WO2017113148A1 - 检测急性早幼粒细胞白血病相关融合基因的试剂盒 - Google Patents

检测急性早幼粒细胞白血病相关融合基因的试剂盒 Download PDF

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WO2017113148A1
WO2017113148A1 PCT/CN2015/099764 CN2015099764W WO2017113148A1 WO 2017113148 A1 WO2017113148 A1 WO 2017113148A1 CN 2015099764 W CN2015099764 W CN 2015099764W WO 2017113148 A1 WO2017113148 A1 WO 2017113148A1
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dna
seq
nucleotide sequence
stranded dna
sequencing
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PCT/CN2015/099764
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French (fr)
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王秀莉
刘伟
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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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention relates to a kit and a detection method capable of simultaneously detecting a plurality of acute promyelocytic leukemia-associated fusion genes, and belongs to the technical field of molecular detection.
  • APL Acute promyelocyte leukemia
  • APL is a special type of acute leukemia with specific genes and karyotype changes. The clinical manifestations are dangerous, and bleeding and embolism are easy to cause death during the onset and treatment.
  • APL has become one of the curable leukemias due to the clinical application of all-trans retinoic acid (ATRA) and arsenic.
  • APL is easy to see in young and middle-aged people, with an average age of onset of 39 years.
  • Epidemiological studies have confirmed that the incidence of APL in foreign countries accounts for 5.0% to 23.8% of leukemia in the same period, and 6.2% to 40.2% of acute myeloid leukemia (AML). Many domestic researchers reported that the incidence rate accounted for 3.3% to 21.2% of acute leukemia in the same period.
  • detection of fusion genes is one of the most specific and sensitive methods for the diagnosis of APL, and is the most reliable indicator for APL treatment options, efficacy analysis, prognosis analysis and recurrence prediction.
  • the object of the present invention is to provide a novel second-generation sequencing-based APL-related fusion gene detection method, detection kit, and method for constructing a second-generation sequencing DNA library for the above detection method, and a library kit.
  • the present invention includes:
  • a method for constructing a second generation sequencing DNA library for APL-associated fusion gene detection comprising:
  • Step A End-repairing the cDNA desired to be sequenced to obtain a blunt-ended DNA fragment obtained by reverse transcription of total RNA extracted from human blood or bone marrow samples;
  • Step B adding a blunt-end DNA fragment to the 3' end and adding A to obtain a DNA fragment having a 3' end and A;
  • Step C adding a DNA fragment of the 3' end to A to obtain a linker DNA fragment
  • Step D performing the first PCR amplification on the ligated DNA fragment to obtain a first amplification product
  • Step E performing nested PCR amplification of the first amplification product to obtain a second amplification product
  • step C an annealing product of a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 2 is used as the linker;
  • a single-stranded DNA having a nucleotide sequence such as SEQ ID NOS: 51 to 62 and a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 6 are used as PCR amplification primers;
  • step E a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NOS: 31 to 42, a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NO: 4, and a nucleotide sequence such as SEQ ID NO:
  • the single-stranded DNA shown in :6 was used as a PCR amplification primer.
  • a method for detecting APL-associated fusion genes comprising the steps of:
  • Step A End-repairing the cDNA desired to be sequenced to obtain a blunt-ended DNA fragment obtained by reverse transcription of total RNA extracted from human blood or bone marrow samples;
  • Step B adding a blunt-end DNA fragment to the 3' end and adding A to obtain a DNA fragment having a 3' end and A;
  • Step C adding a DNA fragment of the 3' end to A to obtain a linker DNA fragment
  • Step D performing the first PCR amplification on the ligated DNA fragment to obtain a first amplification product
  • Step E performing nested PCR amplification of the first amplification product to obtain a second amplification product
  • Step F performing second generation sequencing on the second amplification product, and performing bioinformatics analysis based on the sequencing result;
  • step C an annealing product of a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 2 is used as the linker;
  • a single-stranded DNA having a nucleotide sequence such as SEQ ID NOS: 51 to 62 and a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 6 are used as PCR amplification primers;
  • step E a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NOS: 31 to 42, a single-stranded DNA (index primer) having a nucleotide sequence as shown in SEQ ID NO: 4, and a nucleotide sequence are used.
  • Single-stranded DNA (common primer) as shown in SEQ ID NO: 6 was used as a PCR amplification primer.
  • step of purifying the product is further included between the step C and the step D, between the step D and the step E, and/or after the step E.
  • the APL-related fusion gene comprises PLZF-RAR ⁇ , NuMA-RAR ⁇ , NPM1-RAR ⁇ , St ⁇ t5b-RAR ⁇ , F1P1L1-RAR ⁇ , PRKAR1A-RAR ⁇ , BCOR appearing in atypical APL.
  • -RAR ⁇ and PML-RAR ⁇ present in typical APL.
  • a kit for constructing a second generation sequencing DNA library for APL-associated fusion gene detection comprising reagents for second generation sequencing DNA library construction, the reagents for second generation sequencing DNA library construction comprising:
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NOS: 31-42;
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NO: 4;
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NO: 6;
  • the nucleotide sequence is a single-stranded DNA as shown in SEQ ID NOS: 51 to 62.
  • kit of clause 8 further comprising reagents for total RNA extracted from human blood or bone marrow samples, and reverse transcription of the total RNA to obtain a first strand of cDNA and a second strand of cDNA.
  • the reagent for constructing the second generation DNA sequencing library further comprises at least one or more selected from the group consisting of T4 DNA polymerase, Klenow fragment, Klenow Buffer, DNA ligase buffer, DNA ligase, Taq enzyme, dNTP, T4 polynucleotide kinase, and T4 polynucleotide kinase buffer.
  • the kit according to Item 9 wherein the reagent for the total RNA extracted from human blood or bone marrow samples and which is reverse transcribed from the total RNA to obtain the first strand of the cDNA and the second strand of the cDNA includes At least one or more selected from the group consisting of Trizol reagent, chloroform, isopropanol, ethanol, RNase-free double distilled water, random primer, reverse transcriptase, reverse transcription reaction buffer, RNase Inhibitor, dNTP, DNA polymerase, and ribonuclease H.
  • a kit for APL-associated fusion gene detection comprising:
  • the reagent for constructing the second generation sequencing DNA library comprises:
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NOS: 31-42;
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NO: 4;
  • nucleotide sequence such as the single-stranded DNA shown in SEQ ID NO: 6;
  • the nucleotide sequence is a single-stranded DNA as shown in SEQ ID NOS: 51 to 62.
  • kit of clause 13 further comprising reagents for total RNA extracted from human blood or bone marrow samples and which are reverse transcribed from the total RNA to obtain a first strand of cDNA and a second strand of cDNA.
  • the reagent for constructing the second generation DNA sequencing library further comprises at least one or more selected from the group consisting of T4 DNA polymerase, Klenow fragment, Klenow Buffer, DNA ligase buffer, DNA ligase, Taq enzyme, dNTP, T4 polynucleotide kinase, and T4 polynucleotide kinase buffer.
  • the reagent for total RNA extracted from human blood or bone marrow samples and reverse-transcribed from the total RNA to obtain a first strand of cDNA and a second strand of cDNA comprises At least one or more selected from the group consisting of Trizol reagent, chloroform, isopropanol, ethanol, RNase-free double distilled water, random primer, reverse transcriptase, reverse transcription reaction buffer, RNase Inhibitor, dNTP, DNA polymerase, and ribonuclease H.
  • the reagent for performing sequencing on the second generation sequencing DNA library comprises at least one or more selected from the group consisting of: a resynthesis reagent, a linearized P7 linker , linearized P5 linker, DNA polymerase, dNTP, rinsing hybridization/buffer, 100% formamide (mass/volume), Read 2 primers for sequencing, Index i7 sequencing primers, Read 1 primers for sequencing, Hiseq Rapid PE Flow Cell, water, and enhanced light sensitivity/photographic reagents.
  • a plurality of APL-related fusion genes can be simultaneously detected based on the second-generation sequencing technology, and the molecular detection basis is provided for the diagnosis of acute promyelocytic leukemia (APL) and the determination of minimal residual disease (MRD).
  • APL acute promyelocytic leukemia
  • MRD minimal residual disease
  • the present invention provides a method for constructing a second generation sequencing DNA library for APL-related fusion gene detection (the method of building a library of the present invention), the method comprising:
  • Step A End-repairing the cDNA desired to be sequenced to obtain a blunt-ended DNA fragment obtained by reverse transcription of total RNA extracted from human blood or bone marrow samples;
  • Step B adding a blunt-end DNA fragment to the 3' end and adding A to obtain a DNA fragment having a 3' end and A;
  • Step C adding a DNA fragment of the 3' end to A to obtain a linker DNA fragment
  • Step D performing PCR amplification on the ligated DNA fragment to obtain an amplification product
  • step C an annealing product of a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 2 is used as the linker (P5 linker). ;
  • a single-stranded DNA (GSP1 primer pool) having a nucleotide sequence as shown in SEQ ID NOS: 51 to 62 and a single-stranded DNA (common primer) having a nucleotide sequence as shown in SEQ ID NO: 6 are used.
  • GSP1 primer pool having a nucleotide sequence as shown in SEQ ID NOS: 51 to 62 and a single-stranded DNA (common primer) having a nucleotide sequence as shown in SEQ ID NO: 6 are used.
  • a single-stranded DNA (GSP2 primer pool) having a nucleotide sequence as shown in SEQ ID NOS: 31 to 42 and a single-stranded DNA (Index primer) having a nucleotide sequence as shown in SEQ ID NO: 4 are used.
  • a single-stranded DNA (common primer) having a nucleotide sequence as shown in SEQ ID NO: 6 as a PCR amplification primer.
  • APL refers to acute promyelocyte leukemia.
  • the APL-associated fusion gene is a specific marker for acute promyelocytic leukemia. By adopting the present invention, it can be detected theoretically as long as it is a fusion with RAR ⁇ or a break point, including but not limited to: PLZF-RAR ⁇ , NuMA-RAR ⁇ , NPM1-RAR ⁇ , St ⁇ t5b- appearing in atypical APL. RAR ⁇ , F1P1L1-RAR ⁇ , PRKAR1A-RAR ⁇ , BCOR-RAR ⁇ , and PML-RAR ⁇ appearing in typical APL.
  • the method for constructing a cell of the present invention further comprises the steps of: extracting total RNA from a human blood or bone marrow sample, and reverse transcription of the total RNA from the first strand of the cDNA and the second strand of the cDNA before the step A .
  • the specific method used in this step is not particularly limited, and those generally used by those skilled in the art can be used.
  • total RNA can be extracted by using a Trizol reagent according to a standard operation procedure, and then DNase digestion and mRNA purification are not performed directly. Reverse transcription was performed using random primers.
  • the steps A and B can be carried out by a conventional method in the art.
  • an annealed product of a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 2 is used as a linker to add a linker.
  • This step can be carried out in a corresponding reaction system using a DNA ligase such as T4 DNA ligase.
  • PCR amplification was carried out using each of the above single-stranded DNAs as PCR amplification primers.
  • This step can be carried out in a corresponding reaction system using a DNA polymerase (for example, Extaq DNA polymerase).
  • the step of purifying the product is further included between the step C and the step D, after the step D and the step E, and/or after the step E.
  • the purification step can be carried out by methods generally employed in the art, such as magnetic bead purification.
  • kits for constructing a second-generation sequencing DNA library for APL-related fusion gene detection (the library of the present invention) Kits, which are useful in practicing the methods of constructing the invention, comprising reagents for constructing a second generation sequencing DNA library, the reagents for constructing a second generation sequencing DNA library comprising:
  • the single-stranded DNA having the nucleotide sequence as shown in SEQ ID NOS: 31 to 42 and the single-stranded DNA shown in SEQ ID NO: 4 and the nucleotide sequence as shown in SEQ ID NO: 6 Chain DNA;
  • the nucleotide sequence is a single-stranded DNA represented by SEQ ID NOS: 51 to 62 and a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NO: 6.
  • the reagent for constructing the second generation DNA sequencing library further comprises at least one or more selected from the group consisting of T4 DNA polymerase, Klenow fragment, Klenow buffer, DNA ligase buffer, DNA Ligase, Taq enzyme, dNTP, T4 polynucleoside Acid kinase, and T4 polynucleotide kinase buffer.
  • the library kit of the present invention may further comprise reagents for total RNA extracted from human blood or bone marrow samples, and reverse transcription of the total RNA to obtain a first strand of cDNA and a second strand of cDNA.
  • the reagent for total RNA extracted from human blood or bone marrow samples and reverse-transcribed from the total RNA to obtain a first strand of cDNA and a second strand of cDNA comprises at least one selected from the group consisting of or Two or more: Trizol reagent, chloroform, isopropanol, ethanol, RNase-free double distilled water, random primer, reverse transcriptase, reverse transcription reaction buffer, RNase inhibitor, dNTP, DNA polymerase, and ribose Nuclease H.
  • APL cell leukemia
  • MRD minimal residual disease
  • the present invention provides an APL-related fusion gene detection method (detection method of the present invention), the method comprising the steps of:
  • Step A End-repairing the cDNA desired to be sequenced to obtain a blunt-ended DNA fragment obtained by reverse transcription of total RNA extracted from human blood or bone marrow samples;
  • Step B adding a blunt-end DNA fragment to the 3' end and adding A to obtain a DNA fragment having a 3' end and A;
  • Step C adding a DNA fragment of the 3' end to A to obtain a linker DNA fragment
  • Step D performing the first PCR amplification on the ligated DNA fragment to obtain a first amplification product
  • Step E performing nested PCR amplification of the first amplification product to obtain a second amplification product
  • Step F performing second generation sequencing on the second amplification product, and performing bioinformatics analysis based on the sequencing result;
  • step C an annealing product of a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as SEQ ID NO: 2 is used as the linker;
  • a single-stranded DNA having a nucleotide sequence such as SEQ ID NOS: 51 to 62 and a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 6 are used as PCR amplification primers;
  • step E a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NOS: 31 to 42, a single-stranded DNA (index primer) having a nucleotide sequence as shown in SEQ ID NO: 4, and a nucleotide sequence are used.
  • Single-stranded DNA (common primer) as shown in SEQ ID NO: 6 was used as a PCR amplification primer.
  • the detecting method of the present invention can add the step F to the building method of the present invention.
  • the second amplification product is subjected to second generation sequencing, and bioinformatics analysis is performed based on the sequencing result.
  • second generation sequencing can be performed using conventional methods in the art, for example, second generation sequencing can be performed using an Illumina platform (e.g., HiSeq 2500 or NextSeq 500).
  • bioinformatics analysis is performed based on the sequencing result, which can be performed, for example, by obtaining the reads obtained by sequencing.
  • the detection method of the present invention can be carried out, for example, using a kit, and therefore, in another aspect, the present invention provides a kit for detection of APL-related fusion gene (test kit of the present invention) comprising:
  • the reagent for constructing the second generation sequencing DNA library comprises:
  • the single-stranded DNA having the nucleotide sequence as shown in SEQ ID NOS: 31 to 42 and the single-stranded DNA shown in SEQ ID NO: 4 and the nucleotide sequence as shown in SEQ ID NO: 6 Chain DNA;
  • the nucleotide sequence is a single-stranded DNA represented by SEQ ID NOS: 51 to 62 and a single-stranded DNA having a nucleotide sequence as shown in SEQ ID NO: 6.
  • the detection kit of the present invention can add an agent for performing on-the-sequence sequencing of the constructed second-generation sequencing DNA library based on the library kit of the present invention.
  • the reagent for sequencing the second-generation sequencing DNA library including but not limited to at least one or more selected from the group consisting of: a resynthesis reagent, a linearized P7 linker, a linearized P5 linker, and a DNA polymerization Enzyme, dNTP, rinsing hybridization/buffer, 100% formamide (mass/volume), Read 2 primers for sequencing, Index i7 sequencing primers, Read 1 primers for sequencing, Hiseq Rapid PE Flow Cell, water, And enhanced light sensitivity / photographic reagents.
  • each reagent or device is preferably packaged separately, but may be mixed and packaged without affecting the practice of the present invention.
  • the library was constructed with 250 ng of total RNA, and a single sample was single reaction.
  • Sample code RNR130626 (sample 1, clinical patient), RNR130627 (sample 2, clinical patient), HC14CS00067 (sample 3, healthy person) 250ng total RNA without DNase digestion and mRNA capture purification, direct random primer reverse transcription, experimental procedure as follows:
  • the synthesized cDNA-stranded product was recovered with Ampure XP Beads (1.8 ⁇ ), and EB was dissolved, and the EB volume was 21 ⁇ L.
  • the 5 x First strand buffer reagent was from Invitrogen.
  • the 5 ⁇ Second strand buffer reagent was from Invitrogen.
  • the synthesized cDNA double-stranded product was recovered with Ampure XP Beads (1.8 ⁇ ), and EB was dissolved, and the EB volume was 37 ⁇ L.
  • the reaction was carried out for 30 minutes at 20 ° C in a thermomixer, and the reaction product was recovered with Ampure XP Beads (1.8 ⁇ ), and EB was dissolved, and the EB volume was 33 ⁇ L.
  • the reaction was carried out at a thermomixer at 37 ° C for 30 minutes.
  • the reaction product was recovered with Ampure XP Beads (1.8 ⁇ ), and EB was dissolved, and the EB volume was 20 ⁇ L.
  • the reaction was carried out for 15 minutes at 20 ° C in a thermomixer, and the reaction product was recovered with Ampure XP Beads (1.8 ⁇ ), and EB was dissolved, and the EB volume was 20 ⁇ L.
  • the Adapter is an annealing product of the nucleotide sequence shown by SEQ ID NO: 1 and the nucleotide sequence shown by SEQ ID NO: 2.
  • HiFi DNA Polymerase Mix is from kapa biosystems.
  • Step 7 Purification of the magnetic beads is the same as step 7.
  • HiFi DNA Polymerase Mix is from kapa biosystems.
  • Sample 1 was positive for the NPM1-RAR ⁇ fusion gene, indicating that it may be an APL patient.
  • Sample 2 was positive for the PML-RAR ⁇ fusion gene, indicating that it may be an APL patient.
  • Sample 3 did not detect the fusion gene, indicating that it may be a normal person.
  • nucleotide sequences referred to in the description are as follows.
  • SEQ ID NO: 1 5'-GATCGGAAGAGCACACGTCT-3';
  • SEQ ID NO: 2 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3';
  • CTGCTCAGGTGTGTGCTGGCA CCAAAGAGGGCAGGCTA 53 GATGGTGTGCTATATCCACTA 54 CTTGCAGCCCTCACAGG 55 CCACTTCAAAGCACTTCTGCA 56 CGTAGTGTATTTGCCCAGCTG 57 CAGGATGTCCAGGCAGGC 58 CTCCGCAGATGAGGCAGATG 59 CCTTGGCGCTGATGCTTCG 60 ATCCAGTAGATCTCAGTGGAAAG 61 TCATGGTGGGTGCAGGCA 62 AGCAATGGCTTGTGAGTTCTG
  • 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.
  • a detection method for constructing a second generation sequencing DNA library for the above detection method based on the second generation sequencing technology, which can simultaneously detect a plurality of APL-related fusion genes, and a library reagent box.

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Abstract

提供了一种检测急性早幼粒细胞白血病相关融合基因的试剂盒。具体而言，提供了一种基于二代测序技术的、可同时检测急性早幼粒白血病（APL）相关的多种RARα融合基因的检测试剂盒、基因检测方法，以及用于上述检测方法的二代测序DNA文库的构建方法、建库试剂盒。

Description

检测急性早幼粒细胞白血病相关融合基因的试剂盒 技术领域
本发明涉及可同时检测多种急性早幼粒细胞白血病相关融合基因的试剂盒及检测方法，属于分子检测技术领域。
背景技术
急性早幼粒细胞白血病(Acute promyelocyte leukemia，APL)是一种有着特异基因与染色体核型改变的特殊类型急性白血病。临床表现凶险，起病及治疗过程中容易发生出血和栓塞而引起死亡。近二十年来，由于全反式维甲酸(ATRA)及砷剂的临床应用，APL已成为可以治愈的白血病之一。APL易见于中青年人，平均发病年龄为39岁，流行病学研究证实国外APL发病率占同期白血病的5.0％～23.8％，占急性髓系白血病(AML)的6.2％～40.2％。国内多位学者报道发病率占同期急性白血病的3.3％～21.2％。
在基因检测方面，检测融合基因是诊断APL的最特异、敏感的方法之一，也是APL治疗方案选择、疗效分析、预后分析和复发预测最可靠的指标。
在APL相关融合基因检测方面，需要首先提取血液或骨髓中的总RNA并反转录得到cDNA，然后针对每种融合基因设计特异Taqman探针进行实时荧光定量PCR扩增检测，或者利用多重巢式RT-PCR定性检测多种融合基因。前一种方法的缺点是检测通量受限且样本需求量大；后一种方法的缺点是流程繁琐、耗时长。
因此，迫切希望开发出新的APL相关融合基因的检测方法。
发明内容
本发明的目的在于提供一种新的基于二代测序的APL相关融合基因检测方法、检测试剂盒，以及构建用于上述检测方法的二代测序DNA文库的方法、建库试剂盒。
即，本发明包括：
1.一种用于APL相关融合基因检测的二代测序DNA文库的构建方法，该方法包括：
步骤A：将希望进行测序的cDNA进行末端修复，得到平末端DNA片段，该cDNA是由从人血液或骨髓样本中提取的总RNA反转录得到的；
步骤B：将平末端DNA片段进行3′端加A，得到3′端加A的DNA片段；
步骤C：将所述3′端加A的DNA片段进行加接头，得到加接头DNA片段；以及
步骤D：将所述加接头DNA片段进行第一次PCR扩增，得到第一次扩增产物；
步骤E：将所述第一次扩增产物进行巢式PCR扩增，得到第二次扩增产物；
其中，
所述步骤C中使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为所述接头；
所述步骤D中使用核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物；
所述步骤E中使用核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物。
2.根据项1所述的方法，其中，在所述步骤A之前还包括下述步骤：
从人血液或骨髓样本中提取的总RNA，并由该总RNA反转录得到cDNA第一链和cDNA第二链。
3.根据项1所述的方法，其中，在所述步骤C与步骤D之间、步骤D与步骤E之间、和/或所述步骤E之后还包括对产物进行纯化的步骤。
4.一种APL相关融合基因检测方法，该方法包括下述步骤：
步骤A：将希望进行测序的cDNA进行末端修复，得到平末端DNA片段，该cDNA是由从人血液或骨髓样本中提取的总RNA反转录得到的；
步骤B：将平末端DNA片段进行3′端加A，得到3′端加A的DNA片段；
步骤C：将所述3′端加A的DNA片段进行加接头，得到加接头DNA片段；以及
步骤D：将所述加接头DNA片段进行第一次PCR扩增，得到第一次扩增产物；
步骤E：将所述第一次扩增产物进行巢式PCR扩增，得到第二次扩增产物；以及
步骤F：对所述第二次扩增产物进行二代测序，并基于测序结果进行生物信息学分析；
其中，
所述步骤C中使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为所述接头；
所述步骤D中使用核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物；
所述步骤E中使用核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA(index引物)以及核苷酸序列如SEQ ID NO：6所示的单链DNA(公共引物)作为PCR扩增引物。
5.根据项4所述的方法，其中，在所述步骤A之前还包括下述步骤：
从人血液或骨髓样本中提取的总RNA，并由该总RNA反转录得到cDNA第一链和cDNA第二链。
6.根据项4所述的方法，其中，在所述步骤C与步骤D之间、步骤D与步骤E之间、和/或所述步骤E之后还包括对产物进行纯化的步骤。
7.根据项4所述的方法，其中，所述APL相关融合基因包括非典型APL中出现的PLZF-RARα、NuMA-RARα、NPM1-RARα、Stαt5b-RARα、F1P1L1-RARα、PRKAR1A-RARα、BCOR-RARα和典型APL中出现的PML-RARα。
8.一种用于APL相关融合基因检测的二代测序DNA文库构建的试剂盒，其包含用于二代测序DNA文库构建的试剂，所述用于二代测序DNA文库构建的试剂包含：
核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
核苷酸序列如SEQ ID NO：31～42所示的单链DNA；
核苷酸序列如SEQ ID NO：4所示的单链DNA；
核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
核苷酸序列如SEQ ID NO：51～62所示的单链DNA。
9.根据项8所述的试剂盒，其还包含用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂。
10.根据项8所述的试剂盒，其中，所述用于构建二代DNA测序文库的试剂还包括选自下组中的至少一种或两种以上：T4 DNA聚合酶、Klenow片段、Klenow缓冲液、DNA连接酶缓冲液、DNA连接酶、Taq酶、dNTP、T4多聚核苷酸激酶、以及T4多聚核苷酸激酶缓冲液。
11.根据项9所述的试剂盒，其中，所述用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂包括选自下组中的至少一种或两种以上：Trizol试剂、氯仿、异丙醇、乙醇、无RNA酶的双蒸水、随机引物、反转录酶、反转录反应缓冲液、RNA酶抑制剂、dNTP、DNA聚合酶以及核糖核酸酶H。
12.一种用于APL相关融合基因检测的试剂盒，其包含：
用于构建二代测序DNA文库的试剂，以及
用于对二代测序DNA文库进行上机测序的试剂；
其中，所述用于构建二代测序DNA文库的试剂包含：
核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
核苷酸序列如SEQ ID NO：31～42所示的单链DNA；
核苷酸序列如SEQ ID NO：4所示的单链DNA；
核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
核苷酸序列如SEQ ID NO：51～62所示的单链DNA。
13.根据项12所述的试剂盒，其还包含用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂。
14.根据项12所述的试剂盒，其中，所述用于构建二代DNA测序文库的试剂还包括选自下组中的至少一种或两种以上：T4 DNA聚合酶、Klenow片段、Klenow缓冲液、DNA连接酶缓冲液、DNA连接酶、Taq酶、dNTP、T4多聚核苷酸激酶、以及T4多聚核苷酸激酶缓冲液。
15.根据项12所述的试剂盒，其中，所述用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂包括选自下组中的至少一种或两种以上：Trizol试剂、氯仿、异丙醇、乙醇、无RNA酶的双蒸水、随机引物、反转录酶、反转录反应缓冲液、RNA酶抑制剂、dNTP、DNA聚合酶以及核糖核酸酶H。
16.根据项12所述的试剂盒，其中，所述对二代测序DNA文库进行上机测序的试剂包括选自下组中的至少一种或两种以上：再合成试剂、线性化P7接头、线性化P5接头、DNA聚合酶、dNTP、冲洗杂交液/缓冲液、100％甲酰胺(质量/体积)、用于测序的Read 2引物、Index i7测序引物、用于测序的Read 1引物、Hiseq Rapid PE Flow Cell、水、以及增强光感度/照相用试剂。
17.根据项8～11中任一项所述的试剂盒，其用于实施项1～3中任一项所述的方法。
18.根据项12～16中任一项所述的试剂盒，其用于实施项4～7中任一项所述的方法。
发明效果
根据本发明，可以基于二代测序技术同时检测多种APL相关融合基因，为急性早幼粒细胞白血病(APL)的诊断、微小残留病(MRD)的判断提供分子检测基础。
发明的具体实施方式
在一个方面中，本发明提供一种用于APL相关融合基因检测的二代测序DNA文库的构建方法(本发明的建库方法)，该方法包括：
步骤A：将希望进行测序的cDNA进行末端修复，得到平末端DNA片段，该cDNA是由从人血液或骨髓样本中提取的总RNA反转录得到的；
步骤B：将平末端DNA片段进行3′端加A，得到3′端加A的DNA片段；
步骤C：将所述3′端加A的DNA片段进行加接头，得到加接头DNA片段；以及
步骤D：将所述加接头DNA片段进行PCR扩增，得到扩增产物；
其中，
所述步骤C中使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为所述接头(P5接头)；
所述步骤D中使用核苷酸序列如SEQ ID NO：51～62所示的单链DNA(GSP1引物池)以及核苷酸序列如SEQ ID NO：6所示的单链DNA(公共引物)作为PCR扩增引物；
所述步骤E中使用核苷酸序列如SEQ ID NO：31～42所示的单链DNA(GSP2引物池)、核苷酸序列如SEQ ID NO：4所示的单链DNA(Index引物)以及核苷酸序列如SEQ ID NO：6所示的单链DNA(公共引物)作为PCR扩增引物。
在本说明书中，APL是指急性早幼粒细胞白血病(Acute promyelocyte leukemia)。APL相关融合基因是急性早幼粒细胞白血病特异性标志。通过采用本发明，理论上只要是与RARα发生融合或存在断裂点的突变，都能检出，包括但不限于：非典型APL中出现的PLZF-RARα、NuMA-RARα、NPM1-RARα、Stαt5b-RARα、F1P1L1-RARα、PRKAR1A-RARα、BCOR-RARα和典型APL中出现的PML-RARα。
优选地，本发明的建库方法在所述步骤A之前还包括下述步骤：从人血液或骨髓样本中提取总RNA，并由该总RNA反转录得到cDNA第一链和cDNA第二链。对于该步骤中采用的具体方法没有特殊限制，可以采用本领域技术人员通常采用的那些方法，例如，可以使用Trizol试剂按标准操作流程提取总RNA，然后不进行DNase消化及mRNA钓取纯化，直接用随机引物进行反转录。
所述步骤A及步骤B可以通过本技术领域的常规方法来进行。
在所述步骤C中，使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为接头来进行加接头。该步骤可以使用DNA连接酶(例如T4 DNA连接酶)在相应的反应体系中进行。
在所述步骤D、E中，使用上述各单链DNA作为PCR扩增引物来进行PCR扩增。该步骤可以使用DNA聚合酶(例如Ex taq DNA聚合酶)在相应的反应体系中进行。
优选地，在所述步骤C与步骤D之间、所述步骤D与步骤E、和/或所述步骤E之后还包括对产物进行纯化的步骤。所述纯化步骤可以按本技术领域通常采用的方法进行，例如进行磁珠纯化。
本发明的建库方法可以例如使用试剂盒来实施，因此，在另一个方面中，本发明提供一种用于APL相关融合基因检测的二代测序DNA文库构建的试剂盒(本发明的建库试剂盒)，其可用于实施本发明的建库方法，其包含用于构建二代测序DNA文库的试剂，所述用于构建二代测序DNA文库的试剂包含：
核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA。
优选地，所述用于构建二代DNA测序文库的试剂还包括选自下组中的至少一种或两种以上：T4 DNA聚合酶、Klenow片段、Klenow缓冲液、DNA连接酶缓冲液、DNA连接酶、Taq酶、dNTP、T4多聚核苷 酸激酶、以及T4多聚核苷酸激酶缓冲液。
优选地，本发明的建库试剂盒还可以包含用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂。优选地，所述用于从人血液或骨髓样本中提取的总RNA、并由该总RNA反转录得到cDNA第一链和cDNA第二链的试剂包括选自下组中的至少一种或两种以上：Trizol试剂、氯仿、异丙醇、乙醇、无RNA酶的双蒸水、随机引物、反转录酶、反转录反应缓冲液、RNA酶抑制剂、dNTP、DNA聚合酶以及核糖核酸酶H。
通过对所得的PCR第二次扩增产物进行二代测序，可以知晓样本中是否存在APL相关融合基因中的任意一种或两种以上，从而进行APL相关融合基因的检测，为急性早幼粒细胞白血病(APL)的诊断、微小残留病(MRD)的判断提供分子检测基础。需要说明的是，本发明的建库方法本身并不包含任何检测或诊断步骤。
在另一方面中，本发明提供一种APL相关融合基因检测方法(本发明的检测方法)，该方法包括下述步骤：
步骤A：将希望进行测序的cDNA进行末端修复，得到平末端DNA片段，该cDNA是由从人血液或骨髓样本中提取的总RNA反转录得到的；
步骤B：将平末端DNA片段进行3′端加A，得到3′端加A的DNA片段；
步骤C：将所述3′端加A的DNA片段进行加接头，得到加接头DNA片段；以及
步骤D：将所述加接头DNA片段进行第一次PCR扩增，得到第一次扩增产物；
步骤E：将所述第一次扩增产物进行巢式PCR扩增，得到第二次扩增产物；以及
步骤F：对所述第二次扩增产物进行二代测序，并基于测序结果进行生物信息学分析；
其中，
所述步骤C中使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为所述接头；
所述步骤D中使用核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物；
所述步骤E中使用核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA(index引物)以及核苷酸序列如SEQ ID NO：6所示的单链DNA(公共引物)作为PCR扩增引物。
即，本发明的检测方法可以在本发明的建库方法的基础上增加所述步骤F。在所述步骤F中，对所述第二次扩增产物进行二代测序，并基于测序结果进行生物信息学分析。这里，二代测序可以采用本技术领域的常规方法来进行，例如，二代测序可以利用Illumina平台(例如HiSeq 2500或NextSeq 500)进行。然后，基于测序结果进行生物信息学分析，所述生物信息学分析例如可以这样进行：获取测序获得的reads 中与RARα基因外显子或其片段连接区域的核苷酸序列的信息，然后将该核苷酸序列信息与参考基因组核苷酸序列信息进行比对，由此确定RARα基因是否发生了基因融合以及与之融合的基因(参考基因组hg19)。
本发明的检测方法可以例如使用试剂盒来实施，因此，在另一个方面中，本发明提供一种用于APL相关融合基因检测的试剂盒(本发明的检测试剂盒)，其包含：
用于构建二代测序DNA文库的试剂，以及
用于对二代测序DNA文库进行上机测序的试剂；
其中，所述用于构建二代测序DNA文库的试剂包含：
核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA。
即，本发明的检测试剂盒可以在本发明的建库试剂盒的基础上，增加用于对所构建的二代测序DNA文库进行上机测序的试剂。作为所述对二代测序DNA文库进行上机测序的试剂，包括但不限于选自下组中的至少一种或两种以上：再合成试剂、线性化P7接头、线性化P5接头、DNA聚合酶、dNTP、冲洗杂交液/缓冲液、100％甲酰胺(质量/体积)、用于测序的Read 2引物、Index i7测序引物、用于测序的Read 1引物、Hiseq Rapid PE Flow Cell、水、以及增强光感度/照相用试剂。
在本发明的建库试剂盒及检测试剂盒中，各试剂或装置优选单独包装，但在不影响本发明的实施的前提下，也可以混合包装。
实施例
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。
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。
以250ng总RNA起始建库，单一样本单一反应。样本编码RNR130626(样本1，临床患者)、RNR130627(样本2，临床患者)、HC14CS00067(样本3，健康人)250ng总RNA未经DNase消化及mRNA钓取纯化，直接随机引物反转录，实验流程如下：
1.总RNA提取
从以上3份样本(200μL)中提取总RNA，完全按照Trizol试剂提取标准操作流程进行RNA提取，无需DNase消化及mRNA调取纯化，直接随机引物反转录。
2.反转录第一链的合成
1)向1μg总RNA(2μL)中加入1.0μL N6 primer(0.1μg/μL)，12μL无RNAase的水。
2)在65℃，5分钟变性RNA二级结构后，立即置于冰上。
3)在0.2mLPCR管中按照下列的配比准备反应混合物：
Figure PCTCN2015099764-appb-000001
4)混匀后在PCR仪上按照以下程序进行反应：
Figure PCTCN2015099764-appb-000002
反应结束后，将合成的cDNA一链产物用Ampure XP Beads(1.8×)进行回收，EB溶解，EB体积为21μL。
5×First strand buffer试剂来自于Invitrogen公司。
3.反转录第二链的合成
1)将合成的cDNA一链产物置于冰上共20μL
2)按照下列的配比准备反应混合物：
Figure PCTCN2015099764-appb-000003
Figure PCTCN2015099764-appb-000004
5×Second strand buffer试剂来自于Invitrogen公司。
混合均匀后，置于16℃Thermomixer上2小时(300rpm间歇振动15秒，静止2分钟)。
反应结束后，将合成的cDNA二链产物用Ampure XP Beads(1.8×)进行回收，EB溶解，EB体积为37μL。
4.末端修复
按照下列的配比准备反应混合物：
Figure PCTCN2015099764-appb-000005
在thermomixer，20℃条件下反应30分钟，将反应产物用Ampure XP Beads(1.8×)进行回收，EB溶解，EB体积为33μL。
10×PNK buffer来自Enzymatics公司
5.cDNA3′末端加“A”碱基
按照下列的配比准备反应混合物
Figure PCTCN2015099764-appb-000006
在thermomixer37℃条件下反应30分钟。将反应产物用Ampure XP Beads(1.8×)进行回收，EB溶解，EB体积为20μL。
10×blue buffer来自Enzymatics公司。
6.Adapter连接
按照下列的配比准备反应混合物
Figure PCTCN2015099764-appb-000007
在thermomixer，20℃条件下反应15分钟，将反应产物用Ampure XP Beads(1.8×)进行回收，EB溶解，EB体积为20μL。
所述Adapter为SEQ ID NO：1所示核苷酸序列与SEQ ID NO：2所示核苷酸序列的退火产物。
2×Rapid buffer来自Enzymatics公司。
7.第一轮PCR
1)按照下列的配比准备反应混合物。
2)将反应管置于冰上，加入以下成分，反复吹打68次。
Figure PCTCN2015099764-appb-000008
3)按下表进行PCR反应：
Figure PCTCN2015099764-appb-000009
Figure PCTCN2015099764-appb-000010
4)磁珠纯化
按照磁珠纯化标准流程，加入40μL
Figure PCTCN2015099764-appb-000011
XP beads到50μL反应液中。
最后用20μL 10mM Tris-HCl洗脱。
HiFi DNA Polymerase Mix来自kapa biosystems公司。
8.第二轮PCR(巢式PCR)
1)按照下列的配比准备反应混合物。
2)将反应管置于冰上，加入以下成分，反复吹打6-8次。
Figure PCTCN2015099764-appb-000012
3)按下表进行PCR反应：
Figure PCTCN2015099764-appb-000013
4)磁珠纯化同步骤7。HiFi DNA Polymerase Mix来自kapa biosystems公司。
9.文库纯化后，用安捷伦2100生物分析仪检测合格。
10.文库QPCR定量结果
Figure PCTCN2015099764-appb-000014
11.Hiseq2500 PE100上机测序数据量0.5G data。
12.数据分析结果
样本1为NPM1-RARα融合基因阳性，说明其可能为APL患者。
Figure PCTCN2015099764-appb-000015
样本2为PML-RARα融合基因阳性，说明其可能为APL患者。
Figure PCTCN2015099764-appb-000016
样本3未检测到融合基因，说明其可能为正常人。
本说明中涉及的核苷酸序列如下。
SEQ ID NO:1：5'-GATCGGAAGAGCACACGTCT-3'；
SEQ ID NO:2：5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'；
RARαGSP2引物池
SEQ ID NO 核苷酸序列(5'-3')
31 CCTCTCAGCTCGATTCAG
32 GTCAAGCAGTTCTGTGAGCTG
33 CAGGAGCTGCTGTTGCTGG
34 ACTTGTCCTGACAGACAAAGC
35 GTCACCTTGTTGATGATGCAGTT
36 CGTCAGCGTGTAGCTCTCAGAG
37 GCAGCTGCTTGGCGAACTC
38 AAGCCAGCGTTGTGCATCTG
39 GCTTCCGCACGTAGACCTTTAG
40 CCCATCTACTGTCCAGTG
41 CCAGCTCTGGATCCAGCTC
42 TGTGTCCATGTGGCGTG
(Index-X)SEQ ID NO:4：5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'
RARαGSP1引物池
SEQ ID NO 核苷酸序列(5'-3')
51 CTGCTCAGGTGTGTGCTGGCA
52 CCAAAGAGGGCAGGCTA
53 GATGGTGTGCTATATCCACTA
54 CTTGCAGCCCTCACAGG
55 CCACTTCAAAGCACTTCTGCA
56 CGTAGTGTATTTGCCCAGCTG
57 CAGGATGTCCAGGCAGGC
58 CTCCGCAGATGAGGCAGATG
59 CCTTGGCGCTGATGCTTCG
60 ATCCAGTAGATCTCAGTGGAAAG
61 TCATGGTGGGTGCAGGCA
62 AGCAATGGCTTGTGAGTTCTG
(公共引物)SEQ ID NO:6:
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
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。
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。
工业实用性
根据本发明，提供一种基于二代测序技术的、可同时检测多种APL相关融合基因的检测方法、检测试剂盒，以及构建用于上述检测方法的二代测序DNA文库的方法、建库试剂盒。

Claims (10)

  1. 一种用于APL相关融合基因检测的二代测序DNA文库的构建方法，该方法包括：
    步骤A：将希望进行测序的cDNA进行末端修复，得到平末端DNA片段，该cDNA是由从人血液或骨髓样本中提取的总RNA反转录得到的；
    步骤B：将平末端DNA片段进行3′端加A，得到3′端加A的DNA片段；
    步骤C：将所述3′端加A的DNA片段进行加接头，得到加接头DNA片段；以及
    步骤D：将所述加接头DNA片段进行第一次PCR扩增，得到第一次扩增产物；
    步骤E：将所述第一次扩增产物进行巢式PCR扩增，得到第二次扩增产物；
    其中，
    所述步骤C中使用核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA的退火产物作为所述接头；
    所述步骤D中使用核苷酸序列如SEQ ID NO：51～62所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物；
    所述步骤E中使用核苷酸序列如SEQ ID NO：31～42所示的单链DNA、核苷酸序列如SEQ ID NO：4所示的单链DNA以及核苷酸序列如SEQ ID NO：6所示的单链DNA作为PCR扩增引物。
  2. 根据权利要求1所述的方法，其中，在所述步骤A之前还包括下述步骤：
    从人血液或骨髓样本中提取的总RNA，并由该总RNA反转录得到cDNA第一链和cDNA第二链。
  3. 根据权利要求1所述的方法，其中，在所述步骤C与步骤D之间、步骤D与步骤E之间、和/或所述步骤E之后还包括对产物进行纯化的步骤。
  4. 根据权利要求1所述的方法，其中，所述APL相关融合基因包括非典型APL中出现的PLZF-R4Ra，、NuMA-RARa、NPMl-RARa、Stat5b-RARa、FlPlLl-RARa、PRKARlA-RARa、BCOR-RARa和典型APL中出现的PML-RARa。
  5. 一种用于APL相关融合基因检测的二代测序DNA文库构建的试剂盒，其包含用于构建二代测序DNA文库的试剂，所述用于构建二代测序DNA文库的试剂包含：
    核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
    核苷酸序列如SEQ ID NO：31～42所示的单链DNA；
    核苷酸序列如SEQ ID NO：4所示的单链DNA；
    核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
    核苷酸序列如SEQ ID NO：51～62所示的单链DNA。
  6. 根据权利要求5所述的试剂盒，其中，所述用于构建二代DNA测序文库的试剂还包括选自下组中 的至少一种或两种以上：T4DNA聚合酶、Klenow片段、Klenow缓冲液、DNA连接酶缓冲液、DNA连接酶、Taq酶、dNTP、T4多聚核苷酸激酶、以及T4多聚核苷酸激酶缓冲液。
  7. 根据权利要求5或6所述的试剂盒，其用于实施权利要求1～4中任一项所述的方法。
  8. 一种用于APL相关融合基因检测的试剂盒，其包含：
    用于构建二代测序DNA文库的试剂，以及
    用于对二代测序DNA文库进行上机测序的试剂；
    其中，所述用于构建二代测序DNA文库的试剂包含：
    核苷酸序列如SEQ ID NO：1所示的单链DNA与核苷酸序列如SEQ ID NO：2所示的单链DNA，或者它们的退火产物；
    核苷酸序列如SEQ ID NO：31～42所示的单链DNA；
    核苷酸序列如SEQ ID NO：4所示的单链DNA；
    核苷酸序列如SEQ ID NO：6所示的单链DNA；以及
    核苷酸序列如SEQ ID NO：51～62所示的单链DNA。
  9. 根据权利要求8所述的试剂盒，其中，所述用于构建二代DNA测序文库的试剂还包括选自下组中的至少一种或两种以上：T4DNA聚合酶、Klenow片段、Klenow缓冲液、DNA连接酶缓冲液、DNA连接酶、Taq酶、dNTP、T4多聚核苷酸激酶、以及T4多聚核苷酸激酶缓冲液。
  10. 根据权利要求8所述的试剂盒，其中，所述对二代测序DNA文库进行上机测序的试剂包括选自下组中的至少一种或两种以上：再合成试剂、线性化P7接头、线性化P5接头、DNA聚合酶、dNTP、冲洗杂交液/缓冲液、100％甲酰胺(质量/体积)、用于测序的Read 2引物、Index i7测序引物、用于测序的Read 1引物、Hiseq RapidPE Flow Cell、ddH2O、以及增强光感度/照相用试剂。
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CN112941635A (zh) * 2020-12-30 2021-06-11 上海迪赢生物科技有限公司 一种提高文库转化率的二代测序建库试剂盒及其方法
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CN114277147A (zh) * 2021-12-29 2022-04-05 杭州艾迪康医学检验中心有限公司 一种检测白血病融合基因BCOR/RARa的试剂和试剂盒

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