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WO2025105837A1 - Procédé de détection multiplex à grande échelle de gènes mutants du cancer du poumon à l'aide d'arn et kit pour détection multiplex à grande échelle de gènes mutants du cancer du poumon l'utilisant - Google Patents

Procédé de détection multiplex à grande échelle de gènes mutants du cancer du poumon à l'aide d'arn et kit pour détection multiplex à grande échelle de gènes mutants du cancer du poumon l'utilisant Download PDF

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WO2025105837A1
WO2025105837A1 PCT/KR2024/017991 KR2024017991W WO2025105837A1 WO 2025105837 A1 WO2025105837 A1 WO 2025105837A1 KR 2024017991 W KR2024017991 W KR 2024017991W WO 2025105837 A1 WO2025105837 A1 WO 2025105837A1
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primer
seq
egfr
eml4
alk
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Korean (ko)
Inventor
손성훈
박홍만
박일현
이병철
최보혜
이채원
김동균
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Genecast Co Ltd
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Genecast Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method for large-scale multiplex detection of lung cancer mutant genes using RNA and a large-scale multiplex detection kit for lung cancer mutant genes using the same, and more specifically, to a gene mutation detection method capable of simultaneously and multiplexing 44 types of EGFR gene mutations, 1 type of BRAF gene mutation, 1 type of KRAS gene mutation, 10 types of EML4-ALK gene fusion mutations and 12 types of ROS1 gene rearrangement mutations using RNA, and a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations using a primer set and a composition.
  • Lung cancer is a common cancer worldwide and a major cause of cancer-related deaths. According to the National Cancer Center's cancer statistics in Korea, lung cancer ranks 4th in cancer incidence, with more than 25,000 patients newly diagnosed with lung cancer each year. Although the 5-year survival rate has improved since the 2000s compared to the 1990s, the 5-year relative survival rate for lung cancer still remains at 28.2%, and in cases with distant metastasis, the 5-year observation survival rate in Korea is very low at 6.1%.
  • KRAS gene mutation is the second most frequently found mutation gene after EGFR in Asian lung cancer patients, and with the recent development of Lumacras as a targeted treatment for KRAS G12C mutation non-small cell lung cancer with a poor prognosis, the importance of diagnosing whether or not there is a KRAS G12C mutation is also increasing.
  • Patent Document 1 discloses a lung cancer diagnostic kit that simultaneously detects mutations in EGFR, KRAS, BRAF, and PIK3CA genes, but there is no known kit that can simultaneously and multiplely detect a total of 68 lung cancer-related gene mutations in EGFR (44 types), BRAF (1 type), KRAS (1 type), ROS1 (12 types), and EML4-ALK (10 types) genes using RNA.
  • the inventors of the present invention have developed a kit capable of simultaneously and multiple times detecting each of the 68 types of lung cancer gene mutations using mutation-specific primers after synthesizing and amplifying cDNA using a specific oligomer from mRNA, so that all of the 68 types of lung cancer gene mutations can be detected even from a small amount of sample, and have completed the present invention.
  • Patent Document 1 Republic of Korea Publication Patent No. 10-2015-0102468
  • the purpose of the present invention is to provide a primer set for synthesizing and amplifying cDNA containing each of EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA.
  • Another object of the present invention is to provide a primer set for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations and a composition for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations comprising the same.
  • Another object of the present invention is to provide a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, comprising a primer set and a probe that specifically bind to EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, respectively, and a method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA using the kit.
  • Another object of the present invention is to provide a composition and kit for diagnosing lung cancer, which comprise primer sets that specifically bind to cDNAs containing 44 types of EGFR gene mutations, cDNAs containing 1 type of BRAF gene mutation, cDNAs containing 1 type of KRAS gene mutation, cDNAs containing 10 types of EML4-ALK gene mutations, and cDNAs containing 12 types of ROS1 gene mutations.
  • the present invention provides a primer set for synthesizing and amplifying cDNAs each including EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, including primer sets of SEQ ID NOs: 1 to 34.
  • the present invention provides a primer set for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes, comprising primer sets of SEQ ID NOs: 37 to 40, 43 to 71, 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132, and a composition for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes, comprising the same.
  • the present invention provides a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, comprising a primer set for synthesizing and amplifying cDNAs each including the aforementioned EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations and a primer set for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations.
  • composition or kit may additionally comprise at least one probe selected from the group consisting of SEQ ID NOs: 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133.
  • composition or kit may additionally include a blocking primer of sequence number 72.
  • the present invention provides a method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA using the kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations described above.
  • the method may include the following steps (a) to (d):
  • the EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations detected by the above method may include 44 EGFR gene mutations, 1 BRAF gene mutation, 1 KRAS gene mutation, 10 EML4-ALK gene mutations and 12 ROS1 gene mutations described in Tables 1 to 5 below.
  • a blocking primer having sequence number 72 may be additionally processed in step (c).
  • the polymerase chain reaction of step (c) can be performed by an allele-specific polymerase chain reaction or a real-time polymerase chain reaction.
  • the method may additionally include a step (e) of confirming the amplification result by the polymerase chain reaction by measuring the Ct (cycle threshold) value.
  • the method can be applied to diagnosing lung cancer or predicting the drug response of lung cancer patients.
  • the present invention provides a composition for diagnosing lung cancer, comprising a primer set that specifically binds to each of cDNAs including 44 types of EGFR gene mutations described in Table 1, cDNAs including 1 type of BRAF gene mutation described in Table 2, cDNAs including 1 type of KRAS gene mutation described in Table 3, cDNAs including 10 types of EML4-ALK gene mutations described in Table 4, and cDNAs including 12 types of ROS1 gene mutations described in Table 5.
  • the primer set included in the lung cancer diagnostic composition may be a primer set of SEQ ID NOs: 37 to 40, 43 to 71, 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132.
  • the lung cancer diagnostic composition may additionally include at least one probe selected from the group consisting of sequence numbers 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130, and 133.
  • the lung cancer diagnostic composition may additionally contain a blocking primer of sequence number 72.
  • the lung cancer diagnostic composition may additionally include a primer set for synthesizing and amplifying cDNAs including 44 types of EGFR gene mutations, cDNAs including 1 type of BRAF gene mutation, cDNAs including 1 type of KRAS gene mutation, cDNAs including 10 types of EML4-ALK gene mutations, and cDNAs including 12 types of ROS1 gene mutations.
  • the primer set for synthesizing and amplifying the cDNA may be at least one primer set selected from the group consisting of SEQ ID NOs: 1 to 34.
  • the present invention also provides a lung cancer diagnostic kit comprising the various forms of lung cancer diagnostic compositions described above.
  • the kit according to the present invention synthesizes and amplifies cDNA using a specific oligomer from mRNA, and then uses a primer set specific for single sequence mutations in the EGFR, BRAF, and KRAS genes and EML4-ALK and ROS1 gene recombination mutations to simultaneously detect a total of 68 gene mutations, making it possible to detect lung cancer-related gene mutations at once from a small amount of sample.
  • Figure 1 is a schematic diagram showing an mRNA-based lung cancer-related gene mutation detection method of the present invention.
  • Figure 3 illustrates a method for performing mmRT-qPCR when the mRNA-based lung cancer-related gene mutation detection method of the present invention is implemented as a massive multiplex real-time quantitative polymerase chain reaction (mmRT-qPCR).
  • Figures 4a to 4c illustrate the amplicon design of each gene target.
  • Figures 5a to 5l show the results of performing mmRT-qPCR using a kit for detecting EGFR, BRAF, KRAS, EML4-ALK, and ROS1 gene mutations according to the present invention using the method illustrated in Figure 3.
  • a kit capable of simultaneously and multipleally detecting a total of 68 lung cancer-related gene mutations in the EGFR (44 types), BRAF (1 type), KRAS (1 type), ROS1 (12 types), and EML4-ALK (10 types) genes using RNA has not been developed. Accordingly, the inventors of the present invention have sought a solution to the above-mentioned problem by developing a kit capable of simultaneously and multiplely detecting each of the 68 lung cancer gene mutations using mutation-specific primers after synthesizing and amplifying cDNA from mRNA using a specific oligomer, as shown in Fig. 1, so that all of the 68 types of various lung cancer gene mutations can be detected even from a small amount of sample.
  • the first aspect of the present invention relates to a primer set for synthesizing and amplifying cDNAs each including EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, and a primer set for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations.
  • primer refers to a polynucleotide that can act as an initiator of nucleic acid synthesis in the template-directed direction when placed under conditions that initiate polynucleotide elongation. Primers can also be used in a variety of other oligonucleotide-mediated synthetic processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes. Primers are typically single-stranded oligonucleotides (e.g., oligodeoxyribonucleotides). The appropriate length of a primer varies depending on the intended use, typically in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides.
  • primer pair means a set of primers comprising a 5'-sense primer that hybridizes complementarily to the 5'-end of the nucleic acid sequence to be amplified, and a 3'-antisense primer that hybridizes to the 3'-end of the sequence to be amplified.
  • the primers may be labeled, if desired, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
  • useful labels include: 32 P, a fluorescent dye, an electron-dense reagent, an enzyme (commonly used in ELISA assays), biotin, or a protein to which haptens and antisera or monoclonal antibodies may be used.
  • the primer set for synthesizing and amplifying cDNAs each including the EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations may include the primer set of SEQ ID NOs: 1 to 34.
  • sequence numbers 1 to 4 are primer sets for synthesizing and amplifying cDNA including EGFR gene mutation, wherein a first primer set including a forward primer of sequence number 1 and a reverse primer of sequence number 2, a second primer set including a forward primer of sequence number 1 and a reverse primer of sequence number 3, and a third primer set including a forward primer of sequence number 1 and a reverse primer of sequence number 4 reverse transcribe and pre-amplify the target region of EGFR mRNA, as shown in FIG. 4A, respectively, to generate three types of EGFR amplicons (556 bp, 587 bp, and 618 bp).
  • Sequence numbers 5 to 7 are primer sets for synthesizing and amplifying cDNA including a BRAF gene mutation, wherein a fourth primer set including a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a fifth primer set including a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 7, respectively, reverse transcribe and pre-amplify the target region of BRAF mRNA to generate two types of BRAF amplicons (130 bp and 166 bp), as shown in FIG. 4A.
  • Sequence numbers 8 to 11 are primer sets for synthesizing and amplifying cDNA including a KRAS gene mutation, wherein a sixth primer set including a forward primer of SEQ ID NO: 8 and a reverse primer of SEQ ID NO: 9, a seventh primer set including a forward primer of SEQ ID NO: 8 and a reverse primer of SEQ ID NO: 10, and an eighth primer set including a forward primer of SEQ ID NO: 8 and a reverse primer of SEQ ID NO: 11 each reverse transcribe and pre-amplify a target region of KRAS mRNA, as shown in FIG. 4A, to generate three types of EGFR amplicons (146 bp, 172 bp, and 194 bp).
  • Sequence numbers 12 to 18 are primer sets for synthesizing and amplifying cDNA including EML4-ALK gene mutation, a ninth primer set including a forward primer of SEQ ID NO: 12 and a reverse primer of SEQ ID NO: 18, a tenth primer set including a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 18, an eleventh primer set including a forward primer of SEQ ID NO: 14 and a reverse primer of SEQ ID NO: 18, a twelfth primer set including a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 18, a thirteenth primer set including a forward primer of SEQ ID NO: 16 and a reverse primer of SEQ ID NO: 18, and a fourteenth primer set including a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18, respectively, reverse transcribe and pre-amplify the target region of EML4-ALK mRNA to produce 10 kinds of EML4-ALK amplicons (
  • SEQ ID NOS: 19 to 34 are primer sets for synthesizing and amplifying cDNA including a ROS1 gene mutation, a 15th primer set including a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 22, a 16th primer set including a forward primer of SEQ ID NO: 20 and a reverse primer of SEQ ID NO: 22, a 17th primer set including a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22, an 18th primer set including a forward primer of SEQ ID NO: 23 and a reverse primer of SEQ ID NO: 26, a 19th primer set including a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 26, a 20th primer set including a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26, a 21st primer set including a forward primer of SEQ ID NO: 27 and a reverse primer of SEQ ID NO: 32, a 22nd primer set including a forward
  • ROS1 amplicons (118 bp, 191 bp, 126 bp, 215 bp, 204 bp, 205 bp, 158 bp, 125 bp, 141 bp, 112 bp, 139 bp, and 167 bp).
  • the primer sets for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations may include primer sets of SEQ ID NOs: 37 to 40, 43 to 71, 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132.
  • the primer set for detecting the G719X mutation of the EGFR gene may include the primer set of SEQ ID NOs: 37 to 40. More specifically, the primer set for detecting the G719S mutation of the EGFR gene may include the reverse primer of SEQ ID NO: 37 and the forward primer of SEQ ID NO: 40, the primer set for detecting the G719C mutation of the EGFR gene may include the reverse primer of SEQ ID NO: 38 and the forward primer of SEQ ID NO: 40, and the primer set for detecting the G719A mutation of the EGFR gene may include the reverse primer of SEQ ID NO: 39 and the forward primer of SEQ ID NO: 40. In this case, the reverse primers may specifically bind to the G719S, G719C, and G719A mutations of the EGFR gene, respectively.
  • a primer set for detecting 29 types of Ex19Del mutations of the EGFR gene includes a primer set including a reverse primer of SEQ ID NO: 43 and a forward primer of SEQ ID NO: 73 capable of sequentially detecting the 29 types of mutations, a primer set including a reverse primer of SEQ ID NO: 44 and a forward primer of SEQ ID NO: 73, a primer set including a reverse primer of SEQ ID NO: 45 and a forward primer of SEQ ID NO: 73, a primer set including a reverse primer of SEQ ID NO: 46 and a forward primer of SEQ ID NO: 73, a primer set including a reverse primer of SEQ ID NO: 47 and a forward primer of SEQ ID NO: 73, a primer set including a reverse primer of SEQ ID NO: 48 and a forward primer of SEQ ID NO: 73, a primer set including a reverse primer of SEQ ID NO: 49 and a forward primer of SEQ ID NO: 73, a primer set including a primer set
  • a primer set for detecting the S768I mutation of the EGFR gene may include a reverse primer of SEQ ID NO: 75 and a forward primer of SEQ ID NO: 76.
  • the reverse primer may specifically bind to the S768I mutation of the EGFR gene.
  • a primer set for detecting five Ex20Ins mutations of the EGFR gene may include a primer set of SEQ ID NOs: 78 to 83.
  • the primer set for detecting five types of Ex20Ins mutations of the EGFR gene may be a primer set including a reverse primer of SEQ ID NO: 78 and a forward primer of SEQ ID NO: 83 capable of sequentially detecting the five types of mutations, a primer set including a reverse primer of SEQ ID NO: 79 and a forward primer of SEQ ID NO: 83, a primer set including a reverse primer of SEQ ID NO: 80 and a forward primer of SEQ ID NO: 83, a primer set including a reverse primer of SEQ ID NO: 81 and a forward primer of SEQ ID NO: 83, and a primer set including a reverse primer of SEQ ID NO: 82 and a forward primer of SEQ ID NO: 83.
  • the reverse primers may specifically bind to five types of Ex20Ins mutations of the EGFR gene.
  • a primer set for detecting the T790M mutation of the EGFR gene may include a reverse primer of SEQ ID NO: 85 and a forward primer of SEQ ID NO: 86.
  • the reverse primer may specifically bind to the T790M mutation of the EGFR gene.
  • a primer set for detecting two types of C797S mutations (2390>C and 2389>A) of the EGFR gene may include a primer set of SEQ ID NOs: 89 to 91. More specifically, the primer set for detecting two types of C797S mutations of the EGFR gene may be a primer set including a forward primer of SEQ ID NO: 89 and a reverse primer of SEQ ID NO: 91 capable of sequentially detecting the two types of mutations, and a primer set including a forward primer of SEQ ID NO: 90 and a reverse primer of SEQ ID NO: 91. In this case, the forward primer may specifically bind to two types of T790M mutations of the EGFR gene.
  • a primer set for detecting the L858R mutation of the EGFR gene may include a forward primer of SEQ ID NO: 93 and a reverse primer of SEQ ID NO: 94.
  • the forward primer may specifically bind to the L858R mutation of the EGFR gene.
  • a primer set for detecting the L861Q mutation of the EGFR gene may include a reverse primer of SEQ ID NO: 96 and a forward primer of SEQ ID NO: 97.
  • the reverse primer may specifically bind to the L861Q mutation of the EGFR gene.
  • a primer set for detecting the G12C mutation of the KRAS gene may include a forward primer of SEQ ID NO: 99 and a reverse primer of SEQ ID NO: 100.
  • the forward primer may specifically bind to the G12C mutation of the KRAS gene.
  • a primer set for detecting the V600E1 mutation of the BRAF gene may include a forward primer of SEQ ID NO: 102 and a reverse primer of SEQ ID NO: 103.
  • the forward primer may specifically bind to the V600E1 mutation of the BRAF gene.
  • a primer set for detecting 10 EML4-ALK gene mutations may include a primer set of SEQ ID NOs: 105 to 111. More specifically, a primer set for detecting EML4-ALK Variant 1 may include a forward primer of SEQ ID NO: 105 and a reverse primer of SEQ ID NO: 111, a primer set for detecting EML4-ALK Variant 2 may include a forward primer of SEQ ID NO: 106 and a reverse primer of SEQ ID NO: 111, a primer set for detecting EML4-ALK Variants 3a and 3b may include a forward primer of SEQ ID NO: 107 and a reverse primer of SEQ ID NO: 111, a primer set for detecting EML4-ALK Variants 5a and 5b may include a forward primer of SEQ ID NO: 108 and a reverse primer of SEQ ID NO: 111, a primer set for detecting EML4-ALK Variants 5a and 5b may include a forward primer of SEQ ID NO:
  • a primer set for detecting 12 ROS1 gene mutations may include primer sets of SEQ ID NOs: 124 to 129, 131 and 132.
  • the primers for detecting 12 types of ROS1 gene mutations may be a forward primer of SEQ ID NO: 124 and a reverse primer of SEQ ID NO: 129, a forward primer of SEQ ID NO: 125 and a reverse primer of SEQ ID NO: 129, a forward primer of SEQ ID NO: 126 and a reverse primer of SEQ ID NO: 129, a forward primer of SEQ ID NO: 127 and a reverse primer of SEQ ID NO: 129, a forward primer of SEQ ID NO: 128 and a reverse primer of SEQ ID NO: 129, a forward primer of SEQ ID NO: 131 and a reverse primer of SEQ ID NO: 132, which can sequentially detect the 12 types of mutations.
  • compositions for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes comprising primer sets of the above-mentioned SEQ ID NOs: 37 to 40, 43 to 71 and 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132.
  • the composition may additionally contain at least one probe selected from the group consisting of sequence numbers 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133.
  • the term "probe” means a substance that specifically detects a specific substance, site, condition, etc., and in the present invention, it may be DNA, RNA, PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), or a mixture thereof that can complementarily bind to EGFR, BRAF, KRAS, EML4-ALK, and ROS1 genes.
  • the probe of the present invention can be chemically synthesized using a phosphoramidite solid support method or other well-known methods. In addition, it can be modified by methylation, capping, etc. using a known method. In addition, the probe can have a length of 5 to 1000 bp, and can vary depending on the scale and location of the mutation, and is not limited in its length.
  • each of the probes may be labeled with a fluorescent substance at the 5'-end and a quencher at the 3'-end.
  • the fluorescent substance may be FAM, Quasar 670 or CY5, but is not limited thereto.
  • the quencher may be BHQ-1 or BHQ-2, but is not limited thereto.
  • the composition may additionally include a blocking primer of SEQ ID NO: 72.
  • the blocking primer of SEQ ID NO: 72 may be used together with a primer set and probe for detecting 29 types of Ex19Del mutations of the EGFR gene.
  • blocking primer includes nucleotides of a base sequence complementary to the base sequence of the wild-type gene corresponding to the mutation site among the base sequences of the wild-type gene in the gene in the sample. Therefore, it binds to the wild-type gene and prevents the binding of the general primer, thereby inhibiting the amplification of the wild-type gene.
  • the general primer can specifically bind to the gene having the mutation and amplify the gene, thereby playing a role in increasing the detection sensitivity and specificity of the mutant gene.
  • one end of the blocking primer may include a nucleotide having the same base sequence as a terminal portion adjacent to the mutation site of a primer adjacent to the mutation site. If the primer adjacent to the mutation site is a forward primer, the one end is the 5' end, and if the primer adjacent to the mutation site is a reverse primer, the one end is the 3' end.
  • the nucleotide having the same base sequence as a terminal portion adjacent to the mutation site of the primer adjacent to the mutation site refers to a nucleotide in a portion overlapping with the primer adjacent to the mutation site, and in the case of a gene having a mutation, nucleotides are continuously amplified at the terminal portion adjacent to the mutation site of this primer by binding of the primer adjacent to the mutation site, but in the case of a wild-type gene, one end of the blocking primer is competitively bound instead of the terminal portion of the primer adjacent to the mutation, so the wild-type gene may not be amplified.
  • the terminal of the blocking primer may be modified to block amplification by PCR. If the primer adjacent to the mutation site is a forward primer, the other terminal is the 3' terminal, and if it is a reverse primer, the other terminal is the 5' terminal.
  • the terminal modification is a C3-18 spacer [a structure in which 3-18 carbons are connected in sequence; For example, it may be performed by attaching one or more of a C3-spacer (a structure in which three carbons are connected in series), a C6-spacer (a structure in which six carbons are connected in series), a C12-spacer (a structure in which twelve carbons are connected in series), a C18-spacer (a structure in which eighteen carbons are connected in series), biotin, di-deoxynucleotide triphosphate (ddNTP), ethylene glycol, an amine, or a phosphate.
  • ddNTP di-deoxynucleotide triphosphate
  • a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations using RNA comprising:
  • a primer set for synthesizing and amplifying cDNAs each containing EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations comprising primer sets of the above-mentioned sequence numbers 1 to 34, and
  • a primer set specifically binding to mutations in the EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes each comprising a primer set of the above-mentioned sequence numbers 37 to 40, 43 to 71, 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132.
  • the kit may additionally include at least one probe selected from the group consisting of sequence numbers 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133.
  • the kit may additionally include a blocking primer of sequence number 72.
  • a composition comprising (i) a primer set of SEQ ID NO: 1 to SEQ ID NO: 34 and/or (ii) a primer set of SEQ ID NOs: 37 to 40, 43 to 71 and 73, 75, 76, 78 to 83, 85, 86, 89 to 91, 93, 94, 96, 97, 99, 100, 102, 103, 105 to 111, 114 to 117, 119 to 122, 124 to 129, 131 and 132 for the manufacture of a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations is provided.
  • the composition may additionally comprise at least one probe selected from the group consisting of SEQ ID NOs: 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133.
  • the composition may additionally comprise a blocking primer of SEQ ID NO: 72.
  • the primer sets and probes for detecting the three G719X mutations of the EGFR gene can be provided as one Master Mix (MMX).
  • MMX Master Mix
  • the primer sets of SEQ ID NOs: 37 and 40 and the probe of SEQ ID NO: 41 for detecting the G719S mutation of the EGFR gene, the primer sets of SEQ ID NOs: 38 and 40 and the probe of SEQ ID NO: 41 for detecting the G719C mutation of the EGFR gene, and the primer sets of SEQ ID NOs: 39 and 40 and the probe of SEQ ID NO: 41 for detecting the G719A mutation of the EGFR gene are provided as MMX1.
  • the primer set and probe for detecting 29 kinds of Ex19Del mutations of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 43 to 71 and 73, the blocking primer of SEQ ID NO: 72, and the probe of SEQ ID NO: 74 for detecting 29 kinds of Ex19Del mutations of the EGFR gene are provided as MMX2.
  • the primer set and probe for detecting the S768I mutation of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 75 and 76 and the probe of SEQ ID NO: 77 for detecting the S768I mutation are provided as MMX3.
  • the primer set and probe for detecting five Ex20Ins mutations of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 78 to 83 and the probe of SEQ ID NO: 84 for detecting five Ex20Ins mutations of the EGFR gene are provided as MMX4.
  • the primer set and probe for detecting the T790M mutation of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 85 and 86 and the probes of SEQ ID NOs: 87 and 88 for detecting the T790M mutation of the EGFR gene are provided as MMX5.
  • the primer set and probe for detecting the C797S mutation of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 89 to 91 and the probe of SEQ ID NO: 92 for detecting the C797S mutation of the EGFR gene are provided as MMX6.
  • the primer set and probe for detecting two types of L858R mutations of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 93 and 94 and the probe of SEQ ID NO: 95 for detecting two types of L858R mutations of the EGFR gene are provided as MMX7.
  • the primer set and probe for detecting the L861Q mutation of the EGFR gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 96 and 97 and the probe of SEQ ID NO: 98 for detecting the L861Q mutation of the EGFR gene are provided as MMX8.
  • the primer set and probe for detecting the G12C mutation of the KRAS gene and the primer set and probe for detecting the V600E1 mutation of the BRAF gene can be provided as one MMX.
  • the primer set of SEQ ID NOs: 99 and 100 and the probe of SEQ ID NO: 101 for detecting the G12C mutation of the KRAS gene and the primer set of SEQ ID NOs: 102 and 103 and the probe of SEQ ID NO: 104 for detecting the V600E1 mutation of the BRAF gene are provided as MMX9.
  • a primer set and a probe for detecting EML4-ALK gene mutations can be provided as one MMX.
  • a primer set of SEQ ID NOs: 105 to 111 and probes of SEQ ID NOs: 112 and 113 for detecting EML4-ALK Variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, 7 and 8a are provided as MMX10.
  • primer sets and probes for detecting 12 types of ROS1 gene mutations can be provided as one or more MMXs.
  • a primer set of SEQ ID NO: 114 and SEQ ID NO: 117 and a probe of SEQ ID NO: 118 for detecting CD74 ex6-ROS1 ex34 a primer set of SEQ ID NO: 115 and SEQ ID NO: 117 and a probe of SEQ ID NO: 118 for detecting SDC4 ex2-ROS1 ex34
  • a primer set of SEQ ID NO: 116 and SEQ ID NO: 117 and a probe of SEQ ID NO: 118 for detecting EZR ex10-ROS1 ex34 a primer set of SEQ ID NO: 119 and SEQ ID NO: 122 and a probe of SEQ ID NO: 123 for detecting TPM3 ex8-ROS1 ex35
  • a primer set of SEQ ID NO: 120 and SEQ ID NO: 122 and a probe of SEQ ID NO: 123 for detecting TPM3
  • a primer set of sequence number 122 and a probe of sequence number 123 are provided as MMX11.
  • a primer set of SEQ ID NO: 124 and SEQ ID NO: 129 and a probe of SEQ ID NO: 130 for detecting CD74 ex6-ROS1 ex32 a primer set of SEQ ID NO: 125 and SEQ ID NO: 129 and a probe of SEQ ID NO: 130 for detecting SLC34A2 ex4-ROS1 ex32
  • a primer set of SEQ ID NO: 127 and SEQ ID NO: 129 and a probe of SEQ ID NO: 130 for detecting SDC4 ex2-ROS1 ex32 a primer set of SEQ ID NO: 128 and SEQ ID NO: 129 and a probe of SEQ ID NO: 130 for detecting SDC
  • the MMX1 to MMX12 may be arranged according to the MMX configured for each target to be detected in a 96-well plate configuration through RT-qPCR.
  • a positive control (PC) for setting a threshold for each target, an NTC for checking for contamination, and an internal control (IC) for checking the total amount of RNA used may be arranged in the 96-well plate.
  • the MMX1 to MMX9, the PC, and the NTC may be arranged as shown in FIG. 2, but are not limited thereto.
  • the kit of the present invention can be used for research use only (RUO) or in-vitro diagnostic (IVD).
  • the kit of the present invention may be a PCR kit, and in addition to a primer set for synthesizing and amplifying cDNA including each of the aforementioned EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, and a primer set and probe for detecting each of the EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, the kit may further include a reagent for isolating RNA from a sample, a reagent for synthesizing cDNA from the isolated RNA, a hybridization reagent, and reagents necessary for an amplification reaction of DNA.
  • Reagents required for the above amplification reaction include, for example, an appropriate amount of DNA polymerase (e.g., thermostable DNA polymerase obtained from Thermosquatiucs (Taq), Thermophilus (Tth), Thermosylphilus (Tth), Thermosylphilus filiformis, Thermosyl flavus, Thermococcus litoralis or Phyrococcus furiosis (Pfu)), a DNA polymerase cofactor (Mg 2+ ), a buffer solution, dNTPs (dATP, dCTP, dGTP and dTTP) and water (dH 2 O).
  • DNA polymerase e.g., thermostable DNA polymerase obtained from Thermosquatiucs (Taq), Thermophilus (Tth), Thermosylphilus (Tth), Thermosylphilus filiformis, Thermosyl flavus, Thermococc
  • the buffer solution includes, but is not limited to, an appropriate amount of Triton X-100, dimethylsufoxide (DMSO), Tween 20, nonidet P40, PEG 6000, formamide, and bovine serum albumin (BSA).
  • DMSO dimethylsufoxide
  • Tween 20 nonidet P40, PEG 6000, formamide
  • BSA bovine serum albumin
  • the kit of the present invention may be manufactured with a plurality of separate packages or compartments containing the reagent components.
  • the kit of the present invention can be applied to various methods for amplifying genes.
  • the PCR kit can be applied to general PCR (1st generation PCR), real-time PCR (2nd generation PCR), digital PCR (3rd generation PCR), or mass array (MassARRAY).
  • the digital PCR may be a cast PCR (Competitive allele-specific TaqMan PCR) or a droplet digital PCR (Droplet digital PCR; ddPCR), and more specifically, may be an allele-specific cast PCR or an allele-specific droplet digital PCR, but is not limited thereto.
  • the above “cast PCR” is a method for detecting and quantifying rare mutations in samples containing large amounts of normal wild-type gDNA, which can produce superior specificity over traditional allele-specific PCR by combining allele-specific TaqMan ® qPCR with an allele-specific MGB blocker to suppress non-specific amplification from the wild-type allele.
  • droplet digital PCR is a system that divides a 20 ⁇ l PCR reaction into 20,000 droplets, amplifies them, and then counts the target DNA. Depending on whether the target DNA is amplified in the droplet, it receives and counts it as a digital signal as a positive droplet (1) and a negative droplet (0), calculates the number of copies of the target DNA through the Poisson distribution, and finally confirms the result as the number of copies per ⁇ l of sample. It can be used in cases such as detecting rare mutations, amplifying very small amounts of genes, and simultaneous confirmation of mutation types.
  • mass array is a multiplexing analysis method that can be applied to various genetic studies, such as genotyping, using a MALDI-TOF mass spectrometer. It can be used in cases where a large number of samples and targets need to be analyzed quickly at a low cost, or where customized analysis is desired only for specific targets.
  • the kit of the present invention can employ AS-PCR (Allele-specific PCR) and real-time PCR technology, and can include specific primers and fluorescent probes for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations in a sample containing cDNA (amplicon).
  • AS-PCR Allele-specific PCR
  • the target mutant DNA matches the base at the 3' end of the primer, and is selectively and efficiently amplified, and then the mutant amplicon can be detected by a fluorescent probe labeled with FAM. Wild-type DNA cannot match the specific primer, and no amplification occurs.
  • a method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA using the kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations described above is provided.
  • the biological sample of step (a) refers to a genetic sample including a genetic mutation site to be detected.
  • it may include DNA, cDNA or RNA, preferably mRNA.
  • it may include any biologically derived sample capable of genetic analysis, including nuclei and/or mitochondria, and may be cells, tissues, organs, body fluids, etc., or endogenous genes or exogenous genes extracted therefrom.
  • the cells, tissues, organs, body fluids, etc. may be collected from a patient of a mammal (e.g., a human, a primate, a rodent, etc.), and the cells may include cells of unicellular animals such as viruses or bacteria.
  • the sample may be extracted from cells of a lung cancer patient, and in the case of a residual tumor test after lung cancer treatment, the genetic sample may be extracted from cancer cells of a patient who has received tumor treatment.
  • the cDNA synthesized in step (b) may have a size of about 100 to 600 bp.
  • the synthesized cDNA may be amplified about 2-19 times through pre-amplification, and may be diluted before performing step c).
  • a blocking primer having sequence number 72 may be additionally processed in step (c).
  • the step (c) can be performed by allele-specific PCR or real-time PCR.
  • the EGFR gene mutation detection method of the present invention may additionally include a step of (d) confirming the amplification result by PCR by measuring the Ct (cycle threshold) value.
  • the Ct (cycle threshold) value is the cycle number at which the fluorescence generated in the reaction exceeds the threshold, and is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles at which a sufficient number of amplicons have accumulated in the reaction.
  • the Ct value is the cycle at which an increase in ⁇ Rn is first detected. Rn is the magnitude of the fluorescence signal generated during PCR at each time point, and ⁇ Rn is the fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the reference dye (normalized reporter signal).
  • the Ct value is also referred to as Cp (crossing point) in LightCycler.
  • the Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This point provides the most useful information about the reaction.
  • the slope of the log-linear step represents the amplification efficiency (Eff) (http:/www.appliedbiosystems.co.kr/).
  • TaqMan probes are typically longer oligonucleotides (e.g., 20-30 nucleotides) than the primers that contain a fluorophore at the 5'-end and a quencher (e.g., TAMRA or non-fluorescent quencher (NFQ)) at the 3'-end.
  • TaqMan probes are designed to anneal to internal sites of PCR products.
  • TaqMan probe specifically hybridizes to template DNA in the annealing step, but fluorescence is inhibited by a quencher on the probe.
  • the TaqMan probe hybridized to the template is degraded by the 5' to 3' nuclease activity of Taq DNA polymerase, releasing the fluorescent dye from the probe, thereby releasing the inhibition by the quencher and causing fluorescence.
  • the 5'-terminus of the TaqMan probe must be located downstream of the 3'-terminus of the extension primer.
  • the 5'-terminus of the TaqMan probe is cleaved by the 5' to 3' nuclease activity of the polymerase, thereby generating a fluorescence signal of the reporter molecule.
  • the reporter molecule and quencher molecule coupled to the TaqMan probe include fluorescent substances and non-fluorescent substances.
  • Any fluorescent reporter molecule and quencher molecule known in the art that can be used in the present invention can be used, and examples thereof are as follows (the numbers in parentheses are the maximum emission wavelengths in nanometers): Cy2 TM (506), YOPRO TM -1 (509), YOYO TM -1 (509), Calcein (517), FITC (518), FluorX TM (519), Alexa TM (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green TM 500 (522), Oregon Green TM 488 (524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531), Calcium Green TM (533), TO-PRO TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIP
  • the non-fluorescent material used in the reporter molecule and quencher molecule coupled to the TaqMan probe may include a minor groove binding (MGB) moiety.
  • MGB minor groove binding
  • TaqMan MGB-conjugate probe as used herein means a TaqMan probe conjugated with MGB at the 3'-end of the probe.
  • MGBs are substances that bind to the minor groove of DNA with high affinity, and include, but are not limited to, netropsin, distamycin, lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6-diamidino-2-phenylindole), dimers, trimers, tetramers and pentamers of CDPI, N-methylpyrrole-4-carbox-2-amide (MPC) and dimers, trimers, tetramers and pentamers thereof.
  • netropsin distamycin, lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6
  • Conjugation of the probe to the MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, the increased stability (i.e., increased degree of hybridization) results in an increased melting temperature (Tm) of the hybrid duplex formed by the MGB-conjugated probe compared to the normal probe.
  • Tm melting temperature
  • the MGB stabilizes the van der Waals force, thereby increasing the melting temperature (Tm) of the MGB-conjugated probe without increasing the probe length, thereby enabling the use of shorter probes (e.g., 21 nucleotides or less) in Taqman real-time PCR under more stringent conditions.
  • the MGB-conjugate probe removes background fluorescence more efficiently. Therefore, the probe of the present invention may be in the form of a TaqMan MGB-conjugate, wherein the length of the probe includes, but is not limited to, 15-21 nucleotides.
  • the EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations may include 44 EGFR gene mutations, 1 BRAF gene mutation, 1 KRAS gene mutation, 10 EML4-ALK gene mutations and 12 ROS1 gene mutations described in Tables 1 to 5.
  • the method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA of the present invention can simultaneously and multiple times detect one or more of the 68 gene mutations listed in the above table.
  • Step (d) of the present invention may include melting temperature analysis using a double strand specific dye.
  • Melting temperature curve analysis can be performed on a real-time PCR instrument, such as the ABI 5700/7000 (96 well format) or ABI 7900 (384 well format) instrument with onboard software (SDS 2.1). Alternatively, melting temperature curve analysis can be performed as an end point analysis.
  • “Dyes that bind to double-stranded DNA” or “double-stranded specific dyes” can be used if they have higher fluorescence when bound to double-stranded DNA than in the unbound state.
  • Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, ethidium bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. These dyes, except EtBr and EvaGreen (Quiagen), have been tested in real-time applications.
  • the EGFR gene mutation detection method of the present invention can be performed by real-time PCR (RT-PCR) or quantitative PCR (qPCR), analysis on an agarose gel after standard PCR, gene mutation-specific amplification or allele-specific amplification through real-time PCR, tetra-primer amplification-refractory mutation system PCR, or isothermal amplification.
  • RT-PCR real-time PCR
  • qPCR quantitative PCR
  • PCR is a technique known to those skilled in the art for amplifying single or multiple copies of DNA or cDNA.
  • Most PCRs use a thermostable DNA polymerase, such as Taq polymerase or Klen Taq.
  • the DNA polymerase uses a single-stranded DNA as a template and enzymatically assembles a new DNA strand from the nucleotides by using oligonucleotides (primers).
  • the amplicons produced by PCR can be analyzed, for example, on an agarose gel.
  • real-time PCR can monitor the process in real time while the PCR is being performed. Therefore, data is collected throughout the PCR process, not just at the end of the PCR.
  • the reaction is characterized by the point in the cycle when amplification is first detected, rather than the amount of target accumulated after a fixed number of cycles.
  • Two methods are mainly used to perform quantitative PCR: dye-based detection and probe-based detection.
  • ASA Allele Specific Amplification
  • ASA real-time PCR-based genetic mutation-specific amplification or allele-specific amplification
  • ASA combines amplification and detection in a single reaction based on the discrimination of matched and mismatched primer/target sequence complexes.
  • the increase in amplified DNA during the reaction can be monitored in real time by an increase in fluorescent signal caused by a dye such as SYBR Green I that fluoresces upon binding to double-stranded DNA.
  • Real-time PCR-based genetic mutation-specific amplification or allele-specific amplification is indicated by a delay or absence of fluorescent signal for mismatched cases. In genetic mutation or SNP detection, this provides information on the presence or absence of the genetic mutation or SNP.
  • the above "Tetra-primer amplification-refractory mutagenesis system PCR” amplifies both wild-type and mutant alleles along with a control fragment in a single-tube PCR reaction.
  • a non-allele-specific control amplicon is amplified by two common (outer) primers flanking the mutation region.
  • the two allele-specific (inner) primers are designed in opposite directions to the common primers and, together with the common primers, can simultaneously amplify both the wild-type and mutant amplicons.
  • the two allele-specific amplicons have different lengths because the mutations are positioned asymmetrically with respect to the common (outer) primers and can be easily separated by standard gel electrophoresis.
  • the control amplicon provides an internal control against false negatives as well as amplification failures, and at least one of the two allele-specific amplicons is always present in the tetra-primer amplification-refractory mutagenesis system PCR.
  • isothermal amplification means that the amplification of nucleic acids is carried out at a lower temperature, without relying on a thermocycler, and preferably without the need for temperature change during amplification.
  • the temperature used in isothermal amplification can be between room temperature (22-24 ° C.) and about 65 ° C., or about 60-65 ° C., 45-50 ° C., 37-42 ° C. or ambient temperature of 22-24 ° C.
  • the product of the isothermal amplification can be detected by gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (enhanced chemiluminescence), a bioanalyzer, which is a chip-based capillary electrophoresis instrument that analyzes RNA, DNA and proteins or turbidity.
  • ELOSA Enzyme linked oligosorbent assay
  • ECL enhanced chemiluminescence
  • bioanalyzer which is a chip-based capillary electrophoresis instrument that analyzes RNA, DNA and proteins or turbidity.
  • the method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from the RNA can be used for diagnosing lung cancer or predicting the drug responsiveness of lung cancer patients.
  • the second aspect of the present invention relates to a composition for diagnosing lung cancer, comprising a primer set that specifically binds to each of DNAs comprising 44 kinds of EGFR gene mutations described in Table 1, DNAs comprising 1 kind of BRAF gene mutation described in Table 2, DNAs comprising 1 kind of KRAS gene mutation described in Table 3, DNAs comprising 10 kinds of EML4-ALK gene mutations described in Table 4, and DNAs comprising 12 kinds of ROS1 gene mutations described in Table 5, and a kit comprising the same.
  • a method for diagnosing lung cancer or predicting the responsiveness of a lung cancer patient to a drug using the composition or kit is provided.
  • the method for diagnosing lung cancer or predicting the drug responsiveness of a lung cancer patient according to the present invention is the same as the method for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes from the aforementioned RNA, and therefore, description thereof is omitted.
  • the cancer may be non-small cell lung cancer.
  • the method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations of the present invention simultaneously and on a large scale detects known lung cancer-related gene mutations, thereby diagnosing lung cancer at an early stage and predicting the prognosis and drug responsiveness of lung cancer patients, thereby providing information for establishing a treatment strategy tailored to each patient, thereby contributing to more effective treatment of patients.
  • prognosis means an act of predicting the course and outcome of a disease in advance. More specifically, prognosis prediction can be interpreted as all acts of predicting the course of a disease after treatment by comprehensively considering the patient's condition, as the course of the disease after treatment may vary depending on the patient's physiological or environmental condition.
  • the above prognosis prediction can be interpreted as an act of predicting the disease-free survival rate or survival rate of a patient by predicting the course and whether the disease is cured after treatment of a specific disease. For example, predicting a "good prognosis" means that the patient's disease-free survival rate or survival rate is high after treatment of the disease, meaning that the patient is likely to be cured, and predicting a "bad prognosis” means that the patient's disease-free survival rate or survival rate is low after treatment of the disease, meaning that the patient is likely to relapse or die due to the disease.
  • disease-free survival rate means the possibility that a patient will survive without recurrence of a specific disease after treatment for that disease.
  • survival rate of the present invention means the possibility that a patient will survive after treatment for a specific disease, regardless of whether the disease relapses.
  • the drugs may include, but are not limited to, tyrosine kinase inhibitors.
  • Primer sets and probes for synthesizing and amplifying cDNA containing EGFR, BRAF, KRAS, EML4-ALK, and ROS1 gene mutations in Table 6 were designed using the OligoAnalyzer Tool (https:/sg.idtdna.com/calc/analyzer) program.
  • EGFR, KRAS, and BRAF 2 to 3 reverse primers were designed to increase reverse transcription efficiency.
  • Primer sets and probes for detecting EGFR, BRAF, KRAS, EML4-ALK, and ROS1 gene mutations in Table 7 were designed using the OligoAnalyzer Tool (https:/sg.idtdna.com/calc/analyzer) program.
  • EGFR_Oligo Mix_1 Sequence (5'-3') Tm(°C) Sequence number G719S Rmt17 CCG AAC GCA CCG GAG CT 66.6 37 G719C Rmt16 CGA ACG CAC CGG AGC A 64.5 38 G719A Rmt18(-3T) TGC CGA ACG CAC CGT AGG 66.6 39 G719X SF4 GCC TCT TAC ACC CAG TGG AGA A 65.4 40 G719X R_FAM AAA CTG AAT TCA AAA AGA TCA AAG TGC TG 64.3 41 EGFR_Oligo Mix_2 Sequence (5'-3') Tm(°C) Sequence number Ex19-20_FAM_R28 TCA CGT AGG CTT CAT CGA GGA TTT CCT T 68.7 42 Ex19del C1 Rmt20 TGT TGG CTT TCG GAG ATG CC 64.9 43 Ex19del C2 Rmt21 TTGG CTT TCG
  • the gDNA of A549 lung-derived cell line with wild-type DNA/RNA of EGFR, BRAF, KRAS, EML4-ALK, and ROS1 and primer sets capable of amplifying the mutant amplification regions were applied to obtain wild-type clones of EGFR exons 18, 19, 20, and 21, BRAF exon 15, KRAS exon 2, EML4 exons 1 to 20, ALK exon 20, ROS1 exons 32, 34, 35, and 36, CD74 exon 6, SDC4 exons 2 and 4, EZR exon 10, TMP3 exon 8, LRIG3 exon 16, CCDC6 exon 5, SLC34A2 exons 4 and 13, and GOPC exon 4. was produced.
  • mutagenesis was performed on 44 EGFR target mutations, 1 BRAF target mutation, 1 KRAS target mutation, 10 EML4-ALK target recombination mutations, and 12 ROS1 target recombination mutations, and each mutant clone was obtained by transforming E. coli DH5 ⁇ cells.
  • the wild-type clones and mutant clones were confirmed by direct base sequence analysis.
  • the wild-type DNA and mutant DNA for each exon extracted through the clones were extracted after producing in vitro transcribed (IVT) RNA using RNA polymerase, and used as standard materials to evaluate the mutation detection performance of EGFR, BRAF, KRAS, EML4-ALK, and ROS1, respectively.
  • IVTT in vitro transcribed
  • samples were prepared by adding 3,000 copies, 300 copies, and 30 copies of each mutant RNA per 100 ng of A549 cell total RNA, and the group to which no mutant RNA was added was used as a control.
  • total RNA 100 ng WT + mut 3,000 A549 cell total RNA (total RNA) 100 ng + corresponding mutant RNA 3,000 copies WT + mut 300 A549 cell total RNA (total RNA) 100 ng + corresponding mutant RNA 300 copies WT + mut 30 A549 cell total RNA (total RNA) 100 ng + 30 copies of corresponding mutant RNA
  • step Cycle temperature hour 1 1 25 °C 2 minutes 2 1 50°C 15 minutes 3 1 95 °C 5 minutes 4 15 95 °C 10 seconds 5 60°C 30 seconds 6 72°C 30 seconds
  • a sufficient reaction mixture containing each component listed in Table 4 was prepared in separate sterile centrifuge tubes, and the reaction master mix was thoroughly mixed by vortexing for 3 seconds and centrifuged briefly.
  • a PCR tube was prepared for each sample as follows. 10 ⁇ l of reverse transcription & pre-amplification master mix was aliquoted into each PCR tube, 2 ⁇ l of reverse transcription & pre-amplification oligo mix was added, 2 ⁇ l of each sample RNA was added, and 6 ⁇ l of nuclease-free distilled water was added and the PCR tube was capped. The PCR tube was centrifuged briefly to collect all the liquid at the bottom of each PCR tube. The PCR tube was placed in the PCR machine, and the PCR machine was set up using the temperature conversion table in Table 5 and PCR was performed.
  • the lid was carefully opened on a biological safety bench and the first dilution was performed with 180 ⁇ l of nuclease-free distilled water. 10 ⁇ l of the first diluted solution was transferred to a tube containing 990 ⁇ l of nuclease-free distilled water and the second dilution was performed, thereby diluting the preamplified DNA by a final 1000-fold.
  • step Cycle temperature hour Data collection 1 1 95 °C 5 minutes - 2 45 95 °C 10 seconds - 3 60°C 30 seconds FAM / CY5 4 72°C 10 seconds -
  • a sufficient reaction mixture containing the 1000-fold diluted preamplified DNA sample and each component listed in Table 6 was prepared in separate sterile centrifuge tubes, and the reaction master mix was thoroughly mixed by vortexing for 3 seconds and centrifuged briefly.
  • a PCR tube was prepared for each sample as follows. 10 ⁇ l of the qualitative analysis PCR master mix was aliquoted into each PCR tube, 9.5 ⁇ l of the 1000-fold diluted preamplified sample was added, 0.5 ⁇ l of ADPS Smart DNA polymerase was added, and the PCR tube lid was capped. The PCR tube was centrifuged briefly to collect all the liquid at the bottom of each PCR tube.
  • the PCR tube was placed in a real-time PCR machine, and the PCR machine was set up using the temperature conversion table in Table 7 and PCR was performed. After the PCR was completed, the FAM/CY5 signal of each sample was analyzed to analyze the data, the Ct value was recorded, and the ⁇ Ct value for each well was calculated as follows.
  • ⁇ Ct value Sample Ct value (FAM of each master mix) - Positive control Ct value (FAM of each master mix)
  • ⁇ Ct value Sample Ct value (CY5 of each master mix) - Positive control Ct value (CY5 of each master mix)
  • the calculated ⁇ Ct value for each well is used to determine whether the mutation you want to detect is present in that tube.
  • the minimum detection limit (LOD) for each target is as shown in Figures 5a to 5l.
  • the copy number that appears first without overlapping the 0 copy amplification curve is set as the minimum detection limit, and it was confirmed that a sample can be determined to contain a target mutation if a Ct value that is about 2 to 3 lower than the 0 copy Ct value is obtained.

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Abstract

La présente invention concerne un procédé de détection multiplex à grande échelle de gènes mutants du cancer du poumon à l'aide d'ARN et un kit pour la détection multiplex à grande échelle de gènes mutants du cancer du poumon l'utilisant et, plus spécifiquement, un procédé de détection multiplex à grande échelle de gènes mutants du cancer du poumon à l'aide d'ARN, permettant de détecter simultanément 44 types de mutations du gène EGFR, 1 type de mutation du gène BRAF, 1 type de mutation du gène KRAS, 10 types de mutations de fusion du gène EML4-ALK et 12 types de réarrangements du gène ROS1, ainsi qu'un kit de détection de mutations du gène EGFR, BRAF, KRAS, EML4-ALK et ROS1 à l'aide d'un ensemble d'amorces et d'une composition.
PCT/KR2024/017991 2023-11-15 2024-11-14 Procédé de détection multiplex à grande échelle de gènes mutants du cancer du poumon à l'aide d'arn et kit pour détection multiplex à grande échelle de gènes mutants du cancer du poumon l'utilisant Pending WO2025105837A1 (fr)

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KR10-2023-0158257 2023-11-15
KR1020230158257A KR20250074714A (ko) 2023-11-15 2023-11-15 Rna를 이용한 폐암 변이 유전자의 대규모 다중 검출 방법 및 이를 이용한 폐암 변이 유전자의 대규모 다중 검출 키트

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WO2025105837A1 true WO2025105837A1 (fr) 2025-05-22

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KR (1) KR20250074714A (fr)
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JP2016515380A (ja) * 2013-03-15 2016-05-30 ライフ テクノロジーズ コーポレーション 肺癌の分類及び実施可能性インデックス
KR20170010331A (ko) * 2017-01-13 2017-01-26 연세대학교 산학협력단 폐암 환자의 생존기간 예측용 키트와 생존기간 예측을 위한 정보 제공 방법
KR20200054430A (ko) * 2018-11-09 2020-05-20 주식회사 셀레믹스 폐암 조직 내 세포 유래 돌연변이를 검출하기 위한 프로브 제조 및 검출 방법
KR20200117909A (ko) * 2019-04-05 2020-10-14 주식회사 제놉시 Cfdna를 이용한 폐암 진단방법
US20200362421A1 (en) * 2013-03-15 2020-11-19 Life Technologies Corporation Classification and actionability indices for cancer

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Publication number Priority date Publication date Assignee Title
KR101720050B1 (ko) 2014-02-28 2017-04-20 재단법인 아산사회복지재단 폐암관련 돌연변이 증폭용 프라이머 세트, 및 상기 돌연변이에 상보적인 프로브를 포함하는, 폐암 진단용 키트

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016515380A (ja) * 2013-03-15 2016-05-30 ライフ テクノロジーズ コーポレーション 肺癌の分類及び実施可能性インデックス
US20200362421A1 (en) * 2013-03-15 2020-11-19 Life Technologies Corporation Classification and actionability indices for cancer
KR20170010331A (ko) * 2017-01-13 2017-01-26 연세대학교 산학협력단 폐암 환자의 생존기간 예측용 키트와 생존기간 예측을 위한 정보 제공 방법
KR20200054430A (ko) * 2018-11-09 2020-05-20 주식회사 셀레믹스 폐암 조직 내 세포 유래 돌연변이를 검출하기 위한 프로브 제조 및 검출 방법
KR20200117909A (ko) * 2019-04-05 2020-10-14 주식회사 제놉시 Cfdna를 이용한 폐암 진단방법

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