WO2025105837A1 - Method for large-scale multiplex detection of lung cancer mutant genes using rna and kit for large-scale multiplex detection of lung cancer mutant genes using same - Google Patents

Abstract

The present invention relates to a method for large-scale multiplex detection of lung cancer mutant genes using RNA and a kit for large-scale multiplex detection of lung cancer mutant genes using same and, more specifically, to a method for large-scale multiplex detection of lung cancer mutant genes using RNA, capable of simultaneously detecting 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 rearrangements, and a kit for detection of EGFR, BRAF, KRAS, EML4-ALK, and ROS1 gene mutations using a primer set and a composition.

Description

Method for large-scale multiplex detection of lung cancer mutant genes using RNA and large-scale multiplex detection kit for lung cancer mutant genes using the same

This application claims priority to Republic of Korea Patent Application No. 10-2023-0158257, filed on November 15, 2023, the entire contents of which are incorporated herein by reference.

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%.

However, it is true that there have been great advances in the treatment of metastatic non-small cell lung cancer since the 2000s. It is the cancer type for which the most diverse and effective targeted therapies have been developed and applied, and immunotherapy is also showing good therapeutic effects and is expanding its application area. Cytotoxic anticancer agents are still being used as basic treatment drugs. Therefore, when deciding on anticancer treatment for stage 4 non-small cell lung cancer patients who are difficult to treat with the purpose of complete cure in the clinical field, the molecular pathological information of the patient's cancer should be confirmed first so that the most effective treatment can be received for each patient, and the treatment should be selected accordingly.

In particular, in non-small cell lung cancer, it is very important to confirm whether or not there is a genetic mutation, because targeted therapies targeting those genetic mutations show good effects in patients with those genetic mutations. Important tests for genetic mutations include mutations in EGFR, ALK, ROS1, and BRAF genes, and tests and treatments for these are covered by insurance. In addition, 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.

Meanwhile, 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.

Against this backdrop, 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.

[Prior art literature]

[Patent Document]

(Patent Document 1) Republic of Korea Publication Patent No. 10-2015-0102468

Accordingly, 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.

To solve the above-described problem, 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.

In addition, 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.

In addition, 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.

In the present invention, the 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.

In the present invention, the composition or kit may additionally include a blocking primer of sequence number 72.

Furthermore, 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.

In the present invention, the method may include the following steps (a) to (d):

(a) a step of extracting RNA from a separated biological sample;

(b) a step of synthesizing and amplifying cDNAs each including EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by treating the primer sets of sequence numbers 1 to 34 and reverse transcriptase;

(c) performing a polymerase chain reaction by treating a primer set of 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 and a probe of sequence numbers 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133 Step; and

(d) A step of detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by confirming the amplification results by the polymerase chain reaction using fluorescence.

In the present invention, 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.

Exxon Mutation EGFR nucleic acid sequence exon 18 G719X 2155G>A 2155G>T 2156G>C Exon 19 Ex19Del 2240_2251del12 2239_2247del9 2238_2255del18 2235_2249del15 2236_2250del15 2239_2253del15 2239_2256del18 2237_2254del18 2240_2254del15 2240_2257del18 2239_2248TTAAGAGAAG>C 2239_2251>C 2237_2255>T 2235_2255>AAT 2237_2252>T 2239_2258>CA 2239_2256>CAA 2237_2253>TTGCT 2238_2252>GCA 2238_2248>GC 2237_2251del15 2236_2253del18 2235_2248>AATTC 2235_2252>AAT 2235_2251>AATTC 2253_2276del24 2237_2257>TCT 2238_2252del15 2233_2247del15 exon 20 S768I 2303G>T T790M 2369C>T Ex20Ins 2307_2308ins9GCCAGCGTG 2319_2320insCAC 2310_2311insGGT 2311_2312ins9GCGTGGACA 2309_2310AC>CCAGCGTGGAT C797S 2390>C 2389>A Exon 21 L858R 2573T>G 2573_2574TG>GT L861Q 2582T>A

codon Mutation BRAF nucleic acid sequence 600 V600E1 1799T>A

codon Mutation KRAS nucleic acid sequence G12 G12C 34G>T

EML4-ALK Variant 1 Variant 2 Variant 3a Variant 3b Variant 4 Variant 5a Variant 5b Variant 6 Variant 7 Variant 8a

ROS1 CD74 ex6-ROS1 ex34 SDC4 ex2-ROS1 ex34 EZR ex10-ROS1 ex34 TPM3 ex8-ROS1 ex35 LRIG3 ex16-ROS1 ex35 CCDC6 ex5-ROS1 ex35 CD74 ex6-ROS1 ex32 SLC34A2 ex4-ROS1 ex32 SLC34A2 ex13-ROS1 ex32 SDC4 ex2-ROS1 ex32 SDC4 ex4-ROS1 ex32 GOPC ex4-ROS1 ex36

In the present invention, a blocking primer having sequence number 72 may be additionally processed in step (c).

In the present invention, the polymerase chain reaction of step (c) can be performed by an allele-specific polymerase chain reaction or a real-time polymerase chain reaction.

In the present invention, 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.

In the present invention, the method can be applied to diagnosing lung cancer or predicting the drug response of lung cancer patients.

In addition, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, the lung cancer diagnostic composition may additionally contain a blocking primer of sequence number 72.

In the present invention, 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.

In the present invention, 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.

FIG. 2 is a diagram illustrating the arrangement of each MMX in a 96-well plate configuration when the primer sets and probes for detecting EGFR, BRAF, KRAS, EML4-ALK, and ROS1 gene mutations according to the present invention are prepared as a qPCR Master Mix (MMX1 to MMX9) configured for each target.

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.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention.

As described above, 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.

Accordingly, 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.

The term "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. Shorter primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. The primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for elongation. In certain embodiments, the term "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. For example, 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.

In the present invention, 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.

Specifically, 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 (168 bp, 237bp, 181bp, 149bp, 182bp, 137bp, 163bp, 154bp, 271bp and 181bp).

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 primer of SEQ ID NO: 28 and a reverse primer of SEQ ID NO: 32 A 23rd primer set comprising a forward primer of SEQ ID NO: 29 and a reverse primer of SEQ ID NO: 32, a 24th primer set comprising a forward primer of SEQ ID NO: 30 and a reverse primer of SEQ ID NO: 32, a 25th primer set comprising a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32, and a 26th primer set comprising a forward primer of SEQ ID NO: 33 and a reverse primer of SEQ ID NO: 34 each reverse transcribe and pre-amplify the target region of EML4-ALK mRNA, as shown in FIG. 4c, to generate 12 kinds of 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).

In the present invention, 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.

Specifically, 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.

29 Ex19Del mutations in the EGFR gene (2240_2251del12, 2239_2247del9, 2238_2255del18, 2235_2249del15, 2236_2250del15, 2239_2253del15, 2239_2256del18, 2237_2254del18, 2240_2254del15, 2240_2257del18, 2239_2248TTAAGAGAAG>C, 2239_2251>C, 2237_2255>T, 2235_2255>AAT, 2237_2252>T, 2239_2258>CA, A primer set for detecting 2239_2256>CAA, 2237_2253>TTGCT, 2238_2252>GCA, 2238_2248>GC, 2237_2251del15, 2236_2253del18, 2235_2248>AATTC, 2235_2252>AAT, 2235_2251>AATTC, 2253_2276del24, 2237_2257>TCT, 2238_2252del15 and 2233_2247del15) may include reverse primers of SEQ ID NOs: 43 to 71 and forward primer of SEQ ID NO: 73. More specifically, 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 reverse primer of SEQ ID NO: 50 and a forward primer of SEQ ID NO: 73, a reverse primer of SEQ ID NO: 51 and a forward primer of SEQ ID NO: A primer set comprising a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 52 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 53 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 54 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 55 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 56 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 57 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 58 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 59 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 60 and a forward primer of SEQ ID NO: 73, a reverse primer of SEQ ID NO: 61 and a forward primer of SEQ ID NO: 73 A primer set comprising a primer, a primer set comprising a reverse primer of SEQ ID NO: 62 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 63 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 64 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 65 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 66 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 67 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 68 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 69 and a forward primer of SEQ ID NO: 73, a primer set comprising a reverse primer of SEQ ID NO: 70 and a forward primer of SEQ ID NO: 73, and a primer comprising a reverse primer of SEQ ID NO: 71 and a forward primer of SEQ ID NO: 73 It can be a set. At this time, the reverse primers may specifically bind to each of the 29 Ex19Del mutations of the EGFR gene.

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. In this case, 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 (2307_2308ins9GCCAGCGTG, 2319_2320insCAC, 2310_2311insGGT, 2311_2312ins9GCGTGGACA, and 2309_2310AC>CCAGCGTGGAT) may include a primer set of SEQ ID NOs: 78 to 83. More specifically, 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. At this time, 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. In this case, 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. In this case, 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. In this case, 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. In this case, 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. In this case, the forward primer may specifically bind to the V600E1 mutation of the BRAF gene.

A primer set for detecting 10 EML4-ALK gene mutations (Variant 1, 2, 3a, 3b, 4, 5a, 5b, 6, 7, and 8a) 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 4 and 7 may include a forward primer of SEQ ID NO: 109 and a reverse primer of SEQ ID NO: 111, and a primer set for detecting EML4-ALK Variant 8a may include a forward primer of SEQ ID NO: 105 and a reverse primer of SEQ ID NO: 111. A primer set for detection may include a forward primer of SEQ ID NO: 110 and a reverse primer of SEQ ID NO: 111.

A primer set for detecting 12 ROS1 gene mutations (CD74 ex6-ROS1 ex34, SDC4 ex2-ROS1 ex34, EZR ex10-ROS1 ex34, TPM3 ex8-ROS1 ex35, LRIG3 ex16-ROS1 ex35, CCDC6 ex5-ROS1 ex35, CD74 ex6-ROS1 ex32, SLC34A2 ex4-ROS1 ex32, SLC34A2 ex13-ROS1 ex32, SDC4 ex2-ROS1 ex32, SDC4 ex4-ROS1 ex32 and GOPC ex4-ROS1 ex36) may include primer sets of SEQ ID NOs: 124 to 129, 131 and 132. More specifically, 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.

In relation to the first aspect of the present invention, a composition for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes is provided, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, the composition may additionally include a blocking primer of SEQ ID NO: 72. At this time, 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.

The above "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. However, 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.

In addition, 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.

In addition, 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.

In relation to the first aspect of the present invention, a kit for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations using RNA is provided, 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.

In the present invention, 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.

In the present invention, the kit may additionally include a blocking primer of sequence number 72.

In connection with the first aspect of the present invention, the use of 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.

In the use of the composition for the manufacture of a kit according to the present invention, 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.

In the use of the composition for the manufacture of a kit according to the present invention, the composition may additionally comprise a blocking primer of SEQ ID NO: 72.

In the present invention, the primer sets and probes for detecting the three G719X mutations of the EGFR gene can be provided as one Master Mix (MMX). In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting 29 kinds of Ex19Del mutations of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting the S768I mutation of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting five Ex20Ins mutations of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting the T790M mutation of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting the C797S mutation of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting two types of L858R mutations of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, the primer set and probe for detecting the L861Q mutation of the EGFR gene can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, 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. In a specific embodiment of the present invention, 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.

In the present invention, a primer set and a probe for detecting EML4-ALK gene mutations can be provided as one MMX. In a specific embodiment of the present invention, 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.

In the present invention, primer sets and probes for detecting 12 types of ROS1 gene mutations can be provided as one or more MMXs. In a specific embodiment of the present invention, 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 LRIG3 ex16-ROS1 ex35, and a primer set of SEQ ID NO: 121 and SEQ ID NO: 122 and a probe of SEQ ID NO: 123 for detecting CDC6 ex5-ROS1 ex35. A primer set of sequence number 122 and a probe of sequence number 123 are provided as MMX11. In another specific embodiment of the present invention, 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: 126 and SEQ ID NO: 129 and a probe of SEQ ID NO: 130 for detecting SLC34A2 ex13-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 SDC4 ex4-ROS1 ex32, and a probe of SEQ ID NO: 130 for detecting GOPC ex4-ROS1 ex36. The reverse primers of SEQ ID NO: 131 and 132 and the probe of SEQ ID NO: 133 are provided as MMX12.

According to a specific embodiment of the present invention, 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. At this time, 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. For example, 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). In addition, 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).

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.

According to one embodiment of the present invention, 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).

In the PCR kit of the present invention, 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.

The above "droplet digital PCR" is a system that divides a 20 ㎕ 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 ㎕ 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.

The above "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). During nucleic acid amplification, 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.

In relation to the first aspect of the present invention, 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.

Specifically, the method may include the following steps (a) to (d):

(a) a step of extracting RNA from a separated biological sample;

(b) a step of synthesizing and amplifying cDNAs each including EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by treating the primer sets of sequence numbers 1 to 34 and reverse transcriptase;

(c) performing a polymerase chain reaction by treating a primer set of sequence numbers 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 probe of sequence numbers SEQ ID NOs: 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133. Steps to be performed; and

(d) A step of detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by confirming the amplification results by the polymerase chain reaction using fluorescence.

In the present invention, the biological sample of step (a) refers to a genetic sample including a genetic mutation site to be detected. For example, it may include DNA, cDNA or RNA, preferably mRNA. Specifically, 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. In particular, in the case of lung cancer, 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.

In the present invention, 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).

In the present invention, a blocking primer having sequence number 72 may be additionally processed in step (c).

In the present invention, 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 ΔRn is first detected. Rn is the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn 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/).

Meanwhile, 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. The excited fluorophore transfers energy to a nearby quencher rather than fluorescing (FRET = Frster or fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94(20): 10756-61(1997)). Therefore, when the probe is normal, no fluorescence is emitted. 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. During the extension reaction, 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. At this time, the 5'-terminus of the TaqMan probe must be located downstream of the 3'-terminus of the extension primer. That is, when the 3'-terminus of the extension primer is extended by template-dependent nucleic acid polymerase, 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), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3 ^TM (570), Alexa ^TM 546 (570), TRITC (572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594 (615), Texas Red (615), Nile Red (628), YO-PRO ^TM -3 (631), YOYO ^TM -3 (631), R-phycocyanin (642), CPhycocyanin (648), TO-PRO ^TM -3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5 ^TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), VIC (546), BHQ-1 (534), BHQ-2 (579), BHQ-3 (672), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), and Quasar 705 (610). The numbers in parentheses are the maximum emission wavelengths in nanometers.

Suitable reporter-quencher pairs are described in many references: Pesce et al., editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2 ^nd EDITION (Academic Press, New York, 1971); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg., 1996; US Pat. Nos. 3,996,345 and 4,351,760.

Additionally, 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. The term "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.

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. Thus, 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.

In addition, 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.

In the present invention, 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.

For example, 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.

The above "standard 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.

The above "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. In real-time 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.

The above "Allele Specific Amplification (ASA)" is an amplification technique in which PCR primers are designed to distinguish templates that differ by a single nucleotide residue.

The above "real-time PCR-based genetic mutation-specific amplification or allele-specific amplification" is a highly efficient method for detecting genetic mutations or SNPs. Unlike most other methods for detecting genetic mutations or SNPs, no preliminary amplification of the target genetic material is required. 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. As a result, 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.

The above "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.

In the present invention, 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.

Accordingly, 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.

In relation to the second aspect of the present invention, 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.

In the present invention, 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.

In the present invention, the term "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.

The term "disease-free survival rate" of the present invention means the possibility that a patient will survive without recurrence of a specific disease after treatment for that disease.

The term "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.

When the method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations of the present invention is used for predicting the responsiveness of lung cancer patients to drugs, the drugs may include, but are not limited to, tyrosine kinase inhibitors.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Example 1]

Preparation of primer sets and probes

1-1. Preparation of primer sets for cDNA synthesis and amplification

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. For EGFR, KRAS, and BRAF, 2 to 3 reverse primers were designed to increase reverse transcription efficiency.

Primer name Sea Sequence (5'-3') Tm(℃) Sequence number EGFR_ex18_out_F1 18 GTG GAG CCT CTT ACA CCC 61.1 1 EGFR_ex21_out_R1 18 GCA CTT TGC CTC CTT CTG 60.4 2 EGFR_ex21_out_R2 13 TCC AAT GCC ATC C 49.8 3 EGFR_ex21_out_R4 15 CTG GTG GGT ATA GAT 49.3 4 BRAF_ex13~17_out_F1 21 TTC TTC ATG AAG ACC TCA CAG 59.7 5 BRAF_ex13~17_out_R1 19 CTG ATG ACT TCT GGT GCC 59.5 6 BRAF_ex13~17_out_R4 15 TCT GAC TGA AAG CTG 51.6 7 KRAS_ex1~2_out_F1 19 GAG AGG CCT GCT GAA AAT G 60.6 8 KRAS_ex2~3_out_R1 20 CTT GCT TCC TGT AGG AAT CC 59.8 9 KRAS_ex2~3_out_R3 15 GAG ACA GGT TTC TCC 51.5 10 KRAS_ex2~3_out_R4 16 CTG TGT CGA GAA TAT C 49.9 11 EML4-ALK_V1,6_out_F1 21 GGA GTC ATG CTT ATA TGG AGC 60.1 12 EML4-ALK_V2_out_F1 19 CAT CAC ACA CCT TGA CTG G 60.1 13 EML4-ALK_V3a,b_out_F1 18 CCA AAA CTG CAG ACA AGC 59.1 14 EML4-ALK_V4,7_out_F1 18 CTG GAG GAG GGA AAG ACA 60.0 15 EML4-ALK_V5a,b_out_F1 19 CTC TGA AGA TCA TGT GGC C 59.7 16 EML4-ALK_V8a,b_out_F1 18 CAT CCA AGT GGC ACA GTG 60.4 17 EML4-ALK_out_R1 20 GTT GTA GTC GGT CAT GAT GG 60.0 18 CD74 ex6-ex34_out_F1 17 GGA GCA AAA GCC CAC TG 60.4 19 SDC4 ex2-ex34_out_F1 21 CTC CTA GAA GGC CGA TAC TTC 61.0 20 EZR ex10-ex34_out 18 GCT GCA GGA CTA TGA GGA 60.4 21 ROS1 ex34_out_R1 19 CCA AAG GTC AGT GGG ATT G 60.3 22 TPM3 ex8-ex35_out_F1 19 GTG CTG AGT TTG CTG AGA G 60.4 23 LRIG3 ex16-ex35_out_F1 19 GTC ACA TCT TCA GGT GCT G 60.2 24 CCDC6 ex5-ex35_out_F1 19 GAA TGA AGT GGA ACG GCT G 60.8 25 ROS1 ex35_out_R1 21 GAT GGC CAA AGC TAC ATA CTG 60.8 26 CD74 ex6-ex32_out_F1 18 GAG CAA AAG CCC ACT GAC 60.7 27 SLC34A2 ex4-ex32_out_F1 17 CGT GTG CTC CCT GGA TA 60.3 28 SLC34A2 ex13-ex32_out_F1 18 AGG ATG TCC CTG TCA AGG 60.4 29 SDC4 ex2-ex32_out_F1 20 GAT GAC TTT GAG CTG TCT GG 60.3 30 SDC4 ex4-ex32_out_F1 19 GTG TCA ATG TCC AGC ACT G 60.7 31 ROS1 ex32_R1_out 19 CTT CAG CTT TCT CCC ACT G 59.9 32 GOPC ex4-ex36_out_F1 19 CGA GAC TAG CTG CCA AGT A 60.6 33 ROS1 ex36_out_R1 18 GAT GTC CAC TGC TGT TCC 59.8 34 GusB_out_F1 19 TCC CAC CTA GAA TCT GCT G 60.5 35 GusB_out_R1 21 GCT GCA TAG TTA GAG TTG CTC 60.7 36

Using the primer sets in Table 6 above, amplicons for each gene target were designed as shown in Figures 4a to 4c.

1-2. Production of allele-specific primer sets and probes

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(℃) 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(℃) 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 GAG ATG ATT CC 62.5 44 Ex19del C3 Rmt20 GTT GGC TTT CGG AGA TTC CT 62.2 45 Ex19del C4 Rmt24 GCTTTCG GAG ATG TTG CTT CCT TG 65.8 46 Ex19del C5 Rmt21 CCT TGT TGG CTT TCT GTT CCT 62.9 47 Ex19del C6 Rmt21 CCTTG TTG GCT TTC GGA TCC T 64.6 48 Ex19del C7 Rmt21 TGGCT TTC GGA GAT GTT TTG A 62.5 49 Ex19del C8 Rmt20 TGG CTT TCG GAG ATG TCT TG 62.1 50 Ex19del C9 Rmt21 TCCTT GTT GGC TTT CGG TTC C 65.0 51 Ex19del C10 Rmt2(20) TGTTG GCT TTC GGA GCC TTG 65.1 52 Ex19del C11 Rmt20 TTGTT GGC TTT CGG AGA TTC 60.7 53 Ex19del C12 Rmt21 TTCCT TGT TGG CTT TCG ATT C 61.3 54 Ex19del C13 Rmt21 GCT TTC GGA GAT GTT GGT TCC 63.2 55 Ex19del C14 Rmt20 GTTGG CTT TCG GAG ATG GTT 62.7 56 Ex19del C15 Rmt20 CTTG TTG GCT TTC GGA ACC T 62.9 57 Ex19del C16 Rmt23 TTCCT TGT TGG CTT TCG GAA TTT 63.8 58 Ex19del C17 Rmt20 GTT GGC TTT CGG AGA TAC CT 61.6 59 Ex19del C18 Rmt3(21) TTTC CTT GTT GGC TTT CGG TT 63.3 60 Ex19del C19 Rmt21 TGTT GGC TTT CGG AGA AGC AA 64.9 61 Ex19del C20 Rmt21 TGTTG GCT TTC GGA GAT TGC T 64.7 62 Ex19del C21 Rmt21 TTGGC TTT CGG AGA TGT TGG C 65.3 63 Ex19del C22 Rmt20 TGT TGG CTT TCG GAG ACT TG 62.6 64 Ex19del C23 Rmt22 TGGC TTT CGG AGA TGT TGG AAT 64.5 65 Ex19del C24 Rmt22 GTT GGC TTT CGG AGA TAT TTT G 60.1 66 Ex19del C25 Rmt22 TGTTG GCT TTC GGA GAT GGA AT 64.5 67 Ex19del C26 Rmt2(19) TAG GCT TCA TCG AGG GTT G 61.0 68 Ex19del C27 Rmt20 TCC TTG TTG GCT TTC GAG AC 62.3 69 Ex19del C28 Rmt2(20) TTGT TGG CTT TCG GAG ATT C 60.7 70 Ex19del C29 Rmt21 GCTT TCG GAG ATG TTG CGA TA 62.7 71 Ex19del_BR2 GCT TTC GGA GAT GTT GCT TCT CTT AAT TCC TTG A/3phos/ 69.5 72 Ex19del SF1 CGG CAC GGT GTA TAA GGG 61.1 73 Ex19Del R_FAM CTC TGG ATC CCA GAA GGT GAG AAA G 65.6 74 EGFR_Oligo Mix_3 Sequence (5'-3') Tm(℃) Sequence number S768I Rmt14+C CGG GGT TGT CCA CGA 60.4 75 S768I,Ex20lns SF1 CAA CAT CTC CGA AAG CCA 59.7 76 Ex19-20_FAM_R28 TCA CGT AGG CTT CAT CGA GGA TTT CCT T 68.7 77 EGFR_Oligo Mix_4 Sequence (5'-3') Tm(℃) Sequence number 20Ins C1 Rmt2(15)-4A CCA CGC TGG CAA CGC 63.5 78 20Ins C2 Rmt3(15) GGC ACA CGT GGT GGG 73.3 79 20Ins C3 Rmt2(16) CAC ACG TGG GGG TTA C 62.5 80 20Ins C4 Rmt2(16)-3T TGT CCA CGC TGT TCA C 58.9 81 20Ins C5 Rmt2(16)-3T ATC CAC GCT GGC TAC G 62.5 82 S768I,Ex20lns SF1 CAA CAT CTC CGA AAG CCA 59.7 83 Ex19-20_FAM_R28 TCA CGT AGG CTT CAT CGA GGA TTT CCT T 68.7 84 EGFR_Oligo Mix_5 Sequence (5'-3') Tm(℃) Sequence number T790M Rmt(15) AGG GCA TGA GCT GCA 60.4 85 T790M SF5 ACC CCC ACG TGT GCC G 66.9 86 [MX2] T790M IR4_FAM GTG ATG AGC TGC ACG G 59.7 87 [MX2] T790M IR2(-8T)_FAM GTG ATG AGT TGC ACG GTG G 63.0 88 EGFR_Oligo Mix_6 Sequence (5'-3') Tm(℃) Sequence number C797S C1 Fmt19 AGC TCA TGC CCT TCG GCT C 66.4 89 C797S C2 Fmt18 AGC TCA TGC CCT TCG GCA 66.3 90 C797S SR2 CAG GTA CTG GGA GCC AAT ATT GTC 64.4 91 T790M_C797S FAM_F CTC CTG GAC TAT GTC CGG GAA CAC 67.2 92 EGFR_Oligo Mix_7 Sequence (5'-3') Tm(℃) Sequence number L858R Fmt21-2A CAA GAT CAC AGA TTT TGG ACG 59.3 93 L858R OR1 TTG CCT CCT TCT GCA TGG TAT TC 65.1 94 L858R_FAM CCA AAC TGC TGG GTG CGG AAG AG 69.0 95 EGFR_Oligo Mix_8 Sequence (5'-3') Tm(℃) Sequence number L861Q Rmt17 CTC TTC CGC ACC CAG CT 63.6 96 L861Q SF1 CGT ACT GGT GAA AAC ACC G 60.6 97 L861Q FAM_R CAG CAT GTC AAG ATC ACA GAT TTT GGG CT 68.8 98 KRAS,BRAF_Oligo Mix_1 Sequence (5'-3') Tm(℃) Sequence number G12C Fmt (17) TGT GGT AGT TGG AGC TT 57.6 99 G12_13 SR5 GTT GGA TCA TAT TCG TCC ACA AA 61.3 100 KRAS G12_13 FAM CAA GAG TGC CTT GAC GAT ACA GCT A 65.8 101 V600E Fmt(17+A) ATT TGG TCT AGC TAC AGA 54.7 102 BRAF SR1 GAT CCA GAC AAC TGT TCA AAC TG 61.7 103 BRAF_VIC AAA TCT CGA TGG AGT GGG TCC CAT CA 68.8 104 EML4-ALK_Oligo Mix_1 Sequence (5'-3') Tm(℃) Sequence number V1_Fusion_F1 GGA GCA AAA CTA CTG TAG AGC 60.3 105 V2_Fusion_F6 TGT CTA ACT CGG GAG ACT ATG 60.2 106 V3a_Fusion_F4+1 GCA GAC AAG CAT AAA GAT GTC A 61.2 107 V5ab_Fusion_F2 GTG GCC TCA GTG AAA AAA TCA G 62.0 108 V4,7_Fusion_F1 CTG TGG GAT CAT GAT CTG AA 59.2 109 V8a_Fusion_F4 GTG GCC ATA GGA ACG CA 61.6 110 qPCR_R1_modified GAG CTT GCT CAG CTT GTA CT 62.1 111 EML4-ALK_FAM_Probe_R1 AGG GCT CTG CAG CTC CAT CTG CAT G 71.5 112 V4_Fusion_Probe TAG AGA TAT GCT GGA TGA GCC CTG 65.0 113 ROS1_Oligo Mix_1 Sequence (5'-3') Tm(℃) Sequence number CD74 ex6_F3 ACTGACGCTCCACCGAA 62.9 114 SDC4 ex2_F2 ACCAGACGATGAGGATGT 59.9 115 EZR ex10_F10 TGAGGAGAAGACAAAGAAGGC 61.8 116 ROS1 ex34_R1 TGGGATTGTAACAACCAGAAAT 60.6 117 ROS1 ex34_P6_FAM TTGGATACCAGAAACAAGTTTCATACTTA 63.5 118 TPM3 ex8_F9 TAGCCAAGCTGGAAAAGACA 61.7 119 LRIG3 ex16_F12 TTACCACAACATGACAGTAGT 59.3 120 CCDC6 ex5_F1 AGTGGAACGGCTGAAGAAGCA 66.2 121 ROS1 ex35_R3 TGCATAGCAGGCATTAGC 59.9 122 ROS1 ex35_P3_VIC TCGTTTATAAGCACTGTCACCC 62.3 123 ROS1_Oligo Mix_2 Sequence (5'-3') Tm(℃) Sequence number CD74 ex6_F3_17 ACTGACGCTCCACCGAA 62.9 124 SLC34A2 ex4_F11 TGGATATTCTTAGTAGCGCCTT 61.2 125 SLC34A2 ex13_F11 ACCTTTGATAACATAACCATTAGC 59.7 126 SDC4 ex2_F6 TGTCTGGCTCTGGAGATCT 61.8 127 SDC4 ex4_F11 AGCAACATCTTTGAGAGAACG 60.7 128 ROS1 ex32_R7 TCTCCCACTGTATTGAATTTTTAC 59.6 129 ROS1 exon32_P6_FAM AGGCATTCCCAAATTACTAGAAGGGA 65.7 130 GOPC ex4_F12 AGCTGCCAAGTACTTGGATAA 61.6 131 ROS1 ex36_R3_17 TCCACTTCCCAGCAAGA 60.0 132 ROS1 exon36_P4_VIC TCCCGAGGGAAGGCAGGAAG 67.4 133

[Example 2]

Preparation of mutant RNA samples

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. Using the produced wild-type DNA, 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 DH5α 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.

As shown in Table 8, 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.

group Sample WT A549 cell total RNA (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

[Example 3]

Detection of EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations using mRNA

Specific cDNA synthesis and amplification experimental conditions are as shown in Tables 9 and 10 below.

2X Reverse & Preamp Master Mix 10 μl 2X Reverse Transcription & Preamplification Oligo Mix (Oligois in Table 1) 2 μl Samples from each group in Table 3 2 μl Distilled water with unidentified nuclease 6 μl Total amount 20 μl

step Cycle temperature hour 1 1 25 ℃ 2 minutes 2 1 50℃ 15 minutes 3 1 95 ℃ 5 minutes 4
15 95 ℃ 10 seconds 5 60℃ 30 seconds 6 72℃ 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 μl of reverse transcription & pre-amplification master mix was aliquoted into each PCR tube, 2 μl of reverse transcription & pre-amplification oligo mix was added, 2 μl of each sample RNA was added, and 6 μl 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.

After centrifuging the PCR tube that completed the cDNA synthesis and preamplification steps, the lid was carefully opened on a biological safety bench and the first dilution was performed with 180 μl of nuclease-free distilled water. 10 μl of the first diluted solution was transferred to a tube containing 990 μl of nuclease-free distilled water and the second dilution was performed, thereby diluting the preamplified DNA by a final 1000-fold.

Next, the specific genetic mutation detection experimental conditions are as shown in Tables 11 and 12 below.

2X Qualitative Analysis PCR Master Mix
(Oligomers in Table 2) 10 μl 1000-fold diluted preamplified sample 9.5 μl ADPS Smart DNA Polymerase (1 U/ μl) 0.5 μl Total amount 20 μl

step Cycle temperature hour Data collection 1 1 95 ℃ 5 minutes - 2
45 95 ℃ 10 seconds - 3 60℃ 30 seconds FAM / CY5 4 72℃ 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 μl of the qualitative analysis PCR master mix was aliquoted into each PCR tube, 9.5 μl of the 1000-fold diluted preamplified sample was added, 0.5 μl 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 ΔCt value for each well was calculated as follows.

ΔCt value = Sample Ct value (FAM of each master mix) - Positive control Ct value (FAM of each master mix)

ΔCt value = Sample Ct value (CY5 of each master mix) - Positive control Ct value (CY5 of each master mix)

The calculated ΔCt value for each well is used to determine whether the mutation you want to detect is present in that tube.

As a result, 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.

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

Primer sets for synthesizing and amplifying cDNAs containing EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, respectively: A forward primer of sequence number 1 and a reverse primer of sequence numbers 2 to 4; A forward primer of sequence number 5 and a reverse primer of sequence numbers 6 to 7; A forward primer of sequence number 8 and a reverse primer of sequence numbers 9 to 11; Forward primers of SEQ ID NOs: 12 to 17 and reverse primers of SEQ ID NO: 18; A forward primer of SEQ ID NO: 19 to 21 and a reverse primer of SEQ ID NO: 22; A forward primer of SEQ ID NO: 23 to 25 and a reverse primer of SEQ ID NO: 26; A forward primer of SEQ ID NO: 27 to 31 and a reverse primer of SEQ ID NO: 32; and A forward primer of sequence number 33 and a reverse primer of sequence number 34. A primer set for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes, comprising primer sets of 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. A composition for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes, comprising the primer set of the second clause. A composition for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes, further comprising 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, in the third paragraph. Kit for detection of EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations using RNA, including: A primer set for synthesizing and amplifying cDNAs each containing EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations, comprising a primer set of sequence number 1 to sequence number 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 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. A method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA using the kit of Article 5. In the 6th paragraph, a method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA, comprising the following steps: (a) a step of extracting RNA from a separated biological sample; (b) a step of synthesizing and amplifying cDNAs each including EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by treating the primer sets of sequence numbers 1 to 34 and reverse transcriptase; (c) a step of performing a polymerase chain reaction by treating 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 probes of SEQ ID NOs: 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130 and 133; and (d) A step of detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations by confirming the amplification results by the polymerase chain reaction using fluorescence. A method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA, wherein the EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations include 44 EGFR gene mutations described in Table 1 below, 1 BRAF gene mutation described in Table 2 below, 1 KRAS gene mutation described in Table 3 below, 10 EML4-ALK gene mutations described in Table 4 below and 12 ROS1 gene mutations described in Table 5 below. [Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

A method for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes from RNA, wherein in step (c), a blocking primer having sequence number 72 is additionally processed. A method for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes from RNA, wherein the polymerase chain reaction of step (c) is performed by allele-specific polymerase chain reaction or real-time polymerase chain reaction. A method for detecting mutations in EGFR, BRAF, KRAS, EML4-ALK and ROS1 genes from RNA, further comprising the step of (e) confirming the amplification result by the polymerase chain reaction by measuring the Ct (cycle threshold) value. A method for detecting EGFR, BRAF, KRAS, EML4-ALK and ROS1 gene mutations from RNA according to any one of claims 6 to 11, which is applied to diagnosing lung cancer or predicting the responsiveness of lung cancer patients to drugs. 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 below, cDNAs including 1 type of BRAF gene mutation described in Table 2 below, cDNAs including 1 type of KRAS gene mutation described in Table 3 below, cDNAs including 10 types of EML4-ALK gene mutations described in Table 4 below, and cDNAs including 12 types of ROS1 gene mutations described in Table 5 below. [Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

A composition for diagnosing lung cancer, wherein the primer set in claim 13 comprises primer sets of 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. A composition for diagnosing lung cancer, further comprising at least one probe selected from the group consisting of sequence numbers SEQ ID NOs: 41, 42, 74, 77, 84, 87, 88, 92, 95, 98, 101, 104, 112, 113, 118, 123, 130, and 133 and a blocking primer of sequence number 72, in claim 13. A composition for diagnosing lung cancer, further comprising a primer set for synthesizing and amplifying cDNAs comprising the 44 types of EGFR gene mutations, cDNAs comprising the 1 type of BRAF gene mutation, cDNAs comprising the 1 type of KRAS gene mutation, cDNAs comprising the 10 types of EML4-ALK gene fusion mutations, and cDNAs comprising the 12 types of ROS1 gene rearrangement mutations in claim 13. A composition for diagnosing lung cancer, wherein in claim 16, the primer set is at least one primer set selected from the group consisting of the following sequence numbers 1 to 34. A kit for diagnosing lung cancer, comprising a composition according to any one of claims 13 to 17.

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