WO2017084027A1 - Kit for prognostic stratification of acute myelocytic leukemia and testing method therefor - Google Patents

Kit for prognostic stratification of acute myelocytic leukemia and testing method therefor Download PDF

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WO2017084027A1
WO2017084027A1 PCT/CN2015/094788 CN2015094788W WO2017084027A1 WO 2017084027 A1 WO2017084027 A1 WO 2017084027A1 CN 2015094788 W CN2015094788 W CN 2015094788W WO 2017084027 A1 WO2017084027 A1 WO 2017084027A1
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primer
gene
fragment
seq
nucleotide sequence
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PCT/CN2015/094788
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French (fr)
Chinese (zh)
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王秀莉
洪燕
玄兆伶
李大为
梁峻彬
陈重建
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安诺优达基因科技(北京)有限公司
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Publication of WO2017084027A1 publication Critical patent/WO2017084027A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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

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  • the invention relates to a kit and a detection method which can be used for prognosis stratification of acute myeloid leukemia, and belongs to the technical field of molecular detection.
  • AML Acute Myelocytic Leukemia
  • Cytogenetic changes have greatly affected the treatment and prognosis of patients. With the continuous advancement of science and technology, more and more prognostic factors have been identified: (1) gene mutation; (2) cytogenetic diagnosis; (3) Clinical manifestations, including age, WBC, and clinical manifestations; (4) rate of decline in minimal residual disease (MRD) after treatment. Among them, gene mutation is one of the key factors affecting prognosis.
  • Non-Patent Documents 1 to 4 The most common mutations in genes are CEBPA, FLT3-ITD, and NMP1 gene mutations, which occur at about 15%, 35%, and 20%, respectively [Non-Patent Documents 1 to 4]. Due to the prevalence of multiple gene multi-mutation coexistence in AML cases, systematic detection of multi-gene/multi-site mutations is more conducive to prognostic stratification of AML, and also helps to determine treatment options and predict patient response to treatment. .
  • kits and assays for prognostic stratification of acute myeloid leukemia that are capable of rapidly and accurately detecting polygenic/multiple site mutations.
  • the present invention includes:
  • a kit for prognosis stratification of acute myeloid leukemia comprising an ITD region for detecting a FLT3 gene a primer set of 1 or a fragment thereof, a primer set 2 for detecting an NPM1 gene or a fragment thereof, and a primer set 3 for detecting a CEBPA gene or a fragment thereof;
  • Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
  • the fluorescent molecule is selected from the group consisting of FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7.
  • each primer set for detecting a gene of a similar size or a fragment thereof is labeled with a different fluorescent molecule.
  • the primer set 1 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2;
  • the primer set 2 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 4;
  • the primer set 3 is the following primer set 31 to 34:
  • a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
  • a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
  • a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
  • Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
  • a method for simultaneously detecting a gene mutation of an ITD region, an NPM1 gene, and a CEBPA gene of a FLT3 gene of a subject which comprises the kit according to any one of items 1 to 4, comprising:
  • A using the primer set 1 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the ITD region of the FLT3 gene of the sample or a fragment thereof, to obtain an amplification product A;
  • the method according to item 5 further comprising: comparing the ITD region of the FLT3 gene or a fragment thereof, the NPM1 gene or a fragment thereof, and the CEBPA gene or a fragment thereof based on the result of the multiplex fluorescent capillary gel electrophoresis If the size of one or more amplification products is the same as the size of the corresponding wild-type gene or a fragment thereof, such as the two, it is determined that the subject's gene has not been mutated, and if the two are inconsistent, the test is determined to be a test. The gene has been mutated.
  • the multi-gene multi-mutation with significant clinical guiding significance for the prognosis stratification of AML can be simultaneously detected, and the entire detection process from sample preparation to report is completed within one working day, and the detection result is fast and accurate. It can effectively provide rapid guidance on the prognosis of AML.
  • One of the advantages of the present invention over the prior art is that it is fast. Traditionally, it takes at least 3 days to complete the prognostic stratification of AML by one-generation sequencing; it takes at least one week to complete the prognostic stratification of AML by second-generation sequencing; and the method of the present invention is used to complete the prognostic stratification of AML. It takes less than a day to complete, which is extremely important for clinical testing.
  • another advantage of the present invention is that it is accurate, and the specific prognosis results detected by the present invention are consistent with the results of actual clinician feedback.
  • Figure 1 is the result of testing a male AML patient using the method of the examples.
  • Figure 2 is the result of testing a female AML patient using the method of the examples.
  • the present invention provides a kit for prognosis stratification of acute myeloid leukemia (kit of the invention) comprising a primer set 1 for detecting an ITD region of a FLT3 gene or a fragment thereof, for use in a primer set 2 for detecting the NPM1 gene or a fragment thereof, and a primer set 3 for detecting the CEBPA gene or a fragment thereof;
  • Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
  • for detection refers to the use of a designated gene or a fragment thereof from a subject comprising the gene or a fragment thereof.
  • the source sample is amplified by PCR and the amplification product is given a fluorescent label that can be used for multiplex capillary gel electrophoresis.
  • the "subject” is a person who has already suffered from acute myeloid leukemia, preferably a person who has developed acute myeloid leukemia and has received clinical treatment and needs a prognostic evaluation.
  • Fluorescent molecular labeling of the nucleotide sequence can be carried out by methods known to those skilled in the art, which are typically labeled at the 5' or 3' end of the upstream primer and/or downstream primer.
  • fluorescent molecule include FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7, but are not limited thereto.
  • Capillary electrophoresis also known as high performance capillary electrophoresis (HPCE)
  • HPCE high performance capillary electrophoresis
  • capillary gel electrophoresis is a zone electrophoresis in which a gel is transferred to a capillary as a support for separation.
  • Gel is a solid dispersion system with porosity, similar to the role of molecular sieves. The separation is separated by the gel force in the capillary, according to the volume of each molecule. The larger the volume is first Isolated, suitable for the analysis of biological macromolecules.
  • Capillary gel electrophoresis can identify oligonucleotides with one base difference and can be used to isolate DNA fragments. Fluorescence capillary gel electrophoresis distinguishes nucleotide sequences of different sizes by detecting fluorescent signals from fluorescent molecules labeled on nucleotide sequences. In the present specification, multiplex capillary capillary gel electrophoresis refers to simultaneous detection of fluorescent capillary gel electrophoresis from a plurality of fluorescent molecules.
  • each primer set for detecting genes of similar sizes or fragments thereof.
  • each gene and its fragment can be more significantly distinguished by fluorescent capillary gel electrophoresis.
  • the size is similar means that the difference is within ⁇ 30 bp.
  • nucleotide sequences of the ITD region, the NPM1 gene and the CEBPA gene of the human FLT3 gene are known, and amplification of primers for genes of known nucleotide sequences is well known to those skilled in the art, for example, according to, for example, "Molecule The Guide to Cloning Experiments (J. Sambrook. J., Huang Peitang et al., 3rd edition, 2005) is designed to be designed, or using computer software (such as Primer Premier 6.0 developed by Premier). Design.
  • PCR polymerase chain reaction
  • the PCR reaction procedure is generally a step of denaturation, annealing, extension, and the like.
  • the primer set 1 consists of an upstream primer having a nucleotide sequence such as SEQ ID NO: 1 and a downstream primer having a nucleotide sequence such as SEQ ID NO: 2;
  • Group 2 consists of an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4;
  • the primer set 3 is the following primer set 31 to 34:
  • a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
  • a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
  • a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
  • Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
  • each of the obtained amplification products shows a "high quality" peak in the multiplex fluorescent capillary gel electrophoresis measurement.
  • “high quality” means that the amplification peaks for each purpose are single, and no peaks appear.
  • the kit of the present invention may further comprise, in addition to the above primer sets 1 to 3, a method for extracting genomic DNA for use in a PCR amplification reaction for extracting total RNA for reverse transcription. And one or more of reagents or devices for multiplex capillary gel electrophoresis.
  • the kit of the present invention can simultaneously detect a gene mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene in a subject.
  • the mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene of the subject is simultaneously detected using the kit of the present invention, it can be carried out as follows:
  • Step A Using the primer set 1 as a primer and using the genomic DNA of the sample from the subject as a template, the ITD region of the FLT3 gene of the sample or a fragment thereof is amplified to obtain an amplification product A.
  • Step B Using the primer set 2 as a primer, the genomic DNA of the sample from the subject is used as a template to amplify the NPM1 gene of the sample or a fragment thereof to obtain an amplification product B.
  • Step C Using the primer set 3 as a primer, the cDNA obtained by reverse transcription of total RNA from a sample of the subject is used as a template to amplify the CEBPA gene of the sample or a fragment thereof to obtain an amplification product C.
  • the steps A, B, and C can be performed in any order or simultaneously.
  • sample from the subject may be, for example, any tissue or cells from the subject, preferably whole blood or bone marrow.
  • Obtaining genomic DNA from a sample from a subject can be carried out by methods well known to those skilled in the art, for example, using a commercially available kit. Furthermore, obtaining total RNA from a sample from a subject, and reverse transcription of the total RNA to obtain cDNA can be carried out by a method known to those skilled in the art, for example, using a commercially available kit.
  • step D is carried out: the above amplification product A, amplification product B and amplification product C are mixed to obtain a mixed sample, and the mixed sample is subjected to multiplex fluorescent capillary gel electrophoresis.
  • the size of the amplification product of one or more (preferably all) of the gene or fragment thereof and the CEBPA gene or a fragment thereof is identical to the size of the corresponding wild-type gene or a fragment thereof, such as both, and is determined to be the subject's
  • the gene did not mutate, and if the two were inconsistent, it was determined that the subject had a mutation in the gene.
  • the size of the ITD region of the wild-type FLT3 gene is 365 bp
  • the size of the wild-type NPM1 gene fragment is 283 bp
  • the fragment of the wild-type CEBPA gene is four, and the sizes thereof are 309 bp, 345 bp, 444 bp, and 416 bp.
  • the kit of the present invention can be used for prognosis stratification of acute myeloid leukemia, that is, in the case where the result is that the FLT3-ITD is not mutated and at least one of NMP1 or CEBPA is mutated, the prognosis of the patient can be determined.
  • the prognosis of the patient can be determined to be moderate; in the judgment result, the mutation of FLT3-ITD and the mutation of NMP1 and CEBPA are not mutated. In the case of this, it can be determined that the patient's prognosis is poor.
  • F or FS represents an upstream primer
  • R or RS represents a downstream primer
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C extension for 5 minutes.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C final extension for 5 minutes.
  • wild-type NPM1 gene fragment size is 283 bp
  • wild-type FLT3 gene ITD region fragment size is 365 bp
  • wild-type CEPBA gene fragment has four, its size is 309 bp, 345 bp, 444 bp And 415 bp. Whether or not an insertion or deletion mutation occurs in the gene to be detected is determined based on the size of the fragment corresponding to the peak.
  • the test results are shown in Figure 1. It can be seen that the fragment of the amplification product of the NPM1 gene is large. The small size was 283 bp, indicating that the detection gene was not mutated; the size of the four amplified fragments of CEBPA gene were 309 bp, 345 bp, 444 bp and 416 bp, both of which were wild type, indicating that the CEBPA detection gene was not mutated; FLT3 gene amplification product The size is 383 bp, indicating that the gene has an 18 bp insertion mutation. In this sample, only the insertion mutation occurred in FLT3, and the CEBPA and NPM1 genes did not mutate, indicating that the male AML sample had a poor prognosis.
  • the test results are shown in Figure 2. It can be seen that the size of the amplified product of NPM1 gene is 287 bp, indicating that the detection gene has a 4 bp insertion mutation; the four amplified fragments of CEBPA gene are 309 bp, 345 bp, 444 bp and 416 bp, respectively, all of which are wild-type fragments. , indicating that the CEBPA detection gene did not mutate; the size of the amplification product of the ITT region of the FLT3 gene was 365 bp, which was wild-type, indicating that the FLT3 of the sample did not change. In this sample, only the insertion mutation occurred in NPM1, and the CEBPA and FLT3 genes did not mutate, indicating that the prognosis of the female AML sample is better.
  • any technical feature or combination of technical features described in the specification as a component of a certain technical solution may also be applied to the embodiments that can be implemented without obscuring the gist of the present invention.
  • Other technical solutions; and, while being able to implement and not clearly deviating from the gist of the present invention, the technical features described as the constituent parts of the different technical solutions may be combined in any manner to constitute other technical solutions.
  • the present invention also encompasses the technical solutions obtained by the combination in the above case, and these technical solutions are equivalent to those described in the present specification.
  • kits and a detection method for prognostic stratification of acute myeloid leukemia capable of rapidly detecting polygenic/multiple site mutations.

Abstract

A kit for prognostic stratification of acute myelocytic leukemia (AML) and testing method therefor, capable of quickly testing multigene/multisite mutation. The kit can completely test mutated sites with high test sensitivity, can complete the whole experiment process from sample preparation to report production within one day, can quickly obtain an accurate result, and can implement high degree of coverage. In addition, the kit can provide effective and quick guidance for prognostic stratification of AML.

Description

一种用于急性髓细胞白血病预后分层的试剂盒及检测方法Kit and detection method for prognosis stratification of acute myeloid leukemia 技术领域Technical field
本发明涉及可用于急性髓细胞白血病预后分层的试剂盒及检测方法，属于分子检测技术领域。The invention relates to a kit and a detection method which can be used for prognosis stratification of acute myeloid leukemia, and belongs to the technical field of molecular detection.
背景技术Background technique
急性髓细胞白血病(Acute Myelocytic Leukemia，简称AML)是一种具有显著异质性的血液恶性克隆性疾病。即使AML患者预后良好，也仍有40％的患者复发，5年以上长期生存率不足50％，该类患者同时具有c-kit突变复发率高达70％，一旦复发，预后极差，异基因造血干细胞移植(allo-HSCT)也不能改善其预后。因此，尽早识别高危复发人群，采用包括造血干细胞移植在内的手段对这些患者进行抢先治疗，有可能改善预后。细胞遗传学改变很大程度上影响了患者的治疗和预后，随着科技的不断进步，越来越多的预后相关因素被确认：(1)基因突变；(2)细胞遗传学诊断；(3)临床表现，包括年龄、WBC以及临床表现等；(4)治疗后微小残留病灶(MRD)的下降速度等。其中，基因突变是影响预后的关键因素之一。Acute Myelocytic Leukemia (AML) is a blood malignant clonal disease with significant heterogeneity. Even if the prognosis of patients with AML is good, 40% of patients still have recurrence, and the long-term survival rate is less than 50% for more than 5 years. This type of patient also has a recurrence rate of c-kit mutation of up to 70%. Once relapsed, the prognosis is extremely poor, allogeneic hematopoiesis. Stem cell transplantation (allo-HSCT) also does not improve its prognosis. Therefore, early identification of high-risk relapse populations, preemptive treatment of these patients, including hematopoietic stem cell transplantation, may improve prognosis. Cytogenetic changes have greatly affected the treatment and prognosis of patients. With the continuous advancement of science and technology, more and more prognostic factors have been identified: (1) gene mutation; (2) cytogenetic diagnosis; (3) Clinical manifestations, including age, WBC, and clinical manifestations; (4) rate of decline in minimal residual disease (MRD) after treatment. Among them, gene mutation is one of the key factors affecting prognosis.
基因突变中最常见的是CEBPA、FLT3-ITD和NMP1的基因突变，其发生率分别约为15％、35％和20％[非专利文献1～4]。由于在AML病例中普遍存在多基因多突变共存的现象，因而***地检测多基因/多位点的突变更有助于AML的预后分层，也有助于确定治疗方案和预测病人对治疗的反应。The most common mutations in genes are CEBPA, FLT3-ITD, and NMP1 gene mutations, which occur at about 15%, 35%, and 20%, respectively [Non-Patent Documents 1 to 4]. Due to the prevalence of multiple gene multi-mutation coexistence in AML cases, systematic detection of multi-gene/multi-site mutations is more conducive to prognostic stratification of AML, and also helps to determine treatment options and predict patient response to treatment. .
此外，临床检验往往要求在短时间内(例如一个工作日)完成从样品制备到出具报告的整个检验流程。In addition, clinical testing often requires the entire inspection process from sample preparation to reporting to be completed in a short period of time (eg, one business day).
因此，需要能够快速地、准确地检测多基因/多位点突变的、用于急性髓细胞白血病预后分层的试剂盒及检测方法。Therefore, there is a need for kits and assays for prognostic stratification of acute myeloid leukemia that are capable of rapidly and accurately detecting polygenic/multiple site mutations.
发明内容Summary of the invention
鉴于上述现有技术中存在的问题，本发明的目的在于提供一种能够快速地检测多基因/多位点突变的、用于急性髓细胞白血病预后分层的试剂盒及检测方法。In view of the above problems in the prior art, it is an object of the present invention to provide a kit and a detection method for prognostic stratification of acute myeloid leukemia capable of rapidly detecting polygenic/multiple site mutations.
即，本发明包括：That is, the present invention includes:
1.一种用于急性髓细胞白血病预后分层的试剂盒，其包括用于检测FLT3基因的ITD区 或其片段的引物组1、用于检测NPM1基因或其片段的引物组2、以及用于检测CEBPA基因或其片段的引物组3；A kit for prognosis stratification of acute myeloid leukemia, comprising an ITD region for detecting a FLT3 gene a primer set of 1 or a fragment thereof, a primer set 2 for detecting an NPM1 gene or a fragment thereof, and a primer set 3 for detecting a CEBPA gene or a fragment thereof;
所述每个引物组均包括上游引物和下游引物，且所述上游引物和下游引物中的至少一个是经过荧光分子标记的。Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
2.根据项1所述的试剂盒，其中，所述荧光分子选自FAM、HEX、TET、VIC、Cy3、Cy5和ROX7。2. The kit of clause 1, wherein the fluorescent molecule is selected from the group consisting of FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7.
3.根据项1或2所述的试剂盒，其中，对于用于检测大小相近的基因或其片段的各引物组，采用不同的荧光分子进行标记。3. The kit according to item 1 or 2, wherein each primer set for detecting a gene of a similar size or a fragment thereof is labeled with a different fluorescent molecule.
4.根据项1～3中任一项所述的试剂盒，其中，4. The kit according to any one of items 1 to 3, wherein
所述引物组1由核苷酸序列如SEQ ID NO：1所示的上游引物和核苷酸序列如SEQ ID NO：2所示的下游引物组成；The primer set 1 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2;
所述引物组2由核苷酸序列如SEQ ID NO：3所示的上游引物和核苷酸序列如SEQ ID NO：4所示的下游引物组成；和The primer set 2 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 4;
所述引物组3是下述引物组31～34：The primer set 3 is the following primer set 31 to 34:
引物组31，其由核苷酸序列如SEQ ID NO：5所示的上游引物和核苷酸序列如SEQ ID NO：6所示的下游引物组成；a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
引物组32，其由核苷酸序列如SEQ ID NO：7所示的上游引物和核苷酸序列如SEQ ID NO：8所示的下游引物组成；a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
引物组33，其由核苷酸序列如SEQ ID NO：9所示的上游引物和核苷酸序列如SEQ ID NO：10所示的下游引物组成；和a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
引物组34，其由核苷酸序列如SEQ ID NO：11所示的上游引物和核苷酸序列如SEQ ID NO：12所示的下游引物组成。Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
5.一种同时检测受试者的FLT3基因的ITD区、NPM1基因和CEBPA基因的基因突变的方法，其使用项1～4中任一项所述的试剂盒，其包括：A method for simultaneously detecting a gene mutation of an ITD region, an NPM1 gene, and a CEBPA gene of a FLT3 gene of a subject, which comprises the kit according to any one of items 1 to 4, comprising:
A.以所述引物组1作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的FLT3基因的ITD区或其片段，得到扩增产物A；A. using the primer set 1 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the ITD region of the FLT3 gene of the sample or a fragment thereof, to obtain an amplification product A;
B.以所述引物组2作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的NPM1基因或其片段，得到扩增产物B；B. using the primer set 2 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the NPM1 gene of the sample or a fragment thereof to obtain an amplification product B;
C.以所述引物组3作为引物，以由来自受试者的样本的总RNA反转录得到的cDNA作为模板，扩增样本的CEBPA基因或其片段，得到扩增产物C；C. using the primer set 3 as a primer, using the cDNA obtained by reverse transcription of total RNA from a sample of the subject as a template, amplifying the CEBPA gene of the sample or a fragment thereof to obtain an amplification product C;
D.将上述扩增产物A、扩增产物B和扩增产物C混合，得到混合试样，并对该混合试 样进行多重荧光毛细管凝胶电泳。D. mixing the above amplification product A, amplification product B and amplification product C to obtain a mixed sample, and the mixed test Multiplex fluorescent capillary gel electrophoresis was performed.
6.根据项5所述的方法，其还包括：基于所述多重荧光毛细管凝胶电泳的结果，比较所述FLT3基因的ITD区或其片段、NPM1基因或其片段和CEBPA基因或其片段中的一个或多个的扩增产物的大小与相应的野生型基因或其片段的大小，如两者一致，则判定为受试者的基因未发生突变，如两者不一致，则判定为受试者的该基因发生了突变。6. The method according to item 5, further comprising: comparing the ITD region of the FLT3 gene or a fragment thereof, the NPM1 gene or a fragment thereof, and the CEBPA gene or a fragment thereof based on the result of the multiplex fluorescent capillary gel electrophoresis If the size of one or more amplification products is the same as the size of the corresponding wild-type gene or a fragment thereof, such as the two, it is determined that the subject's gene has not been mutated, and if the two are inconsistent, the test is determined to be a test. The gene has been mutated.
7.根据项6所述的方法，其中，所述野生型FLT3基因的ITD区的大小为365bp，所述野生型NPM1基因片段的大小为283bp，所述野生型CEBPA基因的片段有四个，其大小分别为309bp、345bp、444bp和416bp；且当所述扩增产物的大小与相应的野生型基因的片段的大小相差±1bp以下时，认定为两者一致。7. The method according to Item 6, wherein the size of the ITD region of the wild type FLT3 gene is 365 bp, the size of the wild type NPM1 gene fragment is 283 bp, and the fragment of the wild type CEBPA gene is four. The sizes are 309 bp, 345 bp, 444 bp, and 416 bp, respectively; and when the size of the amplified product differs from the size of the fragment of the corresponding wild-type gene by ±1 bp or less, it is considered that the two are identical.
8.根据项5～7中任一项所述的方法，其中，所述样本为全血或骨髓。The method according to any one of items 5 to 7, wherein the sample is whole blood or bone marrow.
发明效果。The effect of the invention.
根据本发明，可以同时检测对AML预后分层的具有明显临床指导意义的多基因多突变，在一个工作日之内就可以完成从样本制备到出具报告的整个检测流程，检测结果快速、准确，可以有效地对AML的预后分层提供快速的指导。According to the present invention, the multi-gene multi-mutation with significant clinical guiding significance for the prognosis stratification of AML can be simultaneously detected, and the entire detection process from sample preparation to report is completed within one working day, and the detection result is fast and accurate. It can effectively provide rapid guidance on the prognosis of AML.
与现有技术相比，本发明的优势之一在于快速。传统上，采用一代测序完成AML预后分层的检测至少需要3天的时间；采用二代测序完成AML预后分层的检测至少需要一周的时间；而采用本发明的方法完成AML预后分层的检测则只需要不到1天的时间即可完成，这对于临床检验而言极为重要。此外，本发明的另一优势在于准确，具体体现在：本发明检测出的预后结果跟实际临床医生反馈的结果是一致的。One of the advantages of the present invention over the prior art is that it is fast. Traditionally, it takes at least 3 days to complete the prognostic stratification of AML by one-generation sequencing; it takes at least one week to complete the prognostic stratification of AML by second-generation sequencing; and the method of the present invention is used to complete the prognostic stratification of AML. It takes less than a day to complete, which is extremely important for clinical testing. In addition, another advantage of the present invention is that it is accurate, and the specific prognosis results detected by the present invention are consistent with the results of actual clinician feedback.
附图说明DRAWINGS
图1为采用实施例的方法对一名男性AML患者进行检测的结果。Figure 1 is the result of testing a male AML patient using the method of the examples.
图2为采用实施例的方法对一名女性AML患者进行检测的结果。Figure 2 is the result of testing a female AML patient using the method of the examples.
发明的具体实施方式DETAILED DESCRIPTION OF THE INVENTION
在一个方面中，本发明提供一种用于急性髓细胞白血病预后分层的试剂盒(本发明的试剂盒)，其包括用于检测FLT3基因的ITD区或其片段的引物组1、用于检测NPM1基因或其片段的引物组2、以及用于检测CEBPA基因或其片段的引物组3；In one aspect, the present invention provides a kit for prognosis stratification of acute myeloid leukemia (kit of the invention) comprising a primer set 1 for detecting an ITD region of a FLT3 gene or a fragment thereof, for use in a primer set 2 for detecting the NPM1 gene or a fragment thereof, and a primer set 3 for detecting the CEBPA gene or a fragment thereof;
所述每个引物组均包括上游引物和下游引物，且所述上游引物和下游引物中的至少一个是经过荧光分子标记的。Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
在这里，“用于检测”是指：用于将指定基因或其片段从包含该基因或其片段的受试者来 源的样本中通过PCR扩增出来，并对扩增产物赋予能够用于多重荧光毛细管凝胶电泳测定的荧光标记。As used herein, "for detection" refers to the use of a designated gene or a fragment thereof from a subject comprising the gene or a fragment thereof. The source sample is amplified by PCR and the amplification product is given a fluorescent label that can be used for multiplex capillary gel electrophoresis.
在本说明中，所述“受试者”是已经罹患急性髓细胞白血病的人，优选是已经罹患急性髓细胞白血病且接受了临床治疗、需要进行预后评估的人。In the present description, the "subject" is a person who has already suffered from acute myeloid leukemia, preferably a person who has developed acute myeloid leukemia and has received clinical treatment and needs a prognostic evaluation.
对核苷酸序列进行荧光分子标记可以采用本领域技术人员已知的方法来进行，所述荧光分子通常被标记在所述上游引物和/或下游引物的5′端或3′端。作为所述荧光分子，可以列举出FAM、HEX、TET、VIC、Cy3、Cy5和ROX7等，但不限于此。Fluorescent molecular labeling of the nucleotide sequence can be carried out by methods known to those skilled in the art, which are typically labeled at the 5' or 3' end of the upstream primer and/or downstream primer. Examples of the fluorescent molecule include FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7, but are not limited thereto.
毛细管电泳(capillary electrophoresis，CE)又称高效毛细管电泳(high performance capillary electrophoresis，HPCE)，是一类以毛细管为分离通道、以高压直流电场为驱动力的新型液相分离技术。毛细管电泳的分离模式有很多种，其中毛细管凝胶电泳是将凝胶转移到毛细管中作为支撑物进行分离的区带电泳。凝胶是一种固态的分散的体系，具有多孔性，类似于分子筛的作用，被分离物在通过装入毛细管内的凝胶力，按照各自分子的体积大小逐一分离，分子体积大的首先被分离出来，适用于生物大分子的分析。毛细管凝胶电泳可以识别一个碱基差异的寡核苷酸，可用于分离DNA片段。荧光毛细管凝胶电泳通过检测来自核苷酸序列上标记的荧光分子的荧光信号，从而区分不同大小的核苷酸序列。在本说明书中，多重荧光毛细管凝胶电泳是指同时检测来自多种荧光分子的荧光毛细管凝胶电泳。Capillary electrophoresis (CE), also known as high performance capillary electrophoresis (HPCE), is a new type of liquid phase separation technology with capillary as separation channel and high voltage DC electric field as driving force. There are many separation modes for capillary electrophoresis, in which capillary gel electrophoresis is a zone electrophoresis in which a gel is transferred to a capillary as a support for separation. Gel is a solid dispersion system with porosity, similar to the role of molecular sieves. The separation is separated by the gel force in the capillary, according to the volume of each molecule. The larger the volume is first Isolated, suitable for the analysis of biological macromolecules. Capillary gel electrophoresis can identify oligonucleotides with one base difference and can be used to isolate DNA fragments. Fluorescence capillary gel electrophoresis distinguishes nucleotide sequences of different sizes by detecting fluorescent signals from fluorescent molecules labeled on nucleotide sequences. In the present specification, multiplex capillary capillary gel electrophoresis refers to simultaneous detection of fluorescent capillary gel electrophoresis from a plurality of fluorescent molecules.
在本发明中，优选对于用于检测大小相近的基因或其片段的各引物组，采用不同的荧光分子进行标记。由此，可以通过荧光毛细管凝胶电泳更显著地区分各基因及其片段。这里，“大小相近”是指相差±30bp以内。In the present invention, it is preferred to use different fluorescent molecules for labeling each primer set for detecting genes of similar sizes or fragments thereof. Thus, each gene and its fragment can be more significantly distinguished by fluorescent capillary gel electrophoresis. Here, "the size is similar" means that the difference is within ±30 bp.
人类FLT3基因的ITD区、NPM1基因和CEBPA基因的核苷酸序列是已知的，针对已知核苷酸序列的基因设计其扩增引物是本领域技术人员熟知的，例如可以按照例如《分子克隆实验指南》(J.萨姆布鲁克(Sambrook.J.)等著，黄培堂等译，第3版，2005)的教导来进行设计，或者利用计算机软件(例如Premier公司开发的Primer Premier 6.0)来进行设计。The nucleotide sequences of the ITD region, the NPM1 gene and the CEBPA gene of the human FLT3 gene are known, and amplification of primers for genes of known nucleotide sequences is well known to those skilled in the art, for example, according to, for example, "Molecule The Guide to Cloning Experiments (J. Sambrook. J., Huang Peitang et al., 3rd edition, 2005) is designed to be designed, or using computer software (such as Primer Premier 6.0 developed by Premier). Design.
PCR(聚合酶链式反应)扩增是本领域技术人员熟知的，其一般通过一定的PCR反应程序(温度循环)来实现。所述PCR反应程序一般变性、退火、延伸等步骤。PCR (polymerase chain reaction) amplification is well known to those skilled in the art and is typically accomplished by a certain PCR reaction procedure (temperature cycling). The PCR reaction procedure is generally a step of denaturation, annealing, extension, and the like.
在一个优选的实施方式中，所述引物组1由核苷酸序列如SEQ ID NO：1所示的上游引物和核苷酸序列如SEQ ID NO：2所示的下游引物组成；所述引物组2由核苷酸序列如SEQ ID NO：3所示的上游引物和核苷酸序列如SEQ ID NO：4所示的下游引物组成；且In a preferred embodiment, the primer set 1 consists of an upstream primer having a nucleotide sequence such as SEQ ID NO: 1 and a downstream primer having a nucleotide sequence such as SEQ ID NO: 2; Group 2 consists of an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4;
所述引物组3是下述引物组31～34：The primer set 3 is the following primer set 31 to 34:
引物组31，其由核苷酸序列如SEQ ID NO：5所示的上游引物和核苷酸序列如SEQ ID NO：6所示的下游引物组成； a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
引物组32，其由核苷酸序列如SEQ ID NO：7所示的上游引物和核苷酸序列如SEQ ID NO：8所示的下游引物组成；a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
引物组33，其由核苷酸序列如SEQ ID NO：9所示的上游引物和核苷酸序列如SEQ ID NO：10所示的下游引物组成；和a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
引物组34，其由核苷酸序列如SEQ ID NO：11所示的上游引物和核苷酸序列如SEQ ID NO：12所示的下游引物组成。Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
本发明人发现，在使用上述SEQ ID NO：1～12所示的引物进行PCR扩增的情况下，所得各扩增产物在多重荧光毛细管凝胶电泳测定中均显示出“高品质”的峰。这里，“高品质”是指：每个目的扩增峰都单一，没有杂峰出现。The present inventors have found that in the case of PCR amplification using the primers shown in the above SEQ ID NOS: 1 to 12, each of the obtained amplification products shows a "high quality" peak in the multiplex fluorescent capillary gel electrophoresis measurement. . Here, "high quality" means that the amplification peaks for each purpose are single, and no peaks appear.
优选地，本发明的试剂盒中除了包含上述引物组1～3以外，还可以包含用于提取基因组DNA的、用于PCR扩增反应的、用于提取总RNA的、用于反转录的、以及用于多重荧光毛细管凝胶电泳的试剂或装置中的一种或多种。Preferably, the kit of the present invention may further comprise, in addition to the above primer sets 1 to 3, a method for extracting genomic DNA for use in a PCR amplification reaction for extracting total RNA for reverse transcription. And one or more of reagents or devices for multiplex capillary gel electrophoresis.
优选地，本发明的试剂盒可同时检测受试者中的FLT3基因的ITD区、NPM1基因和CEBPA基因的基因突变。在使用本发明的试剂盒同时检测受试者的FLT3基因的ITD区、NPM1基因和CEBPA基因的基因突变的情况下，可以如下进行：Preferably, the kit of the present invention can simultaneously detect a gene mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene in a subject. In the case where the mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene of the subject is simultaneously detected using the kit of the present invention, it can be carried out as follows:
步骤A：以所述引物组1作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的FLT3基因的ITD区或其片段，得到扩增产物A。Step A: Using the primer set 1 as a primer and using the genomic DNA of the sample from the subject as a template, the ITD region of the FLT3 gene of the sample or a fragment thereof is amplified to obtain an amplification product A.
步骤B：以所述引物组2作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的NPM1基因或其片段，得到扩增产物B。Step B: Using the primer set 2 as a primer, the genomic DNA of the sample from the subject is used as a template to amplify the NPM1 gene of the sample or a fragment thereof to obtain an amplification product B.
步骤C：以所述引物组3作为引物，以来自受试者的样本的总RNA反转录得到的cDNA作为模板，扩增样本的CEBPA基因或其片段，得到扩增产物C。Step C: Using the primer set 3 as a primer, the cDNA obtained by reverse transcription of total RNA from a sample of the subject is used as a template to amplify the CEBPA gene of the sample or a fragment thereof to obtain an amplification product C.
所述步骤A、步骤B和步骤C可以以任意顺序进行或同时进行。The steps A, B, and C can be performed in any order or simultaneously.
这里，对于来自受试者的样本没有特殊限制，可以是例如来自受试者的任何组织或细胞，优选全血或骨髓。Here, there is no particular limitation on the sample from the subject, and may be, for example, any tissue or cells from the subject, preferably whole blood or bone marrow.
从来自受试者的样本获得基因组DNA可以采用本领域技术人员公知的方法，例如可以使用商品化的试剂盒来进行。此外，从来自受试者的样本获得总RNA、以及将所述总RNA反转录获得cDNA可以采用本领域技术人员公知的方法，例如可以使用商品化的试剂盒来进行。Obtaining genomic DNA from a sample from a subject can be carried out by methods well known to those skilled in the art, for example, using a commercially available kit. Furthermore, obtaining total RNA from a sample from a subject, and reverse transcription of the total RNA to obtain cDNA can be carried out by a method known to those skilled in the art, for example, using a commercially available kit.
然后，进行步骤D：将上述扩增产物A、扩增产物B和扩增产物C混合，得到混合试样，并对该混合试样进行多重荧光毛细管凝胶电泳。Then, step D is carried out: the above amplification product A, amplification product B and amplification product C are mixed to obtain a mixed sample, and the mixed sample is subjected to multiplex fluorescent capillary gel electrophoresis.
基于所述多重荧光毛细管凝胶电泳的结果，比较所述FLT3基因的ITD区或其片段、NPM1 基因或其片段和CEBPA基因或其片段中的一个或多个(优选全部)的扩增产物的大小与相应的野生型基因或其片段的大小，如两者一致，则判定为受试者的基因未发生突变，如两者不一致，则判定为受试者的该基因发生了突变。其中，所述野生型FLT3基因的ITD区的大小为365bp，所述野生型NPM1基因片段的大小为283bp，所述野生型CEBPA基因的片段有四个，其大小分别为309bp、345bp、444bp和416bp。当所述扩增产物的大小与相应的野生型基因或其片段的大小相差±1bp以下时，认定为两者一致；当所述扩增产物的大小与相应的野生型基因或其片段的大小相差大于±1bp时，认定为两者不一致。Comparing the ITD region of the FLT3 gene or a fragment thereof, NPM1 based on the result of the multiplex fluorescent capillary gel electrophoresis The size of the amplification product of one or more (preferably all) of the gene or fragment thereof and the CEBPA gene or a fragment thereof is identical to the size of the corresponding wild-type gene or a fragment thereof, such as both, and is determined to be the subject's The gene did not mutate, and if the two were inconsistent, it was determined that the subject had a mutation in the gene. Wherein, the size of the ITD region of the wild-type FLT3 gene is 365 bp, the size of the wild-type NPM1 gene fragment is 283 bp, and the fragment of the wild-type CEBPA gene is four, and the sizes thereof are 309 bp, 345 bp, 444 bp, and 416 bp. When the size of the amplification product differs from the size of the corresponding wild-type gene or a fragment thereof by ±1 bp or less, it is determined that the two are identical; when the size of the amplification product is related to the size of the corresponding wild-type gene or fragment thereof When the difference is greater than ±1 bp, it is considered that the two are inconsistent.
上述所述FLT3基因的ITD区、NPM1基因和CEBPA基因的突变情况与急性髓细胞白血病患者的预后的关系见下表：The relationship between the mutation of the ITD region, the NPM1 gene and the CEBPA gene of the FLT3 gene described above and the prognosis of patients with acute myeloid leukemia is as follows:
Figure PCTCN2015094788-appb-000001
Figure PCTCN2015094788-appb-000001
因此，本发明的试剂盒可以用于急性髓细胞白血病预后分层，即，在判定结果为FLT3-ITD未发生突变而NMP1或CEBPA至少一者发生了突变的情况下，可以判定该患者的预后好；在判定结果为FLT3-ITD发生突变而NMP1或CEBPA至少一者发生了突变的情况下，可以判定该患者的预后中等；在判定结果为FLT3-ITD发生突变而NMP1和CEBPA均未发生突变的情况下，可以判定该患者的预后差。Therefore, the kit of the present invention can be used for prognosis stratification of acute myeloid leukemia, that is, in the case where the result is that the FLT3-ITD is not mutated and at least one of NMP1 or CEBPA is mutated, the prognosis of the patient can be determined. In the case where the result of the mutation is FLT3-ITD mutation and at least one of NMP1 or CEBPA is mutated, the prognosis of the patient can be determined to be moderate; in the judgment result, the mutation of FLT3-ITD and the mutation of NMP1 and CEBPA are not mutated. In the case of this, it can be determined that the patient's prognosis is poor.
实施例Example
以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。The present invention will be further described in detail below with reference to the embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
实施例中所用实验方法如无特殊说明，均为常规方法。实施例中所用材料、试剂等如无特殊说明，均可从商业途径得到。The experimental methods used in the examples are all conventional methods unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.
实施例1 荧光标记的引物的合成Example 1 Synthesis of Fluorescently Labeled Primers
以下荧光标记引物由上海捷瑞公司合成。The following fluorescently labeled primers were synthesized by Shanghai Jierui.
Figure PCTCN2015094788-appb-000002
Figure PCTCN2015094788-appb-000002
Figure PCTCN2015094788-appb-000003
Figure PCTCN2015094788-appb-000003
表中，F或FS表示上游引物，R或RS表示下游引物。In the table, F or FS represents an upstream primer, and R or RS represents a downstream primer.
实施例2 PCR及多重荧光毛细管凝胶电泳检测Example 2 PCR and multiplex fluorescent capillary gel electrophoresis
1)提取血液中的总RNA1) Extract total RNA from the blood
a.取200μL血液，加入1mLTrizol(Invitrogen公司)，剧烈震荡混匀，室温放置10分钟；a. Take 200 μL of blood, add 1 mL of Trizol (Invitrogen), mix vigorously and shake, and let stand for 10 minutes at room temperature;
b.向a中加入200μL氯仿，剧烈混匀；室温放置5分钟； b. Add 200 μL of chloroform to a, mix vigorously; place at room temperature for 5 minutes;
c.12000rpm，4℃离心15分钟；c. 12000 rpm, centrifugation at 4 ° C for 15 minutes;
d.将上清吸到另一个新的1.5mL离心管中；d. suck the supernatant into another new 1.5 mL centrifuge tube;
e.向d中加入等倍体积的异丙醇，室温放置30分钟；e. Add an equal volume of isopropanol to d, and leave it at room temperature for 30 minutes;
f.12000rpm，4℃离心15分钟；弃上清；f. 12000 rpm, centrifugation at 4 ° C for 15 minutes; discard the supernatant;
g.用500μL75％的乙醇洗涤沉淀，洗涤两遍；g. Wash the precipitate with 500 μL of 75% ethanol and wash twice;
h.用20μLddH2O(无RNA酶)溶解沉淀；h. Dissolve the pellet with 20 μL of ddH 2 O (no RNase);
i.电泳检测提取RNA的完整性，并用Qubit或者Nanodrop测定RNA的浓度。i. Electrophoretic detection of RNA integrity and determination of RNA concentration using Qubit or Nanodrop.
2)反转录得到cDNA2) Reverse transcription to obtain cDNA
a.取1μg提取的RNA，65℃10分钟后，立即置于冰上5分钟；a. 1 μg of the extracted RNA, immediately after 10 minutes at 65 ° C, placed on ice for 5 minutes;
b.向a中加入1μL随机引物，同时加入以下反应成分：b. Add 1 μL of random primer to a while adding the following reaction components:
Figure PCTCN2015094788-appb-000004
Figure PCTCN2015094788-appb-000004
c.42℃反应1小时；c. 42 ° C reaction for 1 hour;
d.反应结束后，65℃10分钟使酶失活。得到的cDNA用于后续PCR反应。d. After the reaction was completed, the enzyme was inactivated at 65 ° C for 10 minutes. The resulting cDNA was used for subsequent PCR reactions.
3)利用2)中的cDNA做模板，使用4对引物通过多重PCR扩增CEBPA基因的完整表达框。3) Using the cDNA in 2) as a template, the complete expression cassette of the CEBPA gene was amplified by multiplex PCR using 4 pairs of primers.
4)反应体系：4) Reaction system:
Figure PCTCN2015094788-appb-000005
Figure PCTCN2015094788-appb-000005
PCR反应条件：94℃预变性5分钟，(95℃变性1分钟，55℃退火30秒，72℃延伸30秒)30个循环，72℃延伸5分钟。 PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C extension for 5 minutes.
5)提取血液因组DNA，具体步骤如下：5) Extract the blood group DNA, the specific steps are as follows:
a.向200μL血液中加入600μLDNA提取液；a. Add 600 μL of DNA extract to 200 μL of blood;
b.加蛋白酶K至终浓度为100μg/mL，50-56℃消化2小时；b. Add proteinase K to a final concentration of 100 μg / mL, digest at 50-56 ° C for 2 hours;
c.加入300μLTris-饱和酚，反转数次，充分混匀。10000rpm，15分钟，4℃离心，吸取上清；c. Add 300 μL of LTris-saturated phenol, reverse several times, and mix well. Centrifuge at 10,000 rpm for 15 minutes at 4 ° C to absorb the supernatant;
d.重复上一步骤；d. repeat the previous step;
e.加入200μL氯仿，反转数次，充分混匀。10000rpm4℃离心15分钟，吸取上清；e. Add 200 μL of chloroform, invert several times, and mix well. Centrifuge at 10,000 rpm for 4 minutes at 4 ° C, and aspirate the supernatant;
f.加入与上清等体积的异丙醇(或2倍体积的无水乙醇)，反转数次，充分混匀。室温下放置30分钟；f. Add an equal volume of isopropanol (or 2 volumes of absolute ethanol) to the supernatant, invert several times, and mix well. Leave at room temperature for 30 minutes;
g.12000rpm，4℃离心15分钟，弃上清；g. 12000 rpm, centrifuge at 15 ° C for 15 minutes, discard the supernatant;
h.用75％乙醇漂洗沉淀1-2次，晾干；h. Rinse the precipitate 1-2 times with 75% ethanol and dry it;
i.加入50-100mLTE/H2O溶解(根据起始样品量可以调整最终溶解体积)。i. Add 50-100 m LTE/H 2 O to dissolve (the final dissolved volume can be adjusted according to the starting sample amount).
6)利用5)中的基因组DNA为模板，使用两重PCR扩增FLT3-ITD和NMP1基因6) Using the genomic DNA in 5) as a template, double-PCR amplification of FLT3-ITD and NMP1 genes
7)反应体系：7) Reaction system:
Figure PCTCN2015094788-appb-000006
Figure PCTCN2015094788-appb-000006
PCR反应条件：94℃预变性5分钟，(95℃变性1分钟，55℃退火30秒，72℃延伸30秒)30个循环，72℃终延伸5分钟。PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, (denaturation at 95 ° C for 1 minute, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds) 30 cycles, 72 ° C final extension for 5 minutes.
8)将3)和7)中的PCR产物混合进行多重毛细管电泳检测。8) Mix the PCR products in 3) and 7) for multicapillary electrophoresis.
根据基因突变判读结果，判读方法：野生型NPM1基因片段大小为283bp；野生型FLT3基因的ITD区的片段大小为365bp；野生型CEPBA基因的片段有四个，其大小分别为309bp，345bp，444bp和415bp。根据峰对应的片段的大小来判断待检测基因中是否发生***或者缺失突变。According to the results of gene mutation interpretation, the interpretation method: wild-type NPM1 gene fragment size is 283 bp; wild-type FLT3 gene ITD region fragment size is 365 bp; wild-type CEPBA gene fragment has four, its size is 309 bp, 345 bp, 444 bp And 415 bp. Whether or not an insertion or deletion mutation occurs in the gene to be detected is determined based on the size of the fragment corresponding to the peak.
9)对AML的预后进行指导。9) Guide the prognosis of AML.
对于一个男性AML患者，检测结果如图1所示。可知：NPM1基因的扩增产物的片段大 小为283bp，表明该检测基因未发生突变；CEBPA基因的4个扩增片段的大小分别为309bp、345bp、444bp和416bp，均为野生型，表明CEBPA检测基因未发生突变；FLT3基因扩增产物的大小为383bp，表明该基因发生了18bp的***突变。该样本只有FLT3发生了***突变，而CEBPA和NPM1基因均未发生突变，表明该男性AML样本的预后较差。For a male AML patient, the test results are shown in Figure 1. It can be seen that the fragment of the amplification product of the NPM1 gene is large. The small size was 283 bp, indicating that the detection gene was not mutated; the size of the four amplified fragments of CEBPA gene were 309 bp, 345 bp, 444 bp and 416 bp, both of which were wild type, indicating that the CEBPA detection gene was not mutated; FLT3 gene amplification product The size is 383 bp, indicating that the gene has an 18 bp insertion mutation. In this sample, only the insertion mutation occurred in FLT3, and the CEBPA and NPM1 genes did not mutate, indicating that the male AML sample had a poor prognosis.
对于一个女性AML患者，检测结果如图2所示。可知：NPM1基因的扩增产物的大小为287bp，表明该检测基因发生了4bp的***突变；CEBPA基因的4个扩增片段的大小分别为309bp、345bp、444bp和416bp，均为野生型大小片段，表明CEBPA检测基因未发生突变；FLT3基因的ITD区的扩增产物的大小为365bp，为野生型，表明该样本的FLT3未发生改变。该样本只有NPM1发生了***突变，而CEBPA和FLT3基因均未发生突变，表明该女性AML样本的预后较好。For a female AML patient, the test results are shown in Figure 2. It can be seen that the size of the amplified product of NPM1 gene is 287 bp, indicating that the detection gene has a 4 bp insertion mutation; the four amplified fragments of CEBPA gene are 309 bp, 345 bp, 444 bp and 416 bp, respectively, all of which are wild-type fragments. , indicating that the CEBPA detection gene did not mutate; the size of the amplification product of the ITT region of the FLT3 gene was 365 bp, which was wild-type, indicating that the FLT3 of the sample did not change. In this sample, only the insertion mutation occurred in NPM1, and the CEBPA and FLT3 genes did not mutate, indicating that the prognosis of the female AML sample is better.
还需要说明的是，在可实施且不明显违背本发明的主旨的前提下，在本说明书中作为某一技术方案的构成部分所描述的任一技术特征或技术特征的组合同样也可以适用于其它技术方案；并且，在可实施且不明显违背本发明的主旨的前提下，作为不同技术方案的构成部分所描述的技术特征之间也可以以任意方式进行组合，来构成其它技术方案。本发明也包含在上述情况下通过组合而得到的技术方案，并且这些技术方案相当于记载在本说明书中。It should also be noted that any technical feature or combination of technical features described in the specification as a component of a certain technical solution may also be applied to the embodiments that can be implemented without obscuring the gist of the present invention. Other technical solutions; and, while being able to implement and not clearly deviating from the gist of the present invention, the technical features described as the constituent parts of the different technical solutions may be combined in any manner to constitute other technical solutions. The present invention also encompasses the technical solutions obtained by the combination in the above case, and these technical solutions are equivalent to those described in the present specification.
上述说明示出并描述了本发明的优选实施例，如前所述，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域技术人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。The above description shows and describes a preferred embodiment of the present invention. As described above, it should be understood that the present invention is not limited to the form disclosed herein, and should not be construed as being Combinations, modifications, and environments are possible, and can be modified by the teachings of the above teachings or related art within the scope of the inventive concept described herein. The modifications and variations made by those skilled in the art are intended to be within the scope of the appended claims.
工业实用性Industrial applicability
根据本发明，提供一种能够快速地检测多基因/多位点突变的、用于急性髓细胞白血病预后分层的试剂盒及检测方法。 According to the present invention, there is provided a kit and a detection method for prognostic stratification of acute myeloid leukemia capable of rapidly detecting polygenic/multiple site mutations.

Claims (8)

  1. 一种用于急性髓细胞白血病预后分层的试剂盒，其包括用于检测FLT3基因的ITD区或其片段的引物组1、用于检测NPM1基因或其片段的引物组2、以及用于检测CEBPA基因或其片段的引物组3；A kit for prognosis stratification of acute myeloid leukemia, comprising a primer set for detecting an ITD region of a FLT3 gene or a fragment thereof, a primer set 2 for detecting an NPM1 gene or a fragment thereof, and for detecting Primer set 3 of the CEBPA gene or a fragment thereof;
    所述每个引物组均包括上游引物和下游引物，且所述上游引物和下游引物中的至少一个是经过荧光分子标记的。Each of the primer sets includes an upstream primer and a downstream primer, and at least one of the upstream primer and the downstream primer is labeled with a fluorescent molecule.
  2. 根据权利要求1所述的试剂盒，其中，所述荧光分子选自FAM、HEX、TET、VIC、Cy3、Cy5和ROX7。The kit according to claim 1, wherein the fluorescent molecule is selected from the group consisting of FAM, HEX, TET, VIC, Cy3, Cy5, and ROX7.
  3. 根据权利要求2所述的试剂盒，其中，对于用于检测大小相近的基因或其片段的各引物组，采用不同的荧光分子进行标记。The kit according to claim 2, wherein each of the primer sets for detecting genes of similar sizes or fragments thereof is labeled with a different fluorescent molecule.
  4. 根据权利要求1所述的试剂盒，其中，The kit according to claim 1, wherein
    所述引物组1由核苷酸序列如SEQ ID NO：1所示的上游引物和核苷酸序列如SEQ ID NO：2所示的下游引物组成；The primer set 1 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2;
    所述引物组2由核苷酸序列如SEQ ID NO：3所示的上游引物和核苷酸序列如SEQ ID NO：4所示的下游引物组成；和The primer set 2 is composed of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 4;
    所述引物组3是下述引物组31～34：The primer set 3 is the following primer set 31 to 34:
    引物组31，其由核苷酸序列如SEQ ID NO：5所示的上游引物和核苷酸序列如SEQ ID NO：6所示的下游引物组成；a primer set 31 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6;
    引物组32，其由核苷酸序列如SEQ ID NO：7所示的上游引物和核苷酸序列如SEQ ID NO：8所示的下游引物组成；a primer set 32 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8;
    引物组33，其由核苷酸序列如SEQ ID NO：9所示的上游引物和核苷酸序列如SEQ ID NO：10所示的下游引物组成；和a primer set 33 consisting of an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 10;
    引物组34，其由核苷酸序列如SEQ ID NO：11所示的上游引物和核苷酸序列如SEQ ID NO：12所示的下游引物组成。Primer set 34 consists of an upstream primer as shown in SEQ ID NO: 11 and a downstream primer as shown in SEQ ID NO: 12.
  5. 一种同时检测受试者的FLT3基因的ITD区、NPM1基因和CEBPA基因的基因突变的方法，其使用权利要求1～4中任一项所述的试剂盒，其包括：A method for simultaneously detecting a gene mutation of an ITD region, an NPM1 gene, and a CEBPA gene of a subject's FLT3 gene, using the kit of any one of claims 1 to 4, comprising:
    A.以所述引物组1作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的FLT3基因的ITD区或其片段，得到扩增产物A；A. using the primer set 1 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the ITD region of the FLT3 gene of the sample or a fragment thereof, to obtain an amplification product A;
    B.以所述引物组2作为引物，以来自受试者的样本的基因组DNA作为模板，扩增样本的NPM1基因或其片段，得到扩增产物B；B. using the primer set 2 as a primer, using the genomic DNA of the sample from the subject as a template, amplifying the NPM1 gene of the sample or a fragment thereof to obtain an amplification product B;
    C.以所述引物组3作为引物，以由来自受试者的样本的总RNA反转录得到的cDNA作 为模板，扩增样本的CEBPA基因或其片段，得到扩增产物C；C. Using the primer set 3 as a primer, the cDNA obtained by reverse transcription of total RNA from a sample of the subject is used as a primer. As a template, amplifying the CEBPA gene of the sample or a fragment thereof to obtain an amplification product C;
    D.将上述扩增产物A、扩增产物B和扩增产物C混合，得到混合试样，并对该混合试样进行多重荧光毛细管凝胶电泳。D. The above amplification product A, amplification product B and amplification product C are mixed to obtain a mixed sample, and the mixed sample is subjected to multiplex fluorescent capillary gel electrophoresis.
  6. 根据权利要求5所述的方法，其还包括：基于所述多重荧光毛细管凝胶电泳的结果，比较所述FLT3基因的ITD区或其片段、NPM1基因或其片段和CEBPA基因或其片段中的一个或多个的扩增产物的大小与相应的野生型基因或其片段的大小，如两者一致，则判定为受试者的基因未发生突变，如两者不一致，则判定为受试者的该基因发生了突变。The method according to claim 5, further comprising: comparing the ITD region of the FLT3 gene or a fragment thereof, the NPM1 gene or a fragment thereof, and the CEBPA gene or a fragment thereof based on the result of the multiplex fluorescent capillary gel electrophoresis If the size of one or more amplification products is consistent with the size of the corresponding wild-type gene or a fragment thereof, such as both, it is determined that the subject's gene has not been mutated, and if the two are inconsistent, the subject is determined to be a subject. The gene has been mutated.
  7. 根据权利要求6所述的方法，其中，所述野生型FLT3基因的ITD区的大小为365bp，所述野生型NPM1基因片段的大小为283bp，所述野生型CEBPA基因的片段有四个，其大小分别为309bp、345bp、444bp和416bp；且当所述扩增产物的大小与相应的野生型基因的片段的大小相差±1bp以下时，认定为两者一致。The method according to claim 6, wherein the size of the ITD region of the wild type FLT3 gene is 365 bp, the size of the wild type NPM1 gene fragment is 283 bp, and the fragment of the wild type CEBPA gene has four, The sizes were 309 bp, 345 bp, 444 bp, and 416 bp, respectively; and when the size of the amplified product differed from the size of the fragment of the corresponding wild-type gene by ±1 bp or less, it was confirmed that the two were identical.
  8. 根据权利要求5～7中任一项所述的方法，其中，所述样本为全血或骨髓。 The method according to any one of claims 5 to 7, wherein the sample is whole blood or bone marrow.
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