WO2023185560A1 - 一种检测alk融合基因的引物和探针组合物、试剂盒及方法 - Google Patents

一种检测alk融合基因的引物和探针组合物、试剂盒及方法 Download PDF

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WO2023185560A1
WO2023185560A1 PCT/CN2023/082930 CN2023082930W WO2023185560A1 WO 2023185560 A1 WO2023185560 A1 WO 2023185560A1 CN 2023082930 W CN2023082930 W CN 2023082930W WO 2023185560 A1 WO2023185560 A1 WO 2023185560A1
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fusion gene
alk fusion
detecting
alk
probe
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彭玲
刘辉超
芮立
黄士栩
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上海信诺佰世医学检验有限公司
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of molecular biology gene detection technology, and specifically relates to a primer and probe composition, a kit and a method for detecting ALK fusion genes.
  • the ALK gene is located on chromosome 2p23. Under normal circumstances, human ALK can be transcribed to produce an mRNA of 6222bp, consisting of 29 exons, encoding a type I transmembrane protein ALK of 200KDa consisting of 1620 amino acid sequences. , this protein is a receptor tyrosine kinase (receptortyrosinekinase, RTK) and a member of the RTK insulin superfamily.
  • RTK receptor tyrosine kinase
  • the complete ALK has a typical three-part structure of RTK, namely extracellular domain, lipophilic transmembrane domain and intracytoplasmic tyrosine kinase.
  • ALK protein is expressed in 60% to 85% of primary systemic ALCL, in addition to being expressed in a very small number of diffuse large B-cell lymphomas. It is a relatively specific immune immune system for primary systemic ALCL. Phenotypic characteristics.
  • ALCL is a T-cell non-Hodgkin lymphoma in which ALK gene abnormalities are common. Up to 50% of adult patients with ALCL are ALK-positive, and up to 90% of pediatric patients with ALCL are ALK-positive. NPM1-ALK is the most common translocation in ALCL, present in 75% to 80% of ALK-positive ALCL patients. In addition, TPM3-ALK is present in 12% to 18% of ALCL patients. Other fusion proteins appeared less frequently (all within 2%), including TFG-ALK, CLTC1-ALK and TRAF1-ALK.
  • the current examinations for ALCL mainly include pathological examination, immune phenotype examination, blood routine, bone marrow routine, flow cytometry, imaging examination, etc.
  • pathological examination mainly include pathological examination, immune phenotype examination, blood routine, bone marrow routine, flow cytometry, imaging examination, etc.
  • RT-PCR fluorescence quantitative PCR
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • FISH In situ hybridization
  • FISH is known as the gold standard for ALK fusion gene detection because of its relatively high sensitivity and specificity for ALK fusion gene detection.
  • the FISH detection method also has its own problems. The main problem of FISH detection is that it cannot determine the fusion type of ALK and must be completed by experienced pathologists. Misinterpretation of some cases near the threshold and cases with atypical signals can easily lead to false negative or false positive results.
  • IHC Immunohistochemistry detects through the binding reaction of antigen and antibody, combined with signal cascade amplification.
  • Roche's Ventana IHC kit can achieve a consistency rate of 94 to 100% with FISH test results.
  • the interpretation standard is that clusters of brown-yellow strongly stained granules appear in the cytoplasm of tumor cells, and the cell membrane is negative for staining.
  • Both the European Union and China have approved Ventana IHC for the detection of ALK rearrangements. Due to tumor heterogeneity, the expression intensity of target proteins is not uniform. Due to the possibility of protein and RNA degradation, FFPE sections should be stored for longer than 3 months. Ventana IHC testing is not recommended.
  • Real-time fluorescence quantitative PCR refers to a method that adds fluorescent groups to the PCR reaction system, uses fluorescence signal accumulation to monitor the entire PCR process in real time, and finally uses a standard curve to quantitatively analyze the unknown template. It is simple and feasible to detect fusion mutations by reverse-transcribing the RNA of the sample with pre-designed specific primer probes, and the fusion type of ALK can be determined. This detection method requires high-quality RNA samples. Most clinical samples are paraffin samples. RNA is seriously degraded, which affects the detection rate and leads to false negative results. PCR primers can only detect known fusion genotypes, and known Ct values cannot accurately quantify the initial copy number of the template. The quantitative results rely on the standard curve, and the sensitivity can reach 1%, but it cannot meet the needs of high-sensitivity detection.
  • the purpose of the present invention is to overcome the shortcomings of the existing technology and provide a primer and probe composition, a kit and a method for detecting ALK fusion genes.
  • the present invention proposes highly specific primers and probe sequences for detecting ALK fusion genes NPM1-ALK, TPM3-ALK, and TRAF1-ALK.
  • An object of the present invention is to provide a primer and probe composition for detecting ALK fusion genes.
  • a primer and probe composition for detecting ALK fusion gene including a primer pair and probe for detecting NPM1-ALK fusion gene, a primer pair and probe for detecting TPM3-ALK fusion gene, and a primer pair and probe for detecting TRAF1-
  • the sequence of the primer pair used to detect the TPM3-ALK fusion gene is shown in SEQ ID NO: 4-5, and the sequence of the primer pair used to detect the TRAF1-ALK fusion gene is shown in SEQ ID NO: 7-8.
  • the probe sequence for detecting NPM1-ALK fusion gene, the probe sequence for detecting TPM3-ALK fusion gene, and the probe sequence for detecting TRAF1-ALK fusion gene are respectively as SEQ ID NO: 3, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO:13; the ABL internal reference gene primer pair sequence is as shown in SEQ ID NO:10-11, and the probe sequence is as SEQ ID NO: 12 shown.
  • the probe sequence for detecting NPM1-ALK fusion gene the probe sequence for detecting TPM3-ALK fusion gene and the 5' end of the probe sequence for detecting TRAF1-ALK fusion gene
  • the ABL internal reference gene probe sequence is labeled with a FAM reporter group and the 3' end with a MGB fluorescent group; the 5' end of the ABL internal reference gene probe sequence is labeled with a HEX reporter group and the 3' end is labeled with a BHQ1 fluorescence quenching group.
  • Another object of the present invention is to provide a kit for detecting ALK fusion genes.
  • a kit for detecting ALK fusion gene includes the above-mentioned primer and probe composition for detecting ALK fusion gene.
  • kit also includes positive standards, negative standards, ddPCR Supermix and ddH 2 O.
  • the final object of the present invention is to provide a method for detecting ALK fusion genes.
  • a method for detecting ALK fusion genes for non-diagnostic purposes including the following steps:
  • step S1 the reverse transcription reaction program is as follows: 30°C, 10min, 42°C, 50min, 95°C, 5min, 4°C, and incubation.
  • the concentration of the primer and probe composition for detecting ALK fusion gene is 10 ⁇ , wherein the sequence of the primer pair for detecting NPM1-ALK fusion gene, the primer pair for detecting The concentrations of the primer pair sequence of TPM3-ALK fusion gene, the primer pair sequence for detecting TRAF1-ALK fusion gene and the ABL internal reference gene primer pair sequence are all 6-9 ⁇ M; the primer pair sequence for detecting NPM1-ALK fusion The concentrations of the probe sequence of the gene, the probe sequence for detecting the TPM3-ALK fusion gene, the probe sequence for detecting the TRAF1-ALK fusion gene and the ABL internal reference gene probe sequence are all 3 ⁇ 8 ⁇ M.
  • step S3 the specific method of using the ddPCR mixture to make PCR micro-reaction droplets is as follows: add the ddPCR mixture into the droplet generator to generate 15,000 to 20,000 micro-reaction droplets.
  • step S3 the PCR amplification reaction procedure is as follows: pre-denaturation at 93-97°C for 3-10 minutes, denaturation at 93-95°C for 20-40s, annealing at 55-60°C for 50-70s, a total of 35-45 times. Cycle, keep warm at 10 ⁇ 12°C.
  • PCR amplification reaction procedure is as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 60 s, a total of 40 cycles, and incubation at 12°C.
  • the present invention has the following advantages:
  • the instrument has a high degree of automation and the experimental results can be accurately judged.
  • Figure 1 is a diagram showing the test results of peripheral blood sample numbered 1 in Example 2 of the present invention.
  • Figure 2 is a diagram showing the test results of peripheral blood sample numbered 2 in Example 2 of the present invention.
  • Figure 3 is a graph showing the test results of peripheral blood sample numbered 3 in Example 2 of the present invention.
  • Figure 4 is a diagram showing the test results of peripheral blood sample numbered 4 in Example 2 of the present invention.
  • Figure 5 is a diagram showing the test results of peripheral blood sample numbered 5 in Example 2 of the present invention.
  • Figure 6 is a diagram showing the test results of peripheral blood sample numbered 6 in Example 2 of the present invention.
  • Figure 7 is a graph showing the test results of the bone marrow sample numbered 7 in Example 2 of the present invention.
  • Figure 8 is a graph showing the test results of the bone marrow sample numbered 8 in Example 2 of the present invention.
  • Figure 9 is a graph showing the detection results of the bone marrow sample numbered 9 in Example 2 of the present invention.
  • ALK anaplastic lymphoma kinase
  • the fusion transcript sequence containing the breakpoints was spliced to approximately 500 bp.
  • Primer Premier5.0 was used to design the upstream and downstream sequences of the ALK fusion gene NPM1-ALK, TPM3-ALK, and TRAF1-ALK fusion gene amplification.
  • Two sets of upstream and downstream primers were designed, as well as the conserved region of the ABL internal reference gene transcript.
  • the amplified fragment contains the breakpoint region and the length of the amplified fragment is about 100 to 150 bp.
  • Primer Express3.0.1 was used to design two specific probes for each of NPM1-ALK, TPM3-ALK, and TRAF1-ALK fusion genes.
  • the 5' end was labeled with FAM fluorescent group and the 3' end was labeled with MGB fluorescent group. group; design a universal specific probe, and label the 5' end with the FAM fluorophore and the 3' end with the BHQ1 fluorophore; at the same time, design a probe for the ABL internal reference gene, with the 5' end labeled with the HEX fluorophore. group, and the 3' end is labeled with a BHQ1 fluorescence quenching group.
  • primer-probe set designed above was specifically screened on NCBI-blast through orthogonal experiments. Primer-probe sequences that can align the target fragment and have no homology with other genes are combinations with good specificity. .
  • the primer and probe sets completed by the above screening passed the sensitivity and specificity tests, and the sensitivity template selection Artificially prepared pseudoviruses are selected, and RNA samples extracted from healthy people are selected as templates for specificity testing, which are reverse transcribed into cDNA as templates to select a combination with high sensitivity and good specificity.
  • the two sets of probes passed the test of sensitivity and specificity.
  • the probes are located on exon 20 of the ALK gene and have good sensitivity and specificity. They can accurately detect three fusion genes.
  • the sequences of the primers and probes that have been screened to detect fusion mutations have been tested. As shown in Table 1 below:
  • the total volume of the reaction system is 20 ⁇ L, and the insufficient volume is made up with enzyme-free water.
  • the final concentration of the ddPCR enzyme master mix is 1 ⁇ . Adjust the ratio of each primer and probe concentration in the reaction system to the optimal concentration to maximize the sensitivity and specificity of the reaction system. Then configure the primers with a concentration of 10 ⁇ ALK fusion gene. And probe composition, the optimal concentration of each primer and probe is shown in Table 2 below:
  • the templates prepared for the sensitivity reference are artificially prepared pseudoviruses.
  • NPM1-ALK, TPM3-ALK, TRAF1-ALK templates and ABL internal reference gene templates are mixed in certain proportions respectively, and the prepared fusion ratios are 1%, 0.1%, 0.01%, and 0.001 % mixed solution, the template copy number of the internal reference gene is ⁇ 10 6 , and reverse transcribed into cDNA as a positive template.
  • the negative control is a healthy human RNA sample, and reverse transcribed into cDNA as a negative template.
  • the Biorad QX200 droplet generator is used to prepare and generate droplets. Each well generates about 20,000 droplets, which are transferred to a 96-well PCR deep well plate for droplet PCR reaction.
  • the reaction program is: pre-denaturation at 95°C for 5 minutes, denaturation at 94°C. 30s, annealing at 60°C for 60s, a total of 40 cycles, and heat preservation at 12°C.
  • the fluorescence signal is collected by a digital PCR instrument and analyzed to obtain NPM1-ALK, TPM3-ALK and TRAF1-ALK gene fusion mutations, and the judgment results are given.
  • the ddPCR system was used to detect the sensitivity and lowest detection rate of the present invention.
  • the theoretical values of the above positive control sample mixture were 1%, 0.1%, 0.01%, and 0.001% respectively, the actual measured result ratios are as shown in Table 4 below. Show:
  • the sensitivity test results of the ALK fusion gene detection kit are consistent with the theoretical values.
  • the sensitivity test is good, the negative control fusion copy number is zero, and the specificity is good; when NPM1-ALK, TPM3-ALK, TRAF1 -When the positive sample with mixed ALK copy number and ABL copy number is 0.01%, the ddPCR system can also stably detect the corresponding fusion type, and the positive coincidence rate is 100%.
  • 1.5 mL of peripheral blood and bone marrow from 9 human ALCL patients were taken into heparin-anticoagulated blood collection tubes, and 9 patients were subjected to fluorescent quantitative PCR detection to clarify the type of ALK fusion gene, or the absence of NPM1-ALK, TPM3-ALK, TRAF1-ALK 3 fusion types; mark the sample well and ensure that the label information is correct, and store it at 4°C.
  • RNA is extracted from human peripheral blood and bone marrow using the Trizol method, and the RNA concentration is detected using the NanoDrop2000 ultra-micro spectrophotometer.
  • the 260/280 ratio satisfies 1.7 to 2.0. It is recommended that the extracted RNA be tested immediately, otherwise the sample should be stored below -70°C. Avoid repeated freezing and thawing during storage.
  • the extracted RNA from 9 cases was subjected to reverse transcription reaction using TaKaRa PrimeScriptTM 1st Strand cDNA Synthesis Kit (Cat. No.: 6110A).
  • the reverse transcription reaction program is as follows: 30°C, 10min, 42°C, 50min, 95°C, 5min, 4°C, heat preservation.
  • Biorad QX200 droplet generator was used to prepare and generate droplets. Approximately 20,000 droplets were generated in each well and transferred to a 96-well PCR deep well plate for droplet PCR reaction. The reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 60 s, a total of 40 cycles, and insulation at 12°C.
  • the ddPCR detection system for ALK gene fusion mutations provided by the present invention has good specificity and high sensitivity. Compared with products from other companies on the market, it has a greater degree of sensitivity and accuracy in specific detection. Improvement; the false positive rate of detection is greatly reduced, and the accuracy of detection results is high; at the same time, the detection system provided by the invention can be applied to various biological sample types such as bone marrow and peripheral blood, and one reaction can simultaneously detect three fusion mutation types. , high detection success rate has obvious advantages for the detection of micro-templates, and has high application value in clinical testing.

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Abstract

提供一种检测ALK融合基因的引物和探针组合物、试剂盒及方法。该非诊断目的检测ALK融合基因的方法包括如下步骤：S1、从样品中提取RNA，并进行逆转录，获得cDNA；S2、以cDNA为模板，将待测样品cDNA模板、检测ALK融合基因的引物和探针组合物以及ddPCR Supermix混合，制备ddPCR混合液；S3、用所述ddPCR混合液制作PCR微反应液滴，再进行PCR扩增反应；S4、通过数字PCR仪对PCR扩增反应后的产物进行荧光信号的采集，根据荧光信号的类型，从而判断出ALK融合基因类型。该检测ALK融合基因NPM1-ALK、TPM3-ALK、TRAF1-ALK的引物和探针序列，能够同时实现三种融合基因在同一个反应孔进行检测，实现对ALK相关融合基因的筛查及定量检测。

Description

一种检测ALK融合基因的引物和探针组合物、试剂盒及方法 技术领域
本发明属于分子生物学基因检测技术领域，具体涉及一种检测ALK融合基因的引物和探针组合物、试剂盒及方法。
背景技术
ALK基因位于染色体2p23位点，正常情况下人源的ALK可转录产生大小6222bp的mRNA，由29个外显子构成，编码由1620个氨基酸序列组成的，大小为200KDa的I型穿膜蛋白ALK，该蛋白为一种受体酪氨酸激酶(receptortyrosinekinase，RTK)，是RTK胰岛素超家族的成员。完整的ALK具有典型的RTK三部分结构，即胞外区、亲脂性穿膜区和胞浆内酪氨酸激酶。据文献报道，ALK蛋白除在极少部分弥漫性大B细胞淋巴瘤中表达外，可在60％～85％的原发性系统性ALCL中表达，是原发性系统性ALCL相对特异的免疫表型特征。
在大多数ALCL病例中，ALK是通过染色体重排激活的。ALCL是一种T细胞非霍奇金淋巴瘤，ALK基因异常在该病中较为常见。多达50％的成人ALCL患者为ALK阳性，高达90％的ALCL儿科患者为ALK阳性。NPM1-ALK是ALCL中最常见的易位，存在于75％～80％的ALK阳性ALCL患者中。此外，TPM3-ALK存在于12％～18％的ALCL患者中。其他融合蛋白的出现频率较低(均在2％以内)，包括TFG-ALK、CLTC1-ALK及TRAF1-ALK。
许多学者认为ALCL患者预后与其染色体是否发生易位有关。在儿童和年轻患者的侵袭性NHL中，ALK阳性系统性ALCL治愈可能性最大，预后优于任何其他形式的外周T细胞淋巴瘤。众多研究表明有ALK融合基因的ALCL病例的预后明显好于阴性病例，因此对于临床诊断来说很有必要检测ALCL中有 无ALK融合基因。目前，染色体易位和ALK的表达已经被WHO规定为ALCL的临床诊断指标之一。
目前对ALCL的检查主要有病理检查、免疫表型检查、血常规、骨髓常规、流式细胞检查、影像学检查等。分子病理的检测工作中，ALK基因的融合主要有三种方法，荧光定量PCR法(RT-PCR)，免疫组化法(IHC)和荧光原位杂交法(FISH)。
原位杂交法(FISH)是通过荧光素标记的DNA探针与样本细胞核内的DNA靶序列杂交，从而获得细胞核内染色体或基因状态的信息。FISH试验过程中包含了单链DNA精确的退火温度，荧光标记的DNA探针能够与目标序列结合发出荧光，杂交之后的DNA片段能够直接在荧光显微镜下观察到。当杂交的ALK基因重组探针生成之后，在自然状态下可以观察到2p23 ALK基因区域会成为融合的红色和绿色信号。反之，若2p23 ALK区域发生断裂：红色和绿色信号分开且距离大于两个信号直径的大小、或者是一个单独的红色信号显示为模糊或者分裂的单独信号。FISH因其对ALK融合基因检测敏感度和特异性均相对较高，被称为ALK融合基因检测的金标准。但FISH检测方法也有自身的问题，FISH检测主要问题是无法判断ALK的融合型，必须由经验丰富的病理科医师来完成。对于一些阈值附近的病例和不典型信号病例的错误判读容易造成结果假阴性或假阳性。
免疫组化(IHC)是通过抗原和抗体的结合反应，配合信号级联放大来检测。罗氏公司的Ventana IHC试剂盒可以达到与FISH检测结果94～100％的一致率。判读标准是肿瘤细胞胞浆出现簇状棕黄色强染色颗粒，胞膜着色为阴性。欧盟和中国都已经批准Ventana IHC用于检测ALK重排。由于肿瘤异质性，靶蛋白的表达强度不均一。由于蛋白和RNA降解的可能，FFPE切片存储大于3个月 的不建议Ventana IHC检测。
实时荧光定量PCR(Quantitative Real-time PCR)是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。通过预先设计好的特异性引物探针对样本的RNA进行逆转录来检测融合突变，简便可行，可以确定ALK的融合类型。该检测方法需要高质量的RNA样本，临床大部分是石蜡样本，RNA降解严重，影响检出率，导致假阴性的结果。通过PCR引物只能检测已知的融合基因型，已知Ct值不能准确定量出模板的起始拷贝数，定量结果依赖标准曲线，灵敏度能达到1％，但是不能满足高灵敏度检测的需求。
有鉴于此，有必要开发一种检测ALK融合基因的引物和探针组合物、试剂盒来解决上述问题。
发明内容
本发明的目的在于克服现有技术的不足，提供一种检测ALK融合基因的引物和探针组合物、试剂盒及方法。本发明提出了检测ALK融合基因NPM1-ALK、TPM3-ALK、TRAF1-ALK高特异性引物和探针序列，并通过优化融合基因检测体系和PCR扩增程序，试剂的灵敏度变高，最低检测限试剂性能达到0.01％；同时实现三种融合基因在同一个反应孔进行检测，实现对ALK相关融合基因的筛查及定量检测。
本发明的一个目的在于提供一种检测ALK融合基因的引物和探针组合物。
一种检测ALK融合基因的引物和探针组合物，包括用于检测NPM1-ALK融合基因的引物对和探针、用于检测TPM3-ALK融合基因的引物对和探针、用于检测TRAF1-ALK融合基因的引物对和探针和ABL内参基因引物对和探针；所述用于检测NPM1-ALK融合基因的引物对序列如SEQ ID NO:1-2所示，所述 用于检测TPM3-ALK融合基因的引物对序列如SEQ ID NO:4-5所示，所述用于检测TRAF1-ALK融合基因的引物对序列如SEQ ID NO:7-8所示，所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列、所述用于检测TRAF1-ALK融合基因的探针序列分别如SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9所示或如SEQ ID NO:13所示；所述ABL内参基因引物对序列如SEQ ID NO:10-11所示，探针序列如SEQ ID NO:12所示。
进一步地，所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列和所述用于检测TRAF1-ALK融合基因的探针序列5'端标记有FAM报告基团，3'端标记MGB荧光基团；所述ABL内参基因探针序列5'端标记有HEX报告基团，3'端标记有BHQ1荧光淬灭基团。
本发明的另一个目的在于提供一种检测ALK融合基因的试剂盒。
一种检测ALK融合基因的试剂盒，所述试剂盒包括上述所述的检测ALK融合基因的引物和探针组合物。
进一步地，所述试剂盒还包括阳性标准品、阴性标准品、ddPCR Supermix和ddH2O。
本发明的最后一个目的在于提供一种检测ALK融合基因的方法。
一种非诊断目的检测ALK融合基因的方法，包括如下步骤：
S1、从样品中提取RNA，并进行逆转录，获得cDNA；
S2、以所述cDNA为模板，将待测样品cDNA模板、权利要求1或2所述的用于检测ALK融合基因的引物和探针组合物以及ddPCR Supermix混合，制备ddPCR混合液；
S3、用所述ddPCR混合液制作PCR微反应液滴，再进行PCR扩增反应；
S4、通过数字PCR仪对PCR扩增反应后的产物进行荧光信号的采集，根据 荧光信号的类型，从而判断出ALK融合基因类型。
进一步地，步骤S1中，所述逆转录反应程序如下：30℃、10min，42℃、50min，95℃、5min，4℃、保温。
进一步地，步骤S2中，所述用于检测ALK融合基因的引物和探针组合物的浓度为10×，其中，所述用于检测NPM1-ALK融合基因的引物对序列、所述用于检测TPM3-ALK融合基因的引物对序列、所述用于检测TRAF1-ALK融合基因的引物对序列和所述ABL内参基因引物对序列的浓度均为6～9μM；所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列、所述用于检测TRAF1-ALK融合基因的探针序列和所述ABL内参基因探针序列的浓度均为3～8μM。
进一步地，步骤S3中，用所述ddPCR混合液制作PCR微反应液滴的具体方法如下：将ddPCR混合液加入到微滴发生器中，生成15000～20000个微反应液滴。
进一步地，步骤S3中，所述PCR扩增反应程序如下：93～97℃预变性3～10min，93～95℃变性20～40s，55～60℃退火50～70s，共进行35～45个循环，10～12℃保温。
更进一步地，所述PCR扩增反应程序如下：95℃预变性5min，94℃变性30s，60℃退火60s，共进行40个循环，12℃保温。
与现有技术相比，本发明具有如下优点：
1)能同时实现三种融合基因在同一个反应孔进行检测，实现对ALK相关融合基因的筛查及定量检测；
2)不依赖标准曲线实现绝对定量，消除PCR扩增效率偏差对定量结果的影响，使定量更准确；
3)灵敏度更高，能够达到最低检测限0.01％，满足临床需要；
4)能够提高对抑制物的耐受度，克服抑制物对定量结果的干扰，对于FFPE样本提取质量差的RNA样本，依然能够定量检测；
5)仪器自动化程度高，实验结果判别准确。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例2中编号为1的外周血样本检测结果图；
图2为本发明实施例2中编号为2的外周血样本检测结果图；
图3为本发明实施例2中编号为3的外周血样本检测结果图；
图4为本发明实施例2中编号为4的外周血样本检测结果图；
图5为本发明实施例2中编号为5的外周血样本检测结果图；
图6为本发明实施例2中编号为6的外周血样本检测结果图；
图7为本发明实施例2中编号为7的骨髓样本检测结果图；
图8为本发明实施例2中编号为8的骨髓样本检测结果图；
图9为本发明实施例2中编号为9的骨髓样本检测结果图。
具体实施方式
下面将结合本发明实施例中的附图对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明所使用的常规试剂和设备，如无特殊说明，均可市售获得。
实施例1 ALK融合基因检测体系的建立
1 1.引物、探针的的设计合成及筛选
通过文献报到的约60％-85％左右ALCL病例表达间变性淋巴瘤激酶(anaplasticlymphomakinase，ALK)融合蛋白，ALK融合在间变性淋巴瘤里最常见的类型为NPM1-ALK，约占72％以上，第二常见的类型为TPM3-ALK，约占15％以上，因此，选择常见的三种融合基因类型跨融合基因断点区域进行引物探针设计合成，通过引物探针的筛选得到扩增效率高，特异性好的引物探针组合。序列通过NCBI数据库下载，根据文献报道的常见的融合基因断点和生信结果，拼接包含断点的融合转录本序列约500bp。根据引物设计原则，应用Primer Premier5.0设计ALK融合基因的上下游序列NPM1-ALK、TPM3-ALK、TRAF1-ALK融合基因扩增上下游引物各设计两组，以及ABL内参基因转录本的保守区域设计1组引物，并分析引物的Hairpin、Dimer结构，扩增片段包含断点区域，扩增片段长度约100～150bp。
根据探针设计原则，应用Primer Express3.0.1设计NPM1-ALK、TPM3-ALK、TRAF1-ALK融合基因各2条特异性的探针，5'端标记FAM荧光基团，3'端标记MGB荧光基团；设计1条通用型特异性探针，并且5'端标记FAM荧光基团，3'端标记BHQ1荧光基团；同时，设计1条ABL内参基因的探针，5'端标记HEX荧光基团，3'端标记BHQ1荧光淬灭基团。
上述设计的引物探针组通过正交试验的方法，在NCBI-blast上进行特异性的筛选，能够比对到目的片段且和其他基因没有同源性的引物探针序列为特异性好的组合。
上述筛选完成的引物探针组通过灵敏度和特异性的测试，灵敏度的模板选 择人工制备的假病毒，特异性测试的模板选择健康人提取的RNA样本，经逆转录成cDNA作为模板，选择灵敏度高而且特异性好的组合。
两组探针通过灵敏度和特异性的测试，探针位于ALK基因外显子20上灵敏度特异性均良好，能准确检测三种融合基因，经筛选完成的检测融合变异的引物、探针的序列如下表1所示：
表1 ALK融合基因检测的引物和探针序列
1.2反应体系的优化及建立
反应体系总体积为20μL，不足的体积用无酶水补足。ddPCR酶预混液终浓度为1×，调整反应体系中各引物、探针浓度的比例到最适浓度，使反应体系的灵敏度和特异性最好，然后配置成浓度为10×ALK融合基因的引物和探针组合物，各引物、探针最适浓度如下表2所示：
表2各引物、探针最适浓度

1.3 ALK融合基因检测试剂盒的灵敏度检测及最低检出率实验
灵敏度参考品制备的模板为人工制备的假病毒，NPM1-ALK、TPM3-ALK、TRAF1-ALK模板和ABL内参基因模板分别按一定比例混合，制备融合比例为1％、0.1％、0.01％、0.001％混合液，内参基因的模板拷贝数≥106，逆转录成cDNA作为阳性模板，阴性对照为健康人RNA样本，逆转录成cDNA作为阴性模板。
按照上述引物探针和反应体系配制ddPCR的体系，加入上述1％、0.1％、0.01％、0.001％阳性模板，阴性对照各5μL，反应体系如下表3所示：
表3反应体系
应用Biorad QX200微滴发生器制备生成微滴，每孔生成约20000个微滴，转移至96孔PCR深孔板，进行微滴式PCR反应，反应程序为：95℃预变性5min，94℃变性30s，60℃退火60s，共进行40个循环，12℃保温。
通过数字PCR仪采集荧光信号，分析得到检测模板中NPM1-ALK、 TPM3-ALK、TRAF1-ALK基因融合变异情况，给出判定结果。
用ddPCR系统对本发明的灵敏度和最低检出率进行检测，当上述阳性对照样本混合液理论的值分别为1％、0.1％、0.01％、0.001％时，实际测得的结果比例如下表4所示：
表4灵敏度和最低检出率测试结果
由表中的结果可以看出，ALK融合基因检测试剂盒的灵敏度检测结果与理论值相符，灵敏度检测良好，阴性对照融合拷贝数为零，特异性好；当NPM1-ALK、TPM3-ALK、TRAF1-ALK拷贝数与ABL拷贝数混合的阳性样本为0.01％时，ddPCR系统也可稳定检测出相应融合型别，且阳性符合率为100％。
实施例2临床样本的检测
取9例人ALCL患者外周血和骨髓1.5mL置于肝素抗凝的采血管中，并且9例患者进行荧光定量PCR检测，明确了ALK融合基因类型，或者不含NPM1-ALK、TPM3-ALK、TRAF1-ALK 3种融合类型；做好样本标记并确保标签信息无误，4℃保存。
人外周血和骨髓用Trizol法提取出RNA，使用NanoDrop2000超微量分光光度计检测RNA浓度，260/280比值满足1.7～2.0；提取的RNA建议立即进行检测，否则应在-70℃以下保存，样本保存期间避免反复冻融。
提取的9例RNA用TaKaRa PrimeScriptTM 1st Strand cDNA Synthesis Kit(货号：6110A)进行逆转录反应。所述逆转录反应程序如下：30℃、10min， 42℃、50min，95℃、5min，4℃、保温。
配制ddPCR反应体系，加入上述9例cDNA模板，上样量5μL，总体积20μL。应用Biorad QX200微滴发生器制备生成微滴，每孔生成约20000个微滴，转移至96孔PCR深孔板，进行微滴式PCR反应。反应程序为：95℃预变性5min，94℃变性30s，60℃退火60s，共进行40个循环，12℃保温。
QX200微滴荧光信号收集系统收集FAM和HEX的荧光信号，对PCR微反应液进行定量分析，得到样本中融合基因的绝对含量a以及内参基因总RNA绝对含量b；融合比例X的计算公式为：X＝a/(a+b)*100％；9例样本的检测结果如下表5和6所示：
表5 9例样本的检测结果(NALK-TP1、TALK-TP2和TRALK-TP3 3条探针)
表6 9例样本的检测结果(ALK-TP4通用型探针)

从表中的结果可以看出，9例样本中，检出4例阳性样本，其余样本为阴性，微滴式数字PCR和荧光定量PCR检出结果一致性为100％；另外，使用ALK-TP4通用型探针，得到的结果几乎与NALK-TP1、TALK-TP2和TRALK-TP3 3条探针检测结果一致。
具体检测的结果图见图1～9。
综上所述，本发明提供的ALK基因融合变异的ddPCR检测体系，特异性好，灵敏性高，相比市场上其他公司的产品在特异性检测中灵敏度、准确性方面均有较大程度的提高；检测的假阳性率大幅度降低，检测结果准确度高；同时本发明提供的检测体系，可以应用于骨髓、外周血等多种生物样本类型，同时一个反应可以同步检测三种融合突变类型，检测成功率高针对微量模板的检测具有明显的优势，在临床检验中具有较高的应用价值。
以上实施例，仅为本发明的具体实施方式，用以说明本发明的技术方案，而非对其限制，本发明的保护范围并不局限于此，尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，其依然可以对前述实施例所记载的技术方案进行修改或可轻易想到变化，或者对其中部分技术特征进行等同替换；而这些修改、变化或者替换，并不使相应技术方案的本质脱离本发明实施例技术方案的精神和范围，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种检测ALK融合基因的引物和探针组合物，其特征在于，包括用于检测NPM1-ALK融合基因的引物对和探针、用于检测TPM3-ALK融合基因的引物对和探针、用于检测TRAF1-ALK融合基因的引物对和探针和ABL内参基因引物对和探针；所述用于检测NPM1-ALK融合基因的引物对序列如SEQ ID NO:1-2所示，所述用于检测TPM3-ALK融合基因的引物对序列如SEQ ID NO:4-5所示，所述用于检测TRAF1-ALK融合基因的引物对序列如SEQ ID NO:7-8所示，所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列、所述用于检测TRAF1-ALK融合基因的探针序列分别如SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9所示或如SEQ ID NO:13所示；所述ABL内参基因引物对序列如SEQ ID NO:10-11所示，探针序列如SEQ ID NO:12所示。
  2. 根据权利要求1所述的检测ALK融合基因的引物和探针组合物，其特征在于，所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列和所述用于检测TRAF1-ALK融合基因的探针序列5'端标记有FAM报告基团，3'端标记MGB荧光基团；所述ABL内参基因探针序列5'端标记有HEX报告基团，3'端标记有BHQ1荧光淬灭基团。
  3. 一种检测ALK融合基因的试剂盒，其特征在于，所述试剂盒包括权利要求1或2所述的检测ALK融合基因的引物和探针组合物。
  4. 根据权利要求3所述的检测ALK融合基因的试剂盒，其特征在于，所述试剂盒还包括阳性标准品、阴性标准品、ddPCR Supermix和ddH2O。
  5. 一种非诊断目的检测ALK融合基因的方法，其特征在于，包括如下步骤：
    S1、从样品中提取RNA，并进行逆转录，获得cDNA；
    S2、以所述cDNA为模板，将待测样品cDNA模板、权利要求1或2所述 的用于检测ALK融合基因的引物和探针组合物以及ddPCR Supermix混合，制备ddPCR混合液；
    S3、用所述ddPCR混合液制作PCR微反应液滴，再进行PCR扩增反应；
    S4、通过数字PCR仪对PCR扩增反应后的产物进行荧光信号的采集，根据荧光信号的类型，从而判断出ALK融合基因类型。
  6. 根据权利要求5所述的非诊断目的检测ALK融合基因的方法，其特征在于，步骤S1中，所述逆转录反应程序如下：30℃、10min，42℃、50min，95℃、5min，4℃、保温。
  7. 根据权利要求5所述的非诊断目的检测ALK融合基因的方法，其特征在于，步骤S2中，所述用于检测ALK融合基因的引物和探针组合物的浓度为10×，其中，所述用于检测NPM1-ALK融合基因的引物对序列、所述用于检测TPM3-ALK融合基因的引物对序列、所述用于检测TRAF1-ALK融合基因的引物对序列和所述ABL内参基因引物对序列的浓度均为6～9μM；所述用于检测NPM1-ALK融合基因的探针序列、所述用于检测TPM3-ALK融合基因的探针序列、所述用于检测TRAF1-ALK融合基因的探针序列和所述ABL内参基因探针序列的浓度均为3～8μM。
  8. 根据权利要求5所述的非诊断目的检测ALK融合基因的方法，其特征在于，步骤S3中，用所述ddPCR混合液制作PCR微反应液滴的具体方法如下：将ddPCR混合液加入到微滴发生器中，生成15000～20000个微反应液滴。
  9. 根据权利要求5所述的非诊断目的检测ALK融合基因的方法，其特征在于，步骤S3中，所述PCR扩增反应程序如下：93～97℃预变性3～10min，93～95℃变性20～40s，55～60℃退火50～70s，共进行35～45个循环，10～12℃保温。
  10. 根据权利要求9所述的非诊断目的检测ALK融合基因的方法，其特征在于，所述PCR扩增反应程序如下：95℃预变性5min，94℃变性30s，60℃退火60s，共进行40个循环，12℃保温。
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CN114807124B (zh) * 2022-04-01 2023-09-12 上海信诺佰世医学检验有限公司 一种检测alk融合基因的引物和探针组合物、试剂盒及方法
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102575287A (zh) * 2009-05-15 2012-07-11 因赛特遗传学公司 用于诊断和治疗癌症的涉及alk融合物的方法和组合物
CN112852967A (zh) * 2021-03-23 2021-05-28 上海真固生物科技有限公司 一种基于ddPCR定量检测ALK、RET和ROS1融合基因的试剂盒
CN114807124A (zh) * 2022-04-01 2022-07-29 上海信诺佰世医学检验有限公司 一种检测alk融合基因的引物和探针组合物、试剂盒及方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382896A (zh) * 2011-12-06 2012-03-21 苏州苏大赛尔免疫生物技术有限公司 Npm1基因突变检测方法及其试剂盒
US20210095334A1 (en) * 2017-02-10 2021-04-01 The Rockefeller University Methods for cell-type specific profiling to identify drug targets
CN107022610B (zh) * 2017-04-01 2019-02-05 常州桐树生物科技有限公司 肿瘤驱动基因的检测芯片及其应用
CN111471770A (zh) * 2020-05-07 2020-07-31 南京实践医学检验有限公司 一种基于多重荧光rt-pcr检测白血病融合基因的试剂盒及方法
CN112094915B (zh) * 2020-11-17 2021-02-19 苏州科贝生物技术有限公司 一种肉瘤融合基因和/或突变联合检测引物组及试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102575287A (zh) * 2009-05-15 2012-07-11 因赛特遗传学公司 用于诊断和治疗癌症的涉及alk融合物的方法和组合物
CN112852967A (zh) * 2021-03-23 2021-05-28 上海真固生物科技有限公司 一种基于ddPCR定量检测ALK、RET和ROS1融合基因的试剂盒
CN114807124A (zh) * 2022-04-01 2022-07-29 上海信诺佰世医学检验有限公司 一种检测alk融合基因的引物和探针组合物、试剂盒及方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AGARWAL INDU, SABATINI LINDA, ALIKHAN MIR B.: "Diagnostic Capability of Next-Generation Sequencing Fusion Analysis in Identifying a Rare CASE of TRAF1-ALK-Associated Anaplastic Large Cell Lymphoma", FRONTIERS IN ONCOLOGY, vol. 10, 8 May 2020 (2020-05-08), pages 730, XP093097050, DOI: 10.3389/fonc.2020.00730 *
DAMM-WELK CHRISTINE, LOVISA FEDERICA, CONTARINI GIORGIA, LÜDERSEN JETTE, CARRARO ELISA, KNÖRR FABIAN, FÖRSTER JAN, ZIMMERMANN MART: "Quantification of Minimal Disease by Digital PCR in ALK-Positive Anaplastic Large Cell Lymphoma: A Step towards Risk Stratification in International Trials?", CANCERS, vol. 14, no. 7, 27 March 2022 (2022-03-27), pages 1703, XP093097034, DOI: 10.3390/cancers14071703 *
DATABASE Nucleotide 17 October 2007 (2007-10-17), ANONYMOUS : "Human nucleophosmin-anaplastic lymphoma kinase fusion protein (NPM/ALK -Nucleotide -NCBI", XP093097047, retrieved from NCBI Database accession no. U04946.1 *
DATABASE Nucleotide 22 March 2020 (2020-03-22), ANONYMOUS : "Homo sapiens TNF receptor associated factor 1/ALK receptor tyrosine ki -Nucleotide -NCBI", XP093097041, retrieved from NCBI Database accession no. MT163661.1 *
DRIEUX FANNY, RUMINY PHILIPPE, SATER VINCENT, MARCHAND VINCIANE, FATACCIOLI VIRGINIE, LANIC MARIE-DELPHINE, VIENNOT MATHIEU, VIAIL: "Detection of Gene Fusion Transcripts in Peripheral T-Cell Lymphoma Using a Multiplexed Targeted Sequencing Assay", THE JOURNAL OF MOLECULAR DIAGNOSTICS, AMERICAN SOCIETY FOR INVESTIGATIVE PATHOLOGY AND THE ASSOCIATION FOR MOLECULAR PATHOLOGY, vol. 23, no. 8, 1 August 2021 (2021-08-01), pages 929 - 940, XP093097029, ISSN: 1525-1578, DOI: 10.1016/j.jmoldx.2021.04.013 *
MELCHERS RUTGER C., WILLEMZE REIN, VAN DE LOO MEREL, VAN DOORN REMCO, JANSEN PATTY M., CLEVEN ARJEN H.G., SOLLEVELD NIENKE, BEKKEN: "Clinical, Histologic, and Molecular Characteristics of Anaplastic Lymphoma Kinase-positive Primary Cutaneous Anaplastic Large Cell Lymphoma", AMERICAN JOURNAL OF SURGICAL PATHOLOGY., RAVEN PRESS, NEW YORK, NY., US, vol. 44, no. 6, 1 June 2020 (2020-06-01), US , pages 776 - 781, XP009549269, ISSN: 0147-5185, DOI: 10.1097/PAS.0000000000001449 *
READING N SCOTT, JENSON STEPHEN D, SMITH JEFFREY K, LIM MEGAN S, ELENITOBA-JOHNSON S J: "Consultations in Molecular Diagnostics 5Ј-(RACE) Identification of Rare ALK Fusion Partner in Anaplastic Large Cell Lymphoma", JOURNAL OF MOLECULAR DIAGNOSTICS, vol. 5, no. 2, 1 May 2003 (2003-05-01), pages 136 - 140, XP093097032 *
于冉 (YU, RAN): "ALK基因在肿瘤中的改变及意义 (Non-official translation: Alterations and Significance of ALK Gene in Tumors)", 产业与科技论坛 (INDUSTRIAL & SCIENCE TRIBUNE), vol. 20, no. 7, 31 December 2021 (2021-12-31), pages 49 - 50 *

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