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WO2024000778A1 - Produit de détection de séquence de bases pour le diagnostic in-vitro de la malformation artérioveineuse cérébrale - Google Patents

Produit de détection de séquence de bases pour le diagnostic in-vitro de la malformation artérioveineuse cérébrale Download PDF

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Publication number
WO2024000778A1
WO2024000778A1 PCT/CN2022/115220 CN2022115220W WO2024000778A1 WO 2024000778 A1 WO2024000778 A1 WO 2024000778A1 CN 2022115220 W CN2022115220 W CN 2022115220W WO 2024000778 A1 WO2024000778 A1 WO 2024000778A1
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seq
sequence
probe
vitro diagnosis
kras
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Chinese (zh)
Inventor
赵森
王坤
李翛然
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Suzhou Acemapai Umed Co Ltd
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Suzhou Acemapai Umed Co Ltd
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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
    • 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
    • C12Q1/6858—Allele-specific amplification
    • 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 in vitro detection technology, and specifically relates to a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations.
  • bAVM Brain arteriovenous malformation
  • ctDNA is circulating tumor cell DNA, which refers to the DNA fragments secreted by a small number of necrotic tumor cells that may exist in the blood. It is often used for the early diagnosis of some cancers. Previous studies have found that bAVM lesions also secrete ctDNA, but its plasma concentration is much lower than that of other malignant tumors. Therefore, there is an urgent need for a highly sensitive product that can detect ctDNA in vitro to achieve early warning of cerebral arteriovenous malformations.
  • the present invention proposes a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations, which overcomes the shortcomings and defects mentioned in the background technology.
  • the invention points of the present invention are to provide a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations.
  • the product includes a primer-probe composition for detecting base sequences related to cerebral arteriovenous malformations.
  • the nucleotide sequence of the primer includes the upstream primer sequence shown in SEQ ID NO: 1 and the downstream primer sequence shown in SEQ ID NO: 2; the nucleotide sequence of the probe includes the nucleotide sequence that specifically binds to wild-type KRAS.
  • the primer nucleotide sequence is specifically:
  • F primer 5′-gactgaatataaacttgtggta-3′;
  • R primer 5′-gtccacaaaatgattctga-3′.
  • the probe nucleotide sequence is specifically:
  • G12A FAM-5′-actcttgcctacgccagcag-3′-BHQ1.
  • the probe sequence SEQ ID NO: 3 specifically binds to wild-type KRAS
  • SEQ ID NO: 4 specifically binds to G12A variant KRAS
  • SEQ ID NO: 5 specifically binds to G12V variant KRAS
  • SEQ ID NO: 6 Binds specifically to G12D variant KRAS.
  • the sequence of the primer probe composition is: primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID Combination of NO: 3, SEQ ID NO: 4; combination of primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 5; primer sequence SEQ ID NO: 1 , SEQ ID NO: 2 and the combination of probe sequence SEQ ID NO: 3, SEQ ID NO: 6.
  • the wild-type KRAS sequence is the nucleotide sequence shown in SEQ ID NO: 7 ;
  • the three mutant KRAS sequences are the nucleotide sequences shown in SEQ ID NO: 8-SEQ ID NO: 10.
  • SEQ ID NO: 8 (g is mutated to c at position 35, i.e. G12A)
  • SEQ ID NO: 9 (g mutates to t at position 35, i.e. G12V)
  • SEQ ID NO: 10 (g at position 35 mutates to a, i.e. G12D)
  • the above-mentioned base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations is a kit.
  • the above-mentioned base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations also includes a PCR reaction solution, a positive quality control product, a negative quality control product and a blank control.
  • the PCR reaction solution includes AccuStart II Taq DNA polymerase, MgCl 2 , dATP, dCTP, dGTP and dTTP components.
  • dATP is deoxyadenosine triphosphate
  • dCTP is deoxycytidine triphosphate
  • dGTP is deoxyguanosine triphosphate
  • dTTP is deoxythymidine triphosphate.
  • the reaction system for PCR amplification is: 5 ⁇ PerfeCTa Multiplex qPCR ToughMix 5 ⁇ l, Fluorescein sodium salt 2.5 ⁇ l, 1 ⁇ l of 20 ⁇ KRAS probe, 2 ⁇ l of sample DNA to be tested, and add ddH 2 O to 25 ⁇ l.
  • the reaction program of PCR amplification is: 95°C, 3min, 1 cycle; 95°C, 15sec, 60°C , 30sec, cycle 45 times.
  • the PCR amplification method of the present invention adopts the ddPCR method, which specifically involves dividing the reaction system containing nucleic acid molecules into tens of thousands of nanoliter droplets. After PCR amplification, each droplet is detected one by one. Droplets with fluorescent signals are interpreted as 1, and droplets without fluorescent signals are interpreted as 0. According to the Poisson distribution principle and the number and proportion of positive droplets, That is, the proportion of samples carrying mutant KRAS and wild-type KRAS can be obtained.
  • the occurrence of cerebral arteriovenous malformations can be determined through the detection of brain arteriovenous malformation-related base sequences detected by the above-mentioned base sequence detection products for in vitro diagnosis of cerebral arteriovenous malformations, and then through the detection of specific gene mutations.
  • the present invention has the following advantages:
  • the invention provides a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations, which has high detection sensitivity and a significantly higher accuracy than before.
  • a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations, which has high detection sensitivity and a significantly higher accuracy than before.
  • Figure 1 shows the detection results of specific gene mutations in brain arteriovenous malformation-related base sequences of patients with cerebral arteriovenous malformations through highly sensitive ddPCR provided by one embodiment of the present invention
  • Figures 1A and 1B show the use of primers The combination of sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 4 detects the results of plasma and tissue KRAS G12A mutation
  • Figure 1C and Figure 1D show the use of primer sequence SEQ ID
  • Figure 1E and Figure 1F show the results of using the primer sequence SEQ ID NO: 1 , SEQ ID NO: 2 and the combination of probe sequences SEQ ID NO: 3, SEQ ID NO: 6 to detect the results of plasma and tissue KRAS G12D mutations.
  • a base sequence detection product for in vitro diagnosis of cerebral arteriovenous malformations including a primer probe composition for detecting brain arteriovenous malformation-related base sequences.
  • the nucleotide sequence of the primers includes as shown in SEQ ID NO: 1
  • the nucleotide sequence of the probe includes the sequence shown in SEQ ID NO: 3 that specifically binds to wild-type KRAS and the sequence that specifically binds to mutant KRAS Combined sequences shown in any one of SEQ ID NO: 4-SEQ ID NO: 6.
  • the primer nucleotide sequence is specifically:
  • F primer 5′-gactgaatataaacttgtggta-3′;
  • R primer 5′-gtccacaaaatgattctga-3′.
  • the probe nucleotide sequence is specifically:
  • G12A FAM-5′-actcttgcctacgccagcag-3′-BHQ1.
  • the probe sequence SEQ ID NO: 3 specifically binds to wild-type KRAS
  • SEQ ID NO: 4 specifically binds to G12A variant KRAS
  • SEQ ID NO: 5 specifically binds to G12V variant KRAS
  • SEQ ID NO: 6 Binds specifically to G12D variant KRAS.
  • the sequence of the primer-probe composition is: the combination of the primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and the probe sequence SEQ ID NO: 3, SEQ ID NO: 4; the primer sequence SEQ ID NO: 1, SEQ ID The combination of NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 5; the combination of primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 6 combination.
  • the wild-type KRAS sequence is the nucleotide sequence shown in SEQ ID NO: 7; the three mutant KRAS sequences are shown in SEQ ID NO: 8-SEQ ID NO: 10 nucleotide sequence.
  • SEQ ID NO: 8 (g is mutated to c at position 35, i.e. G12A)
  • SEQ ID NO: 9 (g mutates to t at position 35, i.e. G12V)
  • SEQ ID NO: 10 (g at position 35 mutates to a, i.e. G12D)
  • the wild-type KRAS sequence SEQ ID NO: 7 is specifically recognized by the probe sequence SEQ ID NO: 3; the mutant KRAS sequence SEQ ID NO: 8 is specifically recognized by the probe sequence SEQ ID NO: 4; the mutant KRAS sequence SEQ ID NO: 8 is specifically recognized by the probe sequence SEQ ID NO: 4.
  • the KRAS sequence SEQ ID NO: 9 is specifically recognized by the probe sequence SEQ ID NO: 5; the mutant KRAS sequence SEQ ID NO: 10 is specifically recognized by the probe sequence SEQ ID NO: 6.
  • the base sequence detection product used for in vitro diagnosis of cerebral arteriovenous malformations can be a kit.
  • the kit also includes PCR reaction solution, positive quality control materials, negative quality control materials and blank control.
  • the PCR reaction solution includes AccuStart II Taq DNA polymerase, MgCl 2 , dATP, dCTP, dGTP and dTTP components.
  • dATP is deoxyadenosine triphosphate
  • dCTP is deoxycytidine triphosphate
  • dGTP is deoxyguanosine triphosphate
  • dTTP is deoxythymidine triphosphate.
  • the reaction system for PCR amplification is: 5 ⁇ PerfeCTa Multiplex qPCR ToughMix 5 ⁇ l, Fluorescein sodium salt 2.5 ⁇ l, 20 ⁇ KRAS probe 1 ⁇ l, sample DNA to be tested 2 ⁇ l, and supplemented with ddH 2 O to 25 ⁇ l.
  • the reaction procedures for PCR amplification are: 95°C, 3min, one cycle; 95°C, 15sec, 60°C, 30sec, 45 cycles.
  • This method uses CWBIO TM cfDNA storage tube to collect the peripheral blood of bAVM patients. Circulating Nucleic Acid Kit 55114 kit extracts cfDNA from peripheral blood.
  • the specific method is as follows:
  • step 4 Add 3.2 ml of the Buffer ACL* solution prepared in step 1 (containing 1.0 ⁇ g transfer RNA), tighten the cap and vortex for 30 seconds to mix thoroughly, and immediately proceed to step 5 to start the lysis reaction.
  • step 8 Carefully add all the ACB mixture obtained in step 8 into the extender and turn on the vacuum pump. When all the lysate has passed through the column, turn off the vacuum pump and return the pressure to 0 mbar. Remove the extender carefully.
  • BUFFER AVE needs to be returned to room temperature in advance for use.
  • the dead volume recovered is 5 ⁇ l.
  • This method was used to conduct parallel detection on the peripheral blood and arteriovenous malformation tissues of 13 patients with cerebral arteriovenous malformations, in which:
  • primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 4 detects KRAS G12A mutation
  • primer sequence SEQ ID NO: 1, SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 5 detects KRAS G12V mutation
  • primer sequence SEQ ID NO: 1 SEQ ID NO: 2 and probe sequence SEQ ID NO: 3, SEQ ID NO: 6 detects KRAS G12D mutation
  • the KRAS G12D mutation was detected in the arteriovenous malformation tissue of 26 patients, of which 24 patients had positive peripheral blood in vitro diagnostic results; the KRAS G12V mutation was detected in the arteriovenous malformation tissue of 11 patients.
  • the peripheral blood in vitro diagnostic results of 10 patients were positive;
  • KRAS G12A mutation was detected in the arteriovenous malformation tissue of 7 patients, and the peripheral blood in vitro diagnostic results were all positive; that is to say, a total of 44 patients had arteriovenous malformations.
  • KRAS mutations were detected in tissue samples, and KRAS mutations were detected in peripheral blood in vitro testing samples (plasma) of 42 patients.
  • a detection rate of 95.4% (42/44) can be achieved.
  • the 100% detection accuracy rate greatly reduces the difficulty and harm of detection while maintaining a certain detection rate. It can be widely used as a preliminary screening and early warning detection method for arteriovenous malformations.

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Abstract

La présente invention concerne un produit de détection de séquence de bases pour le diagnostic in vitro de la malformation artérioveineuse cérébrale, comprenant une composition amorce-sonde pour détecter des séquences de bases liées à la malformation artérioveineuse cérébrale. Les séquences nucléotidiques d'une amorce sont représentées dans SEQ ID NO : 1 et SEQ ID NO : 2 ; et les séquences nucléotidiques d'une sonde comprennent une séquence, comme représentée dans SEQ ID NO : 3, se liant particulièrement au KRAS de type sauvage, et une séquence, comme représentée dans l'une quelconque des séquences SEQ ID NO : 4 à SEQ ID NO : 6, se liant particulièrement au KRAS mutant. La présente invention présente les avantages suivants : le produit de détection de séquence de bases pour le diagnostic in vitro de la malformation artérioveineuse cérébrale fourni par la présente invention présente une sensibilité de détection élevée, et la précision est grandement améliorée par comparaison avec l'état de la technique. Grâce à la détection in vitro d'une quantité infime de séquence de bases liées à la malformation artérioveineuse cérébrale, en plus de la détection des mutations génétiques, l'apparition de la malformation artérioveineuse cérébrale peut être clairement prévenue/diagnostiquée in vitro.
PCT/CN2022/115220 2022-06-29 2022-08-26 Produit de détection de séquence de bases pour le diagnostic in-vitro de la malformation artérioveineuse cérébrale Ceased WO2024000778A1 (fr)

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CN202210756411.8 2022-06-29
CN202210756411.8A CN117344002A (zh) 2022-06-29 2022-06-29 一种用于体外诊断脑动静脉畸形的碱基序列检测产品

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018126192A1 (fr) * 2016-12-30 2018-07-05 Children's Medical Center Corporation Map2k1 (mek1) utilisée comme cible thérapeutique pour des malformations artérioveineuses et des troubles associés
CN111568898A (zh) * 2020-04-23 2020-08-25 首都医科大学附属北京天坛医院 洛伐他汀或其药学上可接受的盐用于制备治疗脑动静脉畸形药物的应用
WO2021163464A1 (fr) * 2020-02-12 2021-08-19 The Regents Of The University Of Michigan Traitement et prévention d'une malformation capillaire-malformation artérioveineuse
CN113981082A (zh) * 2021-10-29 2022-01-28 上海交通大学医学院附属第九人民医院 动静脉畸形及相关病症的无创检测方法及装置
CN114196757A (zh) * 2021-12-28 2022-03-18 普瑞斯新(上海)生物医疗科技有限公司 Kras基因突变多重检测引物探针及其试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018126192A1 (fr) * 2016-12-30 2018-07-05 Children's Medical Center Corporation Map2k1 (mek1) utilisée comme cible thérapeutique pour des malformations artérioveineuses et des troubles associés
WO2021163464A1 (fr) * 2020-02-12 2021-08-19 The Regents Of The University Of Michigan Traitement et prévention d'une malformation capillaire-malformation artérioveineuse
CN111568898A (zh) * 2020-04-23 2020-08-25 首都医科大学附属北京天坛医院 洛伐他汀或其药学上可接受的盐用于制备治疗脑动静脉畸形药物的应用
CN113981082A (zh) * 2021-10-29 2022-01-28 上海交通大学医学院附属第九人民医院 动静脉畸形及相关病症的无创检测方法及装置
CN114196757A (zh) * 2021-12-28 2022-03-18 普瑞斯新(上海)生物医疗科技有限公司 Kras基因突变多重检测引物探针及其试剂盒

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