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WO2018014520A1 - Amorces, sondes et kit destinés à être utilisés pour détecter les mutations du gène c-kit - Google Patents

Amorces, sondes et kit destinés à être utilisés pour détecter les mutations du gène c-kit Download PDF

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Publication number
WO2018014520A1
WO2018014520A1 PCT/CN2017/000445 CN2017000445W WO2018014520A1 WO 2018014520 A1 WO2018014520 A1 WO 2018014520A1 CN 2017000445 W CN2017000445 W CN 2017000445W WO 2018014520 A1 WO2018014520 A1 WO 2018014520A1
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WIPO (PCT)
Prior art keywords
kit
seq
mutation
gene
detection
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Ceased
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PCT/CN2017/000445
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English (en)
Chinese (zh)
Inventor
莫敏俐
丁凤
陈钊
李晖
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Jiaxing Accb Diagnostics Ltd
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Jiaxing Accb Diagnostics Ltd
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Priority claimed from CN201610562811.XA external-priority patent/CN107630090A/zh
Priority claimed from CN201610562719.3A external-priority patent/CN107630089A/zh
Priority claimed from CN201610654357.0A external-priority patent/CN107723360A/zh
Priority claimed from CN201610654356.6A external-priority patent/CN107739757A/zh
Application filed by Jiaxing Accb Diagnostics Ltd filed Critical Jiaxing Accb Diagnostics Ltd
Publication of WO2018014520A1 publication Critical patent/WO2018014520A1/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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a primer, a probe and a related kit for detecting a mutation of a C-KIT gene.
  • C-KIT is a protooncogene located on chromosome 4q11-12 with a total length of about 80 kb.
  • the C-KIT-encoded C-KIT protein is a member of the type III receptor tyrosine kinase family, a transmembrane protein with a molecular weight of approximately 145 kD, comprising an intracellular tyrosine kinase domain, a transmembrane domain, and ligand binding. Site extracellular region.
  • the ligand binds to it, it activates its own tyrosine protein kinase activity, and activates the downstream signal transduction pathway through a series of reactions, thereby regulating cell growth and proliferation.
  • Gastrointestinal stromal tumors are the most common mesenchymal tumors of the digestive tract. Studies have shown that the C-KIT gene mutation rate in GIST patients is about 90%.
  • the inventors of the present invention found four new mutation sites (mutations 1-4) located in the C-KIT gene associated with gastrointestinal stromal tumors in the routine detection of gastrointestinal stromal tumors (GIST).
  • the inventors of the present invention have developed a rapid, highly sensitive, and easy-to-use detection kit and related detection methods for these mutation sites, which can be used for the prognosis of GIST chemotherapy.
  • the 1689-1718 position was replaced by GGGTCT (base mutation: 1689_1718>GGGTCT, protein mutation: I563_P573>MGL).
  • Primers and probes directed to mutation 1 include the following sequences:
  • Reverse primer AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 2)
  • the 1703-1724 position was replaced by T (base mutation: 1703_1724>T, protein mutation: Y568_Q575>L).
  • Primers and probes for mutation 2 include the following sequences:
  • Primers and probes directed to mutation 3 include the following sequences:
  • 1737-1738 was inserted into 24 bases ACCCCCACCCAACTTCCCTACGAC, and the corresponding amino acid sequence 579-580 was inserted into TPTQLPYD (base mutation: 1737_1738ins24, protein mutation: D579_H580insTPTQLPYD).
  • Primers and probes directed to mutation 4 include the following sequences:
  • Reverse primer AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 11)
  • kits for detecting a mutation of a C-KIT gene comprising the primer shown in SEQ ID No. 1-2 and the probe shown in SEQ ID No. 3, The primer shown in SEQ ID No. 4-5 and the probe shown in SEQ ID No. 6, the primer shown in SEQ ID No. 7-8, and the probe shown in SEQ ID No. 9, and/or SEQ The primer shown in ID No. 10-11 and the probe shown in SEQ ID No. 12.
  • the kit of the present invention may further comprise an internal reference gene and an internal control gene depending on the PCR method employed.
  • Another aspect of the invention provides a method of detecting a C-KIT gene mutation comprising the steps of:
  • the detection sample includes fresh pathological tissue, paraffin-embedded tissue, whole blood and plasma;
  • the present invention employs specific primer and probe technologies to specifically detect C-KIT gene mutations. This method has high sensitivity, high specificity and fast detection speed.
  • Figure 1 is a PCR diagram of a negative sample (wild type).
  • Figure 2 is a PCR diagram of a positive sample for detecting mutation 1.
  • Figure 3 is a PCR diagram of a positive sample for detecting mutation 2.
  • Figure 4 is a PCR diagram of a positive sample for detecting mutation 3.
  • Figure 5 is a PCR diagram of a positive sample for detecting mutation 4.
  • Taq DNA polymerase uses deoxynucleotides (dNTPs) as a substrate, and internal reference gene (Internal Reference, IR) and C-KIT gene mutant genes are in vitro. Amplification. The detection was carried out by fluorescence PCR, and the fluorescence was released by specific probe hydrolysis, and the progress of the PCR reaction was monitored to determine the mutation of the C-KIT gene.
  • dNTPs deoxynucleotides
  • IR Internal Reference, IR
  • C-KIT gene mutant genes are in vitro. Amplification. The detection was carried out by fluorescence PCR, and the fluorescence was released by specific probe hydrolysis, and the progress of the PCR reaction was monitored to determine the mutation of the C-KIT gene.
  • the kit of the present invention is separately provided with an internal reference gene detection system.
  • the internal reference gene is a housekeeping gene that is different from the C-KIT gene to be examined. By detecting the amplification of the internal reference gene (FAM channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating DNA purity, poor concentration, or containing PCR inhibitors and the like to cause PCR detection failure.
  • the kit of the present invention simultaneously sets an internal control (IC) detection system in the C-KIT gene mutation detection system. Both systems react simultaneously in the same PCR tube.
  • the internal control gene is also a housekeeping gene that is different from the gene C-KIT to be detected.
  • the probe that recognizes the C-KIT gene mutation template is modified to a FAM fluorescent group, and the probe that recognizes the internal control gene template is modified to a HEX fluorescent group.
  • HEX channel By detecting the internal control gene amplification (HEX channel), it can be analyzed whether the DNA to be detected can be normally amplified, thereby eliminating the possibility of PCR detection failure caused by missing reagents or samples, and samples containing PCR inhibitors.
  • the FAM and HEX channels in the negative control (NC) should be amplified without a typical S-shaped curve.
  • the typical S-shaped curve is in the exponential phase and straight line.
  • Period and platform period) or no Ct value, FAM, HEX channel detection in positive control products (PC) should have amplification (typical S-shaped curve) and Ct value ⁇ 28; otherwise, the experiment is invalid, repeat experiment .
  • the optimized primers and probes are as follows:
  • Reverse primer AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 2)
  • Reverse primer AAGTCACTGTTATGTGTACCCAA (SEQ ID No: 11)
  • the test sample may be fresh pathological tissue, paraffin embedded tissue, whole blood, plasma, and peritoneal effusion.
  • the following is only an example of a paraffin-embedded tissue sample.
  • the patient did not pass the tyrosine kinase inhibitor (tyrosine kinase) prior to sample collection.
  • the paraffin-embedded tissue samples should not be stored for more than 12 months at room temperature, and the extracted DNA samples should be stored under -20 °C freezing conditions for a period of not more than 6 months.
  • the composition of the kit is shown in Table 1.
  • the kit does not contain nucleic acid extraction components, and DNA extraction of paraffin-embedded tissue samples is performed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Cat. No. 56404) kit.
  • the composition of the test reagent and the mutation site of the C-KIT gene to be detected are shown in Table 2.
  • the IR detection reagent contains only the internal reference gene detection system (FAM channel), and the C-KIT detection reagent also contains the C-KIT gene mutation detection system (FAM channel) and the internal control gene detection system (HEX channel).
  • the specific sequence of the internal reference gene and the internal control gene can be easily determined by a person skilled in the art according to experimental conditions or provided by Beijing Yakambo Biotechnology Co., Ltd.
  • test reagent and positive control PC
  • the test reagent and the positive control product PC were briefly centrifuged and placed on ice.
  • the IR detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3, and the C-KIT detection reagent and Taq DNA polymerase were mixed at a volume ratio of 17:0.3.
  • the IR detection reagent and C-KIT detection reagent were dispensed into the eight tubes at 17.3/well.
  • PC blow and mix, cover the tube cover and centrifuge briefly. Note: Do not use a vortex oscilloscope when mixing; follow up immediately after mixing.
  • the fluorescence PCR instrument detection channel setting needs to select FAM and HEX channels at the same time (the reference dye is set to “None”).
  • the reaction procedure is set as follows (Table 2):
  • Ct value determination First set the baseline of the Stratagene MX3000P fluorescence PCR instrument: select the fluorescence signal when the "Adaptive baseline” setting is selected, and the threshold setting principle is just above the threshold line just above the normal negative control. The highest point of the NC amplification curve (random noise line) is that the NC control curve of the negative control shows "No Ct". The Ct value of each sample detected at each point is read from the software.
  • Figure 1 is a PCR diagram of a sample with a negative test result (wild-type sample)
  • Figure 2-5 is a PCR chart of a sample with a positive test result (a mutant sample containing mutations 1-4).
  • the fluorescent PCR reaction system of the invention can detect C-KIT gene mutations 1-4, and the detection is convenient, rapid and accurate, and can meet the requirement of rapid detection of C-KIT gene mutation.

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

Abstract

L'invention concerne des amorces, des sondes et un kit destinés à être utilisés pour détecter les mutations du gène c-kit. Les amorces sont exposées dans SEQ ID No: 1-2, 4-5, 7-8 ou 10-11, et les sondes dans SEQ ID No: 3, 6, 9, 12. Le kit peut être utilisé pour effectuer une PCR fluorescente en temps réel qui est capable de détecter des mutations du gène c-kit.
PCT/CN2017/000445 2016-07-18 2017-07-14 Amorces, sondes et kit destinés à être utilisés pour détecter les mutations du gène c-kit Ceased WO2018014520A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN201610562811.XA CN107630090A (zh) 2016-07-18 2016-07-18 用于检测c‑kit基因1703‑1724位突变的引物、探针及试剂盒
CN201610562719.3A CN107630089A (zh) 2016-07-18 2016-07-18 用于检测c-kit基因1689-1718位突变的引物、探针及试剂盒
CN201610562811.X 2016-07-18
CN201610562719.3 2016-07-18
CN201610654357.0A CN107723360A (zh) 2016-08-11 2016-08-11 用于检测c‑kit基因1737‑1738位突变的引物、探针及试剂盒
CN201610654356.6 2016-08-11
CN201610654357.0 2016-08-11
CN201610654356.6A CN107739757A (zh) 2016-08-11 2016-08-11 用于检测c‑kit基因1737‑1738位突变的引物、探针及试剂盒

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WO2018014520A1 true WO2018014520A1 (fr) 2018-01-25

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080003603A1 (en) * 2006-04-27 2008-01-03 Holden Joseph A Human melanoma mutation
CN102274230A (zh) * 2003-11-18 2011-12-14 诺瓦提斯公司 Kit突变形式的抑制剂
CN105112500A (zh) * 2015-06-29 2015-12-02 北京雅康博生物科技有限公司 一种用于检测c-kit基因突变的引物、探针及试剂盒
CN105441577A (zh) * 2016-01-13 2016-03-30 武汉海吉力生物科技有限公司 用于检测人类ckit基因7种突变的探针、引物及试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102274230A (zh) * 2003-11-18 2011-12-14 诺瓦提斯公司 Kit突变形式的抑制剂
US20080003603A1 (en) * 2006-04-27 2008-01-03 Holden Joseph A Human melanoma mutation
CN105112500A (zh) * 2015-06-29 2015-12-02 北京雅康博生物科技有限公司 一种用于检测c-kit基因突变的引物、探针及试剂盒
CN105441577A (zh) * 2016-01-13 2016-03-30 武汉海吉力生物科技有限公司 用于检测人类ckit基因7种突变的探针、引物及试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HE, DAN ET AL.: "wei4chang2dao4 jian1zhi4liu2 c-kit ji1yin1 tu1bian4 de yan2jiu1 jin4zhan3", CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, vol. 20, no. 7, 31 July 2013 (2013-07-31), pages 820 - 825 *
SUN, HAO ET AL.: "Polymorphism Analysis of c-kit and PDGFRA Gene Mutations in Gastrointestinal Stromal Tumors", MEDICAL & PHARMACEUTICAL JOURNAL OF CHINESE PEOPLE'S LIBERATION ARMY, vol. 27, no. 12, 31 December 2015 (2015-12-31), pages 36 - 40 *
ZHANG, XIUMIN ET AL.: "wei4chang2dao4 jian1hi4liu2 KIT ji2 PDGFRAjilyinl tulbian4 de jian3ce4 ji2 fen4xil", CHINESE JOURNAL OF CLINICAL ONCOLOGY, vol. 39, no. 10, 31 December 2012 (2012-12-31), pages 660 - 665 *

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