TW202129008A - Idh mutation detection kit and method thereof - Google Patents

Idh mutation detection kit and method thereof Download PDF

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TW202129008A
TW202129008A TW109138073A TW109138073A TW202129008A TW 202129008 A TW202129008 A TW 202129008A TW 109138073 A TW109138073 A TW 109138073A TW 109138073 A TW109138073 A TW 109138073A TW 202129008 A TW202129008 A TW 202129008A
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徐安
林佩頤
魏大程
陳淑貞
陳華鍵
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香港商行動基因（智財）有限公司
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Abstract

The disclosure relates a kit and a method for detecting an IDH mutation. The kit includes primers, polymerase, and especially a series of properly-designed probe sequences specific for the IDH mutation detection. Moreover, the method used to detect an IDH mutation includes the steps of amplification, and the multiplex detection of IDH mutation, which can identify different types of IDH mutation in biological samples.

Description

檢測異檸檬酸脫氫酶突變的套組及方法Kit and method for detecting mutation of isocitrate dehydrogenase

本申請案主張2019年11月05日提出的美國臨時申請案第62/931,190號的優先權，其全部內容通過引用併入本文。This application claims the priority of U.S. Provisional Application No. 62/931,190 filed on November 05, 2019, the entire content of which is incorporated herein by reference.

本發明涉及分子診斷學、癌症基因體學和分子生物學領域。The invention relates to the fields of molecular diagnostics, cancer genomics and molecular biology.

異檸檬酸脫氫酶（IDH）1/2是一種酵素，由IDH1和IDH2基因轉譯而出。IDH1和IDH2分別位於細胞質（cytoplasm）和線粒體（mitochondria）中。它們是同源二聚體酶，在三羧酸循環（TCA循環）反應中扮演重要腳色。IDH催化異檸檬酸的氧化脫羧，生成α-酮戊二酸（α-KG），並同時由NADP+生成還原的菸鹼醯胺腺嘌呤二核苷酸磷酸（NADPH）。Isocitrate dehydrogenase (IDH) 1/2 is an enzyme that is translated from IDH1 and IDH2 genes. IDH1 and IDH2 are located in cytoplasm and mitochondria, respectively. They are homodimer enzymes and play an important role in the reaction of the TCA cycle (TCA cycle). IDH catalyzes the oxidative decarboxylation of isocitrate to produce α-ketoglutarate (α-KG), and at the same time produces reduced nicotine amide adenine dinucleotide phosphate (NADPH) from NADP+.

近期發現，IDH基因的突變是很常見的。最為人熟知的突變為IDH1突變所引起的單核苷酸變異，其導致該酵素的活性位上的密碼子132處發生胺基酸替換。當IDH基因突變時，可負調控（downregulate）α-酮戊二酸（α-KG）的表達，並將其轉化為2-羥基戊二酸（2-HG），而引起例如DNA全域甲基化（DNA global methylation）和組蛋白甲基化（histone methylation）等表觀遺傳學改變（epigenetic alteration）。它啟動下游訊號路徑，並促進癌症的發展。IDH突變是最重要的基因突變之一，其常出現在許多癌症中，例如多形性膠質母細胞瘤（GBM）和許多血液惡性腫瘤（hematopoietic cancers），包括急性骨髓性白血病（acute myeloid leukemia）、血管免疫母細胞T細胞淋巴瘤（angioimmunoblastic T-cell lymphoma）、軟骨肉瘤（chondrosarcoma）、肝內膽管癌（intrahepatic cholangiocarcinoma）等。It has recently been discovered that mutations in the IDH gene are very common. The most well-known mutation is the single-nucleotide mutation caused by the IDH1 mutation, which results in an amino acid substitution at codon 132 in the active site of the enzyme. When the IDH gene is mutated, it can downregulate the expression of α-ketoglutarate (α-KG) and convert it to 2-hydroxyglutaric acid (2-HG), causing, for example, DNA global methylation Epigenetic alteration such as DNA global methylation and histone methylation. It initiates downstream signaling pathways and promotes the development of cancer. IDH mutations are one of the most important gene mutations, which are often found in many cancers, such as glioblastoma multiforme (GBM) and many hematopoietic cancers, including acute myeloid leukemia , Angioimmunoblastic T-cell lymphoma, chondrosarcoma, intrahepatic cholangiocarcinoma, etc.

IDH突變所誘發的腫瘤往往是新型療法的潛在靶點。具有IDH突變的患者可能對特定的IDH抑制劑或其他治療策略十分敏感，例如：DNA去甲基化劑（DNA demethylating agent）、Bcl-2家族抑制劑（Bcl-2 family inhibitor）、DNA損傷劑（DNA damaging agent）、DLL-3靶向治療、疫苗接種治療或改變代謝的靶點（target for altered metabolism）。因此，檢測IDH突變並確定哪些患者可能從IDH抑制劑或其他療法中獲益是非常重要的。Tumors induced by IDH mutations are often potential targets for new therapies. Patients with IDH mutations may be sensitive to specific IDH inhibitors or other treatment strategies, such as DNA demethylating agents, Bcl-2 family inhibitors, DNA damaging agents (DNA damaging agent), DLL-3 targeted therapy, vaccination therapy or target for altered metabolism. Therefore, it is very important to detect IDH mutations and determine which patients may benefit from IDH inhibitors or other therapies.

許多技術可用於檢測IDH1/2突變，其包括免疫組織化學（IHC）檢測、桑格定序（Sanger sequencing）、焦磷酸定序（pyrosequencing）和解鏈曲線分析（melting curve analysis）。IHC可以檢測IDH突變蛋白的存在，但它需要針對每種突變類型使用不同的專一性抗體。桑格定序可以檢測所有類型的突變，但其不夠敏銳而無法檢測到20%以內的突變等位基因頻率（mutant allele frequency）之變異，而這種變異在癌症樣本中是很常見。雖然焦磷酸定序十分敏銳且可進行定量，但其穩健性仍取決於檢測之設計。解鏈曲線分析可以快速檢測所有類型的突變，但仍需提高其靈敏度。由於IDH1/2中的許多核苷酸置換（transitions）會導致不同的氨基酸被替換，而形成不同的IDH突變類型，因此現今急需具有多重性（multiplexing）、高專一性、有效率和節約成本等優點的IDH突變檢測方法。Many techniques can be used to detect IDH1/2 mutations, including immunohistochemistry (IHC) detection, Sanger sequencing, pyrosequencing, and melting curve analysis. IHC can detect the presence of IDH mutant proteins, but it requires the use of different specific antibodies for each type of mutation. Sanger sequencing can detect all types of mutations, but it is not sensitive enough to detect mutations within 20% of the mutant allele frequency (mutant allele frequency), which is very common in cancer samples. Although pyrophosphate sequencing is very sensitive and quantifiable, its robustness still depends on the design of the test. Melting curve analysis can quickly detect all types of mutations, but its sensitivity still needs to be improved. Because many nucleotide substitutions in IDH1/2 (transitions) will cause different amino acids to be replaced, resulting in different types of IDH mutations, there is an urgent need for multiplexing, high specificity, efficiency and cost savings. Advantages of IDH mutation detection method.

本揭露係關於一種用於檢測檢測異檸檬酸脫氫酶（IDH）基因突變的套組。該套組包含：至少兩種IDH專一性引子對、以及至少兩種探針。此外，IDH專一性引子對中的一IDH專一性正向引子進一步具有該通用引子對（universal primer pair）中的一通用正向引子的核苷酸序列，並且IDH專一性反向引子進一步具有該通用引子對中的一通用反向引子的核苷酸序列。This disclosure relates to a kit for detecting mutations in isocitrate dehydrogenase (IDH) gene. The kit includes: at least two IDH-specific primer pairs and at least two probes. In addition, an IDH-specific forward primer in the IDH-specific primer pair further has the nucleotide sequence of a universal forward primer in the universal primer pair, and the IDH-specific reverse primer further has the nucleotide sequence of a universal forward primer in the universal primer pair. The nucleotide sequence of a universal reverse primer in a universal primer pair.

在一些較佳實施例中，該套組還包含DNA聚合酶。In some preferred embodiments, the kit also includes DNA polymerase.

在一些較佳實施例中，各該至少兩種探針具有選自由SEQ ID NO：1-16及其任一互補序列所組成群組的不同核苷酸序列。In some preferred embodiments, each of the at least two probes has a different nucleotide sequence selected from the group consisting of SEQ ID NO: 1-16 and any complementary sequence thereof.

在一些較佳實施例中，該IDH專一性正向引子或該IDH專一性反向引子係與一可偵測分子相結合。In some preferred embodiments, the IDH-specific forward primer or the IDH-specific reverse primer is combined with a detectable molecule.

在一些較佳實施例中，該可偵測分子係為一螢光分子或用於一呈色反應的一酵素。In some preferred embodiments, the detectable molecule is a fluorescent molecule or an enzyme for a color reaction.

在一些較佳實施例中，各該至少兩種探針係與獨有識別元件（unique identifier）相結合。In some preferred embodiments, each of the at least two probe systems is combined with a unique identifier.

在一些較佳實施例中，該獨有識別元件為一實體條碼並且可於明視野成像中被偵測出。In some preferred embodiments, the unique identification element is a physical barcode and can be detected in bright-field imaging.

在一些較佳實施例中，該套組還包含一對照獨有識別元件並且其未與任何探針相結合。In some preferred embodiments, the kit also includes a control unique identification element and it is not combined with any probe.

在一些較佳實施例中，該至少二種探針係固著於一微陣列板上的不同位置。In some preferred embodiments, the at least two probes are fixed on different positions on a microarray plate.

在一些較佳實施例中，該微陣列板包含一位置指標。In some preferred embodiments, the microarray plate includes a position indicator.

在一些較佳實施例中，該套組還包含一對照探針用以偵測持家基因（housekeeping gene）。In some preferred embodiments, the kit also includes a control probe for detecting housekeeping genes.

另一方面，本文亦提供一種使用前述套組用於檢測異檸檬酸脫氫酶（IDH）基因突變的方法。該方法包含以下步驟：(a)自一生物樣品中獲得去氧核糖核酸（DNA）；(b)使用至少二種IDH專一性引子對擴增該DNA以獲得一第一擴增產物；(c)使用一通用引子對擴增該第一擴增產物以獲得一第二擴增產物；(d)將該第二擴增產物與至少二種探針混合；以及(e)檢測一探針結合產物（probe-bound product）以判定IDH突變的存在。On the other hand, this article also provides a method for detecting isocitrate dehydrogenase (IDH) gene mutations using the aforementioned kit. The method includes the following steps: (a) obtaining deoxyribonucleic acid (DNA) from a biological sample; (b) using at least two IDH-specific primer pairs to amplify the DNA to obtain a first amplified product; (c) ) Use a universal primer pair to amplify the first amplification product to obtain a second amplification product; (d) mix the second amplification product with at least two probes; and (e) detect a probe binding Product (probe-bound product) to determine the presence of IDH mutations.

更進一步地，各該至少二種IDH專一性引子對中的一IDH專一性正向引子進一步具有該通用引子對中的一通用正向引子的核苷酸序列，並且該每個IDH專一性引子對中的一IDH專一性反向引子進一步具有該通用引子對中的一通用反向引子的核苷酸序列。Furthermore, an IDH-specific forward primer in each of the at least two IDH-specific primer pairs further has the nucleotide sequence of a universal forward primer in the universal primer pair, and each IDH-specific primer An IDH-specific reverse primer in the pair further has the nucleotide sequence of a universal reverse primer in the universal primer pair.

在一些較佳實施例中，該至少兩種探針與該第二擴增產物混合於55-70°C、55-65°C、60-65°C、60-70°C。In some preferred embodiments, the at least two probes and the second amplification product are mixed at 55-70°C, 55-65°C, 60-65°C, 60-70°C.

在一些較佳實施例中，該至少兩種探針與該第二擴增產物混合於650-1000 rpm、700-900 rpm、700-800 rpm、700-750 rpm、650-750 rpm。In some preferred embodiments, the at least two probes and the second amplification product are mixed at 650-1000 rpm, 700-900 rpm, 700-800 rpm, 700-750 rpm, 650-750 rpm.

在一些較佳實施例中， 在步驟(d)中各該至少兩種探針係與獨有識別元件相結合。In some preferred embodiments, each of the at least two probe systems is combined with a unique identification element in step (d).

在一些較佳實施例中，在步驟(e)中該至少兩種探針藉由該獨有識別元件被識別出。In some preferred embodiments, the at least two probes are identified by the unique identification element in step (e).

在一些較佳實施例中，該至少二種探針係固著於一微陣列板上。In some preferred embodiments, the at least two probes are fixed on a microarray plate.

在一些較佳實施例中，該生物樣品係源自一固體腫瘤、軟組織肉瘤（soft tissue sarcoma）、或血液腫瘤。In some preferred embodiments, the biological sample is derived from a solid tumor, soft tissue sarcoma (soft tissue sarcoma), or hematological tumor.

在一些較佳實施例中，該生物樣品包含一福馬林固定石蠟包埋（formalin-fixed paraffin-embedded）組織切片、血液、血漿或細胞。In some preferred embodiments, the biological sample comprises a formalin-fixed paraffin-embedded tissue section, blood, plasma or cells.

在一些較佳實施例中，該突變存在代表著生物樣品源自於可能對IDH抑制劑、IDH相關下游信號級聯反應（IDH associated downstream signaling cascade）抑制劑、DNA去甲基化劑、Bcl-2家族抑制劑、DNA損傷劑、DLL-3靶向療法、疫苗接種療法、針對改變代謝的分子、Enasidenib、Ivosidenib或上述任意組合有反應的患者。In some preferred embodiments, the presence of the mutation means that the biological sample is derived from possible IDH inhibitors, IDH associated downstream signaling cascade inhibitors, DNA demethylating agents, Bcl- 2 Patients who respond to family inhibitors, DNA damaging agents, DLL-3 targeted therapy, vaccination therapy, molecules that alter metabolism, Enasidenib, Ivosidenib, or any combination of the above.

除非另有定義，本文中使用的所有技術及科學術語具有與本揭露所屬技術領域中熟習技藝者通常理解的相同含義。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this disclosure belongs.

定義definition

除非上下文另有明確指示，本文中所使用的單數形式「一」、「一個」及「該」包含複數指稱。Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" used in this article include plural referents.

術語 「突變（mutation）」和「變異（variant）」可互換使用，指核酸中一個或幾個核苷酸或鹼基對的替換、***或刪除。The terms "mutation" and "variant" are used interchangeably and refer to the substitution, insertion or deletion of one or several nucleotides or base pairs in a nucleic acid.

本文中所用「連結件（connector）」或「接頭（linker）」係指一分子的一部分或由複數分子所構成複合物的一部分，其將一個分子連接至另一個分子。該連結件或接頭可以透過共價鍵、核酸雜合、或一對分子間的非共價交互作用，例如生物素-鏈親和素(biotin-streptavidin)交互作用等方式發揮作用。在本文中，「連結件」係用於連接一引子與一可偵測分子，例如一螢光分子；「接頭」係用於連接一探針及一可偵測分子或一獨有識別元件（unique identifier），例如一條碼磁珠（barcoded magnetic bead，BMB）。As used herein, "connector" or "linker" refers to a part of a molecule or a part of a complex composed of a plurality of molecules, which connects one molecule to another molecule. The linker or linker can function through covalent bonds, nucleic acid hybridization, or non-covalent interactions between a pair of molecules, such as biotin-streptavidin interactions. In this article, "connector" is used to connect a primer and a detectable molecule, such as a fluorescent molecule; "connector" is used to connect a probe and a detectable molecule or a unique recognition element ( unique identifier), such as a barcoded magnetic bead (BMB).

術語「引子（primer）」係指一合成的單股寡核苷酸，其可用於擴增具有特定長度的一標靶核酸。本文中所用術語「IDH專一性引子（ IDH specific primer）」及「專一性引子（specific primer）」可互換使用，其係指被設計用於擴增一特定野生型（WT）IDH基因以及其突變基因。該IDH專一性引子係成對使用，包含一能夠專一地結合至一野生型（WT）IDH基因以及其突變基因的5’端的一IDH專一性正向引子（IDH-specific forward primer），以及能夠專一地結合至所述野生型（WT）IDH基因以及其突變基因的3’端的一IDH專一性反向引子（IDH -specific reverse primer）。The term "primer" refers to a synthetic single-stranded oligonucleotide that can be used to amplify a target nucleic acid of a specific length. As used herein, the terms "IDH specific primer" and "specific primer" are used interchangeably, and refer to those designed to amplify a specific wild-type (WT) IDH gene and its mutations Gene. The IDH-specific primers are used in pairs, including an IDH-specific forward primer that can specifically bind to the 5'end of a wild-type (WT) IDH gene and its mutant gene, and can An IDH-specific reverse primer (IDH-specific reverse primer) specifically bound to the 3'end of the wild-type (WT) IDH gene and its mutant gene.

本文中所用術語「通用引子（universal primer）」係指被設計用於擴增包含通用引子的核苷酸序列的任何DNA的DNA引子。該通用引子係成對使用，包含一通用正向引子（universal forward primer）及一通用反向引子（universal reverse primer）。The term "universal primer" as used herein refers to a DNA primer designed to amplify any DNA containing the nucleotide sequence of the universal primer. The universal primer is used in pairs, including a universal forward primer and a universal reverse primer.

除非另有定義，術語「探針（probe）」或「IDH突變專一性探針(IDH mutation-specific probe)」係指一合成的單股DNA寡核苷酸，其能夠與源自一種特定IDH突變基因的一區域雜合，該區域包含一突變位點（mutation site）。Unless otherwise defined, the term "probe" or "IDH mutation-specific probe" refers to a synthetic single-stranded DNA oligonucleotide that can be combined with a specific IDH A region of the mutated gene is heterozygous, and the region contains a mutation site.

本揭露提供一種用於檢測IDH突變的方法及套組。該方法包含以下步驟： (a) 自一生物樣品中獲得去氧核糖核酸（DNA）； (b) 使用至少二種IDH專一性引子對擴增該DNA以獲得一第一擴增產物； (c) 使用一通用引子對擴增該第一擴增產物以獲得一第二擴增產物； (d) 將該第二擴增產物與至少二種探針混合，其中該至少二種探針具有選自由SEQ ID NO：1-32；以及 (e) 檢測IDH突變的存在。The present disclosure provides a method and kit for detecting IDH mutations. The method includes the following steps: (a) Obtain deoxyribonucleic acid (DNA) from a biological sample; (b) Use at least two IDH-specific primer pairs to amplify the DNA to obtain a first amplified product; (c) Amplify the first amplification product using a universal primer pair to obtain a second amplification product; (d) mixing the second amplification product with at least two probes, wherein the at least two probes are selected from SEQ ID NO: 1-32; and (e) Detect the presence of IDH mutations.

IDH突變類型有許多種。IDH突變包括IDH1和IDH2基因突變，該兩種IDH突變都可能引起點突變（point mutation），而形成不同的IDH突變類型。本方法能夠檢測下表1中所列的任何IDH突變類型。檢測時需使用專一性的探針接觸每個突變的擴增DNA產物以捕獲擴增產物並進行檢測。在本公開中，表1中揭示了針對每個突變類型所設計的專一性探針序列。There are many types of IDH mutations. IDH mutations include IDH1 and IDH2 gene mutations. Both IDH mutations may cause point mutations and form different types of IDH mutations. This method can detect any of the IDH mutation types listed in Table 1 below. During detection, a specific probe is used to contact the amplified DNA product of each mutation to capture the amplified product and detect it. In this disclosure, the specific probe sequence designed for each mutation type is disclosed in Table 1.

表1：用於檢測的探針序列和IDH突變類型 基因 胺基酸改變 SEQ ID No. 探針序列 (由 5’端 至3’端) IDH1 R132C SEQ ID No. 1 ATCATAGGTTGTCATGCTTA IDH1 R132G SEQ ID No. 2 ATCATAGGTGGTCATGCTTA IDH1 R132S SEQ ID No. 3 ATCATAGGTAGTCATGCTTA IDH1 R132L SEQ ID No. 4 ATCATAGGTCTTCATGCTTA IDH1 R132H SEQ ID No. 5 ATCATAGGTCATCATGCTTA IDH2 R140W SEQ ID No. 6 AGGATGTTCCAGATAGTTCCA IDH2 R140Q SEQ ID No. 7 AGGATGTTCTGGATAGTTCCA IDH2 R140G SEQ ID No. 8 AGGATGTTCCCGATAGTTCCA IDH2 R140L SEQ ID No. 9 AGGATGTTCAGGATAGTTCCA IDH2 R172G SEQ ID No. 10 GGCGTGCCCGCCAATG IDH2 R172K SEQ ID No. 11 GGCGTGCTTGCCAATG IDH2 R172M SEQ ID No. 12 GGCGTGCATGCCAATG IDH2 R172S SEQ ID No. 13 GGCGTGGCTGCCAATG IDH2 R172S SEQ ID No. 14 GGCGTGACTGCCAATG IDH2 R172T SEQ ID No. 15 GGCGTGCGTGCCAATG IDH2 R172W SEQ ID No. 16 GGCGTGCCAGCCAATG IDH1 R132C SEQ ID No. 17 TAAGCATGACAACCTATGAT IDH1 R132G SEQ ID No. 18 TAAGCATGACCACCTATGAT IDH1 R132S SEQ ID No. 19 TAAGCATGACTACCTATGAT IDH1 R132L SEQ ID No. 20 TAAGCATGAAGACCTATGAT IDH1 R132H SEQ ID No. 21 TAAGCATGATGACCTATGAT IDH2 R140W SEQ ID No. 22 TGGAACTATCTGGAACATCCT IDH2 R140Q SEQ ID No. 23 TGGAACTATCCAGAACATCCT IDH2 R140G SEQ ID No. 24 TGGAACTATCGGGAACATCCT IDH2 R140L SEQ ID No. 25 TGGAACTATCCTGAACATCCT IDH2 R172G SEQ ID No. 26 CATTGGCGGGCACGCC IDH2 R172K SEQ ID No. 27 CATTGGCAAGCACGCC IDH2 R172M SEQ ID No. 28 CATTGGCATGCACGCC IDH2 R172S SEQ ID No. 29 CATTGGCAGCCACGCC IDH2 R172S SEQ ID No. 30 CATTGGCAGTCACGCC IDH2 R172T SEQ ID No. 31 CATTGGCACGCACGCC IDH2 R172W SEQ ID No. 32 CATTGGCTGGCACGCC Table 1: Probe sequences and IDH mutation types used for detection Gene Amino acid change SEQ ID No. Probe sequence (from 5'end to 3'end) IDH1 R132C SEQ ID No. 1 ATCATAGGTTGTCATGCTTA IDH1 R132G SEQ ID No. 2 ATCATAGGTGGTCATGCTTA IDH1 R132S SEQ ID No. 3 ATCATAGGTAGTCATGCTTA IDH1 R132L SEQ ID No. 4 ATCATAGGTCTTCATGCTTA IDH1 R132H SEQ ID No. 5 ATCATAGGTCATCATGCTTA IDH2 R140W SEQ ID No. 6 AGGATGTTCCAGATAGTTCCA IDH2 R140Q SEQ ID No. 7 AGGATGTTCTGGATAGTTCCA IDH2 R140G SEQ ID No. 8 AGGATGTTCCCGATAGTTCCA IDH2 R140L SEQ ID No. 9 AGGATGTTCAGGATAGTTCCA IDH2 R172G SEQ ID No. 10 GGCGTGCCCGCCAATG IDH2 R172K SEQ ID No. 11 GGCGTGCTTGCCAATG IDH2 R172M SEQ ID No. 12 GGCGTGCATGCCAATG IDH2 R172S SEQ ID No. 13 GGCGTGGCTGCCAATG IDH2 R172S SEQ ID No. 14 GGCGTGACTGCCAATG IDH2 R172T SEQ ID No. 15 GGCGTGCGTGCCAATG IDH2 R172W SEQ ID No. 16 GGCGTGCCAGCCAATG IDH1 R132C SEQ ID No. 17 TAAGCATGACAACCTATGAT IDH1 R132G SEQ ID No. 18 TAAGCATGACCACCTATGAT IDH1 R132S SEQ ID No. 19 TAAGCATGACTACCTATGAT IDH1 R132L SEQ ID No. 20 TAAGCATGAAGACCTATGAT IDH1 R132H SEQ ID No. 21 TAAGCATGATGACCTATGAT IDH2 R140W SEQ ID No. 22 TGGAACTATCTGGAACATCCT IDH2 R140Q SEQ ID No. 23 TGGAACTATCCAGAACATCCT IDH2 R140G SEQ ID No. 24 TGGAACTATCGGGAACATCCT IDH2 R140L SEQ ID No. 25 TGGAACTATCCTGAACATCCT IDH2 R172G SEQ ID No. 26 CATTGGCGGGCACGCC IDH2 R172K SEQ ID No. 27 CATTGGCAAGCACGCC IDH2 R172M SEQ ID No. 28 CATTGGCATGCACGCC IDH2 R172S SEQ ID No. 29 CATTGGCAGCCACGCC IDH2 R172S SEQ ID No. 30 CATTGGCAGTCACGCC IDH2 R172T SEQ ID No. 31 CATTGGCACGCACGCC IDH2 R172W SEQ ID No. 32 CATTGGCTGGCACGCC

在一些實施例中，本方法只需要一個單一反應來檢測一種類型的IDH突變。在另一個實施例中，本方法還可以利用單一反應進行多重反應（multiplex reaction）以檢測多種類型的IDH突變。現今些許技術平臺可以執行上述任務，例如：微陣列（microarray）、基因晶片（gene chip）、微珠（microbeads）、奈米顆粒（nanoparticles）、膜片（membranes）或微流體裝置（microfluidic devices）。 在一些實施例中，多種突變類型的檢測是在具有選自SEQ ID No.1-32組成的多個不同探針的多重反應中所進行的。在一些實施例中，探針由一個單一的匯集試劑（pooled reagent）提供。在一些實施例中，探針由獨立的複數個試劑提供。在一些實施例中，探針被固著於微流體裝置、微陣列、基因晶片或不同的微珠上的不同位置上。在一些實施例中，引子被固著於微流體裝置、微陣列、基因晶片、膜片或不同的微珠上的不同位置。In some embodiments, this method requires only a single reaction to detect one type of IDH mutation. In another embodiment, the method can also use a single reaction to perform a multiplex reaction to detect multiple types of IDH mutations. Today, a few technology platforms can perform the above tasks, such as: microarrays, gene chips, microbeads, nanoparticles, membranes, or microfluidic devices . In some embodiments, the detection of multiple mutation types is performed in multiple reactions with multiple different probes selected from SEQ ID No. 1-32. In some embodiments, the probe is provided by a single pooled reagent. In some embodiments, the probe is provided by a plurality of independent reagents. In some embodiments, the probes are affixed to different locations on the microfluidic device, microarray, gene chip, or different microbeads. In some embodiments, the primers are affixed to different locations on the microfluidic device, microarray, gene chip, membrane, or different microbeads.

在一些實施例中，本方法使用螢光（fluorescence）、化學發光（chemiluminescence）、色素（pigment）或其它比色信號強度（colorimetric signal intensity）來檢測IDH突變。在一些實施例中，螢光、化學發光、色素或其他比色信號強度用於區分一突變類型的有無。在一些實施例中，IDH專一性引子藉由生物素或其他官能團與鏈親和素-PE（streptavidin-PE）或其他螢光、化學發光、色素或其他比色基團標記/連接。在一些實施例中，探針藉由生物素或其他功能基團與鏈親和素-PE或其他螢光、化學發光、色素或其他比色基團標記/連接。在一些實施例中，步驟(b)使用能夠進行螢光偶聯和檢測的標記的去氧核苷酸。在一些實施例中，引子、探針或去氧核苷酸藉由酵素反應與螢光、化學發光、色素或比色基團連接。In some embodiments, the method uses fluorescence, chemiluminescence, pigment, or other colorimetric signal intensity to detect IDH mutations. In some embodiments, fluorescence, chemiluminescence, pigment or other colorimetric signal intensity is used to distinguish the presence or absence of a mutation type. In some embodiments, IDH-specific primers are labeled/linked with streptavidin-PE (streptavidin-PE) or other fluorescent, chemiluminescent, pigment, or other colorimetric groups by biotin or other functional groups. In some embodiments, the probe is labeled/linked to streptavidin-PE or other fluorescent, chemiluminescent, pigment, or other colorimetric groups via biotin or other functional groups. In some embodiments, step (b) uses labeled deoxynucleotides capable of fluorescent coupling and detection. In some embodiments, primers, probes, or deoxynucleotides are linked to fluorescent, chemiluminescent, pigment, or colorimetric groups through enzyme reactions.

在一些實施例中，在本方法的步驟(d)中，IDH突變的探針標記之擴增產物還與獨有識別元件（unique identifier）相結合。在一些實施例中，該獨有識別元件是條碼、實體條碼、條碼微珠、條碼磁珠、核酸序列或分子條碼。在一些實施例中，條碼可以藉由明視野成像來進行檢測。在一些實施例中，獨有識別元件直接或藉由接頭（linker）與探針相結合。在一些實施例中，獨有識別元件藉由化學偶聯反應（即共價鍵）連接到探針。在一些實施例中，獨有識別元件藉由聚合物接頭連接到探針。在一些實施例中，獨有識別元件使用核酸序列藉由雜合連接到探針。In some embodiments, in step (d) of the method, the amplified product labeled with the IDH mutant probe is also combined with a unique identifier. In some embodiments, the unique identification element is a barcode, a physical barcode, a barcode bead, a barcode magnetic bead, a nucleic acid sequence, or a molecular barcode. In some embodiments, barcodes can be detected by bright-field imaging. In some embodiments, the unique identification element is combined with the probe directly or via a linker. In some embodiments, the unique recognition element is connected to the probe by a chemical coupling reaction (ie, a covalent bond). In some embodiments, the unique identification element is connected to the probe via a polymer linker. In some embodiments, the unique recognition element is linked to the probe by hybridization using a nucleic acid sequence.

在一些實施例中，探針和擴增產物之間的接觸是藉由核酸雜合進行的。在一些實施例中，雜合步驟是藉由熱混合（thermomixing）進行的。 雜合步驟是在單一溫度下進行的，其混合條件為探針與它們的同源擴增產物雜合。表1中的探針被設計為提供與其他適合於多重反應的探針相似的效率。 在一些實施方案中，雜合溫度在55-65℃、55-70℃、60-65℃或60-70℃之間。在一些實施例中，雜合步驟在熱攪拌器（thermomixer）中進行，其旋轉速度在700-1000 rpm、700-900 rpm、700-800 rpm、700-750 rpm或650-750 rpm之間。In some embodiments, the contact between the probe and the amplification product is by nucleic acid hybridization. In some embodiments, the hybridization step is performed by thermomixing. The hybridization step is performed at a single temperature, and the mixing condition is that the probes hybridize with their homologous amplification products. The probes in Table 1 are designed to provide similar efficiencies to other probes suitable for multiple reactions. In some embodiments, the hybridization temperature is between 55-65°C, 55-70°C, 60-65°C, or 60-70°C. In some embodiments, the hybridization step is performed in a thermomixer with a rotation speed between 700-1000 rpm, 700-900 rpm, 700-800 rpm, 700-750 rpm, or 650-750 rpm.

在一些實施例中，本方法中使用的生物樣品包括福馬林固定的石蠟包埋切片、無細胞核酸、血液、血漿或細胞。在一些實施例中，本方法包括從生物樣品中提取DNA的步驟。在一些實施例中，生物樣品來源於癌症患者。在一些實施例中，生物樣品來源於固體腫瘤（solid tumor）、軟組織肉瘤（soft tissue sarcoma）或血液腫瘤（hematological cancer）。在一些實施例中，生物樣品來源於急性髓性白血病（acute myeloid leukemia）、惡性膠質瘤（malignant glioma）、少突膠質瘤（oligodendroglioma）、星形細胞瘤（astrocytoma）、白血病（leukemia）、膽管癌（bile duct carcinoma）、乳腺癌（breast carcinoma）、結直腸腺癌（colorectal adenocarcinoma）和非小細胞肺癌（non-small cell lung carcinoma）患者。In some embodiments, the biological samples used in the method include formalin-fixed paraffin-embedded sections, cell-free nucleic acids, blood, plasma, or cells. In some embodiments, the method includes the step of extracting DNA from a biological sample. In some embodiments, the biological sample is derived from a cancer patient. In some embodiments, the biological sample is derived from solid tumor, soft tissue sarcoma, or hematological cancer. In some embodiments, the biological sample is derived from acute myeloid leukemia, malignant glioma, oligodendroglioma, astrocytoma, leukemia, bile duct Patients with bile duct carcinoma, breast carcinoma, colorectal adenocarcinoma and non-small cell lung carcinoma.

在本揭露中還提供一種用於檢測異檸檬酸脫氫酶（IDH）突變的套組。該套組的成分包含：（a）一個與突變的5'-端結合的正向引子和一個與突變的3'-端結合的反向引子、（b）DNA聚合酶、以及（c）至少一種探針具有選自由SEQ ID NO：1-32所組成群組的核苷酸序列。The disclosure also provides a kit for detecting the mutation of isocitrate dehydrogenase (IDH). The components of the kit include: (a) a forward primer bound to the 5'-end of the mutation and a reverse primer bound to the 3'-end of the mutation, (b) DNA polymerase, and (c) at least A probe has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-32.

在一些實施例中，該套組用於使用表1中所列出的該突變類型的同源探針以檢測一種或多種IDH突變類型。在一些實施例中，藉由多重反應檢測多種突變類型，該反應包括選自SEQ ID No.1-32所組成群組的多個不同探針。在該實施例中，該套組盒包括一組探針，而每個探針具有選自SEQ ID No.1-32所組成群組的序列。In some embodiments, the kit is used to use the homologous probes of the mutation type listed in Table 1 to detect one or more IDH mutation types. In some embodiments, multiple types of mutations are detected by multiple reactions including multiple different probes selected from the group consisting of SEQ ID Nos. 1-32. In this embodiment, the kit includes a set of probes, and each probe has a sequence selected from the group consisting of SEQ ID No. 1-32.

在一些實施例中，一個或多個IDH突變-引子被生物素或能夠連接到鏈親和素-PE或其它螢光基團的其它官能團標記。在一些實施例中，探針被生物素或能夠連接到鏈親和素-PE或其它螢光基團的其它官能團標記。在一些實施例中，該套組還包含能夠進行螢光偶聯和檢測的標記的去氧核苷酸。In some embodiments, one or more IDH mutation-primers are labeled with biotin or other functional groups capable of linking to streptavidin-PE or other fluorescent groups. In some embodiments, the probe is labeled with biotin or other functional groups capable of linking to streptavidin-PE or other fluorescent groups. In some embodiments, the kit also includes labeled deoxynucleotides capable of fluorescent coupling and detection.

在一些實施例中，該套組還包含獨有識別元件（unique identifier）。 在一些實施例中，獨有識別元件是條碼、實體條碼、條碼微珠、條碼磁珠、核酸序列或分子條碼。在一些實施例中，條碼可以藉由明視野成像來進行檢測。在一些實施例中，獨有識別元件直接或藉由接頭與探針連接。在一些實施例中，獨有識別元件藉由化學耦合產生共價鍵而連接到探針。在一些實施例中，獨有識別元件藉由聚合物接頭（polymer linker）連接到探針。在一些實施例中，獨有識別元件藉由核酸序列透過雜合反映連接到探針。在一些實施例中，該套組還包含不與任何探針連接的對照獨有識別元件。In some embodiments, the set also includes a unique identifier. In some embodiments, the unique identification element is a barcode, a physical barcode, a barcode bead, a barcode magnetic bead, a nucleic acid sequence, or a molecular barcode. In some embodiments, barcodes can be detected by bright-field imaging. In some embodiments, the unique identification element is connected to the probe directly or via a connector. In some embodiments, the unique identification element is connected to the probe by chemical coupling to create a covalent bond. In some embodiments, the unique identification element is connected to the probe via a polymer linker. In some embodiments, the unique recognition element is linked to the probe through a hybrid reaction through a nucleic acid sequence. In some embodiments, the kit also includes a control unique identification element that is not connected to any probe.

在一些實施方案中，該套組還包含對照生物樣品。在一些實施例中，該套組包含IDH突變的陽性對照樣品。在一些實施方案中，該套組包括具有IDH基因野生型序列的陰性對照樣品。在一些實施例中，對照樣品是寡核苷酸、DNA、RNA、FFPE切片、血液、血漿或細胞。In some embodiments, the kit also includes a control biological sample. In some embodiments, the kit includes a positive control sample for IDH mutations. In some embodiments, the kit includes a negative control sample with a wild-type sequence of the IDH gene. In some embodiments, the control sample is oligonucleotide, DNA, RNA, FFPE section, blood, plasma, or cells.

在一些實施例中，IDH突變的存在代表生物樣品來源為一患者，該患者可能對IDH抑制劑、IDH相關下游信號級聯反應抑制劑、DNA去甲基化劑、Bcl-2家族抑制劑、DNA損傷劑、DLL-3靶向治療、疫苗接種治療、針對改變代謝的分子或上述任一組合產生反應。在一些實施例中，IDH突變的存在代表生物樣品來源於可能對Enasidenib或Ivosidenib有反應的患者。In some embodiments, the presence of an IDH mutation indicates that the source of the biological sample is a patient, and the patient may be resistant to IDH inhibitors, IDH-related downstream signaling cascade inhibitors, DNA demethylating agents, Bcl-2 family inhibitors, DNA damaging agents, DLL-3 targeted therapy, vaccination therapy, a response to a molecule that alters metabolism, or any combination of the above. In some embodiments, the presence of an IDH mutation indicates that the biological sample is derived from a patient who may be responsive to Enasidenib or Ivosidenib.

實施例Example

實施例1：以探針-BMB雜合試驗檢測IDH1單核苷酸變異（single nucleotide variant(s)；SNV）Example 1: Detection of IDH1 single nucleotide variants (single nucleotide variant(s); SNV) by probe-BMB hybrid test

IDH1基因中涉及多個單核苷酸變異。為了在一次反應中同時檢測和區分潛在的IDH1單核苷酸變異，因而開發了IDH1單核苷酸變異檢測方法（即探針-BMB（條碼磁珠）雜合法）。此外，下文將揭露IDH1專一性寡核苷酸和相應的IDH1單核苷酸變異之專一性探針與BMB偶聯的IDH1單核苷酸變異檢測方法之相關設計細節。Multiple single nucleotide variations are involved in the IDH1 gene. In order to simultaneously detect and distinguish potential IDH1 single nucleotide variations in one reaction, the IDH1 single nucleotide variation detection method (ie probe-BMB (barcode magnetic bead) hybridization method) was developed. In addition, the following will disclose the relevant design details of the IDH1 single nucleotide variation detection method coupled with IDH1 specific oligonucleotides and corresponding IDH1 single nucleotide variation specific probes and BMB.

為了檢測IDH1單核苷酸變異，因此藉由聚合酶鏈反應（PCR）檢測法擴增基因組DNA（gDNA）。具體而言，藉由多重探針-BMBs對PCR產物進行信號檢測。在IDH1單核苷酸變異檢測之前，將IDH1專一性探針與BMBs進行偶聯，生成探針-BMBs。然後，透過以下步驟進行IDH1單核苷酸變異檢測。(1)用IDH專一性引子對對gDNA中IDH1單核苷酸變異的區域進行PCR擴增；(2)將擴增及標記的gDNA與探針-BMBs混合；(3)檢測探針-BMBs的螢光信號，以判定IDH1突變類型。上述該檢測整體流程如圖1中所示。In order to detect IDH1 single nucleotide variation, genomic DNA (gDNA) was amplified by polymerase chain reaction (PCR) detection method. Specifically, the PCR products are detected by multiple probes-BMBs. Before IDH1 single nucleotide variation detection, IDH1 specific probe is coupled with BMBs to generate probe-BMBs. Then, perform IDH1 single nucleotide variation detection through the following steps. (1) Use IDH-specific primers to perform PCR amplification on the region of IDH1 single nucleotide variation in gDNA; (2) Mix the amplified and labeled gDNA with probe-BMBs; (3) Detection probe-BMBs To determine the type of IDH1 mutation. The above-mentioned overall process of the detection is shown in Figure 1.

IDH1突變專一性探針之製備Preparation of IDH1 mutation specific probe

更具體地而言，上述探針是專門針對5個不同的IDH1單核苷酸變異所設計的，探針可以在一個反應中同時區分不同的IDH1單核苷酸變異。表2揭示了用於檢測的IDH1單核苷酸變異的序列。為了在表2中劃線的位點進行雜合，每個探針被設計成具有表3中列出的序列。IDH1單核苷酸變異專一性探針由IDT(Integrated DNA Technologies，Inc，Coralville，IA)合成、純化並在5'端用胺基進行修飾。隨後，藉由胺基-羧酸基之間的鍵結將專一性探針與不同的BMB偶聯。具有不同ID的BMBs用於在多重反應中以區分來自不同探針的不同突變信號。More specifically, the above-mentioned probes are specifically designed for 5 different IDH1 single nucleotide variants, and the probe can distinguish different IDH1 single nucleotide variants at the same time in one reaction. Table 2 reveals the sequence of IDH1 single nucleotide variation used for detection. In order to hybridize at the sites underlined in Table 2, each probe was designed to have the sequence listed in Table 3. The IDH1 single-nucleotide variant-specific probe was synthesized and purified by IDT (Integrated DNA Technologies, Inc, Coralville, IA) and modified with an amine group at the 5'end. Subsequently, specific probes were coupled to different BMBs through the bond between the amine group and the carboxylic acid group. BMBs with different IDs are used in multiple reactions to distinguish different mutation signals from different probes.

使用IDH1專一性引子對進行PCR擴增PCR amplification using IDH1 specific primer pair

此外，在檢測突變的第一步中，用IDH1專一性引物對對目標gDNA樣品進行PCR檢測擴增。為了替代具有IDH1單核苷酸變異的臨床樣品，IDT公司合成了5個具有表2中SEQ ID No. 33-37序列的寡核苷酸（稱為IDH1突變寡聚物（oligo））作為陽性對照樣品，以代表不同的IDH1單核苷酸變異。由IDT合成並純化相同的IDH1專一性引子對來擴增5個不同的IDH1單核苷酸變異。引子對的反向引子在5'-端用生物素修飾，以便隨後與鏈親和素-藻紅素（SA-PE）共軛物(Thermo Fisher Scientific)相互作用。PCR係依據製造商的說明使用高保真度Platinum Taq DNA聚合酶(Thermo Fisher Scientific)在Veriti™ 96孔熱循環儀(Thermo Fisher Scientific)上進行40個熱循環。In addition, in the first step of detecting mutations, IDH1 specific primers are used to detect and amplify the target gDNA samples by PCR. In order to replace clinical samples with IDH1 single nucleotide variants, IDT has synthesized 5 oligonucleotides with the sequence of SEQ ID No. 33-37 in Table 2 (called IDH1 mutant oligos (oligo)) as positive Control samples to represent different IDH1 single nucleotide variations. The same IDH1 specific primer pair was synthesized and purified by IDT to amplify 5 different IDH1 single nucleotide variants. The reverse primer of the primer pair was modified with biotin at the 5'-end for subsequent interaction with the streptavidin-phycoerythrin (SA-PE) conjugate (Thermo Fisher Scientific). The PCR system used high-fidelity Platinum Taq DNA polymerase (Thermo Fisher Scientific) to perform 40 thermal cycles on a Veriti™ 96-well thermal cycler (Thermo Fisher Scientific) according to the manufacturer's instructions.

探針雜合及訊號檢測Probe hybridization and signal detection

將各IDH1突變寡聚物的擴增產物分別與5種探針-BMB混合在96孔盤中的同一孔中以進行雜合。該雜合係在40-45°C下進行及約700 rpm的搖動下進行10-30分鐘。雜合後，將SA-PE的螢光綴合物添加至該孔洞以便與擴增產物的生物素相結合，並且清洗該探針-BMB以去除未結合的物質。此外，向該孔洞中添加未攜帶探針的另外的BMB(識別碼為0)以作為陰性對照組。最後，利用配備了能夠同時進行明視野與螢光成像的相機的BioCode 2500分析儀(Applied BioCode Inc.，台北，台灣)去讀取BMB的條碼及檢測BMB的螢光訊號。The amplification products of each IDH1 mutant oligomer were mixed with 5 kinds of probe-BMB in the same well in a 96-well plate for hybridization. The hybrid system is performed at 40-45°C and shaking at about 700 rpm for 10-30 minutes. After hybridization, a fluorescent conjugate of SA-PE is added to the hole to bind to the biotin of the amplified product, and the probe-BMB is washed to remove unbound substances. In addition, another BMB (identification code 0) that does not carry the probe is added to the hole to serve as a negative control group. Finally, a BioCode 2500 analyzer (Applied BioCode Inc., Taipei, Taiwan) equipped with a camera capable of performing bright-field and fluorescent imaging at the same time was used to read the BMB's barcode and detect the BMB's fluorescent signal.

表4顯示了BMB7、BMB14、BMB15、BMB21、BMB106、BMB0的螢光強度。依據表4，指示一種特定的標靶-探針雜合(即一種特定的IDH1單核苷酸變異類型存在)的特定BMB的螢光強度顯著高於非專一性訊號的螢光強度。此結果說明單次擴增標靶-探針-BMB試驗可用於檢測及區別多種IDH1單核苷酸變異類型。Table 4 shows the fluorescence intensity of BMB7, BMB14, BMB15, BMB21, BMB106, and BMB0. According to Table 4, the fluorescence intensity of a specific BMB indicating a specific target-probe hybrid (that is, the presence of a specific IDH1 single nucleotide variant type) is significantly higher than the fluorescence intensity of the non-specific signal. This result shows that a single amplification target-probe-BMB test can be used to detect and distinguish multiple IDH1 single nucleotide variants.

表2：合成的IDH1單核苷酸變異寡核苷酸及相應的探針序列 編號 寡聚物編號 染色體位置 基因 胺基酸改變 寡聚物及探針序列 1 SEQ ID No. 33 Chr2: 209113066-209113184 IDH1 R132C AATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGACAACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 2 SEQ ID No. 34 Chr2: 209113066-209113184 IDH1 R132G AATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGACCACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 3 SEQ ID No. 35 Chr2: 209113066-209113184 IDH1 R132S AATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGACTACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 4 SEQ ID No. 36 Chr2: 209113066-209113184 IDH1 R132L AATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGAAGACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 5 SEQ ID No. 37 Chr2: 209113066-209113184 IDH1 R132H AATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGATGACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA Table 2: Synthetic IDH1 single nucleotide variant oligonucleotides and corresponding probe sequences serial number Oligomer number Chromosome position Gene Amino acid change Oligomer and probe sequence 1 SEQ ID No. 33 Chr2: 209113066-209113184 IDH1 R132C AATCACATTATTGCCAACATGACTTACTTGATCCCCA TAAGCATGACAACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 2 SEQ ID No. 34 Chr2: 209113066-209113184 IDH1 R132G AATCACATTATTGCCAACATGACTTACTTGATCCCCA TAAGCATGACCACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 3 SEQ ID No. 35 Chr2: 209113066-209113184 IDH1 R132S AATCACATTATTGCCAACATGACTTACTTGATCCCCA TAAGCATGACTACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 4 SEQ ID No. 36 Chr2: 209113066-209113184 IDH1 R132L AATCACATTATTGCCAACATGACTTACTTGATCCCCA TAAGCATGAAGACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA 5 SEQ ID No. 37 Chr2: 209113066-209113184 IDH1 R132H AATCACATTATTGCCAACATGACTTACTTGATCCCCA TAAGCATGATGACCTATGAT GATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGA

表3：對應的寡聚物、探針和BMB之資訊 寡聚物編號 探針編號 BMB識別碼 雜合溫度 (°C) SEQ ID No. 33 SEQ ID No. 1 7 62 SEQ ID No. 34 SEQ ID No. 2 14 62 SEQ ID No. 35 SEQ ID No. 3 15 62 SEQ ID No. 36 SEQ ID No. 4 21 62 SEQ ID No. 37 SEQ ID No. 5 106 62 Table 3: Information on corresponding oligomers, probes and BMB Oligomer number Probe number BMB identification code Hybrid temperature (°C) SEQ ID No. 33 SEQ ID No. 1 7 62 SEQ ID No. 34 SEQ ID No. 2 14 62 SEQ ID No. 35 SEQ ID No. 3 15 62 SEQ ID No. 36 SEQ ID No. 4 twenty one 62 SEQ ID No. 37 SEQ ID No. 5 106 62

表4：第一批的信號強度 BMB識別碼 寡聚物編號 0 7 14 15 21 106 SEQ ID No. 33 6 2904 234 258 116 97 SEQ ID No. 34 3 142 3060 166 55 55 SEQ ID No. 35 7 400 156 2667 85 95 SEQ ID No. 36 4 114 90 86 2897 152 SEQ ID No. 37 8 81 40 85 199 2565 Table 4: Signal strength of the first batch BMB identification code oligomer number 0 7 14 15 twenty one 106 SEQ ID No. 33 6 2904 234 258 116 97 SEQ ID No. 34 3 142 3060 166 55 55 SEQ ID No. 35 7 400 156 2667 85 95 SEQ ID No. 36 4 114 90 86 2897 152 SEQ ID No. 37 8 81 40 85 199 2565

實施例2：以二次擴增標靶-探針雜合試驗檢測IDH1/2單核苷酸變異Example 2: The detection of IDH1/2 single nucleotide variation by the secondary amplification target-probe hybrid test

二次擴增靶向探針雜合測定法是另一種為在單一反應中同時檢測多個可能的IDH1/2單核苷酸變異而設計的方法。圖2揭示了該檢測法的整體過程，其包括以下步驟。(1)從生物樣品中獲得基因組DNA(gDNA)；(2)利用IDH1/2專一性引子對gDNA（即目標gDNA）的IDH1/2單核苷酸變異區域進行PCR擴增，得到目標gDNA的第一擴增產物；(3)利用通用引子對對第一擴增產物進行PCR擴增，得到目標gDNA的第二擴增產物；(4)與擴增後的目標gDNA進行探針雜合，檢測探針結合產物。以下是該檢測方法的一個具體實施例。The secondary amplification targeting probe hybrid assay is another method designed to simultaneously detect multiple possible IDH1/2 single nucleotide variations in a single reaction. Figure 2 reveals the overall process of the detection method, which includes the following steps. (1) Obtain genomic DNA (gDNA) from biological samples; (2) Use IDH1/2 specific primers to perform PCR amplification on the IDH1/2 single nucleotide variant region of gDNA (ie target gDNA) to obtain the target gDNA The first amplified product; (3) PCR amplify the first amplified product with a universal primer pair to obtain the second amplified product of the target gDNA; (4) Probe hybridization with the amplified target gDNA, The probe binding product is detected. The following is a specific embodiment of the detection method.

基因組DNA(gDNA) 萃取Genomic DNA (gDNA) extraction

依據製造商的說明書利用RecoverAll總核酸分離套組（Cat No: AM1975，Ambient Technologies）從癌症患者的FFPE組織樣本中提取gDNA。The RecoverAll Total Nucleic Acid Isolation Kit (Cat No: AM1975, Ambient Technologies) was used to extract gDNA from FFPE tissue samples of cancer patients according to the manufacturer's instructions.

使用IDH1/2專一性引子對進行PCR擴增。Use IDH1/2 specific primer pair for PCR amplification.

本檢測中使用的IDH1/2專一性引子對中的每個引子被設計為具有兩個片段。 一個片段，稱為IDH1/2專一性片段，用於結合IDH1/2專一性序列的5'-端或3'-端。另一個片段，稱為通用片段，其具有在第二輪PCR中所使用的通用引子的核苷酸序列。該通用片段相對於IDH1/2專一性片段總是在上游，或在5'位置(圖2)。通用引子可以是表5所列引子的任一者，其中每個通用引子可以用作通用正向引子或通用反向引子。Each primer in the IDH1/2 specific primer pair used in this test is designed to have two fragments. A fragment, called IDH1/2 specific fragment, is used to bind the 5'-end or 3'-end of the IDH1/2 specific sequence. The other fragment, called the universal fragment, has the nucleotide sequence of the universal primer used in the second round of PCR. This universal fragment is always upstream relative to the IDH1/2 specific fragment, or at the 5'position (Figure 2). The universal primer can be any of the primers listed in Table 5, and each universal primer can be used as a universal forward primer or a universal reverse primer.

表5 通用引子編號 引子序列 ( 由5’ 端至3’ 端) SEQ ID NO U01 GTTTTCCCAGTCACGACGT 78 U02 GCAAATGGCATTCTGACATCC 79 U03 GCGGATAACAATTTCACACAGG 80 U04 CGTCCATGCCGAGAGTG 81 U05 CTTTATGTTTTTGGCGTCTTCCA 82 U06 GACTGGTTCCAATTGACAAGC 83 U07 GCGTGAATGTAAGCGTGAC 84 U08 TGTAAAACGACGGCCAGT 85 U09 AAGGGTCTTGCGAAGGATAG 86 U10 GGGTTATGCTAGTTATTGCTCAG 87 table 5 Universal primer number Primer sequence ( 'end to the 3' end of 5) SEQ ID NO U01 GTTTTCCCAGTCACGACGT 78 U02 GCAAATGGCATTCTGACATCC 79 U03 GCGGATAACAATTTCACACAGG 80 U04 CGTCCATGCCGAGAGTG 81 U05 CTTTATGTTTTTGGCGTCTTCCA 82 U06 GACTGGTTCCAATTGACAAGC 83 U07 GCGTGAATGTAAGCGTGAC 84 U08 TGTAAAACGACGGCCAGT 85 U09 AAGGGTCTTGCGAAGGATAG 86 U10 GGGTTATGCTAGTTATTGCTCAG 87

進行IDH1/2專一性PCR時，將7 μL水加入10 μL的gDNA產物中，其後將所得混合物(17 μL)等分為1至8組匯集物（pool）。匯集物的數目係取決於引子效能。更具體而言，首先測定IDH1及IDH2專一性引子對的引子效率，然後將效率相近的IDH1及IDH2專一性引子對混合以形成單一引子匯集物。該每個單一引子匯集物將包含1至20個IDH1/2專一性引子對，並添加到一組gDNA匯集物中。其後，依據製造商說明書使用多重PCR套組(貨號：206143，Qiagen)在Veriti™96孔熱循環儀(Thermo Fisher Scientific)上對各組gDNA進行擴增15至30個熱循環，由此生成10 μL的第一個擴增產物。When IDH1/2 specific PCR is performed, 7 μL of water is added to 10 μL of gDNA product, and then the resulting mixture (17 μL) is equally divided into 1 to 8 pools. The number of pools depends on the efficiency of the primer. More specifically, firstly, the primer efficiency of IDH1 and IDH2 specific primer pairs is measured, and then IDH1 and IDH2 specific primer pairs with similar efficiencies are mixed to form a single primer pool. Each single primer pool will contain 1 to 20 IDH1/2 specific primer pairs and will be added to a set of gDNA pools. After that, a multiple PCR kit (Cat. No.: 206143, Qiagen) was used to amplify each group of gDNA on a Veriti™ 96-well thermal cycler (Thermo Fisher Scientific) for 15 to 30 thermal cycles according to the manufacturer’s instructions. 10 μL of the first amplification product.

使用通用引子對進行PCR擴增PCR amplification using universal primer pairs

由於每個IDH1/2專一性引子在5’端包含一通用引子的核苷酸序列，藉由使用通用引子對的PCR能夠進一步擴增該第一擴增產物。該通用引子對包含具有選自SEQ ID NO：78-87之序列的一通用正向引子，以及具有選自SEQ ID NO：78-87之序列的一通用反向引子。該通用反向引子被生物素修飾。進行第二次PCR時，將前述每組第一擴增產物的每一組稀釋100倍於最終反應混合物中，並且依據製造商說明書使用Platinum SuperFi II PCR預混液(Cat No：12368010，Invitrogen)在Veriti™96孔熱循環儀(Thermo Fisher Scientific)上對各組第一擴增產物進行擴增25個熱循環，由此得到10 μL的第二擴增產物。Since each IDH1/2 specific primer contains the nucleotide sequence of a universal primer at the 5'end, the first amplified product can be further amplified by PCR using a universal primer pair. The universal primer pair includes a universal forward primer having a sequence selected from SEQ ID NO: 78-87, and a universal reverse primer having a sequence selected from SEQ ID NO: 78-87. The universal reverse primer is modified with biotin. When performing the second PCR, dilute each of the aforementioned first amplification products 100 times in the final reaction mixture, and use the Platinum SuperFi II PCR master mix (Cat No: 12368010, Invitrogen) in accordance with the manufacturer’s instructions. The first amplification product of each group was amplified for 25 thermal cycles on the Veriti™ 96-well thermal cycler (Thermo Fisher Scientific), thereby obtaining 10 μL of the second amplification product.

探針雜合及訊號檢測Probe hybridization and signal detection

將前述所有第二擴增產物匯集並混合以產生一混合物。將該混合物置於96孔PCR盤（貨號：P46-4TI-1000 /C，4titude）。該第二擴增產物在96°C變性5分鐘，再移入經預阻斷處理的孔洞（pre-blocked wells），各孔洞中預先印製一個探針點陣列，包括IDH1/2單核苷酸變異專一性探針的點、對照探針的點、及錨定探針的點。標靶-探針雜合係在50°C伴隨振盪進行15分鐘。雜合後，將孔洞冷卻並清洗二次。隨後，將含有鏈親和素-鹼性磷酸酶綴合物的緩衝液添加到該孔洞中，使生物素-鏈親和素得以交互作用，然後加入鹼性磷酸酶的受質，以便有色產物在探針-標靶雜合體所在的位置生成。透過使用照相機對孔洞拍照以及識別有色斑點在該孔洞中的位置，可判定指示一種特定IDH1/2單核苷酸變異存在的特定雜合。有色斑點位置的分析可以由電腦進行。All the aforementioned second amplification products are pooled and mixed to produce a mixture. Place the mixture on a 96-well PCR plate (Cat. No.: P46-4TI-1000/C, 4titude). The second amplified product was denatured at 96°C for 5 minutes, and then moved into the pre-blocked wells. Each hole was pre-printed with an array of probe spots, including IDH1/2 mononucleotide The points of the variant-specific probes, the points of the control probes, and the points of the anchor probes. The target-probe hybrid system was shaken at 50°C for 15 minutes. After hybridization, the holes are cooled and cleaned twice. Subsequently, the buffer containing the streptavidin-alkaline phosphatase conjugate is added to the hole to allow the biotin-streptavidin to interact, and then the substrate for alkaline phosphatase is added so that the colored products can be probed. The needle-target hybrid is generated at the location. By taking a picture of the hole with a camera and identifying the position of the colored spot in the hole, it is possible to determine the specific heterozygosity that indicates the presence of a specific IDH1/2 single-nucleotide variant. The analysis of the position of the colored spots can be performed by a computer.

無without

本技術領域之熟習技藝者憑藉以下對較佳實施方式的詳細說明並配合所附圖式將清楚理解本發明，在該圖式中：Those skilled in the art will clearly understand the present invention by virtue of the following detailed description of the preferred embodiments and the accompanying drawings, in which:

圖1係依據本發明一實施例的IDH突變檢測方法的示意圖。Fig. 1 is a schematic diagram of an IDH mutation detection method according to an embodiment of the present invention.

圖2係依據本發明一實施例的IDH1/2單核苷酸變異（SNVs）檢測方法的示意圖； 該檢測係基於二次擴增標靶－探針雜合試驗（double-amplified target-probe hybridization assay）。Figure 2 is a schematic diagram of a method for detecting IDH1/2 single nucleotide variants (SNVs) according to an embodiment of the present invention; the detection is based on double-amplified target-probe hybridization assay).

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Claims (24)

一種用於檢測異檸檬酸脫氫酶（IDH）突變的套組，包含： 至少兩種IDH專一性引子對； 一通用引子對，其中各該至少兩種IDH專一性引子對中的一IDH專一性正向引子進一步具有該通用引子對中的一通用正向引子的核苷酸序列，並且各該至少兩種IDH專一性引子對中的一IDH專一性反向引子進一步具有該通用引子對中的一通用反向引子的核苷酸序列；以及 至少兩種探針。A kit for detecting mutations in isocitrate dehydrogenase (IDH), including: At least two IDH-specific primer pairs; A universal primer pair, wherein one IDH-specific forward primer in each of the at least two IDH-specific primer pairs further has the nucleotide sequence of a universal forward primer in the universal primer pair, and each of the at least two An IDH-specific reverse primer in the IDH-specific primer pair further has the nucleotide sequence of a universal reverse primer in the universal primer pair; and At least two probes. 如請求項1所述之套組，進一步包含一DNA聚合酶。The kit according to claim 1, further comprising a DNA polymerase. 如請求項1所述之套組，其中各該至少兩種探針具有選自由SEQ ID NO：1-16及其任一互補序列所組成群組的不同核苷酸序列。The kit according to claim 1, wherein each of the at least two probes has a different nucleotide sequence selected from the group consisting of SEQ ID NO: 1-16 and any complementary sequence thereof. 如請求項1所述之套組，其中該IDH專一性正向引子或該IDH專一性反向引子係與一可偵測分子相結合。The kit according to claim 1, wherein the IDH-specific forward primer or the IDH-specific reverse primer is combined with a detectable molecule. 如請求項4所述之套組，其中該可偵測分子係為一螢光分子或用於一呈色反應的一酵素。The kit according to claim 4, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. 如請求項1所述之套組，其中各該至少兩種探針係與獨有識別元件相結合。The kit according to claim 1, wherein each of the at least two probes is combined with a unique identification element. 如請求項6所述之套組，其中該獨有識別元件為一實體條碼並且可於明視野成像中被偵測出。The kit according to claim 6, wherein the unique identification element is a physical barcode and can be detected in bright-field imaging. 如請求項6所述之套組更進一步包含一對照獨有識別元件並且其未與任何探針相結合。The kit according to claim 6 further includes a control unique identification element and it is not combined with any probe. 如請求項1所述之套組，其中該至少二種探針係固著於一微陣列板上的不同位置。The kit according to claim 1, wherein the at least two probes are fixed on different positions on a microarray plate. 如請求項1所述之套組，其中該微陣列板包含一位置指標。The kit according to claim 1, wherein the microarray plate includes a position indicator. 如請求項1所述之套組更進一步包含一對照探針用以偵測持家基因。The kit according to claim 1 further includes a control probe for detecting housekeeping genes. 一種用於檢測異檸檬酸脫氫酶（IDH）突變的方法，包含以下步驟： (a) 自一生物樣品中獲得去氧核糖核酸（DNA）； (b) 使用至少二種IDH專一性引子對擴增該DNA以獲得一第一擴增產物； (c) 使用一通用引子對擴增該第一擴增產物以獲得一第二擴增產物； (d) 將該第二擴增產物與至少二種探針混合；以及 (e) 檢測一探針結合產物以判定IDH突變的存在， 其中各該至少二種IDH專一性引子對中的一IDH專一性正向引子進一步具有該通用引子對中的一通用正向引子的核苷酸序列，並且該每個IDH專一性引子對中的一IDH專一性反向引子進一步具有該通用引子對中的一通用反向引子的核苷酸序列。A method for detecting mutations in isocitrate dehydrogenase (IDH), including the following steps: (a) Obtain deoxyribonucleic acid (DNA) from a biological sample; (b) Use at least two IDH-specific primer pairs to amplify the DNA to obtain a first amplified product; (c) Amplify the first amplification product using a universal primer pair to obtain a second amplification product; (d) mixing the second amplification product with at least two kinds of probes; and (e) Detection of a probe binding product to determine the presence of IDH mutations, Wherein each of the at least two IDH-specific primer pairs has an IDH-specific forward primer further having the nucleotide sequence of a universal forward primer in the universal primer pair, and each of the IDH-specific primer pairs An IDH-specific reverse primer further has the nucleotide sequence of a universal reverse primer in the universal primer pair. 如請求項12所述之方法，其中各該至少兩種探針具有選自由SEQ ID NO：1-16及其任一互補序列所組成群組的不同核苷酸序列。The method according to claim 12, wherein each of the at least two probes has a different nucleotide sequence selected from the group consisting of SEQ ID NO: 1-16 and any complementary sequence thereof. 如請求項12所述之方法，其中該IDH專一性正向引子或該IDH專一性反向引子係與一可偵測分子相結合。The method according to claim 12, wherein the IDH-specific forward primer or the IDH-specific reverse primer is combined with a detectable molecule. 如請求項14所述之方法，其中該可偵測分子係為一螢光分子或用於一呈色反應的一酵素。The method according to claim 14, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. 如請求項12所述之方法，其中該至少兩種探針與該第二擴增產物混合於55-70°C。The method according to claim 12, wherein the at least two probes and the second amplification product are mixed at 55-70°C. 如請求項12所述之方法，其中該至少兩種探針與該第二擴增產物混合於650-1000 rpm。The method according to claim 12, wherein the at least two probes and the second amplification product are mixed at 650-1000 rpm. 如請求項12所述之方法，其中各該至少兩種探針係與獨有識別元件相結合。The method according to claim 12, wherein each of the at least two probes is combined with a unique identification element. 如請求項18所述之方法，其中在步驟(e)中該至少兩種探針藉由該獨有識別元件被識別出。The method according to claim 18, wherein the at least two probes are identified by the unique identification element in step (e). 如請求項18所述之方法，其中該獨有識別元件為一實體條碼並且可於明視野成像中被偵測出。The method according to claim 18, wherein the unique identification element is a physical barcode and can be detected in bright-field imaging. 如請求項12所述之方法，其中該至少二種探針係固著於一微陣列板上。The method according to claim 12, wherein the at least two probes are fixed on a microarray plate. 如請求項12所述之方法，其中該生物樣品係源自一固體腫瘤、軟組織肉瘤（soft tissue sarcoma）、或血液腫瘤。The method according to claim 12, wherein the biological sample is derived from a solid tumor, soft tissue sarcoma (soft tissue sarcoma), or hematological tumor. 如請求項12所述之方法，其中該生物樣品包含一福馬林固定石蠟包埋（formalin-fixed paraffin-embedded）組織切片、血液、血漿或細胞。The method according to claim 12, wherein the biological sample comprises a formalin-fixed paraffin-embedded tissue section, blood, plasma or cells. 如請求項12所述之方法，其中該突變存在代表著生物樣品源自於可能對IDH抑制劑、IDH相關下游信號級聯反應抑制劑、DNA去甲基化劑、Bcl-2家族抑制劑、DNA損傷劑、DLL-3靶向療法、疫苗接種療法、針對改變代謝的分子、Enasidenib、Ivosidenib或上述任意組合有反應的患者。The method according to claim 12, wherein the presence of the mutation indicates that the biological sample is derived from possible IDH inhibitors, IDH-related downstream signal cascade reaction inhibitors, DNA demethylating agents, Bcl-2 family inhibitors, Patients who respond to DNA damaging agents, DLL-3 targeted therapy, vaccination therapy, molecules that alter metabolism, Enasidenib, Ivosidenib, or any combination of the above.
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