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WO2019242753A1 - Paire d'amorces pour détecter la méthylation d'un gène multiplex du cancer du poumon non à petites cellules, et kit de réactifs - Google Patents

Paire d'amorces pour détecter la méthylation d'un gène multiplex du cancer du poumon non à petites cellules, et kit de réactifs Download PDF

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WO2019242753A1
WO2019242753A1 PCT/CN2019/092334 CN2019092334W WO2019242753A1 WO 2019242753 A1 WO2019242753 A1 WO 2019242753A1 CN 2019092334 W CN2019092334 W CN 2019092334W WO 2019242753 A1 WO2019242753 A1 WO 2019242753A1
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primer
lung cancer
small cell
cell lung
reverse primer
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Chinese (zh)
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陈琦
梁昊原
谷东风
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Shenzhen Shengbizhi Science And Technology Development Co Ltd
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Shenzhen Shengbizhi Science And Technology Development 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
    • C12Q1/6876—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to the technical field of EB virus detection, in particular to a primer pair and a kit for detecting multiple gene methylation of non-small cell lung cancer.
  • DNA methylation is an important epigenetic mechanism, one of the key mechanisms for inactivation of tumor suppressor genes, and may be the only mechanism in some cases. Methylation of non-small cell lung cancer with multiple motions has been confirmed, suggesting that non-small cell lung cancer displays a CpG island methylation phenotype. As an early event of tumorigenesis, detection of abnormal methylation of tumor suppressor gene DNA can make molecular diagnosis before clinical manifestations or imaging evidence appears.
  • ctDNA circulating tumor DNA: DNA fragments from the tumor genome that carry certain characteristics (including mutations, deletions, insertions, rearrangements, abnormal copy number, methylation, etc.) in the human blood circulation system. Its low concentration in blood and high fragmentation make it difficult to extract and reduce the sensitivity and specificity of clinical detection.
  • one methylation-specific PCR reaction can only detect the methylation level of a single gene and cannot accurately predict the possibility of lung cancer.
  • the uncertainty of tumor suppressor genes and the limitation of ctDNA in non-small cell lung cancer have caused the detection of methylation markers in single non-small cell lung cancer to meet clinical needs.
  • the main purpose of the present invention is to provide a primer pair and a kit for detecting multiple gene methylation of non-small cell lung cancer, which aims to solve the existing methylation-specific PCR reaction that can only detect single gene methylation and cannot The problem of accurately predicting the likelihood of lung cancer.
  • the present invention provides a primer pair for detecting multiple gene methylation of non-small cell lung cancer, including 6 sets of primer sequences corresponding to 6 genes as methylation markers of non-small cell lung cancer.
  • the methylation markers are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six sets of primer sequences are specifically represented as follows:
  • CALCA forward primer 5'-CGGAATTTTTTCGATTTATAGC-3 ';
  • CALCA reverse primer 5'-AAAACCCTATAAAAACGACGAC-3 ';
  • DLEC1 forward primer 5'-GATTAAGCGATGACGGGATTC-3 ';
  • DLEC1 reverse primer 5'-ACCCGACTAATAACGAAATTAACG-3 ';
  • HOXA9 forward primer 5'-GGTTAATGGGGGCGCGGGCGTC-3 ';
  • HOXA9 reverse primer 5'-TCATATAACAACTTAATAACACCG-3 ';
  • TBX5 forward primer 5'-GGGACGCGTAAAATTTAGAATC-3 ';
  • TBX5 reverse primer 5'-AACACAAAACCGAAAAACGTC-3 ';
  • PITX2 forward primer 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 ';
  • PITX2 reverse primer 5'-AACACCGAAAAATACAATCCG-3 ';
  • RASSF1a forward primer 5'-GTGTTAACGCGTTGCGTATC-3 ';
  • RASSF1a reverse primer 5'-AACCCCGCGAACTAAAAACGA-3 '.
  • the primer pair used for detecting multiple gene methylation of non-small cell lung cancer further includes a set of primer sequences corresponding to a reference marker ⁇ -ACTIN, which is specifically expressed as follows:
  • ⁇ -ACTIN forward primer 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3 ';
  • ⁇ -ACTIN reverse primer 5'-AAAATATACC CTCCCCCATA CC-3 '.
  • the present invention also provides an application of a primer pair for detecting multiple gene methylation of non-small cell lung cancer in preparing a non-small cell lung cancer detection reagent.
  • the present invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, including the primer pair and a PCR reaction system, wherein:
  • the PCR reaction system includes a 1.8 ⁇ PCR solution, 5 mM MgCl2, 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase;
  • the concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer.
  • the present invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, comprising the primer pair and a PCR reaction system, wherein:
  • the PCR reaction system includes a 1.8 ⁇ PCR solution, 5 mM MgCl2, 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase;
  • the concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer, 10nM ⁇ -ACTIN forward primer 10nM ⁇ -ACTIN reverse primer.
  • the DNA polymerase is Taq Platinum DNA polymerase.
  • the present invention also provides an application of a kit for detecting multiple gene methylation of non-small cell lung cancer in a marker for detecting multiple gene methylation of non-small cell lung cancer.
  • the primer pair and kit for detecting multiple gene methylation of non-small cell lung cancer adopt the above technical scheme, and achieve the following technical effects: multiple non-small cell lung cancers can be detected simultaneously in one PCR reaction
  • the presence of methylation markers in small cell lung cancer improves the accuracy, specificity, and sensitivity of non-small cell lung cancer detection.
  • Detection of multiple gene methylation in non-small cell lung cancer by using the primer pair and the kit according to the present invention can solve a methylation-specific PCR reaction that can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer. Possibility, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitation of ctDNA lead to the problem that single non-small cell lung cancer methylation marker detection cannot meet clinical needs.
  • FIG. 1 is a schematic diagram of a first preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention
  • Fig. 2 is a schematic diagram of a second preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention.
  • the invention provides a primer pair for detecting multiple gene methylation of non-small cell lung cancer, comprising 6 sets of primer sequences corresponding to 6 genes as methylation markers of non-small cell lung cancer, the methylation markers Can be used to detect non-small cell lung cancer using blood as a sample.
  • the methylation markers are: CALAC, DLEC1, HOXA9, TBX5, PITX2, RASSF1a.
  • FIG. 1 is a schematic diagram of a first preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention.
  • the primer pair for detecting non-small cell lung cancer multiple gene methylation includes six sets of primer sequences corresponding to six methylation markers, and each methylation marker corresponds to one primer. Yes, each primer pair includes forward primer (MF) and reverse primer (MR), which are specifically expressed as follows:
  • CALCA forward primer 5'-CGGAATTTTTTCGATTTATAGC-3 '(SEQ ID NO.1);
  • DLEC1 reverse primer 5'-ACCCGACTAATAACGAAATTAACG-3 '(SEQ ID NO.4);
  • HOXA9 forward primer 5'-GGTTAATGGGGGCGCGGGCGTC-3 '(SEQ ID NO. 5);
  • HOXA9 reverse primer 5'-TCATATAACAACTTAATAACACCG-3 '(SEQ ID NO.6);
  • TBX5 forward primer 5'-GGGACGCGTAAAATTTAGAATC-3 '(SEQ ID NO.7);
  • TBX5 reverse primer 5'-AACACAAAACCGAAAAACGTC-3 '(SEQ ID NO.8);
  • PITX2 forward primer 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 '(SEQ ID NO.9);
  • PITX2 reverse primer 5'-AACACCGAAAAATACAATCCG-3 '(SEQ ID NO.10);
  • RASSF1a forward primer 5'-GTGTTAACGCGTTGCGTATC-3 '(SEQ ID NO.11);
  • RASSF1a reverse primer 5'-AACCCCGCGAACTAAAAACGA-3 '(SEQ ID NO.12).
  • the present invention in order to verify the correctness of the ctDNA conversion and PCR reaction process in the blood sample to be tested to ensure the accuracy of the detection of non-small cell lung cancer, the present invention will refer to the primer pair corresponding to the marker ⁇ -ACTIN ( Including ⁇ -ACTIN forward primer and ⁇ -ACTIN reverse primer) were added to the PCR reaction system.
  • FIG. 2 is a schematic diagram of a second preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention.
  • the primer pair for detecting non-small cell lung cancer multiple gene methylation according to the present invention includes not only the six sets of primer sequences corresponding to the six methylation markers, but also a reference marker Corresponding set of primer sequences.
  • a set of primer sequences corresponding to a reference marker includes:
  • ⁇ -ACTIN reverse primer 5’-AAAATATACC CTCCCCCATA CC-3 ’(SEQ ID NO.14).
  • the invention also provides the application of the primer pair for detecting non-small cell lung cancer multiple gene methylation in preparing a non-small cell lung cancer detection reagent, for example, preparing a non-small cell lung cancer multiple gene methylation Kit, this non-small cell lung cancer detection reagent can be used to detect the multiple gene methylation level of non-small cell lung cancer, and improve the accuracy, specificity and sensitivity of lung cancer detection.
  • the invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, which can be applied to detect a specific multiple methylation marker of non-small cell lung cancer.
  • the kit includes the six primers corresponding to the six non-small cell lung cancer-related genes as methylation markers and a PCR reaction system, wherein the PCR reaction system includes 1.8 ⁇ PCR solution, 5 mM MgCl 2 , 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase, for example, Taq Platinum DNA polymerase is preferred; the concentration of the primer pair is CALCA forward primer of 40 nM, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20
  • the kit includes 6 sets of primer pairs corresponding to the methylation markers, 1 set of primer pairs corresponding to a reference marker, and a PCR reaction system, wherein: the The PCR reaction system includes 1.8 ⁇ PCR solution, 5mM MgCl2, 0.3nM deoxyribonucleoside triphosphate, and 2.5 units of DNADNA polymerase, for example, Taq Platinum DNA polymerase is preferred; the concentration of each of the primer pairs is 40nM.
  • CALCA forward primer 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 Reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer, 10nM ⁇ -ACTIN forward primer, 10nM ⁇ -ACTIN reverse primer.
  • the present invention also provides an application of a kit for detecting multiple gene methylation of non-small cell lung cancer in detecting a specific multiple methylation marker of non-small cell lung cancer. That is, the kit of the present invention can be used to detect multiple gene methylation markers of non-small cell lung cancer, which is convenient for users to detect non-small cell lung cancer, and can detect the presence of multiple non-small cell lung cancers in a single PCR reaction. Basic markers improve the accuracy, specificity and sensitivity of lung cancer detection.
  • Step 1 Select 6 genes as methylation markers of NSCLC and 1 gene as reference marker; specifically, based on pre-lung cancer research and existing research results, select 6 genes as NSCLC
  • the methylation markers are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a.
  • the gene as a reference marker is different from the gene of the methylation marker, and the reference marker is ⁇ -ACTIN .
  • Step 2 Construct six primer pairs corresponding to the six methylation markers and one primer pair corresponding to the one reference marker; in this embodiment, each methylation marker corresponds to one primer pair, each One primer pair includes a forward primer (MF) and a reverse primer (MR).
  • the forward primer of the reference marker ⁇ -ACTIN is a positive control
  • the sequence of the ⁇ -ACTIN forward primer is: 5′-TTTTTAGGGAGGAGT TGGAAGTAGT-3 ′
  • the reverse primer of the reference marker is a negative control.
  • the sequence of ⁇ -ACTIN reverse primer is: 5'-AAAATATACC CTCCCCCATA CC-3 '.
  • FIG. 2 lists seven sets of primer sequences, including six sets of primer sequences corresponding to six methylation markers and one set of primer sequences corresponding to one reference marker.
  • Step 3 Extract the free ctDNA in the blood sample to be tested.
  • the concentration of ctDNA in the blood of the blood sample is 3-24 times that in the plasma, but the coagulation process is easily contaminated by impurities.
  • CtDNA was extracted from plasma, and ctDNA was purified and transformed.
  • ctDNA is easily decomposed by DNase in the blood. The purification of ctDNA needs to be performed as soon as possible.
  • the purification method can use the industry's common magnetic bead method or spin column method to extract free ctDNA in the blood sample to be tested to remove impurities
  • the ctDNA is purified, and the reagent used for the conversion treatment may be sulfite or bisulfite, that is, the ctDNA is subjected to sulfite or bisulfite conversion treatment.
  • Step 4 Take 4ul (27ng) of the transformed ctDNA and add it to a 25ul PCR reaction system; in this embodiment, the PCR reaction system includes: 1.8 ⁇ PCR solution, 5mM MgCl2, 0.3nM deoxyribonucleoside triphosphate , 6 primers corresponding to 6 methylation markers (40 nM of forward and reverse primers for DLEC1, 20 nM of forward and reverse primers for PITX2, 20 nM of forward and reverse primers for TBX5, CALCA forward primer and reverse primer each 40nM, RASSF1a forward primer and reverse primer each 280nM, HOXA9 forward primer and reverse primer each 120nM), 1 reference marker ⁇ -ACTIN 1 primer (10-M each of the forward primer and the reverse primer of ⁇ -ACTIN) and a 2.5-unit DNA polymerase, for example, Taq Platinum DNA polymerase is preferred).
  • 6 primers corresponding to 6 methylation markers 40 nM of forward and reverse primers for DLEC
  • Step 5 Put the PCR reaction system into a PCR instrument, and perform a PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product.
  • the PCR reaction procedure includes the following steps: (1), lasting for 3 minutes at a temperature of 95 ° C; (2), continuing for 1 minute at a temperature of 94 ° C, for 30 seconds at a temperature of 60 ° C, This step is performed for 4 seconds at a temperature of 65 ° C, and a total of 4 cycles are performed; (3) This step is performed for 1 minute at a temperature of 94 ° C, for 1 minute at a temperature of 56 ° C, and for 45 seconds at a temperature of 65 ° C A total of 36 amplification cycles were performed; (4), at 65 ° C for 4 minutes, as an extension reaction; (5), the PCR reaction was stopped at 4 ° C, and the PCR reaction product was stored at 4 ° C.
  • Step 6 Perform gel electrophoresis on the PCR reaction product. Specifically, perform gel electrophoresis on the PCR reaction product.
  • the gel electrophoresis may be agarose gel electrophoresis, and the concentration may be selected as: The agarose gel has an agarose concentration of 2.5%) or polyacrylamide gel electrophoresis (PAGE).
  • Step 7 Stain the gel after electrophoresis using a staining agent, and perform fluorescence analysis on the gel after staining using a gel imager; specifically, stain the gel after electrophoresis by using a staining agent as the preferred
  • the staining agent can be selected from ethidium bromide (EB), SYBR Green I, GelRed or GoldView staining agent, and a gel imager is used to perform fluorescence analysis on the stained gel and pass the gel imager. Read the fluorescent band in the stained gel.
  • Step 8 Determine whether three or more fluorescent color bands appear in the gel; in this embodiment, whether the gel imager reads three or more fluorescent color bands from the dyed gel, Among them, one fluorescent band is the PCR reaction band of the reference marker ⁇ -ACTIN, and the other fluorescent band is the PCR reaction band of the mutant gene marker.
  • Step 9 If three or more fluorescent color bands appear in the gel, the gel imager determines that the blood sample to be tested contains a non-small cell lung cancer gene mutation DNA fragment.
  • Step 10 If there are 2 or 1 fluorescent bands in the gel, the gel imager determines that the blood sample to be tested does not contain a mutant DNA fragment of a non-small cell lung cancer gene.
  • Step 11 If there is no fluorescent band in the gel, the gel imager determines that the ctDNA conversion and PCR reaction are invalid during the methylation detection of lung cancer characteristics. It is not possible to verify whether the blood sample to be tested contains non-small cell lung cancer. Gene mutation DNA fragment. Therefore, in this embodiment, the reference marker ⁇ -ACTIN forward primer and reverse primer are added to the PCR reaction system, mainly to verify whether the ctDNA conversion and the PCR reaction process are correct to ensure the accuracy of the detection of non-small cell lung cancer.
  • the primer pair and the kit of the present invention By using the primer pair and the kit of the present invention, the presence of multiple non-small cell lung cancer methylation markers can be detected simultaneously in one PCR reaction detection, which improves the accuracy, specificity, and sensitivity of lung cancer detection.
  • the primer pair and the kit of the invention are widely used for detecting methylation of non-small cell lung cancer genes, and solve a methylation-specific PCR reaction that can only detect the methylation level of a single gene and cannot accurately predict lung cancer.
  • the possibility of occurrence, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitations of ctDNA lead to the problem that single non-small cell lung cancer methylation marker detection cannot meet the clinical needs.
  • the primer pair and kit for detecting multiple gene methylation of non-small cell lung cancer adopt the above technical scheme, and achieve the following technical effects: multiple non-small cell lung cancers can be detected simultaneously in one PCR reaction
  • the presence of methylation markers in small cell lung cancer improves the accuracy, specificity, and sensitivity of non-small cell lung cancer detection.
  • Detection of multiple gene methylation in non-small cell lung cancer by using the primer pair and the kit according to the present invention can solve a methylation-specific PCR reaction that can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer. Possibility, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitation of ctDNA lead to the problem that single non-small cell lung cancer methylation marker detection cannot meet clinical needs.
  • AAAACCC TAT AAAAACGACG AC It is the case with the following: twenty two
  • TC The following is the name of the TC: twenty two
  • GTGTTAACGC GTTGCGTATC The following is the name of the GTTGCGTATC: 20
  • AACCCC GCGA ACTAAAAACG A The following is the case with the following: twenty one
  • AAAATATACC CTCCCCCATA CC It is the case of the following: twenty two

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Abstract

L'invention concerne une paire d'amorces pour détecter la méthylation d'un gène multiplex du cancer du poumon non à petites cellules, et un kit de réactifs. La paire d'amorces comprend 6 ensembles de séquences d'amorces correspondant à 6 gènes servant de marqueurs de méthylation du cancer du poumon non à petites cellules. Les marqueurs de méthylation sont respectivement : CALCA, DLEC1, HOXA9, TBX5, PITX2 et RASSF1a; et les 6 ensembles de séquences d'amorces comprennent une amorce sens de CALCA, une amorce antisens de CALCA, une amorce sens de DLEC1, une amorce antisens de DLEC1, une amorce sens de HOXA9, une amorce antisens de HOXA9, une amorce sens de TBX5, une amorce antisens de TBX5, une amorce sens de PITX2, une amorce antisens de PITX2, une amorce sens de RASSF1a et une amorce antisens de RASSF1a. Les paires d'amorces comprennent également un ensemble de séquences d'amorces correspondant à un marqueur représentatif de bêta-actine, comprenant spécifiquement une amorce sens de bêta-actine et une amorce antisens de bêta-actine. Une détection simple par réaction PCR peut être utilisée pour détecter simultanément la présence de multiples marqueurs de méthylation du cancer du poumon non à petites cellules, ce qui améliore la précision, la spécificité et la sensibilité de la détection du cancer du poumon non à petites cellules.
PCT/CN2019/092334 2018-06-22 2019-06-21 Paire d'amorces pour détecter la méthylation d'un gène multiplex du cancer du poumon non à petites cellules, et kit de réactifs Ceased WO2019242753A1 (fr)

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WO2022187246A1 (fr) * 2021-03-01 2022-09-09 National Taiwan University Procédé et kit de surveillance du cancer du poumon non à petites cellules

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CN116121387A (zh) * 2023-01-19 2023-05-16 深圳市人民医院 一种用于结直肠癌检测的组合标志物及其应用

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