WO2011120398A1 - Procédé de détection conjointe de gènes de fusion de leucémie et trousses de diagnostic - Google Patents

Procédé de détection conjointe de gènes de fusion de leucémie et trousses de diagnostic Download PDF

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WO2011120398A1
WO2011120398A1 PCT/CN2011/072139 CN2011072139W WO2011120398A1 WO 2011120398 A1 WO2011120398 A1 WO 2011120398A1 CN 2011072139 W CN2011072139 W CN 2011072139W WO 2011120398 A1 WO2011120398 A1 WO 2011120398A1
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leukemia
sequence
seq
fusion gene
gene
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PCT/CN2011/072139
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邵棠
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江苏迈迪基因生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the technical field of in vitro diagnostic detection, in particular to a liquid phase chip combined detection method for a plurality of fusion genes related to leukemia and a diagnostic kit thereof.
  • Leukemia is a hematopoietic malignant tumor caused by abnormal differentiation of hematopoietic stem cells and malignant hyperplasia. It is one of the most common malignant tumors in China. With the continuous development of leukemia research, many leukemia patients have found specific chromosomal translocations, leading to the production of new fusion genes, which have become molecular and biological markers of different types of leukemia. In 2000, WHO's criteria for leukemia typing further classified some common abnormal genes into the criteria for basic diagnosis of leukemia. Therefore, the detection of leukemia-associated fusion genes at the molecular level can not only provide an important basis for leukemia diagnosis, classification, clinical treatment selection and prognosis, but also provide a basis for detection of minimal residual disease in leukemia. .
  • Common fusion genes in leukemia include BCR-ABL (b2a2) and BCR-ABL (b3a2) in chronic myeloid leukemia (CML); BCR-ABL (e1a2) and E2A- in acute lymphocytic leukemia (ALL).
  • PBX1, TEL-AML1, MLL-AF4 e10/e4, e9/e5, e9/e4
  • CBFB-MYH11 type in acute myeloid leukemia (AML) A, type D), AML1-ETO
  • PML-RARA L form
  • PML-RARA S
  • SIL-TAL1 type II, type III
  • T-ALL T cell acute lymphoblastic leukemia
  • leukemia mainly relies on cell morphology, immunology, cytogenetics, molecular biology and other detection methods for diagnosis and detection.
  • Cell morphology is influenced by subjective factors, and the coincidence rate of clinical diagnosis is only about 70%.
  • Cytogenetic methods such as karyotyping and banding techniques are screened for chromosomal translocations at the genome-wide level, and are susceptible to the detection of minor abnormalities in many chromosomes.
  • Immunological methods have high false positive rates and false negative rates, and early diagnosis is not possible.
  • FISH fluorescence in situ hybridization
  • the liquid phase chip also known as the liquid chip, can perform qualitative and quantitative detection on a small number of samples, and has the outstanding advantages of high throughput, simple operation, good repeatability, high sensitivity, and wide linear range.
  • the system is composed of many microspheres as the main matrix. In the manufacturing process of the microspheres, two different red-classified fluorescences are incorporated. According to the ratio of the two kinds of fluorescence, the spherical matrix is divided into 100 types, which can be marked with 100. Different probe molecules can simultaneously detect up to 100 different target molecules in a sample. Depending on the analyte, the surface of the microsphere can be covalently bound to a variety of nucleic acid detection probes. Fluorescent labeling is added as the hybridization reaction proceeds.
  • Different detection microspheres can be added simultaneously in the same reaction system. This allows for rapid, high-throughput testing with a small number of samples.
  • the microspheres are arranged into a single column and flow through the liquid phase chip detector by microfluidic technology. Each microsphere can be detected by two lasers at the same time, and the red laser excites the red classified fluorescence on the microsphere. , the different reactions are distinguished and characterized; the green laser stimulates the fluorescent label bound to the sample to be tested for quantification.
  • the labeled sample to be tested is combined with the probe on a specific microsphere, The light excited by both lasers can be detected.
  • the average fluorescence intensity on a particular microsphere can be automatically statistically analyzed to determine the type and amount of the analyte.
  • the invention is based on the high-throughput, simple operation, good repeatability, high sensitivity and wide linear range of the liquid phase chip technology, and the combined parallel detection of various fusion genes of leukemia can obtain better clinical detection. application.
  • the technical problem to be solved by the present invention is to provide a combined detection method and a diagnostic kit for a leukemia-associated fusion gene.
  • the method and kit comprise SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4), CBFB-MYH11 (type D)
  • SIL-TAL1 type II, type III
  • MLL-AF4 e9/e5, e9/e4
  • CBFB-MYH11 type D
  • Combined detection of multiple fusion genes can clinically classify T cell acute lymphoblastic leukemia (T-ALL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), and observe the efficacy and prognosis of patients. And dynamic monitoring of small residual diseases.
  • the detection method and the diagnostic kit have the advantages of high sensitivity, high specificity, high accuracy, and rapid detection.
  • a method for detecting a leukemia-associated fusion gene comprising the steps of:
  • each microsphere is covalently bound to three fusions for chromosomal translocation in leukemia a specific nucleic acid probe designed by the mRNA of the gene;
  • upstream and downstream primers were designed for the mRNAs of various fusion genes formed by chromosomal translocations in different leukemia types, and the corresponding products were amplified by reverse transcription PCR for different fusion gene mRNAs;
  • the microspheres described in the above detection methods may have an average diameter of 5.6 ⁇ m, polyphenylene microspheres combined with different fluorescent dyes, ie color-coded microspheres (color-coded
  • the fusion gene formed by the chromosomal translocation in the leukemia is a combination comprising any one or more of the following fusion genes or a combination of other fusion genes: SIL-TAL1 (type II, type III), MLL-AF4 (e9) /e5,e9/e4), CBFB-MYH11(type D).
  • the specific nucleic acid probe covalently bound to the microspheres includes the following sequence (wherein the 5' end contains an amino group modification):
  • SIL-TAL1 type II, type III: 5'-Aminolinker C12AGTTACGCTGCGGTGTGGTC-3', such as SEQ ID As shown in NO.1;
  • MLL-AF4 (e9/e5, e9/e4): 5'-Aminolinker C12TATTGCTGTCAAAGGAGGCGG-3', as SEQ ID NO. 2 Shown
  • CBFB-MYH11(type D) 5'-Aminolinker C12TGTCCTTCTCCGAGCCTCTTCA-3', as shown in SEQ ID NO.
  • any one or a combination of more than one of the above sequences may be used.
  • the upstream and downstream primers designed for mRNAs of different fusion genes include the following sequences (wherein the upstream primer contains a biotin tag at the 5' end):
  • SIL-TAL1 type II, type III: upstream primer 5'-biotinCCTGCAAACAGACCTCAGCTC-3', as SEQ ID NO. 4 Shown; downstream primer 5'-CGAGGAAGAGGATGCACAC-3', as shown in SEQ ID NO.
  • MLL-AF4 (e9/e5, e9/e4): upstream primer 5'-biotinTCCAGAGCAGAGCAAACAG-3', as shown in SEQ ID NO.
  • CBFB-MYH11(type D) upstream primer 5'-biotinGAGGATGCATTAGCACAACAG-3', as shown in SEQ ID NO. 8; downstream primer 5'- GAAGCAACTCCTGGGTGTC -3' , as shown in SEQ ID NO.
  • any one or a combination of more than one of the above sequences may be used.
  • the primer and probe sequences of the internal reference gene Abelson gene are as follows:
  • any one or a combination of more than one of the above sequences may be used.
  • a diagnostic kit for detecting a plurality of fusion genes associated with leukemia comprising a microsphere mixture covalently bound to a leukemia fusion gene mRNA-specific probe, and a microsphere binding to an Abelson gene probe , upstream and downstream primers of leukemia fusion gene mRNA, upstream and downstream primers of Abelson gene, streptavidin-PE, control substance (negative control and positive control).
  • the control contained in the above kit contains a positive control and a negative control, wherein the positive control is a mixed solution containing various fusion gene plasmids (including a plasmid containing the Abelson gene), and the negative control product does not contain the fusion gene and the Abelson gene.
  • the plasmid solution; the microsphere mixture is freely combined according to the needs of different test samples.
  • a diagnostic kit for detecting a plurality of fusion genes associated with leukemia for detecting in vitro samples, for leukemia typing, early diagnosis, therapeutic observation, prognosis, and dynamic monitoring of minimal residual diseases.
  • Applications, as well as applications for early diagnosis, classification, efficacy observation, and prognosis of other related diseases are provided.
  • the detection method and the kit have the advantages of high sensitivity, high specificity, high throughput, good stability, rapid detection and accuracy, and can qualitatively and quantitatively detect the leukemia fusion gene. Can be better applied in clinical testing.
  • Example 2 Liquid-phase chip combined parallel detection method for three kinds of fusion genes related to leukemia
  • the specific detection method includes the following steps:
  • SIL-TAL1 type II , type III
  • MLL-AF4 e9/e5, e9/e4
  • CBFB-MYH11 type D
  • SIL-TAL1 type II, type III: 5'-Aminolinker C12AGTTACGCTGCGGTGTGGTC-3', such as SEQ ID As shown in NO.1;
  • MLL-AF4 (e9/e5, e9/e4): 5'- AminolinkerC12 TATTGCTGTCAAAGGAGGCGG -3', as shown in SEQ ID NO. 2;
  • Abelson gene 5' - AminolinkerC12 CTGAAGGGCTTCTTCCAGAT -3' , as shown in SEQ ID NO.
  • the coupled microspheres were diluted 1:100 with d H 2 O;
  • Microspheres / ⁇ l (4 large microspheres total) ⁇ 2.5 ⁇ 100 (dilution factor).
  • microspheres coupled with an oligonucleotide probe are as follows: SIL-TAL1 (type II, type III) Probe microsphere 11, MLL-AF4 (e9/e5, e9/e4) probe microsphere 15, CBFB-MYH11 (type D) probe microsphere 17, Abelson probe microsphere 21, In a proportional mixture, the final concentration of each microsphere is 1500 / ⁇ l, and it is stored at 2-8 °C in the dark.
  • the clinical samples of leukemia patients 1-15 were separately extracted with RNA:
  • the preparation method was the same as the preparation steps 1-13 of the sample in Example 1.
  • Method for synthesizing the first strand of cDNA The method for synthesizing the first strand of cDNA in the same manner as in Example 1.
  • SIL-TAL1 (type II , type III) : upstream primer 5'-biotinCCTGCAAACAGACCTCAGCTC -3' , as shown in SEQ ID NO. 4; downstream primer 5'- CGAGGAAGAGGATGCACAC -3' as shown in SEQ ID NO. 5;
  • MLL-AF4 (e9/e5, e9/e4): upstream primer 5'-biotin TCCAGAGCAGAGCAAACAG -3' , as shown in SEQ ID NO. 6; downstream primer 5'-CCTTGCTGAGAATTTGAGTG -3', As shown in SEQ ID NO. 7;
  • CBFB-MYH11(type D) upstream primer 5'-biotin GAGGATGCATTAGCACAACAG-3', as shown in SEQ ID NO. 8; downstream primer 5'-GAAGCAACTCCTGGGTGTC-3', As shown in SEQ ID NO.
  • Abelson gene upstream primer 5' -biotin GCGCAAAATGTTGGAGATC -3' , as shown in SEQ ID NO. 10; downstream primer 5'-GGAGCTTTTCACCTTTAGTTATGC-3', such as SEQ ID NO. Shown.
  • Types 1, 5, 9, 10, and 13 are leukemia fusion gene CBFB-MYH11 (type D) positive
  • Type 2, 7, 8, 11, 14, 15 patients were leukemia fusion gene MLL-AF4 (e9/e5, e9/e4) Positive
  • Type 3, 4, 6, and 12 are leukemia fusion gene SIL-TAL1 (type II, type III) Positive
  • the expression of the fusion gene can be obtained by comparing the fluorescence MFI value of the patient positive fusion gene with the fluorescence MFI value of the internal reference Abelson gene.
  • Example 1 Liquid-phase chip combined parallel detection method for two kinds of fusion genes related to leukemia
  • the specific detection method includes the following steps:
  • SIL-TAL1 type II, type III: 5'-Aminolinker C12AGTTACGCTGCGGTGTGGTC -3', as SEQ ID NO. Shown
  • MLL-AF4 (e9/e5, e9/e4): 5'- AminolinkerC12 TATTGCTGTCAAAGGAGGCGG -3', as shown in SEQ ID NO. 2;
  • Abelson gene 5' - AminolinkerC12 CTGAAGGGCTTCTTCCAGAT -3' , as shown in SEQ ID NO.
  • Three kinds of carboxyl microsphere storage suspensions with full-speed vortex numbers 11, 15, 21 are respectively at least 3 min, resulting in a uniform microsphere suspension;
  • the coupled microspheres were diluted 1:100 with d H 2 O;
  • Microspheres / ⁇ l (4 large microspheres total) ⁇ 2.5 ⁇ 100 (dilution factor).
  • microspheres coupled with an oligonucleotide probe are as follows: SIL-TAL1 (type II, type III) Probe microsphere 11, MLL-AF4 (e9/e5, e9/e4) probe microsphere 15, Abelson probe microsphere 21, mixed in equal proportion, the final concentration of various microspheres is 1500 / ⁇ l, 2-8 °C protected from light.
  • the clinical samples of patients with leukemias 1-10 were extracted according to the following steps:
  • Lymphocyte extraction Take 2 ml of fresh whole blood and 1 ml of 3 % sodium citrate, mix and mix 3000 r/min Centrifugation, 10 min, 4 °C, taking the light yellow layer on the blood cell layer is the lymphocyte;
  • RNA sample with 50 ul H 2 O , TE buffer or 0.5% SDS, 55-60 ° C, 5-10 min (H 2 O, TE or 0.5% SDS must be treated with DEPC and high pressure);
  • the multiplex PCR was performed on the mRNA of the above sample No. 1-10 as follows:
  • the tube was incubated at 70 ° C for 5 minutes, then quickly taken out and cooled in ice, and the reaction liquid on the tube wall was collected by a centrifugal centrifuge to the bottom of the tube;
  • SIL-TAL1 (type II , type III) : upstream primer 5'-biotinCCTGCAAACAGACCTCAGCTC -3', as shown in SEQ ID NO. a downstream primer 5'-CGAGGAAGAGGATGCACAC-3', as shown in SEQ ID NO.
  • MLL-AF4 (e9/e5, e9/e4): upstream primer 5'-biotin TCCAGAGCAGAGCAAACAG -3' , as shown in SEQ ID NO. 6; downstream primer 5'-CCTTGCTGAGAATTTGAGTG -3', As shown in SEQ ID NO. 7;
  • Abelson gene upstream primer 5' -biotin GCGCAAAATGTTGGAGATC -3', as shown in SEQ ID NO. 10; downstream primer 5'-GGAGCTTTTCACCTTTAGTTATGC-3', such as SEQ ID NO. Shown.
  • PCR amplification procedure 95 ° C for 10 min; 95 ° C for 30 s, 64-50 ° C for 1 min, 72 ° C 30 Min; the first 14 cycles, once per cycle, the annealing temperature is reduced by 1 ° C, after 21 cycles, the annealing temperature is maintained at 50 ° C; 72 ° C 7 min; 4 ° C insulation.
  • microsphere working solution dilute the coupled microspheres with a 1.5 ⁇ TMAC hybridization solution to a concentration of 150 microspheres/ ⁇ l (Note: 33 ⁇ l of microsphere working solution is required for each reaction);
  • Type 2, 3, 6, 8, and 9 are leukemia fusion gene MLL-AF4 (e9/e5, e9/e4) Positive
  • Type 1 , 4, 5, 7, and 10 are leukemia fusion gene SIL-TAL1 (type II, type III) Positive
  • the expression of the fusion gene can be obtained by comparing the fluorescence MFI value of the fusion gene with the fluorescence MFI value of the internal reference Abelson gene.
  • Example 2 Liquid-phase chip combined parallel detection method for three kinds of fusion genes related to leukemia
  • the specific detection method includes the following steps:
  • SIL-TAL1 type II, type III: 5'-Aminolinker C12AGTTACGCTGCGGTGTGGTC -3', as SEQ ID NO. Shown
  • MLL-AF4 (e9/e5, e9/e4): 5'- AminolinkerC12 TATTGCTGTCAAAGGAGGCGG -3', as shown in SEQ ID NO. 2;
  • Abelson gene 5' - AminolinkerC12 CTGAAGGGCTTCTTCCAGAT -3' , as shown in SEQ ID NO.
  • the coupled microspheres were diluted 1:100 with d H 2 O;
  • Microspheres / ⁇ l (4 large microspheres total) ⁇ 2.5 ⁇ 100 (dilution factor).
  • microspheres coupled with an oligonucleotide probe are as follows: SIL-TAL1 (type II, type III) Probe microsphere 11, MLL-AF4 (e9/e5, e9/e4) probe microsphere 15, CBFB-MYH11 (type D) probe microsphere 17, Abelson probe microsphere 21, In a proportional mixture, the final concentration of each microsphere is 1500 / ⁇ l, and it is stored at 2-8 °C in the dark.
  • RNA samples from leukemia patients 1-15 were extracted separately: The preparation method is the same as the preparation steps 1-13 of the sample in Example 1.
  • Method for synthesizing the first strand of cDNA The method for synthesizing the first strand of cDNA in the same manner as in Example 1.
  • SIL-TAL1 (type II , type III) : upstream primer 5'-biotinCCTGCAAACAGACCTCAGCTC -3' , as shown in SEQ ID NO. 4; downstream primer 5'- CGAGGAAGAGGATGCACAC -3' as shown in SEQ ID NO. 5;
  • MLL-AF4 (e9/e5, e9/e4): upstream primer 5'-biotin TCCAGAGCAGAGCAAACAG -3' , as shown in SEQ ID NO. 6; downstream primer 5'-CCTTGCTGAGAATTTGAGTG -3', As shown in SEQ ID NO. 7;
  • CBFB-MYH11(type D) upstream primer 5'-biotin GAGGATGCATTAGCACAACAG-3', as shown in SEQ ID NO. 8; downstream primer 5'-GAAGCAACTCCTGGGTGTC-3', As shown in SEQ ID NO.
  • Abelson gene upstream primer 5' -biotin GCGCAAAATGTTGGAGATC -3', as shown in SEQ ID NO. 10; downstream primer 5'-GGAGCTTTTCACCTTTAGTTATGC-3', such as SEQ ID NO. Shown.
  • Type 1 5, 9, 10, and 13 patients are leukemia fusion gene CBFB-MYH11 (type D) Positive
  • Type 2, 7, 8, 11, 14, 15 patients were leukemia fusion gene MLL-AF4 (e9/e5, e9/e4) Positive
  • Type 3, 4, 6, and 12 are leukemia fusion gene SIL-TAL1 (type II, type III) Positive
  • the expression of the fusion gene can be obtained by comparing the fluorescent MFI value of the patient-positive fusion gene with the fluorescent MFI value of the internal reference Abelson gene.
  • the detection method and the kit have the advantages of high sensitivity, high specificity, high throughput, good stability, rapid detection and accuracy, and can qualitatively and quantitatively detect the leukemia fusion gene. Can be better applied in clinical testing.

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Abstract

L'invention concerne un procédé de détection conjointe de gènes de fusion de leucémie et des trousses de diagnostic associées. Une fois que des amorces et des sondes pour l'ARNm de gènes de fusion de leucémie SIL-TAL1 (type II, type III), MLL-AF4 (e9/e5, e9/e4) et CBFB-MYH11 (type D) sont conçues, les amorces sont liées de manière covalente aux billes pour former des mélanges sondes-billes, qui sont hybridés avec les produits d'amplification de RT-PCR. Après l'ajout de streptavidine-phycoérythrine, des signaux fluorescents provenant de différentes billes peuvent être détectés et il peut être déterminé si l'échantillon à détecter contient ou non des gènes de fusion de leucémie ainsi que les profils d'expression des gènes de fusion.
PCT/CN2011/072139 2010-03-31 2011-03-30 Procédé de détection conjointe de gènes de fusion de leucémie et trousses de diagnostic WO2011120398A1 (fr)

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CN101955991B (zh) * 2010-03-31 2014-05-28 江苏迈迪基因生物科技有限公司 白血病相关的融合基因的联合检测方法及诊断试剂盒
CN103667453B (zh) * 2013-11-18 2015-06-17 福州艾迪康医学检验所有限公司 检测11q23/MLL融合基因相对表达量的方法、引物和探针
CN104278093B (zh) * 2014-09-28 2016-11-23 南京百捷生物科技有限公司 焦磷酸测序法检测sil-tal1融合基因的引物对及试剂盒
CN107151699A (zh) * 2017-06-01 2017-09-12 武汉艾迪康医学检验所有限公司 检测nup214‑abl1基因相对表达量的试剂盒和方法

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WO2009124901A1 (fr) * 2008-04-07 2009-10-15 Erasmus University Medical Center Rotterdam Procédés et kits de détection de protéines de fusion spécifiques des tumeurs
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480456A (zh) * 2021-12-21 2022-05-13 安徽医科大学第二附属医院 用于检测多种融合基因的标准质粒及检测试剂盒

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