair for specifically amplifying three polymorphic sites on a human fructose diphosphate aldolase B gene, three polymorphisms
  • the sex sites are A150P, A175D, N335K, respectively
  • the primer pair includes a common upstream primer and a typing downstream primer
  • the typing downstream primer includes a wild type specific primer and a mutant specific primer
  • the upstream primer is a common primer, including a fluorescent label and a target sequence specific binding region
  • the downstream primer includes a typing recognition site and a length adjustment region
  • the fluorescent dye is one or more of FAM, HEX, TAMRA, ROX, Cy3, Cy5, JOE, VIC, PETkind.
  • control gene sequences also included are control gene sequences, PCR buffers, hot-start Taq enzymes, ultrapure water, fluorescent molecular weight internal standards, and allelic ladders, including normal and mutant sequences.
  • the appropriate method is selected to extract the sample DNA.
  • Common methods include magnetic bead method, anion exchange resin method and alkali lysis method. If the sample is hemofiltered Paper, FTA card, saliva card, etc., can simplify the steps and directly expand.
  • the amplification system of the PCR reaction is as follows:
  • the amplification procedure for the PCR reaction is as follows:
  • control gene sequence was detected according to the above-mentioned method of use, and three site detection maps of the normal sequence control wild type specific primers as shown in FIGS. 1 and 2 and three site detection maps of the mutant sequence control mutant specific primers were obtained;
  • the No. 1 sample was tested according to the above-mentioned method, and the map shown in Fig. 3 was obtained. It can be seen that the No. 1 sample is a single point mutation of the A150P site.
  • Example 2 The overall flow was the same as in Example 1.
  • the results of the second sample were as shown in FIG. 4, and it was found that the No. 2 sample was a single point mutation of the A175D site.
  • Example 2 The overall flow was the same as in Example 1.
  • the results of the No. 3 sample are shown in Fig. 5. It can be seen that the No. 3 sample is a single point mutation of the N335K site.
  • Example 6 The overall procedure was the same as in Example 1. The results of the sample No. 4 were as shown in Fig. 6. It can be seen that the No. 4 sample was a double site mutation of the A150P and A175D sites.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pathology (AREA)

Abstract

L'invention concerne un procédé et un kit de détection du gène humain de la fructose bisphosphate aldolase B. Le kit de détection comprend une paire d'amorces pour l'amplification spécifique de trois sites polymorphes sur le gène humain de la fructose bisphosphate aldolase B. Les trois sites polymorphes sont respectivement A150P, A175D et N335K. La paire d'amorces comprend une amorce amont consensus et une amorce aval de typage, et l'amorce aval de typage comprend une amorce spécifique de type sauvage et une amorce spécifique mutante. Le kit permet de détecter rapidement et efficacement des génotypes des trois sites polymorphes du gène humain de la fructose bisphosphate aldolase B, et forme un kit rapide, simple et pratique, économique et efficace pour cribler le gène humain de la fructose bisphosphate aldolase B.
PCT/CN2016/107577 2016-08-31 2016-11-29 Procédé et kit de détection du gène humain de la fructose bisphosphate aldolase b Ceased WO2018040320A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610799420.X 2016-08-31
CN201610799420.XA CN106399487B (zh) 2016-08-31 2016-08-31 一种人果糖二磷酸醛缩酶b基因检测方法及试剂盒

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WO2018040320A1 true WO2018040320A1 (fr) 2018-03-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112126685A (zh) * 2020-09-10 2020-12-25 长沙金域医学检验实验室有限公司 一种鉴定aldob基因同时含两个突变位点位置的试剂盒及方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107312834B (zh) * 2017-06-07 2021-02-02 广东华美众源生物科技有限公司 葡萄糖脑苷脂酶基因检测试剂盒及其检测方法
CN107828879B (zh) * 2017-10-19 2021-05-11 广东华美众源生物科技有限公司 一种pkp2基因突变检测试剂盒及其检测方法

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WO2001064957A1 (fr) * 2000-03-02 2001-09-07 Dna Sciences, Inc. Polymorphismes lies aux voies d'acheminement du glucose et de la signalisation de l'insuline
WO2003091454A1 (fr) * 2002-04-26 2003-11-06 Genaissance Pharmaceuticals, Inc. Haplotypes du gene aldob
CN103305618A (zh) * 2013-06-26 2013-09-18 北京迈基诺基因科技有限责任公司 一种遗传代谢疾病基因的筛查方法
CN104232770A (zh) * 2014-09-12 2014-12-24 广东华美众源生物科技有限公司 一种pku基因检测试剂盒
JP2016086678A (ja) * 2014-10-30 2016-05-23 公立大学法人福島県立医科大学 腎がんの悪性度の検査マーカー及び検査方法

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CN103184268A (zh) * 2011-12-28 2013-07-03 协和干细胞基因工程有限公司 检测乙醛脱氢酶2基因多态性的试剂盒及其扩增方法和检测方法
CN104774933A (zh) * 2015-03-30 2015-07-15 深圳市第二人民医院 用于检测人il28b基因多态性的引物、试剂盒及方法

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WO2001064957A1 (fr) * 2000-03-02 2001-09-07 Dna Sciences, Inc. Polymorphismes lies aux voies d'acheminement du glucose et de la signalisation de l'insuline
WO2003091454A1 (fr) * 2002-04-26 2003-11-06 Genaissance Pharmaceuticals, Inc. Haplotypes du gene aldob
CN103305618A (zh) * 2013-06-26 2013-09-18 北京迈基诺基因科技有限责任公司 一种遗传代谢疾病基因的筛查方法
CN104232770A (zh) * 2014-09-12 2014-12-24 广东华美众源生物科技有限公司 一种pku基因检测试剂盒
JP2016086678A (ja) * 2014-10-30 2016-05-23 公立大学法人福島県立医科大学 腎がんの悪性度の検査マーカー及び検査方法

Non-Patent Citations (2)

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Title
DAVIT-SPRAUL ANNE: "Hereditary fructose intolerance: Frequency and spectrum mutations of the aldolase B gene in a large patients cohort from France - Identification of eight new mutations", MOLECULAR GENETICS AND METABOLISM, vol. 94, 31 August 2008 (2008-08-31), pages 443 - 447, XP022833454, DOI: doi:10.1016/j.ymgme.2008.05.003 *
RENÉ SANTER: "The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe", MATERIALS AND METHODS, MUTATION ANALYSIS, vol. 25, no. 6, 30 June 2005 (2005-06-30), pages 1 - 10, XP055603524 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112126685A (zh) * 2020-09-10 2020-12-25 长沙金域医学检验实验室有限公司 一种鉴定aldob基因同时含两个突变位点位置的试剂盒及方法

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CN106399487A (zh) 2017-02-15

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