WO2020145711A1 - Dna polymerase for egfr mutation detection and kit comprising same - Google Patents

Abstract

The present invention relates to a DNA polymerase for EGFR mutation detection and a kit comprising same and, more specifically, to a DNA polymerase capable of detecting 44 somatic mutations in exons 18 to 21 of EGFR gene with high sensitivity, a primer set, a probe, a kit, and a method for detecting an EGFR gene mutation by using the kit.

Description

DNA polymerase for detecting EGFR mutation and kit comprising the same

The present invention relates to a DNA polymerase for detecting EGFR mutations and a kit comprising the same, and more specifically, a DNA polymerase and primer set capable of detecting 44 somatic mutations in exons 18 to 21 of the EGFR gene with high sensitivity. , Probes, kits and methods for detecting EGFR gene mutations using the kits.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% of all lung cancer diagnoses. Patients with epithelial growth factor receptor (EGFR) mutations have high levels of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib (Iressa, AstraZeneca), erlotinib (Tarceba, Roche), and osimmertinib (Tagrisso, AstraZeneca). It is known to exhibit drug response or resistance. G719X, S768I, Ex19 deletion, L858R and L861Q mutations are associated with susceptibility to EGFR TKI, whereas T790M and most Ex20 insertion mutations are associated with reduced EGFR TKI response. Early detection of EGFR gene mutations in lung cancer patients allows predicting drug response before treatment.

So far, one of the most used methods for the detection of EGFR mutations is direct sequencing, which directly identifies the nucleotide sequence of the gene corresponding to exons 18 to 21 of EGFR. This method has the disadvantage that it takes a lot of time compared to real-time polymerase chain reaction or DNA chip analysis, but it is the most accurate analysis in that it is possible to analyze the base of the target site as well as a specific base. It has the advantage of being possible. Accordingly, there is a continuous need for a kit for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations.

On the other hand, Korean Patent Publication No. 10-2015-0102468 discloses a kit for diagnosing lung cancer, which includes a primer set for amplifying a lung cancer-related mutation and a probe complementary to the mutation, but a polymerase for detecting a mutation in the EGFR gene and There is no known reaction buffer to increase its activity.

Accordingly, the present inventors have developed a kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer for increasing its activity. The invention was completed.

An object of the present invention is to provide a DNA polymerase for detecting EGFR gene mutation.

Another object of the present invention is to provide a primer set for detecting EGFR gene mutations.

Another object of the present invention is to provide a probe for detecting an EGFR gene mutation.

Another object of the present invention is to provide a kit for detecting EGFR gene mutations, which includes the DNA polymerase and/or primer set described above.

Another object of the present invention is to provide a method for detecting EGFR gene mutations using the aforementioned kit.

In order to solve the above-described problem, the present invention is a 507th amino acid residue glutamic acid (E) in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K), the 536th amino acid residue arginine (R) is lysine (K) Provided is a DNA polymerase for detecting EGFR gene mutations, including Taq polymerase substituted with valine (V), wherein arginine (R), the 660th amino acid residue, is substituted with

The present invention also provides a primer set for EGFR gene mutation detection, comprising at least one primer selected from the group consisting of SEQ ID NOs: 3 to 54.

The present invention also provides a probe for detecting an EGFR gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.

According to one preferred embodiment of the present invention, the nucleotide sequences of SEQ ID NOs: 55 to 63 are each selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end. Fluorophores of the species may be labeled, and one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 may be labeled at the 3'-end.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue glutamic acid (E) is substituted with lysine (K), the 536th amino acid residue arginine (R) is substituted with lysine (K), and the 660th Taq polymerase in which the amino acid residue arginine (R) is substituted with valine (V); And / or at least one primer selected from the group consisting of SEQ ID NO: 3 to 54; provides a kit for detecting EGFR gene mutation.

According to one preferred embodiment of the present invention, the kit may further include a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.

According to another preferred embodiment of the present invention, the nucleotide sequences of SEQ ID NOs: 55 to 63 are each selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end. One fluorophore may be labeled, and one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 may be labeled at the 3'-end.

According to a preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; And 1 to 7 mM (NH ₄ ) ₂ SO ₄ ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

According to another preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; 1 to 7 mM (NH ₄ ) ₂ SO ₄ ; And 5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

The present invention also provides a method for detecting an EGFR gene mutation comprising the following steps: (a) extracting a nucleic acid from an isolated biological sample; (b) processing the kit of the present invention on the extracted nucleic acid to perform PCR (polymerase chain reaction); And (c) confirming the amplification result by the PCR with fluorescence.

According to a preferred embodiment of the present invention, the PCR may be allele-specific PCR or real-time PCR.

According to another preferred embodiment of the present invention, (d) may further include the step of confirming the amplification result by the PCR by measuring the Ct (cycle threshold) value.

According to a preferred embodiment of the present invention, the EGFR gene mutation includes at least one selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. can do.

According to another preferred embodiment of the present invention, the EGFR gene mutation is a 719th amino acid glycine substitution in exon 18 of EGFR, a deletion in exon 19, a 768th amino acid in exon 20. Serine, 790th amino acid threonine, 797th amino acid cysteine substitution, exon 20 insertion, exon 21 858 amino acid leucine and 861 amino acid leucine substitution. It may include the above.

According to another preferred embodiment of the present invention, the EGFR gene mutation detection method may be for predicting responsiveness to drugs of lung cancer patients.

According to another preferred embodiment of the present invention, the lung cancer may be non-small cell lung cancer.

According to another preferred embodiment of the present invention, the drug may be a tyrosine kinase inhibitor.

According to another preferred embodiment of the present invention, the tyrosine kinase inhibitor may be gefitinib, erlotinib or osimitinib.

According to another preferred embodiment of the present invention, the nucleic acid of step (a) may be extracted from a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy.

The kit of the present invention shows high detection sensitivity (up to 0.01%, 3 mutant copies in 30,000 wild-type copies), high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy. In addition, by simultaneously detecting 44 mutations within exons 18 to 21 of the EGFR gene, it is possible to accurately predict drug reactivity to lung cancer patients against tyrosine kinase inhibitors.

Figure 1 shows the production process of Taq DNA polymerase containing each of the R536K, R660V and R536K/R660V mutations, (a) schematically shows fragment PCR and overlap PCR, (b) is amplified in fragment PCR The result of confirming the product by electrophoresis, and (c) shows the result of confirming the amplified product by electrophoresis by amplifying the entire length by overlap PCR.

Figure 2 is a result of confirming the overlap PCR product of FIG. 1(c) purified by digestion with the restriction enzyme EcoRI/XbaI and then the SAP-treated pUC19 vector for gel extraction.

Figure 3 is a schematic diagram showing fragment PCR and overlap PCR during the preparation of Taq DNA polymerase containing E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V mutations, respectively.

FIG. 4 shows the results of confirming by electrophoresis the overlap PCR product of FIG. 3 purified with the pUC19 vector digested with the restriction enzyme EcoRI/XbaI for gel extraction, and then with SAP.

5A to 5L show the results of detecting mutations with AS-qPCR in the Ex19del C1 to Ex19del C12 mutant plasmid (3, 10 ² and 10 ⁴ copies) template.

6A to 6L show the results of detecting mutations with AS-qPCR in the Ex19del C13 to Ex19del C24 mutant plasmid (3, 10 ² and 10 ⁴ copies) template.

7A to 7G show the results of detecting mutations with AS-qPCR in the Ex19del C25 to Ex19del C31 mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 7h is S768I mutant plasmid (3, 10 ² and 10 ⁴ Copy) shows the result of detecting the mutation with AS-qPCR in the template, and 7i shows the result of detecting the mutation with AS-qPCR in WT.

8 shows the results of detecting mutations with AS-qPCR in the T790M mutant plasmid (0, 3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.

Figure 9a shows the results of detecting the mutation in the G719S mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.

Figure 9b shows the results of detecting the mutation in the G719C mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.

Figure 9c shows the results of detecting the mutation in the G719A mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.

Figure 9d shows the results of detecting the mutation in WT AS-qPCR.

9E shows the results of detecting mutations with AS-qPCR in the L861Q mutant plasmid (0, 3, 10 ² and 10 ⁴ copies) template.

10A to 10E show the results of detecting mutations with AS-qPCR in the Ex20Ins C1 to Ex20Ins C5 mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 10f is the result of detecting mutations from WT to AS-qPCR. 10g shows the results of detecting mutations with AS-qPCR in the L858R mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 10h shows the results of detecting mutations in WT with AS-qPCR. will be.

As described above, there is a continuous need to develop a kit for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations. Thus, the present inventors provide an optimized kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer to increase its activity. The solution to the above problem was sought. The kit of the present invention exhibits high detection sensitivity, high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy. In addition, it features a semi-quantitative analysis, and analysis is possible on all qPCR devices with 4 channels.

Hereinafter, terms used in the present application will be described.

"Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspart Acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan ( Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

Amino acids are typically organic acids, which include substituted or unsubstituted amino groups, substituted or unsubstituted carboxy groups, and one or more side chains or groups, or any analogue of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, Borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thio acid, hydroxylamine, or any combination of these groups.

In the DNA polymerase of the present invention, the term "mutant" refers to a recombinant polypeptide comprising one or more amino acid substitutions compared to the corresponding naturally occurring or unmodified DNA polymerase.

The term “thermal stable polymerase” (referring to a heat stable enzyme) is heat resistant, retains sufficient activity to achieve subsequent polynucleotide elongation reactions and is treated with elevated temperature for the time required to achieve denaturation of the double-stranded nucleic acid Does not irreversibly denature (deactivate) when As used herein, it is suitable for use at temperatures cycling reactions such as PCR. Irreversible denaturation herein refers to permanent and complete loss of enzyme activity. For thermostable polymerases, enzymatic activity refers to catalyzing a combination of nucleotides in a suitable way to form a polynucleotide extension product complementary to the template nucleic acid strand. Thermostable DNA polymerases derived from thermophilic bacteria include, for example: Thermomoto maritima, thermos aquaticus, thermos thermophilus, thermos flavus, thermomod philipformis, thermos species DNA polymerase derived from Sps17, Thermos species Z05, Thermos Caldophyllus, Bacillus caldotenax, Thermomoto Neopolitana, and Thermosippo africanus.

The term “thermal activity” refers to an enzyme that maintains catalytic properties at temperatures (ie 45-80° C.) commonly used for reverse transcription or annealing/extension steps in RT-PCR and/or PCR reactions. Thermostable enzymes are those that are not irreversibly inactivated or denatured when treated at elevated temperatures required for nucleic acid denaturation. The thermoactive enzyme may or may not be thermostable. The thermally active DNA polymerase can be DNA or RNA dependent from thermophilic or mesophilic species, including but not limited to:

The term “nucleotide” is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that exists in the form of a single strand or a double strand, and is not specifically mentioned otherwise. Unless it can contain analogs of natural nucleotides.

The term “nucleic acid” or “polynucleotide” refers to a polymer that can correspond to a DNA or RNA polymer, or analogs thereof. Nucleic acids can be, for example, chromosomal or chromosomal segments, vectors (eg, expression vectors), expression cassettes, naked DNA or RNA polymers, products of polymerase chain reaction (PCR), oligonucleotides, probes, and primers. Or may include it. Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, but are not limited to any particular length. Unless otherwise stated, certain nucleic acid sequences include or encode complementary sequences in addition to any sequence specified.

The term “primer” refers to a polynucleotide that can serve as a starting point for nucleic acid synthesis in the template-direction when placed under conditions where polynucleotide elongation is initiated. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes. Primers are typically single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The appropriate length of the primer will typically depend on the intended use in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides. Short primer molecules generally require a lower temperature to form a sufficiently stable hybridization complex with the template. Primers are not required to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for elongation of the primer. In certain embodiments, the term “primer pair” includes a 5′-sense primer that hybridizes complementarily to the 5′-end of the amplified nucleic acid sequence, and a 3′-antisense primer that hybridizes to the 3′ end of the amplified sequence. Means a set of primers comprising Primers can be labeled, if necessary, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include: ³² P, fluorescent dyes, electron-dense reagents, enzymes (usually used in ELISA assays), biotin, or haptens and proteins in which antiserum or monoclonal antibodies can be used. .

The term “5′-nuclease probe” refers to an oligonucleotide comprising at least one luminescent label moiety used in a 5′-nuclease reaction to target nucleic acid detection. In some embodiments, for example, the 5'-nuclease hydrolyzate probe comprises only a single luminescent moiety (eg, fluorescent dye, etc.). In certain embodiments, the 5'-nuclease probe contains a self-complementary region so that the probe can form a hairpin structure under selective conditions. In some embodiments, the 5'-nuclease hydrolyzate probe comprises two or more labeling moieties, and one of the two labels is released from the oligonucleotide after being separated or degraded to increase the emission intensity. In certain embodiments, the 5'-nuclease hydrolase probe is labeled with two different fluorescent dyes, for example a 5'-terminal reporter dye and a 3'-terminal quencher dye or moiety. In some embodiments, the 5'-nuclease probe is labeled in addition to, or at one or more positions other than the terminal position. When the probe is intact, energy transfer typically occurs between the two phosphors such that the fluorescence emission from the reporter dye is partially extinguished. During the elongation phase of the polymerase chain reaction, for example, a 5'-nucleic acid hydrolase probe bound to the template nucleic acid has an activity such that the fluorescence of the reporter dye is no longer quenched, for example, Taq polymerase or other Degraded by the 5'to 3'-nucleic acid hydrolase activity of the polymerase. In some embodiments, a 5'-nuclease probe can be labeled with two or more different reporter dyes and a 3'-terminal quencher dye or moiety.

The terms "FRET" or "fluorescence resonance energy transfer" or "poster resonance energy transfer" refer to the transfer of energy between two or more chromophores, donor chromophores and receptor chromophores (referred to as quenchers). The donor typically transfers energy to the receptor when the donor is excited by emitting light of a suitable wavelength. Receptors typically re-emit energy transferred in the form of light emitted at different wavelengths. When the receptor is a “cancer” matting agent, it disperses the energy transferred in a form other than light. Whether a particular fluorescent substance acts as a donor or a receptor depends on the properties of other members of the FRET pair. Commonly used donor-receptor pairs include FAM-TAMRA pairs. Commonly used matting agents are DABCYL and TAMRA. Commonly used cancer matting agents include: BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), And BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

The term “conventional” or “natural” when referring to a nucleic acid base, nucleoside triphosphate, or nucleotide refers to naturally occurring polynucleotides described (ie, for DNA, they are dATP, dGTP, dCTP and dTTP). In addition, dITP, and 7-deaza-dGTP are frequently used instead of dGTP and can be used instead of dATP in in vitro DNA synthesis reactions such as sequencing.

The term “unconventional” or “modified” when referring to a nucleic acid base, nucleotide, or nucleotide, is a conventional base, nucleotide, or modification, derivative, or nucleotide that occurs naturally in a particular polynucleotide, or Analogs. Certain unusual nucleotides are modified at the 2'position of the ribose sugar compared to conventional dNTP. Thus, even though naturally occurring nucleotides for RNA are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collective rNTP), since nucleotides have hydroxyl groups at the 2'position of the sugar, this is compared to the absence of dNTP, As used herein, ribonucleotides are unusual nucleotides as substrates for DNA polymerases. As used herein, unusual nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, compounds having a 2',3'- dideoxy structure, referred to as dideoxynucleoside triphosphate. Dideoxynucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. Additional examples of terminator compounds include 2'-PO ₄ analogues of ribonucleotides. Other unusual nucleotides are phosphorothioate dNTP ([[α]-S]dNTP), 5'-[α]-borano-dNTP, [α]-methyl-phosphonate dNTP, and ribonucleosides Triphosphate (rNTP). Uncommon bases include radioactive isotopes such as ³² P, ³³ P, or ³⁵ S; Fluorescent labels; A label for chemiluminescence; Bioluminescent markers; Hapten labels such as biotin; Or it can be labeled with an enzyme label such as streptavidin or avidin. Fluorescent labels can include negatively charged dyes, such as the dyes of the fluorescein family, or neutrally charged dyes, such as the dyes of the rhodamine family, or positively charged dyes, such as the dyes of the cyanine family. Dyes of the fluorescein family include, for example, FAM, HEX, TET, JOE, NAN and ZOE. Rhodamine family dyes include Texas Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen /Molecular Probes (Eugene, OR). The cyanine family dyes include Cy2, Cy3, Cy5, and Cy7, and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

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

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue, glutamic acid (E), is substituted with lysine (K), the 536th amino acid residue, arginine (R), is replaced with lysine (K), and the 660th amino acid residue. Provided is a DNA polymerase for detecting EGFR gene mutations, including Taq polymerase in which phosphorus arginine (R) is substituted with valine (V).

The "Taq polymerase" was a thermophilic DNA polymerase named after the thermophilic bacterium Thermus aquaticus and was first isolated from the bacteria. Thermos Aquaticus is a bacterium inhabiting hot springs and hot water jets, and Taq polymerase has been identified as an enzyme capable of withstanding the protein denaturation conditions (high temperature) required in PCR. The optimum activity temperature of Taq polymerase is 75-80 ℃, has a half-life of 9 hours at 22.5 hours at 92.5 ℃, 40 minutes at 95 ℃, 9 minutes at 97.5 ℃, and replicates 1000 base pair DNA within 10 seconds at 72 ℃ Can. It lacks 3'→5' nucleic acid endonuclease correcting activity, and the error rate is measured in about 1 out of 9,000 nucleotides. For example, using the heat-resistant Taq, PCR can be performed at high temperatures (above 60°C). The amino acid sequence shown in SEQ ID NO: 1 for Taq polymerase is used as a reference sequence.

In the present invention, the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K) in glutamic acid (E), the 536th amino acid residue is substituted with lysine (K) in arginine (R), and the 660th amino acid The Taq polymerase in which the residue is substituted with arginine (R) to valine (V) was designated as “E507K/R536K/R660V”, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 72, respectively.

The present invention also provides a primer set for detecting EGFR gene mutations comprising one or more primers selected from the group consisting of SEQ ID NOs: 3 to 54.

The primer set for detecting EGFR gene mutation of the present invention may be, for example, those described in Tables 16 to 19 of Example 3, but is not limited thereto.

Preferably, the polymerase according to the present invention is particularly excellent in EGFR mutation detection sensitivity when used with the primer sequences of Tables 16 to 19.

The present invention also provides a kit for detecting EGFR gene mutations, which includes the DNA polymerase and/or primer set described above.

The kit of the present invention can be used for research (Research Use Only, RUO) or in-vitro diagnostic (IVD).

The kit of the present invention may be a PCR kit, and may contain any reagents or other elements recognized by those skilled in the art as being used in the primer extension process.

For example, the PCR kit of the present invention includes (a) nucleoside triphosphate; (b) a reagent for quantification that binds double-stranded DNA; (c) polymerase blocking antibodies; (d) one or more control values or control sequences; And (e) one or more templates; may further include one or more selected from the group consisting of.

According to a preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; And 1 to 7 mM (NH ₄ ) ₂ SO ₄ ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

More preferably, the kit comprises 40 to 90 mM KCl; And 1 to 5 mM (NH ₄ ) ₂ SO ₄ ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.0.

According to another preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; 1 to 7 mM (NH ₄ ) ₂ SO ₄ ; And 5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

More preferably, the kit comprises 40 to 90 mM KCl; 1 to 5 mM (NH ₄ ) ₂ SO ₄ ; And 10 to 40 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.0.

The kit of the present invention may further include TrisCl (pH 8.0 to 9.5), MaCl ₂ , Tween 20, and Bovine serum albumin (BSA) in addition to KCl, (NH ₄ ) ₂ SO ₄ , and TMAC, and these components The concentration of can be used by a person skilled in the art to adjust to an appropriate range.

The PCR buffer composition as described above enables reliable gene mutation-specific amplification by remarkably improving the activity of the DNA polymerase of the present invention.

According to a preferred embodiment of the present invention, the PCR kit may 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 cast PCR (Competitive allele-specific TaqMan PCR) or Droplet digital PCR (ddPCR), and more specifically, allele specific cast PCR or allele It may be a specific droplet digital PCR, but is not limited thereto.

The “cast PCR” is a method for detecting and quantifying rare mutations in a sample containing a large amount of normal wild type gDNA, allele-specific TaqMan ^® qPCR to inhibit non-specific amplification from wild type alleles. Combination with gene-specific MGB blockers can produce specificity superior to traditional allele-specific PCR.

The “Droplet digital PCR” is a system for counting target DNA by splitting and amplifying a PCR reaction of 20 μl into 20,000 droplets, and depending on whether or not the target DNA is amplified in the droplet, positive droplets (1) It is counted as a digital signal with a negative drop (0), counts the copy number of the target DNA through Poisson distribution, and finally, the result can be confirmed by the number of copies per µl of the sample. Rare mutation detection, very small amount of gene It can be used when amplification, mutation type, etc. are to be simultaneously confirmed.

The “mass array” is a multiplexing analysis method applicable to various genomic studies such as genotyping using a MALDI-TOF mass spectrometer, and rapidly analyzes multiple samples and targets at a low cost. It can be used when you want to do it, or if you want to do customized analysis only for a specific target.

The EGFR gene mutation detection kit of the present invention may further include a probe, a fluorophore, and/or a quencher. The fluorophore may be VIC, HEX, JOE, FAM, CAL Flour Orange 560, Quasar 670, CY5 EverGreen dye, etc., but is not limited thereto. The probe sequence, the type of the fluorophore and the quencher may be the same as in Table 18 of Example 5, but is not limited thereto.

The kit of the present invention employs AS-PCR (Allele-specific PCR) and real-time PCR technology, and includes a specific primer and a fluorescent probe for detecting EGFR mutation of cfDNA in human plasma samples. During nucleic acid amplification, the targeted mutant DNA matches the base at the 3'end of the primer, is selectively and efficiently amplified, and then the mutant amplicon is detected by a fluorescent probe labeled with FAM or CAL Fluor Orange 560 (CFO560). Wild-type DNA cannot match specific primers, and no amplification occurs. The kit of the present invention may be composed of EGFR Master Mixture 1-4, ADPS ^TM smart DNA polymerase, EGFR positive control and nuclease-free distilled water.

The EGFR gene mutation detection kit of the present invention may be configured as shown in Table 1, for example, but is not limited thereto.

Configuration	main ingredient	amount
EGFR Master Mixture 1	Primers/probes for buffers, EGFR targets and ICs containing dNTPs	240 μL/tube Х1
EGFR Master Mixture 2	Primers/probes for buffers, EGFR targets and ICs containing dNTPs	240 μL/tube Х1
EGFR Master Mixture 3	Primers/probes for buffers, EGFR targets and ICs containing dNTPs	240 μL/tube Х1
EGFR Master Mixture 4	Primers/probes for buffers, EGFR targets and ICs containing dNTPs	240 μL/tube Х1
ADPS TM Smart DNA Polymerase	ADPS TM Smart DNA Polymerase	60 μL/tube Х1
EGFR positive control	Recombinant plasmid blend containing each EGFR mutation	160 μL/tube Х1
Nuclease-free distilled water	PCR grade distilled water	1.0 mL/tube Х1

Exemplary configuration of each EGFR Master Mix in Table 1 is shown in Table 2.

			Final concentration
MMX 1-4	ADPS buffer	TrisCl (pH 8.8)	50 mM
		MgCl 2	2.5 mM
		KCl	60 mM
		(NH 4 ) 2 SO 4	2.5 mM
		TMAC	25 mM
		Tween 20	0.1%
	BSA	0.01%
	dNTP	0.25 mM each
	Primer/Probe	Specific concentration
ROX standard dye	1X

Table 3 shows the detection information of EGFR Master Mix 1-4.

reagent	Fluorescent signal
	FAM	CAL Fluor Orange 560 *	Quasar 670 *
EGFR Master Mixture 1	S768I	Ex19Del	IC **
EGFR Master Mixture 2	T790M	-	IC
EGFR Master Mixture 3	L861Q	G719X	IC
EGFR Master Mixture 4	Ex20Ins	L858R	IC

*Alternative dye: CAL Fluor Orange 560 (VIC/HEX/JOE), Quasar 670 (CY5)**IC: Internal control for PCR

The present invention also provides a method for detecting an EGFR gene mutation comprising the following steps:

(A) extracting the nucleic acid from the isolated biological sample;

(B) processing the kit of the present invention to the extracted nucleic acid to perform a polymerase chain reaction; And

(c) analyzing the size or base sequence of the amplification product as a result of the polymerase chain reaction.

According to a preferred embodiment of the present invention, the PCR may be allele-specific PCR or real-time PCR.

The EGFR gene mutation detection method of the present invention may further include (d) confirming the amplification result by the PCR by measuring a Ct (cycle threshold) value.

The cycle threshold (Ct) value means the number of cycles in which the fluorescence generated in the reaction exceeds a threshold, which 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 in which a sufficient number of amplicons have accumulated in the reaction. The Ct value is the cycle in which the increase in ΔRn was first detected. Rn means the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn means the fluorescence emission intensity (standardized reporter signal) of the reporter dye divided by the fluorescence emission intensity of the reference dye. The Ct value is also referred to as a crossing point (Cp) in LightCycler. The Ct value represents the point in time 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 period provides the most useful information about the reaction. The slope of the log-linear phase represents the amplification efficiency (Eff) (https://www.appliedbiosystems.co.kr/).

On the other hand, TaqMan probes typically include a 5'-terminal fluorophore and a 3'-terminal quencher (e.g., TAMRA or non-fluorescent quencher (NFQ)) primers (e.g., 20- 30 nucleotides). Excited phosphors transfer energy to nearby quenchers rather than fluorescence (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 generated. TaqMan probes are designed to anneal to the inner part of the PCR product.

The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but fluorescence is inhibited by quenching on the probe. During the extension reaction, the TaqMan probe hybridized to the template is decomposed by the 5'to 3'nuclease activity of the Taq DNA polymerase, and the fluorescent dye is released from the probe. At this time, the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'to 3'nuclease activity of this polymerase cuts the 5'-end of the TaqMan probe, thereby Fluorescence signal is generated.

The reporter molecule and quencher molecule bound to the TaqMan probe include fluorescent and non-fluorescent materials. Fluorescent reporter molecules and quencher molecules that can be used in the present invention can use any of those known in the art, and examples thereof are as follows (numbers in parentheses are the maximum emission wavelength 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 number in parentheses is the maximum wavelength of light emitted in nanometers.

Suitable reporter-quencher pairs have been described in many documents: 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.

In addition, the non-fluorescent material used in the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety. The term "TaqMan MGB-conjugate probe" herein refers to a TaqMan probe conjugated with MGB at the 3'-end of the probe.

MGB is a substance that binds to the minor groove of DNA with high affinity, such as netropsin, distamicin, lexitropsin, mitramycin, chromomycin A3, and olibo. Olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6- diamidino-2-phenylindole), CDPI dimers, trimers, tetramers and pentamers, MPC (N-methylpyrrole-4-carbox-2-amide) and dimers thereof, trimers, tetramers and pentamers It does not work.

Conjugation of the probe and MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, increased stability (ie, increased degree of hybridization) results in increased melting temperature (Tm) of the hybrid duplex formed by MGB-conjugated probes compared to normal probes. Thus, MGB stabilizes the van der Waals force, thereby increasing the melting temperature (Tm) of the MGB-conjugate probe without increasing the probe length, resulting in shorter probes (e.g. no more than 21 nucleotides) in Taqman real-time PCR under more stringent conditions. Enables the use of.

In addition, the MGB-conjugated 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 15-21 nucleotides, but is not limited thereto.

According to a preferred embodiment of the present invention, the EGFR gene mutation is selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. It may contain one or more.

Specifically, the EGFR gene mutation is the substitution of the 719th amino acid glycine in exon 18 of EGFR, the deletion in exon 19, the 768th amino acid serine in exon 20, the 790th amino acid threonine, It may include one or more selected from the group consisting of substitution of the cysteine, the 797th amino acid, insertion in exon 20, leucine, which is the 858th amino acid in exon 21, and the replacement of the 861th amino acid, leucine.

For example, the EGFR gene mutation detection method of the present invention can simultaneously detect one or more of the 44 mutations listed in Table 4 below in exons 18, 19, 20 and 21 of EGFR.

Exxon	Mutation	EGFR nucleic acid sequence	COSMIC ID
	Exon 18	G719X	2155G>A	6252
			2155G>T	6253
2156G>C			6239
Exon 19	Ex19Del	2240_2251del12	6210
		2239_2247del9	6218
		2238_2255del18	6220
		2235_2249del15	6223
		2236_2250del15	6225
		2239_2253del15	6254
		2239_2256del18	6255
		2237_2254del18	12367
		2240_2254del15	12369
		2240_2257del18	12370
		2239_2248TTAAGAGAAG>C	12382
		2239_2251>C	12383
		2237_2255>T	12384
		2235_2255>AAT	12385
		2237_2252>T	12386
		2239_2258>CA	12387
		2239_2256>CAA	12403
		2237_2253>TTGCT	12416
		2238_2252>GCA	12419
		2238_2248>GC	12422
		2237_2251del15	12678
		2236_2253del18	12728
		2235_2248>AATTC	13550
		2235_2252>AAT	13551
		2235_2251>AATTC	13552
		2253_2276del24	13556
		2237_2257>TCT	18427
		2238_2252del15	23571
		2233_2247del15	26038
		2232_2249del15	221565
		2234_2248del15	1190791
Exon 20	S768I	2303G>T	6241
	T790M	2369C>T	6240
	Ex20Ins	2307_2308ins9GCCAGCGTG	12376
		2319_2320insCAC	12377
		2310_2311insGGT	12378
		2311_2312ins9GCGTGGACA	13428
		2309_2310AC>CCAGCGTGGAT	13558
Exon 21	L858R	2573T>G	6224
		2573_2574TG>GT	12429
	L861Q	2582T>A	6213

In the EGFR gene mutation detection method of the present invention, the target sequence may be present in the sample of step (a), and includes DNA, cDNA or RNA, preferably genomic DNA. The test sample may be included in an animal, preferably a vertebrate, more preferably a human subject. For example, the biological sample in step (a) may be sputum, blood, saliva, or urine, and the nucleic acid in step (a) is a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy. The EGFR gene mutation detection method of the present invention may include melting temperature analysis using a double-strand specific dye.

Melt temperature curve analysis can be performed in real-time PCR devices such as ABI 5700/7000 (96 well format) or ABI 7900 (384 well format) devices with onboard software (SDS 2.1). Alternatively, melt temperature curve analysis can be performed as an endpoint analysis.

“Dye binding to double-stranded DNA” or “double-strand specific dye” can be used when it has a higher fluorescence when bound to double-stranded DNA than to the unbound state. Examples of such dyes are SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, Etidium 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 includes real-time PCR (RT-PCR) or quantitative PCR (qPCR), analysis on agarose gel after standard PCR, gene mutation specific amplification or allele-specific amplification through real-time PCR, Tetra-primer amplification-refractory mutant systems can be performed by PCR or isothermal amplification.

The "standard PCR" is a technique for amplifying single or several copies of DNA or cDNA known to those skilled in the art. Almost all PCR uses thermostable DNA polymerases such as Taq polymerase or Klen Taq. DNA polymerases use single-stranded DNA as a template and enzymatically assemble new DNA strands from nucleotides by using oligonucleotides (primers). The amplicon generated by PCR can be analyzed, for example, on an agarose gel.

The "real-time PCR" can monitor the process in real time when performing PCR. Therefore, data is collected throughout the PCR process, not when the PCR ends. In real-time PCR, the reaction is characterized by a time point during the cycle when amplification is first detected, rather than the target amount accumulated after a fixed number of cycles. Two methods, mainly dye-based detection and probe-based detection, are used to perform quantitative PCR.

The “allele specific amplification (ASA)” is an amplification technique designed to distinguish PCR primers from different templates with a single nucleotide residue.

The "gene mutation specific amplification or allele-specific amplification through real-time PCR" detects gene mutation or SNP in a very efficient manner. Unlike most other methods for detecting gene mutations or SNPs, preliminary amplification of the target genetic material is not required. ASA combines amplification and detection in a single reaction based on the distinction of matched and mismatched primer/target sequence complexes. The increase in DNA amplified during the reaction can be monitored in real time with an increase in the fluorescence signal caused by dyes such as SYBR Green I, which emit light upon binding to double-stranded DNA. Gene mutation-specific amplification or allele-specific amplification through real-time PCR shows delay or absence of a fluorescent signal for mismatched cases. In detecting genetic variation or SNP, it provides information on the presence or absence of genetic variation or SNP.

The "tetra-primer amplification-refractory mutation system PCR" amplifies both wild type and mutant alleles with control fragments in a single tube PCR reaction. Non-allele specific control amplicons are amplified by two common (outer) primers flanking the mutation region. The two allele specific (inner) primers are designed in the opposite direction to the common primer, and can be amplified both wild-type and mutant amplicons simultaneously with the common primer. Consequently, the two allele-specific amplicons have different lengths and can be easily separated by standard gel electrophoresis because the mutations are located asymmetrically with respect to the common (outer) primer. The control amplicon provides internal control for 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 mutation system PCR.

The "isothermal amplification" does not depend on the thermocycler, and preferably means that the amplification of the nucleic acid takes place at a lower temperature without the need to change the temperature during amplification. The temperature used in isothermal amplification can be between room temperature (22-24 °C) to about 65 °C, or at room temperature of about 60-65 °C, 45-50 °C, 37-42 °C or 22-24 °C. The products of the isothermal amplification results are gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), RNA, DNA, and chip-based capillary electrophoresis devices that analyze protein or turbidity It can be detected with an analyzer (bioanalyzer).

When the EGFR gene mutation detection method of the present invention is performed with qPCR, for example, it may be performed under the conditions of Tables 22 to 24 below.

The DNA polymerase, primer set, probe, and/or kit for detecting EGFR gene mutations of the present invention can be used for predicting reactivity to drugs of lung cancer patients.

Accordingly, the present invention can provide a DNA polymerase, primer set, probe, and/or kit for detecting EGFR gene mutation to predict responsiveness to drugs of lung cancer patients.

The present invention can also provide a method for predicting the reactivity of a lung cancer patient to a drug using the aforementioned DNA polymerase, primer set, probe, and/or kit.

According to another preferred embodiment of the present invention, the cancer may be non-small cell lung cancer.

The EGFR gene mutation detection method of the present invention provides information to diagnose cancer early and establish a treatment strategy for each patient by detecting a known EGFR mutation of Table 4 above, thereby more effectively treating patients Contribute.

The DNA polymerase, primer set and/or kit for detecting EGFR gene mutations of the present invention can be used in the diagnosis, prognosis and drug reactivity prediction methods of all diseases applicable by detecting the mutations listed in Table 4 above.

In the present invention, the term "prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, the prognosis prediction can be interpreted to mean any action that predicts the course of the disease after treatment by comprehensively taking into account the patient's physiological or environmental conditions. Can be.

The prognosis prediction may be interpreted as an action of predicting the disease-free survival rate or survival rate of a patient in advance by predicting the progress and complete cure of the disease after treatment of a specific disease. For example, predicting that "the prognosis is good" means that the patient has a high probability of being treated without disease or having a high survival rate after treatment of the disease, and predicting that the "prognosis is bad" is a disease After treatment, the patient's disease-free survival rate or survival rate is low, indicating that the disease is likely to recur or die from the disease.

The term "no disease survival rate" of the present invention means the possibility that a patient can survive without recurrence of the disease after treatment of the specific disease.

The term "survival rate" of the present invention means the possibility that a patient can survive regardless of whether or not the disease recurs after treatment of a specific disease.

When the EGFR gene mutation detection method of the present invention is used for the purpose of predicting the reactivity to a drug in a lung cancer patient, the drug may be a tyrosine kinase inhibitor, and the type of the tyrosine kinase inhibitor is, for example , Gefitinib, erlotinib or osimmertinib.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for explaining the present invention in more detail, whereby it will be apparent to those skilled in the art that the technical scope of the present invention is not limited to these examples.

[Example 1]

Mutagenesis of Taq polymerase

1-1. Fragment PCR

In this embodiment, Taq DNA polymerase in which the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine to lysine (hereinafter referred to as "R536K"), Taq DNA polymerase in which the 660th amino acid residue is substituted with valine in arginine. (Hereinafter referred to as “R660V”) and Taq DNA polymerase (hereinafter referred to as “R536K/R660V”) in which the 536th amino acid residue is substituted from arginine to lysine and the 660th amino acid residue is substituted from arginine to valine is prepared as follows. Did.

First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutant specific primers listed in Table 5, as shown in Figure 1(a). The reaction conditions are shown in Table 6.

primer	Sequence (5'→3')	Sequence number
Eco-F	GG GGTACC TCA TCA CCC CGG	64
R536K-R	CTT GGT GAG CTC CTT GTA CTG CAG GAT	65
R536K-F	ATC CTG CAG TAC AAG GAG CTC ACC AAG	66
R660V-R	GAT GGT CTT GGC CGC CAC GCG CAT CAG GGG	67
R660V-F	CCC CTG ATG CGC GTG GCG GCC AAG ACC ATC	68
Xba-R	GC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA	69

10xpfu buffer (solvent)	2.5 μl
dNTP (10 mM each)	1 μl
F primer	1 μl
R primer	1 μl
Distilled water	18 μl
pUC19-Taq (10 ng/μl)	1 μl
Pfu polymerase	0.5 μl
30 cycles (Ta=60℃)	25 μl

As a result of confirming the PCR product on the electrophoresis, as shown in Fig. 1 (b), it was confirmed that the band for each fragment was confirmed and the desired fragment was amplified.

1-2. Overlap PCR

Each fragment amplified in 1-1 was used as a template to amplify the full length using primers at both ends (Eco-F and Xba-R primers). The reaction conditions are shown in Tables 7 and 8.

R660V or R536K

10xpfu buffer (solvent)	5 μl
5x Enhancer (Solent)	10 μl
dNTP (10 mM each)	1 μl
Eco-F primer (10 pmol/μl)	2 μl
Xba-R primer (10 pmol/μl)	2 μl
Distilled water	27 μl
Fragment 1 (or Fragment 3)	1 μl
Short 2 (or Short 4)	1 μl
Pfu polymerase	1 μl
40 cycles (Ta=62℃)	50 μl

R536K/R660V

10xpfu buffer (solvent)	5 μl
5x Enhancer (Solent)	10 μl
dNTP (10 mM each)	1 μl
Eco-F primer (10 pmol/μl)	2 μl
Xba-R primer (10 pmol/μl)	2 μl
Distilled water	26 μl
Short 2	1 μl
Short 3	1 μl
Short 5	1 μl
Pfu polymerase	1 μl
40 cycles (Ta=62℃)	50 μl

As a result, it was confirmed that Taq polymerases of "R536K", "R660V" and "R536K/R660V" were amplified as shown in FIG. 1(c).

1-3. Ligation

pUC19 was digested with the restriction enzyme EcoRI/XbaI at 37°C for 4 hours under the conditions of Table 9 below, and then the DNA was purified and the purified DNA was treated with SAP for 1 hour at 37°C under the conditions of Table 10 to prepare a vector. .

10xCutSmart buffer (NEB)	2.5 μl
pUC19 (500 ng/μl)	21.5 μl
EcoRI-HF (NEB)	0.5 μl
XbaI (NEB)	0.5 μl
	25 μl

10xSAP buffer (Roche)	2 μl
Purified DNA	17 μl
SAP (Roche)	1 μl
	20 μl

In the case of inserts, the overlap PCR product of Example 1-2 was purified, digested with restriction enzyme EcoRI/XbaI at 37°C for 3 hours under the conditions of Table 11, and then gel extracted with the prepared vector (FIG. 2 ). ).

10xCutSmart buffer (NEB)	2 μl
Purified PCR product	17 μl
EcoRI-HF (NEB)	0.5 μl
XbaI (NEB)	0.5 μl
	20 μl

After ligation at room temperature (RT) for 2 hours under the conditions of Table 12, E. coli DH5α was transformed to select from the medium containing ampicillin. The plasmid prepared from the obtained colonies was sequenced to obtain Taq DNA polymerase mutants ("R536K", "R660V" and "R536K/R660V") into which the desired mutation was introduced.

	Vector sole	Vector + insert
10x ligase buffer (solvent)	1 μl	1 μl
vector	1 μl	1 μl
insert	-	3 μl
Distilled water	7 μl	4 μl
T4 DNA ligase (solvent)	1 μl	1 μl
	10 μl	10 μl

[Example 2]

Introduction of E507K mutation

2-1. Fragment PCR

Taq polymerase activity of "R536K", "R660V" and "R536K/R660V" prepared in Example 1 was tested to confirm that the activity was poor (data not shown), R536K, R660V, R536K/R660V, respectively In addition, an E507K mutation (substituting the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with glutamic acid for lysine) was introduced, and an E507K mutation was also introduced into the wild-type Taq DNA polymerase (WT) for use as a control. The production method of Taq DNA polymerase introduced with the E507K mutation is the same as in Example 1.

Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutant specific primers listed in Table 13. The reaction conditions are shown in Table 14.

primer	Sequence (5'→3')	Sequence number
Eco-F	GG GGTACC TCA TCA CCC CGG	64
E507K-R	CTT GCC GGT CTT TTT CGT CTT GCC GAT	70
E507K-F	ATC GGC AAG ACG AAA AAG ACC GGC AAG	71
Xba-R	GC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA	69

10xpfu buffer (solvent)	2.5 μl
dNTP (10 mM each)	1 μl
F primer (10 pmol/μl)	1 μl
R primer (10 pmol/μl)	1 μl
Distilled water	18 μl
Template plasmid (10 ng/μl)	1 μl
Pfu polymerase	0.5 μl
30 cycles (Ta=60℃)	25 μl

* Template plasmid: pUC19-Taq (WT) pUC19-Taq (R536K) pUC19-Taq (R660V)

pUC19-Taq (R536K/R660V)

2-2. Overlap PCR

Each fragment amplified in 2-1 was used as a template, and the full length was amplified using primers (Eco-F and Xba-R primers) at both ends. The reaction conditions are shown in Table 15.

10xpfu buffer (solvent)	5 μl
5x Enhancer (Solent)	10 μl
dNTP (10 mM each)	1 μl
Eco-F primer (10 pmol/μl)	2 μl
Xba-R primer (10 pmol/μl)	2 μl
Distilled water	27 μl
Short 6	1 μl
Short 7	1 μl
Pfu polymerase	1 μl
40 cycles (Ta=62℃)	50 μl

2-3. Ligation

pUC19 was digested with the restriction enzyme EcoRI/XbaI at 37°C for 4 hours under the conditions of Table 9, and then the DNA was purified, and the purified DNA was treated with SAP for 1 hour at 37°C under the conditions of Table 10 to prepare a vector. .

In the case of inserts, the overlap PCR product of Example 2-2 was purified, digested with restriction enzyme EcoRI/XbaI at 37° C. for 3 hours under the conditions of Table 11, and then gel extracted with the prepared vector (FIG. 4 ). ).

After ligation at room temperature (RT) for 2 hours under the conditions of Table 12, E. coli was transformed into DH5α or DH10β, and was selected in a medium containing ampicillin. The plasmid prepared from the obtained colonies was sequenced to obtain Taq DNA polymerase mutants introduced with E507K mutations (“E507K/R536K”, “E507K/R660V” and “E507K/R536K/R660V”).

[Example 3]

Production of primer sets

Primer size and Tm value using the OligoAnalyzer Tool (https://sg.idtdna.com/calc/analyzer) program to design primers that generate PCR products for mutations in the EGFR gene but not for the wild-type EGFR gene After designing primers as shown in Tables 16 to 19 after excluding the possibility of formation of a secondary structure by checking whether the primer GC content, whether there is a self-complementary sequence in the primer, etc. Did.

Mutant group	primer	Sequence (5'-3')	Sequence number	Final concentration (nM)
S768I	S768I Rmt14+C	CGG GGT TGT CCA CGA	3	200
	S768I SF2	ACA CTG ACG TGC CTC TCC C	4	200
Ex19del	Ex19del C1 Rmt20	TGT TGG CTT TCG GAG ATG CC	5	50
	Ex19del C2 Rmt21	TTG GCT TTC GGA GAT GAT TCC	6	50
	Ex19del C3 Rmt20	GTT GGC TTT CGG AGA TTC CT	7	40
	Ex19del C4 Rmt24	GCT TTC GGA GAT GTT GCT TCC TTG	8	150
	Ex19del C5 Rmt21	CCT TGT TGG CTT TCT GTT CCT	9	40
	Ex19del C6 Rmt21	CCT TGT TGG CTT TCG GAT CCT	10	40
	Ex19del C7 Rmt21	TGG CTT TCG GAG ATG TTT TGA	11	50
	Ex19del C8 Rmt20	TGG CTT TCG GAG ATG TCT TG	12	50
	Ex19del C9 Rmt21	TCC TTG TTG GCT TTC GGT TCC	13	40
	Ex19del C10 Rmt2(20)	TGT TGG CTT TCG GAG CCT TG	14	50
	Ex19del C11 Rmt20	TTG TTG GCT TTC GGA GAT TC	15	50
	Ex19del C12 Rmt21	TTC CTT GTT GGC TTT CGA TTC	16	50
	Ex19del C13 Rmt21	GCT TTC GGA GAT GTT GGT TCC	17	100
	Ex19del C14 Rmt20	GTT GGC TTT CGG AGA TGG TT	18	100
	Ex19del C15 Rmt20	CTT GTT GGC TTT CGG AAC CT	19	50
	Ex19del C16 Rmt23	TTC CTT GTT GGC TTT CGG AAT TT	20	75
	Ex19del C17 Rmt20	GTT GGC TTT CGG AGA TAC CT	21	50
	Ex19del C18 Rmt3(21)	TTT CCT TGT TGG CTT TCG GTT	22	75
	Ex19del C19 Rmt21	TGT TGG CTT TCG GAG AAG CAA	23	75
	Ex19del C20 Rmt21	TGT TGG CTT TCG GAG ATT GCT	24	50
	Ex19del C21 Rmt21	TTG GCT TTC GGA GAT GTT GGC	25	75
	Ex19del C22 Rmt20	TGT TGG CTT TCG GAG ACT TG	26	50
	Ex19del C23 Rmt22	TGG CTT TCG GAG ATG TTG GAA T	27	100
	Ex19del C24 Rmt22	GTT GGC TTT CGG AGA TAT TTT G	28	50
	Ex19del C25 Rmt22	TGT TGG CTT TCG GAG ATG GAA T	29	100
	Ex19del C26 Rmt19+FL	AAT AAA TCA TAA AAA CTC ACA TCG AGG GTT G	30	100
	Ex19del C27 Rmt20	TCC TTG TTG GCT TTC GAG AC	31	50
	Ex19del C28 Rmt2(20)	TTG TTG GCT TTC GGA GAT TC	32	40
	Ex19del C29 Rmt21	GCT TTC GGA GAT GTT GCG ATA	33	50
	Ex19del C30 Rmt23	GTT GGC TTT CGG AGA TGT TAT AG	34	75
	Ex19del C31 Rmt20	GCT TTC GGA GAT GTT GTG AT	35	100
	19del SF2	TCC TTC TCT CTC TGT CAT AGG G	36	200
EGFR IC	EGFR IC Ex2F	CAG TTG GGC ACT TTT GAA GAT C	37	100
	EGFR IC Ex2R	TCC AAA TTC CCA AGG ACC AC	38	100

Mutant group	primer	Sequence (5'-3')	Sequence number	Final concentration (nM)
T790M	T790M Rmt(15)	AGG GCA TGA GCT GCA	39	240
	T790M SF5	ACC CCC ACG TGT GCC G	40	150
EGFR IC	EGFR IC Ex2F	CAG TTG GGC ACT TTT GAA GAT C	37	100
	EGFR IC Ex2R	TCC AAA TTC CCA AGG ACC AC	38	100

Mutant group	primer	Sequence (5'-3')	Sequence number	Final concentration (nM)
G719X	G719S Rmt17	CCG AAC GCA CCG GAG CT	41	200
	G719C Rmt16	CGA ACG CAC CGG AGC A	42	200
	G719A Rmt18(-3T)	TGC CGA ACG CAC CGT AGG	43	200
	G719X SF4	GCC TCT TAC ACC CAG TGG AGA A	44	200
L861Q	L861Q Rmt17	CTC TTC CGC ACC CAG CT	45	200
	L861Q SF1	CGT ACT GGT GAA AAC ACC G	46	200
EGFR IC	EGFR IC Ex2F	CAG TTG GGC ACT TTT GAA GAT C	37	100
	EGFR IC Ex2R	TCC AAA TTC CCA AGG ACC AC	38	100

Mutant group	primer	Sequence (5'-3')	Sequence number	Final concentration (nM)
Ex20Ins	20Ins C1 Rmt2(15)-4A	CCA CGC TGG CAA CGC	47	400
	20Ins C2 Rmt3(15)	GGC ACA CGT GGT GGG	48	200
	20Ins C3 Rmt2(16)	CAC ACG TGG GGG TTA C	49	300
	20Ins C4 Rmt2(16)-3T	TGT CCA CGC TGT TCA C	50	200
	20Ins C5 Rmt2(16)-3T	ATC CAC GCT GGC TAC G	51	100
	20Ins SF4	GAA GCC ACA CTG ACG TGC C	52	200
L858R	L858R Fmt21-2A	CAA GAT CAC AGA TTT TGG ACG	53	400
	L858R OR2	TGA CCT AAA GCC ACC TCC TTA CT	54	400
EGFR IC	EGFR IC Ex2F	CAG TTG GGC ACT TTT GAA GAT C	37	100
	EGFR IC Ex2R	TCC AAA TTC CCA AGG ACC AC	38	100

[Example 4]

Sample preparation

Mutant plasmid preparation

Using the gDNA of the HEK293T cell line, the wild-type DNA of EGFR, a primer set capable of amplifying the periphery of the target mutation in Table 4 was applied to prepare wild-type clones at exon 18, 19, 20, and 21 sites. Mutagenesis was performed on 44 target mutations using the prepared wild-type DNA, and transformed into E.Coli DH5α cells to obtain each mutant clone. Wild type clones and mutant clones were identified by direct sequencing. Wild-type DNA and mutant DNA for each exon extracted through clones were used as standards to evaluate the performance of the EGFR mutation detection kit.

As shown in Table 20, samples were prepared by adding 10,000 copies, 100 copies, 30 copies, 10 copies, or 3 copies of each mutant plasmid described in Table 4 per 30,000 copies of HEK293T cell genomic DNA. The group was used as a control.

group	sample
WT	HEK293T cell genomic DNA 30,000 copies
WT+mt 10 4	HEK293T cell genomic DNA 30,000 copies + corresponding mutant plasmid 10,000 copies
WT+mt 10 2	HEK293T cell genomic DNA 30,000 copies + corresponding mutant plasmid 100 copies
WT+mt 30	HEK293T cell genomic DNA 30,000 copies + corresponding mutant plasmid 30 copies
WT+mt 10	HEK293T cell genomic DNA 30,000 copies + corresponding mutant plasmid 10 copies
WT+mt 3	HEK293T cell genomic DNA 30,000 copies + corresponding mutant plasmid 3 copies

[Example 5]

EGFR mutation detection

Using the Taq polymerase (SEQ ID NO: 2) and the primer set of Example 3, each containing the "E507K/R536K/R660V" mutation obtained in Example 2 above, the EGFR gene mutations of Table 4 from each group of Table 20 were obtained. Detected. Table 21 shows mutant information targeted by EGFR Master Mixture 1-4, respectively.

MMX	Neon	Mutant group	EGFR nucleic acid sequence (COSMIC ID)
MMX1	FAM	S768I	2303G>T (6241)
	CFO560	Ex19Del	2240_2251del12 (6210), 2239_2247del9 (6218), 2238_2255del18 (6220), 2235_2249del15 (6223), 2236_2250del15 (6225), 2239_2253del15 (6254), 2239_2256del18 (6255), 2237_2254del18 (12367), 2240_2254del15 (370) 2239_2248TTAAGAGAAG>C (12382), 2239_2251>C (12383), 2237_2255>T (12384), 2235_2255>AAT (12385), 2237_2252>T (12386), 2239_2258>CA (12387), 2239_2256>CAA (12403), 2237_2253 >TTGCT (12416), 2238_2252>GCA (12419), 2238_2248>GC (12422), 2237_2251del15 (12678), 2236_2253del18 (12728), 2235_2248>AATTC (13550), 2235_2252>AAT (13551), 2235_2251>AATTC (13552) , 2253_2276del24 (13556), 2237_2257>TCT (18427), 2238_2252del15 (23571), 2233_2247del15 (26038), 2232_2249del15 (221565), 2234_2248del15 (1190791)
MMX2	FAM	T790M	2369C>T (6240)
MMX3	FAM	L861Q	2582T>A (6213)
	CFO560	G719X	2155G>A (6252), 2155G>T (6253), 2156G>C (6239)
MMX4	FAM	Ex20Ins	2307_2308ins9GCCAGCGTG (12376), 2319_2320insCAC (12377), 2310_2311insGGT (12378), 2311_2312ins9GCGTGGACA (13428), 2309_2310AC>CCAGCGTGGAT (13558)
	CFO560	L858R	2573T>G (6224), 2573_2574TG>GT (12429)

Specific qPCR (Applied Biosystems 7500 Fast) experimental conditions are shown in Tables 22 to 24, and the probes were double-labeled as in Table 23.

2X EGFR MMX	10 μl
sample	2 μl
Nuclease-free distilled water	7.5 μl
ADPS Smart DNA polymerase (1 U/ul)	0.5 μl
gun	20 μl

Mutant group	Probe	Sequence (5'-3')	Sequence number	5'fluorophore	3'matting material	Final concentration (nM)
S768I	S768I FAM_R	TCA CGT AGG CTT CCT GGA GGG A	55	FAM	BHQ1	400
Ex19del	19del CFO_R2	CTC TGG ATC CCA GAA GGT GAG AAA G	56	CAL Fluor Orange 560	BHQ1	400
T790M	T790M IR4	GTG ATG AGC TGC ACG G	57	FAM	BHQ1	125
	T790M IR2(-8T)	GTG ATG AGT TGC ACG GTG G	58	FAM	BHQ1	130
G719X	G719X CFO_R2_nova	AAA CTG AAT TCA AAA AGA TCA AAG TGC TG	59	CAL Fluor Orange 560	BHQ1 nova	400
L861Q	L858R FAM_R	AAA CTG AAT TCA AAA AGA TCA AAG TGC TG	60	FAM		BHQ1	50
Ex20Ins	Ex20Ins FAM_R	TCA CGT AGG CTT CCT GGA GGG A	61	FAM	BHQ1	400
L858R	L858R CFO560_F	CCA AAC TGC TGG GTG CGG AAG AG	62	CAL Fluor Orange 560	BHQ1	400
EGFR IC	EGFR IC Ex2 QS670	TCT CAG CCT CCA GAG GAT GTT CAA	63	Quasar 670	BHQ2	200

step	cycle	Temperature	time	Data collection
One	One	95℃	5 minutes	-
		95℃	10 seconds	-
2	50	60℃	30 seconds	FAM /
				CAL Fluor Orange 560 (VIC/HEX/JOE) /
				Quasar670 (CY5)
		72℃	10 seconds	-

Sufficient EGFR reaction mixtures containing each of the components listed in Table 22 were each prepared in separate sterile centrifuge tubes, and the reaction Master Mixture was vortexed for 3 seconds to thoroughly mix and centrifuge briefly. Two PCR tubes were prepared for each sample as follows: 10.0 μL of EGFR reaction mixture was divided for each PCR tube, and 5.0 μL of each sample DNA was added to each sample tube, and then the PCR tube was covered. Nuclease-free distilled water was added to all PCR tubes to 20.0 μL, and PCR strips were briefly centrifuged to collect all liquids at the bottom of each PCR tube. The PCR strip tube was placed in a real-time PCR (real-time PCR) instrument, and PCR was performed after setting the PCR protocol using the cycling parameters in Table 24. After the PCR was completed, FAM/CAL Fluor Orange 560/Quasar 670 Ct values of each sample were recorded to analyze the data, and ΔCt values per well were calculated as follows:

ΔCt value = sample Ct value (FAM of each MMX)-positive control Ct value (FAM of each MMX)

ΔCt value = sample Ct value (each MMX CFO560)-positive control Ct value (CFO560 of each MMX)

The results for each tube were analyzed according to the cutoff ΔCt values in Tables 25 and 26. If the ΔCt value is <cutoff ΔCt value, the sample is judged to be positive, and if there is no fluorescence signal and the Ct value of the internal control is 21.5 <Ct <40, the sample is judged to be negative or below the detection limit (LoD).

Result determination

EGFR MMXs		decision
Internal control Ct value	21.5 ≤ Ct ≤ 40	available
	Ct <21.5 or Ct> 40	invalidity
Sample Ct value	Ct <23.5	invalidity
	Ct> 45 or no signal	voice

Cutoff ΔCt value for each target

Determining valid results		Cutoff ΔCt value	range	decision
ΔCt cutoff value	S768I-FAM	10.0	ΔCt≤10.0	positivity
			ΔCt> 10.0	voice
	Ex19Del-CFO560	14.5	ΔCt≤14.5	positivity
			ΔCt> 14.5	voice
	T790M-FAM	14.5	ΔCt≤14.5	positivity
			ΔCt> 14.5	voice
	L861Q-FAM	13.0	ΔCt≤13.0	positivity
			ΔCt> 13.0	voice
	G719X-CFO560	8.5	ΔCt≤8.5	positivity
			ΔCt≤8.5	voice
	Ex20Ins-FAM	16.0	ΔCt≤16.0	positivity
			ΔCt> 16.0	voice
	L858R-CFO560	10.0	ΔCt≤10.0	positivity
			ΔCt> 10.0	voice

As a result of analyzing the data as described above, as shown in FIGS. 5A to 5L, 6A to 6L, 7A to 7I, 8, 9A to 9E, and 10A to 10H, fluorescence signals were not detected in WT, but the EGFR gene mutation of Table 4 Fluorescence signal was detected in the sample containing, and it was confirmed that EGFR gene mutation can be detected with a high sensitivity of up to 0.01% (3 mutant copies in 30,000 wild-type copies) as shown in Table 27.

Exxon	Mutation	EGFR nucleic acid sequence	COSMIC ID	LoD (copy)	Detection sensitivity (%)
Exon 18	G719X	2155G>A	6252	15	0.05
		2155G>T	6253	10	0.03
		2156G>C	6239	3	0.01
Exon 19	Ex19Del	2240_2251del12	6210	3	0.01
		2239_2247del9	6218	3	0.01
		2238_2255del18	6220	3	0.01
		2235_2249del15	6223	3	0.01
		2236_2250del15	6225	3	0.01
		2239_2253del15	6254	3	0.01
		2239_2256del18	6255	3	0.01
		2237_2254del18	12367	3	0.01
		2240_2254del15	12369	3	0.01
		2240_2257del18	12370	3	0.01
		2239_2248TTAAGAGAAG>C	12382	3	0.01
		2239_2251>C	12383	3	0.01
		2237_2255>T	12384	3	0.01
		2235_2255>AAT	12385	3	0.01
		2237_2252>T	12386	3	0.01
		2239_2258>CA	12387	3	0.01
		2239_2256>CAA	12403	3	0.01
		2237_2253>TTGCT	12416	3	0.01
		2238_2252>GCA	12419	3	0.01
		2238_2248>GC	12422	3	0.01
		2237_2251del15	12678	3	0.01
		2236_2253del18	12728	3	0.01
		2235_2248>AATTC	13550	3	0.01
		2235_2252>AAT	13551	3	0.01
		2235_2251>AATTC	13552	3	0.01
		2253_2276del24	13556	3	0.01
		2237_2257>TCT	18427	3	0.01
		2238_2252del15	23571	3	0.01
		2233_2247del15	26038	3	0.01
		2232_2249del15	221565	3	0.01
		2234_2248del15	1190791	3	0.01
Exon 20	S768I	2303G>T	6241	3	0.01
	T790M	2369C>T	6240	10	0.03
	Ex20Ins	2307_2308ins9GCCAGCGTG	12376	3	0.01
		2319_2320insCAC	12377	3	0.01
		2310_2311insGGT	12378	3	0.01
		2311_2312ins9GCGTGGACA	13428	3	0.01
		2309_2310AC>CCAGCGTGGAT	13558	3	0.01
Exon 21	L858R	2573T>G	6224	3	0.01
		2573_2574TG>GT	12429	3	0.01
	L861Q	2582T>A	6213	3	0.01

The kit of the present invention shows high detection sensitivity (up to 0.01%, 3 mutant copies in 30,000 wild-type copies), high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy. In addition, by simultaneously detecting 44 mutations within exons 18 to 21 of the EGFR gene, it is possible to accurately predict drug reactivity to lung cancer patients against tyrosine kinase inhibitors.

Claims (19)

1. In the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue glutamic acid (E) is replaced with lysine (K), the 536th amino acid residue arginine (R) is replaced with lysine (K), and the 660th amino acid residue arginine ( R) DNA polymerase for detecting EGFR gene mutations, including Taq polymerase substituted with valine (V).
2. A primer set for EGFR gene mutation detection, comprising at least one primer selected from the group consisting of SEQ ID NOs: 3 to 54.
3. A probe for detecting an EGFR gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.
4. The method of claim 3, wherein the nucleotide sequence of SEQ ID NO: 55 to 63, each of the 5'- terminal FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 is selected from the group consisting of one fluorescent material (fluorophore) is labeled, a probe for detecting the EGFR gene mutation, which is labeled with one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 at the 3'-end.
5. In the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue glutamic acid (E) is replaced with lysine (K), the 536th amino acid residue arginine (R) is replaced with lysine (K), and the 660th amino acid residue arginine ( R) Taq polymerase substituted with valine (V); And/or

   At least one primer selected from the group consisting of SEQ ID NOs: 3 to 54;

   Containing, EGFR gene mutation detection kit.
6. The kit for detecting an EGFR gene mutation according to claim 5, further comprising a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.
7. The method of claim 6, wherein the nucleotide sequence of SEQ ID NO: 55 to 63 is a fluorescent substance of one selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-terminal, respectively. A kit for detecting EGFR gene mutation, wherein (fluorophore) is labeled, and one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 is labeled at the 3'-end.
8. The method of claim 5,

   25 to 100 mM KCl; And

   1 to 7 mM (NH ₄ ) ₂ SO ₄ ;

   Included, the final pH is 8.0 to 9.5, further comprising a PCR buffer composition, EGFR gene mutation detection kit.
9. The method of claim 5,

   25 to 100 mM KCl;

   1 to 7 mM (NH ₄ ) ₂ SO ₄ ; And

   A kit for detecting EGFR gene mutations, comprising 5-50 mM of TMAC (Tetra methyl ammonium chloride), and further comprising a PCR buffer composition having a final pH of 8.0 to 9.5.
10. EGFR gene mutation detection method comprising the following steps:

    (A) extracting the nucleic acid from the isolated biological sample;

    (b) performing a polymerase chain reaction (PCR) by treating the kit of any one of claims 5 to 9 with the extracted nucleic acid; And

    (c) confirming the amplification result by the PCR with fluorescence.
11. The method according to claim 10, wherein the PCR is allele-specific PCR or real-time PCR.
12. 11. The method of claim 10, (d) EGFR gene mutation detection method further comprising the step of confirming the amplification result by the PCR by measuring the Ct (cycle threshold) value.
13. The method of claim 10, wherein the EGFR gene mutation is EGFR gene mutation is at least one selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. Including, EGFR gene mutation detection method.
14. 14. The method of claim 13, wherein the EGFR gene mutation is a substitution of the 719th amino acid glycine in exon 18 of EGFR, a deletion in exon 19, the 768th amino acid serine in exon 20, the 790th amino acid. An threonine, a substitution of cysteine at the 797th amino acid, insertion in exon 20, leucine at 858th amino acid in exon 21, and leucine at 861th amino acid; EGFR gene mutation detection method.
15. The method of claim 10, wherein the EGFR gene mutation detection method is for predicting responsiveness to drugs of lung cancer patients.
16. 16. The method of claim 15, wherein the lung cancer is non-small cell lung cancer.
17. 16. The method of claim 15, wherein the drug is a tyrosine kinase inhibitor.
18. 18. The method of claim 17, wherein the tyrosine kinase inhibitor is gefitinib, erlotinib, or osimmertinib.
19. The method of claim 10, wherein the nucleic acid of step (a) is extracted from a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy.

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