WO2021041765A2 - Trousse et procédés pour détecter une fusion de gène ntrk - Google Patents

Trousse et procédés pour détecter une fusion de gène ntrk Download PDF

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Publication number
WO2021041765A2
WO2021041765A2 PCT/US2020/048333 US2020048333W WO2021041765A2 WO 2021041765 A2 WO2021041765 A2 WO 2021041765A2 US 2020048333 W US2020048333 W US 2020048333W WO 2021041765 A2 WO2021041765 A2 WO 2021041765A2
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WO
WIPO (PCT)
Prior art keywords
fusion
ntrk
primer
specific
universal
Prior art date
Application number
PCT/US2020/048333
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English (en)
Other versions
WO2021041765A3 (fr
Inventor
An Hsu
Pei-Yi Lin
Datsen George WEI
Shu-Jen Chen
Hua-Chien Chen
Original Assignee
An Hsu
Lin Pei Yi
Wei Datsen George
Chen Shu Jen
Chen Hua Chien
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 An Hsu, Lin Pei Yi, Wei Datsen George, Chen Shu Jen, Chen Hua Chien filed Critical An Hsu
Publication of WO2021041765A2 publication Critical patent/WO2021041765A2/fr
Publication of WO2021041765A3 publication Critical patent/WO2021041765A3/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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the disclosed method utilizes a set of specifically designed probes, each of which can capture the amplified product including one particular NTRK fusion sequence, to detect all possible NTRK gene fusions. Since the NTRK gene fusion types are numerous, but the exact one or more NTRK fusions in the biological sample are unknown before the detection step, it is important to obtain detectable amounts of the amplified products for all types of NTRK fusions so that any NTRK fusion type can be detected subsequently.
  • Gene fusions may be detected by identifying a fusion junction in a DNA or in an RNA transcript of that DNA.
  • a “fusion type” refers to a unique fusion present in an RNA transcript. In other words, it is considered the same fusion type when the fusions between two specific genes occur at different sites within the same intronic region.
  • a fusion between exon 3 of gene A and exon 5 of gene B may have a DNA fusion region containing a small portion of the intron between exons 3 and 4 of gene A and a large portion of the intron between exons 4 and 5 of gene B.
  • RNA is prepared from a biological sample.
  • the biological sample may be any sample obtained from an animal and a human subject. Examples of the biological samples include a formalin-fixed paraffin-embedded (FFPE) tissue section, blood, plasma, or cells.
  • FFPE formalin-fixed paraffin-embedded
  • the biological sample originates from a cancer patient. In some embodiments, the biological sample originates from a solid tumor, soft tissue sarcoma, or a hematological cancer.
  • the cDNA is amplified with a DNA polymerase and at least two pairs of NTRK fusion-specific primers to obtain an amplified product for probe detection.
  • the amplification may be conducted using a multiplex PCR kit (Cat No: 206143, Qiagen) which includes a DNA polymerase.
  • the NTRK fusion-specific primers may be provided as a regent before use. In some embodiments, all the NTRK fusion-specific primer pairs are pooled together to form a single pooled reagent.
  • step (c) the cDNA is amplified first with the at least two NTRK fusion-specific primer pairs and subsequently with a universal primer pair to obtain the amplified product.
  • the universal primer pair is utilized in the disclosed method, the NTRK fusion-specific forward primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal forward primer in the pair of universal primers, and the NTRK fusion-specific reverse primer in each of the NTRK fusion-specific primer pairs further encompasses the nucleotide sequence of the universal reverse primer in the universal primer pair.
  • Detection of the probe-bound product may be accomplished by detecting the NTRK fusion- specific primers, the universal primers, or the probe in said product.
  • the primers or probes are usually modified to be detectable. They may be modified to have fluorescence or chemiluminescence activity or become chromogenic or colorimetric by being connected directly or indirectly to a detectable molecule.
  • one or both primers in the primer pair are connected to biotin or other compounds capable of binding to a streptavi din-conjugated detectable molecule.
  • each of the four pools of the first amplified products was diluted 100 folds in the final reaction mix and amplified on VeritiTM 96-Well Thermal Cycler (Thermo Fisher Scientific) for 25 thermal cycles using Platinum SuperFi II PCR Master Mix (Cat No: 12368010, Invitrogen) according to the manufacturer’s instructions, yielding a second amplified product in 10 pL.
  • VeritiTM 96-Well Thermal Cycler Thermo Fisher Scientific
  • Platinum SuperFi II PCR Master Mix Cat No: 12368010, Invitrogen

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

Abstract

L'invention concerne une trousse de détection de la fusion de gène de la tyrosine kinase (NTRK) du récepteur de la neurotrophine. La trousse comprend un ensemble de paires d'amorces spécifiques à la fusion NTRK et un ensemble de sondes spécifiques à la fusion NTRK. L'invention concerne également un procédé de détection de la fusion de gène NTRK, consistant à générer un cADN cible amplifié afin qu'il s'hybride avec l'ensemble de sondes spécifiques à la fusion NTRK en une seule réaction, et à détecter le produit lié à une sonde pour identifier toutes les fusions de gène NTRK possibles dans un échantillon biologique.
PCT/US2020/048333 2019-08-28 2020-08-28 Trousse et procédés pour détecter une fusion de gène ntrk WO2021041765A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962893148P 2019-08-28 2019-08-28
US62/893,148 2019-08-28

Publications (2)

Publication Number Publication Date
WO2021041765A2 true WO2021041765A2 (fr) 2021-03-04
WO2021041765A3 WO2021041765A3 (fr) 2021-06-03

Family

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Family Applications (1)

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PCT/US2020/048333 WO2021041765A2 (fr) 2019-08-28 2020-08-28 Trousse et procédés pour détecter une fusion de gène ntrk

Country Status (2)

Country Link
TW (1) TW202122590A (fr)
WO (1) WO2021041765A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561467A (zh) * 2022-02-18 2022-05-31 江苏省中医院 一种met融合基因的检测方法、试剂盒以及探针库

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070218071A1 (en) * 2003-09-15 2007-09-20 Morris David W Novel therapeutic targets in cancer
JP6534930B2 (ja) * 2013-07-26 2019-06-26 公益財団法人がん研究会 Ｎｔｒｋ３融合体の検出法
EP3283645B1 (fr) * 2015-04-17 2019-03-27 Roche Diagnostics GmbH Pcr multiplex pour détecter les fusions de gènes
AU2016362875B2 (en) * 2015-12-01 2022-12-08 LGC Clinical Diagnostics, Inc. Multiplex cellular reference materials

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561467A (zh) * 2022-02-18 2022-05-31 江苏省中医院 一种met融合基因的检测方法、试剂盒以及探针库
CN114561467B (zh) * 2022-02-18 2023-06-09 江苏省中医院 一种met融合基因的检测方法、试剂盒以及探针库

Also Published As

Publication number Publication date
WO2021041765A3 (fr) 2021-06-03
TW202122590A (zh) 2021-06-16

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