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WO2014134790A1 - Kit de réactifs pcr pour le diagnostic de la neurofibromatose - Google Patents

Kit de réactifs pcr pour le diagnostic de la neurofibromatose Download PDF

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Publication number
WO2014134790A1
WO2014134790A1 PCT/CN2013/072211 CN2013072211W WO2014134790A1 WO 2014134790 A1 WO2014134790 A1 WO 2014134790A1 CN 2013072211 W CN2013072211 W CN 2013072211W WO 2014134790 A1 WO2014134790 A1 WO 2014134790A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
primer
primer pair
pcr
pair
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2013/072211
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English (en)
Chinese (zh)
Inventor
贺雄雷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BESTHEAL MEDICAL TREATMENT Co Ltd
GENEHEAL BIOTECHNOLOGY Co Ltd
Original Assignee
BESTHEAL MEDICAL TREATMENT Co Ltd
GENEHEAL BIOTECHNOLOGY Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BESTHEAL MEDICAL TREATMENT Co Ltd, GENEHEAL BIOTECHNOLOGY Co Ltd filed Critical BESTHEAL MEDICAL TREATMENT Co Ltd
Priority to PCT/CN2013/072211 priority Critical patent/WO2014134790A1/fr
Publication of WO2014134790A1 publication Critical patent/WO2014134790A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

  • Neurofibromatosis is a benign peripheral neuropathy [Raphael Rubin, David S. Strayer (2008 Baltimore). Rubin's Pathology: Clinicopathologic Foundation of Medicine (5 ed.). Wolters Kluwer Health: Lippincot Williams & Wilkins. pp. 201-3. ISBN 978-0-7817-9516-6]. Its histologically originated from the connective tissue of the periendron of the peripheral nerve sheath, causing damage to the nervous system and other tissues through growth and compression, often involving organs originating from the ectoderm, such as the nervous system, eyes and skin, etc.
  • NF is an autosomal dominant genetic disease, that is, as long as one copy of the genetic gene is defective, it is enough to cause the disease. It also shows that if one of the parents has the disease, the incidence of the child is 50%. About 50% of the cases in clinical cases are caused by new mutations in the individual's own genes. 50% is family inheritance.
  • NF2 neurofibromatosis type 1
  • NF2 neurofibromatosis type 2
  • Neurofibromatosis type 2 NF2
  • the NF1 pathogenic gene is called the neurofibromatosis protein 1 gene (Neurofibromin 1, NF1 gene) located on the autosome 17qll.2.
  • the gene encodes a neurofibromin protein that negatively regulates the RAS signaling pathway by interacting with the GTPase [John Barone (2008). USMLE Step 1 Lecture Notes: Pathology. KAPLAN Inc. pp. 57.].
  • NF2 neurofibromin 2
  • gene also known as Mer/[gene] located on autosome 22ql2.
  • the gene is also a tumor suppressor gene, but its specific mechanism of action is still unclear.
  • This gene mutation causes NF2 type [Rouleau GA, Merel P, Lutchman M, Sanson M, Zucman J, Marineau C, Hoang-Xuan K, Demczuk S, Desmaze C, Plougastel B (1993). "Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2". Nature 363 (6429): 515-21.
  • the clinical diagnostic criteria for NF1 and NF2 are as follows:
  • Obvious skeletal lesions such as sphenoid dysplasia, long tubular cortical bone, accompanied by pseudoarticular formation;
  • NF2 nerve growth factor 2
  • NF2 nerve growth factor 3
  • the patient has 2 of the following lesions: neurofibromatosis, meningioma, glioma, Schwann cell tumor, adolescent lens posterior capsule opacity.
  • a neurofibroma diagnostic kit comprising at least one of the following primers:
  • PCR primer pair for NF1 gene point mutation/small fragment insertion deletion detection is selected from primer pair 1 ⁇ 58;
  • the PCR primer pair for the NF1 gene LOH detection is selected from the primer pair 75 ⁇ 100; the PCR primer pair for the NF2 gene LOH detection is selected from the primer pair 101 ⁇ 113;
  • the primer pairs 1 to 113 are as follows:
  • the kit contains at least 75 to 113 pairs of primer pairs.
  • the kit contains primer pairs 1 to 113.
  • a method for diagnosing a neurofibromatosis includes the following steps: 1) extracting the whole genome of the test subject and performing PCR amplification using any of the above primer pairs 1 to 113;
  • ENST00000338641 Perform an alignment or compare the results of sequencing with their parental samples to determine whether the subject has a genetic mutation that causes neurofibromatosis.
  • the kit of the invention has the following advantages in detecting NF: 1) short time-consuming, only about 5 days in total time consumption; 2) complete coverage, including major possible defect forms of NF1 and type 2 pathogenic genes; 3) diagnosis Flexible and accurate, it can comprehensively detect two different defects of genes, can also detect NF1 or NF2 genes or even some gene regions, and can also detect different types of gene defects; 4) Low cost, compared with existing NF production The cost of pre-screening is reduced by approximately 90%.
  • the longest transcript ENST00000338641 is selected as the target region, and the transcript has a total of 16 exons encoding 595 amino acids.
  • the present invention takes a total of 74 exons of two genes, and designs PCR amplification primers in the range of 200 bp upstream to 200 bp downstream of each exon.
  • the primers are required to be between 18-25 bp in length; the amplified product completely covers each exon, and at least 20 bp sequence is left upstream and downstream of the exon to ensure the accuracy of the Sanger sequencing read sequence; the primer Tm value is 55-61.
  • the target fragment was PCR amplified by central template method, using 20 ⁇ l system as standard system: 2 ⁇ 1 DNA template, 12.5 pmol primer, 0.1 mM dNTP, lO PCR buffer, 1.5 ⁇ M MgCl 2 , l.OuTaq enzyme.
  • the recovered fragments were sent to Huada Gene Company for sequencing by the Sanger method.
  • the present invention uses the data of 1000 Genome to select 32 and 19 single-nucleotide polymorphism SNPs in the Asian population on the NF1 gene and the NF2 gene, respectively, at the target SNP site.
  • PCR primers were designed from 300 bp upstream to 300 bp downstream. The primers are required to be between 18-25 bp in length; the ends of the amplified product are at least 20 bp from the target SNP site to ensure accurate Sanger sequencing reads; primers have Tm values between 55 and 61; all primer sequences are associated with human whole genome. The sequences were aligned to ensure primer specificity and avoid false amplification; a total of 39 pairs of primers were designed (NF1: 26 pairs, NF2: 13 pairs). Primer synthesis is carried out using conventional synthetic methods. Primer sequences and target SNP site information are shown in Tables 3 and 4.
  • the genomic template DNA preparation method employs a conventional human genomic DNA template preparation method.
  • LOH test a total of 3 genomic DNA samples of the test subject, the tester's father, and the test subject's mother are prepared.
  • the target fragment was PCR amplified by central template method, and the standard system was 20 ⁇ l: 2 ⁇ 1 (50 ⁇ ) DNA template, 12.5pmol primer, 0.1mMdNTP, lOxPCR buffer, 1.5uMMgC12, l.OuTaq enzyme.
  • FIG. 2 is an electropherogram showing the PCR amplification products of SNP rs2952999 and rs2953000.
  • the first, second, and third lanes are mother, father, and testee (son).
  • Figure 3 is a fragmentation peak of the PCR product. As shown in the figure: The tested father and mother are healthy individuals.

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

Abstract

L'invention concerne un kit de réactifs pour le diagnostic de la neurofibromatose. Le kit de réactifs comprend au moins une paire des amorces suivantes : une paire d'amorces PCR pour identifier une mutation ponctuelle/une insertion et délétion de petits fragments du gène NF1 ; une paire d'amorces PCR pour identifier une mutation ponctuelle/insertion et délétion de petits fragments du gène NF2 ; une paire d'amorces PCR pour identifier une LOH du gène NF1 ; et une paire d'amorces PCR pour identifier une LOH du gène NF2.
PCT/CN2013/072211 2013-03-06 2013-03-06 Kit de réactifs pcr pour le diagnostic de la neurofibromatose Ceased WO2014134790A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/072211 WO2014134790A1 (fr) 2013-03-06 2013-03-06 Kit de réactifs pcr pour le diagnostic de la neurofibromatose

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/072211 WO2014134790A1 (fr) 2013-03-06 2013-03-06 Kit de réactifs pcr pour le diagnostic de la neurofibromatose

Publications (1)

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WO2014134790A1 true WO2014134790A1 (fr) 2014-09-12

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Family Applications (1)

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PCT/CN2013/072211 Ceased WO2014134790A1 (fr) 2013-03-06 2013-03-06 Kit de réactifs pcr pour le diagnostic de la neurofibromatose

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018186680A1 (fr) * 2017-04-05 2018-10-11 울산대학교 산학협력단 Composition comprenant un ensemble d'amorces pour pcr longue à base d'adn génomique pour le diagnostic de la neurofibromatose
EP3591076A1 (fr) * 2018-07-02 2020-01-08 Centrum Badan DNA Spolka z Ograniczona Odpowiedzialnoscia Amorces pour la détection d'une mutation germinative prédisposant à la neurofibromatose de type 1 et leurs utilisations
CN113151437A (zh) * 2021-02-01 2021-07-23 浙江理工大学 Nf2基因在制备诊断颅骨发育异常产品中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003074740A1 (fr) * 2002-03-01 2003-09-12 Ravgen, Inc. Analyse rapide de variations dans un genome
WO2007147079A2 (fr) * 2006-06-14 2007-12-21 Living Microsystems, Inc. analyse de celluleS rareS avec recours au fractionnement d'échantillons et à des marqueurs d'adn
WO2012008839A2 (fr) * 2010-07-16 2012-01-19 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Procédé d'analyse d'un échantillon de sang d'un sujet dans le but de détecter la présence d'un marqueur d'une maladie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003074740A1 (fr) * 2002-03-01 2003-09-12 Ravgen, Inc. Analyse rapide de variations dans un genome
WO2007147079A2 (fr) * 2006-06-14 2007-12-21 Living Microsystems, Inc. analyse de celluleS rareS avec recours au fractionnement d'échantillons et à des marqueurs d'adn
WO2012008839A2 (fr) * 2010-07-16 2012-01-19 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Procédé d'analyse d'un échantillon de sang d'un sujet dans le but de détecter la présence d'un marqueur d'une maladie

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018186680A1 (fr) * 2017-04-05 2018-10-11 울산대학교 산학협력단 Composition comprenant un ensemble d'amorces pour pcr longue à base d'adn génomique pour le diagnostic de la neurofibromatose
EP3591076A1 (fr) * 2018-07-02 2020-01-08 Centrum Badan DNA Spolka z Ograniczona Odpowiedzialnoscia Amorces pour la détection d'une mutation germinative prédisposant à la neurofibromatose de type 1 et leurs utilisations
CN113151437A (zh) * 2021-02-01 2021-07-23 浙江理工大学 Nf2基因在制备诊断颅骨发育异常产品中的应用

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