KR101133242B1 - A method for detection of genetic variation using nanoparticle aggregation - Google Patents

Abstract

The present invention relates to a method for detecting a mutation of an epidermal growth factor receptor (EGFR) gene, which is important for predicting the reactivity of a drug in a lung cancer patient, and more particularly, extracting nucleic acid from a cell or tissue of a lung cancer patient. Treatment of oligonucleotide probes complementary to an EGFR (epidermal growth factor receptor) mutant gene to a nucleic acid to cause a hybridization reaction, to treat metal nanoparticles to the hybridization reactant, to a concentration suitable for the metal nanoparticles to cause aggregation It relates to a method of detecting a mutation of the EGFR gene comprising the step of treating the salt solution, and confirming the aggregation of the metal nanoparticles.

Lung cancer, drug sensitivity, nanoparticles, aggregation, EGFR

Description

Genetic mutation detection method using aggregation of nanoparticles {A method for detection of genetic variation using nanoparticle aggregation}

The present invention relates to a method for detecting a mutation of an epidermal growth factor receptor (EGFR) gene, which is important for predicting the reactivity of a drug in a lung cancer patient, and more particularly, extracting a nucleic acid from a cell or tissue of a lung cancer patient. Treatment of oligonucleotide probes complementary to an EGFR (epidermal growth factor receptor) mutant gene to a nucleic acid to cause a hybridization reaction, to treat metal nanoparticles to the hybridization reactant, to a concentration suitable for the metal nanoparticles to cause aggregation It relates to a method of detecting a mutation of the EGFR gene comprising the step of treating the salt solution, and confirming the aggregation of the metal nanoparticles.

Lung cancer is known to be a leading cause of cancer deaths worldwide. In the United States alone, 174,000 people were diagnosed with lung cancer in 2006 alone, of which 162,000 were estimated to have died of lung cancer. In Korea, the mortality rate from lung cancer is one of the most serious diseases among all cancers. Lung cancer is classified into two types of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The non-small cell cancer is again classified into adenocarcinoma, squamous cell carcinoma, large cell carcinoma and adenosquamous carcinoma. It is classified into tissue types such as epithelial cancer, and various clinical forms such as prone site, progression form, speed, and symptoms are varied according to these different types of tissue types and treatment methods are also varied (Brambilla et al., Eur Respir J. 18 (6): 1059-68, 2001).

Most lung cancers cannot be treated by chemotherapy and radiotherapy. Chemotherapy and radiotherapy can be applied by reducing the size of small cell cancer, but it is impossible to expect complete treatment. Non-small cell lung cancer is less effective than chemotherapy because it is less effective than chemotherapy. Surgical removal is the only effective treatment. However, less than 30% of lung cancer patients have tumors that cannot be completely excised at diagnosis, and less than one third of them survive only five years after surgical resection. Therefore, there is a great need for a method for determining the treatment policy of patients by early diagnosis of lung cancer or by predicting the prognosis of drug prescription.

On the other hand, EGFR (epidermal growth factor receptor) is a cell membrane glycoprotein belonging to tyrosine kinase, which is an epithelial receptor in humans, along with erb-B2 (HER2 / neu), erb-B3, and erbB4, and has its own tyrosine kinase activity. When activated, it causes effects such as gene expression, cell proliferation, invasion of surrounding tissues, inhibition of apoptosis and angiogenesis. Overexpression of EGFR has been observed in many epithelial carcinomas, including lung carcinoma. Especially, since EGFR is also observed in the cancer progenitor bronchial epithelium, this is considered to play an important role in the development of lung carcinoma. The EGFR gene encodes a tyrosine kinase receptor to target tyrosine kinase inhibitors (TKIs). Accordingly, gefitinib (Iressa) and erlotinib (Tarceva) have been developed as tyrosine kinase inhibitors useful for the treatment of non-small cell lung carcinoma, which are particularly mutated at specific sites of the tyrosine kinase domain of EGFR. In this case, the effect is reported to be good.

EGFR mutations are mainly observed in women, Asians, nonsmokers, and patients with adenocarcinoma lung cancer, and the presence of these mutations is associated with prognosis and drug responsiveness. Most of the mutations in EGFR are observed in exon 18 to exon 21. Among them, in-frame deletions in exon 19 and L858R point mutations in exon 21 are the most common mutations. Non-small cell lung cancer patients with EGFR mutations were significantly more responsive to TKI agents than non-small cell lung cancer patients without EGFR mutations, and a significant increase in survival was reported. Thus, the presence of EGFR mutations acts as a potent predictor of drug sensitivity in response to TKIs such as gefitinib or erlotinib, so an effective and rapid detection method is required for optimal therapeutic approaches. .

In general, direct sequencing of exon 18 to exon 21 is widely used to detect EGFR mutations in cancer specimens, but it requires a lot of time and cost to amplify. Recently, a mutation detection method using denaturing high-performance liquid chromatography or fluorescence-based detection has been proposed as a method of improving detection sensitivity.

Therefore, the present inventors have made diligent efforts to develop a method that can detect EGFR mutations more effectively and quickly in order to assay drug susceptibility of lung cancer. As a result, the selective aggregation of metal nanoparticles can be used to detect lung cancer cell lines and non-small cell lung cancer. Mutations in exon 19 and exon 21 of the EGFR gene could be detected in genomic DNA isolated from patients' lung cancer tissues. In other words, for mutant DNAs that hybridized with complementary probes at the appropriate salt concentration, the addition of an unmodified gold nanoparticle suspension results in sufficient aggregation of gold nanoparticles and color change in the solution. In the case of wild type DNA that hybridized with, the addition of unmodified suspension of gold nanoparticles showed no significant aggregation and no change in solution color. Accordingly, the present inventors have completed the present invention by clarifying that EGFR mutations can be detected at low cost and high efficiency without using amplification step of DNA by PCR using selective aggregation of gold nanoparticles.

It is an object of the present invention to provide a method of utilizing aggregation of metal nanoparticles to detect mutations in epidermal growth factor receptor (EGFR) genes, which are important for predicting the reactivity of drugs in lung cancer patients.

In one embodiment, the present invention relates to a method for detecting EGFR mutations using aggregation of metal nanoparticles to assay drug susceptibility of lung cancer patients.

More specifically, the present invention

1) extracting nucleic acids from cells or tissues of lung cancer patients,

2) treating the extracted nucleic acid with an oligonucleotide probe complementary to an EGFR (epidermal growth factor receptor) mutant gene to cause a hybridization reaction,

3) treating metal nanoparticles to the hybridization reactant,

4) treating the salt solution at an appropriate concentration to cause the metal nanoparticles to aggregate, and

 5) relates to a method for detecting mutations in the EGFR gene, including the step of checking whether the metal nanoparticles are aggregated.

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

Step 1) is extracting nucleic acids from cells or tissues of lung cancer patients.

Lung cancer is preferably non-small cell lung cancer. The nucleic acid is preferably DNA or RNA, and synthetic DNA or RNA, natural DNA or RNA, or structurally modified DNA or RNA are all included in the scope of the present invention. In addition, the method of the present invention can be applied to cDNA amplified by polymerase chain reaction (PCR), but the method of the present invention is characterized in that the gene mutation can be detected accurately and quickly without the amplification step of the nucleic acid by PCR. do.

Step 2) is a step of generating a hybridization reaction by processing an oligonucleotide probe complementary to an EGFR mutant gene to nucleic acid extracted from cells or tissues of a lung cancer patient.

Mutations of the EGFR gene include mutations, such as deletion, addition, overlapping or inversion, or mutations of genes such as substitution and point mutations, preferably mutations present in exons 18 to exon 21, and more preferably exon 19 In-frame deletions or L858R point mutations in exon 21.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several hundred bases, which can achieve specific binding to a nucleic acid. In the present invention, the probe is preferably designed such that the mutant double-stranded DNA (dsDNA) has a perfectly matched sequence while the wild-type dsDNA has a mismatched sequence.

In a specific embodiment of the present invention, 5-TCGCTATCAARACATCTCCG-3 '(R means A or G), and 5'-ATTTTGGGCGGGCCAAACTG-3', respectively, are used as the exon 19 and exon 21 oligonucleotide probe sequences. It is not. Since the sequences of exon 19 and exon 21 of EGFR are already known, those skilled in the art can design probes that specifically bind to specific regions of these genes based on the sequence information. In addition, the selection and hybridization conditions of suitable probes can be modified based on what is known in the art.

Step 3) is a step of treating the metal nanoparticles to the hybridization reactant.

The metal nanoparticles may be gold nanoparticles, silver nanoparticles, platinum nanoparticles, or a combination thereof, and may have a structure of mixed metal nanoparticles such as a gold core and a silver shell coating the silver core, or a silver core and a gold shell coating the same. It is possible to have Preferably gold nanoparticles, most preferably citrate-reduced gold nanoparticles. In addition, the size of the metal nanoparticles is not limited as long as it is in the range of nano, but may preferably have a diameter of 5nm to 500nm, more preferably 10nm to 30nm.

For colorimetric assays, the metal nanoparticles are preferably provided in the form of a colloidal suspension.

Step 4) is a step of treating the salt solution of the concentration suitable for the metal nanoparticles to agglomerate to the hybridization reactant.

Salt solution, it is preferable to include Na ⁺ between 0.01M to 1M, preferably to more preferably the Na ⁺ between 0.01M to 0.3M. In specific embodiments of the present invention, optimal NaCl concentrations were determined to be 0.0435M and 0.0460M in exon 19 and exon 21 mutant DNA, respectively. However, since this is only one aspect, the person skilled in the art can determine the optimal salt solution and its concentration according to specific experimental conditions or the type of mutation to be detected. The step 3) of treating the metal nanoparticles to the hybridization reactant and the step 4) of treating the salt solution may be performed sequentially or simultaneously.

Step 5) checks whether the metal nanoparticles are agglomerated.

The agglomeration of the metal nanoparticles can be visually confirmed by the agglomeration of the metal nanoparticles through the color change of the solution. In this case, it is desirable to observe the solution color of the aggregated metal nanoparticles (negative control) and / or the solution color of the non-aggregated metal nanoparticles (positive control) to obtain more certain results.

For example, FIG. 2 shows the color change of the gold nanoparticle suspension after 3 hours after the addition of the appropriate concentration of salt in H146, H1975, and H2279 cell lines, in the case of dsDNA (wild type) of H146 cell line inconsistent with probe There was no significant color change compared to the color change of the first citric acid-reduced gold sol, but the solution was perfectly matched with the probe for the H1975 (point mutation of exon 21) and H2279 cell lines (defective mutation of exon 19). It can be seen that the color clearly changes from red to purple, which is the result of the aggregation of gold and nanoparticles. In addition, Figure 4 shows the color change of the gold nanoparticle suspension 3 hours after the addition of the appropriate concentration of salt in eight cancer tissues (E1 to E8) of eight patients with non-small cell lung cancer, a sample hybridized with the exon 19 probe Only E6-E8 hybridized with E3 and exon 21 probes changed color from red to purple, indicating that these samples had mutations.

In addition, the agglomeration of metal nanoparticles may be performed using UV-vis absorption spectroscopy, transmission eletron microscopy (TEM), or quasi-elastic light-scattering (QELS). You can check it. In particular, TEM can be used to monitor the aggregated form of metal nanoparticles, and QELS can be used to monitor the hydrodynamic radius of aggregated metal nanoparticles.

Through specific examples, the inventors have succeeded in detecting mutations of EGFR exon 19 and exon 21 both in lung cancer cell lines as well as in DNA samples actually isolated from cancer tissues of non-small cell lung cancer patients. (FIGS. 2-4). Specifically, the inventors found that in dsDNA (wild type) where gold nanoparticles are not adsorbed, they remain stable like colloids, while selective aggregation occurs due to the addition of dsDNA of perfectly matched sequences (mutants), Mutant DNA could be detected from the color change of the gold nanoparticle suspension. UV-vis spectrophotometry, QELS, and TEM confirmed that selective aggregation of gold nanoparticles can successfully detect mutations in exon 19 and exon 21 of EGFR.

Since the detection method of the present invention utilizes the selective aggregation of gold nanoparticles, EGFR mutations can be detected quickly and accurately at low cost and high efficiency without the amplification step of DNA by PCR. The principle of the detection method of the present invention is schematically shown in FIG.

More specifically, the selective aggregation of metal nanoparticles to dsDNA (mutants) that perfectly match the probe at the optimal salt concentration can be explained as follows.

According to the research of the present inventors, metal nanoparticles are stable like colloids at low salt concentrations, but at high salt concentrations, aggregation occurs due to the blocking of repulsive force between electrical double layers. In addition, single-stranded DNA (ssDNA) readily adsorbs on the surface of the metal nanoparticles to stabilize the metal nanoparticles, preventing the aggregation of the metal nanoparticles under appropriate salt concentrations, whereas the double-stranded DNA (dsDNA) of perfectly matched sequence It has a double helical geometry with a stable phosphate base structure with negative charge, so it is not easily adsorbed on the surface of nanoparticles. However, if the dsDNA has a single base mismatch in the sequence, significant dehybridization occurs and is stabilized by dehybridized ssDNA. Thus, when the appropriate salt is added to block the electrostatic repulsion between the metal nanoparticles, the metal nanoparticle suspension of perfectly matched dsDNA aggregates more easily than the mismatched dsDNA.

The detection of EGFR mutations from genomic DNA of lung cancer patients of the present invention provides useful information for predicting the prognosis of lung cancer treatment for drug prescriptions. In particular, the method of the present invention is useful for predicting the prognosis of tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib.

According to the detection method of the present invention, selective aggregation of gold nanoparticles can be used to detect EGFR mutations quickly and accurately at low cost and high efficiency without the amplification step of DNA by PCR. In addition, by predicting the prognosis of lung cancer treatment for the drug prescription may provide useful information to determine the treatment policy of the patient.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Cell Line and Tissue Samples

NSCLC cell lines NCI-H146, H1975, and H2279 were purchased from the American Type Culture Collection, Manassas, VA, USA, and cultured according to standard procedures. H1975 has an L858R point mutation in exon 21 and H2279 has an exon 19 E746-A750 deletion mutation.

Eight tumor biopsy samples were obtained from histologically confirmed NSCLC patients. Patients donated tissue samples for genetic testing after signing informed consent for surgery approved by the Institutional Review Board (IRB). Genomic DNA samples were extracted from paraffin-embedded, formalin-fixed pieces using the DNAeasy Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

Example 2. Preparation of Gold Nanoparticles and Oligonucleotide Probes

Citrate-reduced gold nanoparticles were synthesized according to the methods of Lee and Meisel (J. Phys. Chem. 86, 3391-3395, 1982). HAuCl ₄ 3H ₂ O (50 mg) purchased from Aldrich was dissolved in double diluted neutralized water (500 mL) and the solution was heated. A 1 wt% trisodium citrate (15 mL) solution was added to the HAuCl ₄ 3H ₂ O solution, stirred vigorously, and the solution was heated for another 30 minutes. The inventors confirmed that the prepared gold nanoparticles were about 18 nm in diameter through quasi-elastic light scattering (QELS). Meanwhile, purified oligonucleotide probes completely complementary to exon 19 deleted mutant DNA and exon 21 point mutant DNA were purchased from Bioneer Inc. (Daejeon, Seoul). Exon 19 and exon 21 oligonucleotide probe sequences are 5-TCGCTATCAARACATCTCCG-3 ′ (R means A or G), and 5′-ATTTTGGGCGGGCCAAACTG-3 ′, respectively.

Example 3 Detection and Direct Sequencing of EGFR Mutations Using Aggregation of Gold Nanoparticles

Deletion mutations in exon 19 and L858R point mutations in exon 21 were selected using gold nanoparticle aggregation. First, the target DNA solution (40ng, 4μL) was denatured for 5 minutes at 98 ℃ in a water bath (water bath). Oligonucleotide probe (10 pmole, 1 μL) was added to the denatured target DNA solution (4 μL) and the mixture was allowed to hybridize for 20 minutes at 51 ° C. (exon 19) or 53.6 ° C. (exon 21) in a constant temperature water bath. Hybridization temperature was determined according to the melting temperature of the purified oligonucleotide probe completely complementary to exon 19 deleted mutant DNA and exon 21 point mutant DNA. Hybridized mixture (5 μL) was added to the gold nanoparticle suspension (0.3 mL). After 10 minutes, distilled water (0.1 mL) and pH 7.2 phosphate buffer (0.05 mL) were added to prevent sudden aggregation. Finally, the inventors added different amounts of NaCl solution to determine the optimal salt concentration to observe the selective aggregation of nanoparticles and the change in the color of the solution that occur extensively in perfectly matched dsDNA (mutant DNA). . Optimal NaCl concentrations were determined to be 0.0435M and 0.0460M in exon 19 and exon 21 mutant DNA, respectively.

In order to separately identify sequences of exon 19 and exon 21 mutant DNA detected by aggregation of gold nanoparticles, the inventors performed direct sequencing. DNA samples extracted from tissue samples and cell lines were amplified by PCR using specific primer pairs (Exon 19: 5-GCAATATCAGCCTTAGGTGCGGCT, R: 5'-CATAGAAAGTGAACATTTAGGATGTG; Exon 21, F: 5'-GCTCAGAGCCTGGCATGAA, R: 5'- CATCCTCCCCTGCATGTGT). Amplification was repeated for 10 minutes at 95 ° C., followed by 35 cycles of 30 seconds at 95 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C. PCR products were purified using QIAgen PCR purification kit (Qiagen, Valencia, CA, USA) and directly sequenced using BigDye terminator version 3.1 (Applied Biosystems, Foster City, CA, USA). The sequencing reaction was denatured at 95 ° C. for 4 minutes, followed by 25 cycles of 10 seconds at 95 ° C., 15 seconds at 50 ° C., and 4 minutes at 60 ° C. The precipitate was sequenced by ABI PRISM 3100 DNA Analyzer (Applied Biosystems).

Example  4. Characterization Characterization )

Monitor aggregation of gold nanoparticles using UV-vis absorption spectroscopy, transmission eletron microscopy (TEM), and quasi-elastic light-scattering (QELS) It was. UV-vis absorption spectra of gold nanoparticle suspensions were measured using a Shimadzu 1650 PC spectrophotometer. The acquisition time of the UV-vis spectrum was about 3-4 minutes. In order to monitor the hydrodynamic radius and morphology of the nanoparticles, Malvern Nano-ZS (QELS) and TEM (JEOL JEM-2100) were used, respectively.

Experiment result

First, we determined the sequence of all DNA samples by direct sequencing (Table 1). Although this method is expensive and time consuming, the results are intended to be used separately to determine detection results based on gold nanoparticle aggregation. As shown in FIG. 1, 15bp deletion (GAATTAAGAGAAGCA) was observed in exon 19 of the H2279 cell line, and leucine (CTG) of exon 21 was replaced with arginine (CGG) in the H1975 cell line. NCI-H146 cell line shows wild-type EGFR sequences in exon 19 and exon 21 (FIG. 1).

Sample ^b Exon 19 Exon 21 PCR-
Sequencing ^a diameter
(nm) PCR-
Sequencing ^a diameter
(nm) H146 W 48.5 W 50.3 H1975 W 45.4 M 328 H2279 M 266 W 43.3 E1 W 29.9 W 44.2 E2 W 27.7 W 71.2 E3 M 164 W 66.6 E4 W 39.3 W 57.6 E5 W 40.5 W 62.7 E6 W 31.3 M 408 E7 W 35.3 M 342 E8 W 31.9 M 287

^a W represents the wild type and M represents the mutant type.

^b DNA samples of H146, H1975, H2297 were extracted from lung cancer cell lines, and E1 to E8 were isolated from lung cancer tissues of 8 NSCLC patients.

To determine the conditions under which gold nanoparticle aggregation can be used to distinguish wild type and mutant DNA, we performed aggregation experiments using unamplified genomic DNA isolated from lung cancer cell lines. 2A shows a typical photograph of a gold nanoparticle suspension when 3 hours after addition of salt at the proper concentration. The probes have a perfectly matched sequence in which the double stranded DNA (dsDNA) of the mutant (ie, H2297 and H1975 cell lines hybridized with exon 19 and exon 21 probes, respectively) has a perfectly matched sequence, whereas the wild type (ie exon 19 or The dsDNA of H146 cell line hybridized with exon 21 probe was designed to have a mismatched sequence. As shown in FIG. 2A, there was no color change for the mismatched dsDNA (wild type) compared to the color change of the initial citric acid-reduced gold sol. However, following the addition of a perfectly-matched dsDNA (mutant), the solution showed a definite color change from red to purple as shown in FIG. 2A, which is a result of the aggregation of gold nanoparticles.

3 compares the UV-vis absorption spectra of gold nanoparticles after addition of salts. Agglomeration of gold nanoparticles is known to be caused by red shift in the surface plasmon band as the distance between particles decreases during the aggregation process. The degree of aggregation can also be measured from integrated extinction between 600 and 800 nm. Figure 3 shows the broadening and redness of the characteristic surface plasmon band at ˜520 nm and shows increased extinction for mutant DNA samples (H2279 and H1975 cell lines) at 600-800 nm. This can be seen as a result of significant aggregation.

In order to confirm the aggregation of the gold nanoparticles, the hydrodynamic radius of the nanoparticles of the gold nanoparticle aggregates after salt addition was measured using QELS. As shown in Table 1 (H146, H2279 and H1975 cell lines), the particle size for the sample of perfectly matched dsDNA (> 260 nm) was larger than that of the mismatch (<50 nm). Clearly, aggregation occurred extensively in mutant DNA samples.

TEM images of the gold nanoparticle suspensions were measured after salt addition to compare the morphology of the gold nanoparticle aggregates (Table 2b). Some aggregates were found in inconsistent dsDNA (wild type) samples, but many gold nanoparticle aggregates were observed in perfectly matched dsDNA (mutants). Thus, it can be seen that selective aggregation of the gold nanoparticles occurs in the perfectly matched sequence, and that the EGFR mutation can be detected quickly by the color change of the gold nanoparticle suspension.

Next, the present inventors applied a method of detecting mutations in cancer tissues of eight patients with non-small cell lung cancer. 4A shows photographs of eight samples at 3 hours after salt addition at the appropriate concentrations. As shown in FIG. 4A, only sample E3 hybridized with exon 19 probe and E6-E8 hybridized with exon 21 probe changed color from red to purple, indicating that these samples had mutations. This is consistent with the results of the direct sequencing method shown in Table 1, it can be confirmed that the method based on gold nanoparticle aggregation has an effectiveness. Therefore, it is clear that mutant DNA and wild type DNA can be easily distinguished by the naked eye from the color change of the gold nanoparticle suspension. As in the experiment with cell line DNA, the QELS results show that the mean particle size increased only in mutant DNA samples due to selective aggregation. In addition, TEM images of the gold nanoparticles shown in FIG. 4B clearly show that significant aggregation of gold nanoparticles occurs extensively in samples of mutant DNA. These results show that selective aggregation of gold nanoparticles can be successfully used to detect mutations in exons 19 and exon 21 of EGFR.

The inventors have found that mutations can be detected in cancer cell lines as well as in actual DNA samples isolated from the tissues of small cell lung cancer patients. Therefore, the detection method of the present invention can be usefully used as a pre-screening tool that can detect mutations in advance before using direct sequencing, and can significantly reduce the time and cost of the current detection method. That has an excellent effect.

1 is a diagram comparing the nucleotide sequence of exon 19 and exon 21 of EGFR in non-small cell lung cancer cell lines NCI-H146, H1975, and H2279. NCI-H146 shows wild-type EGFR sequences in exons 19 and exon 21, indicating that 15 bp deletion (GAATTAAGAGAAGCA) was present in exon 19 of the H2279 cell line and leucine (CTG) was substituted for arginine (CGG) in exon 21 of the H1975 cell line.

FIG. 2 shows (a) color change of the gold nanoparticle suspension and (b) TEM image at 3 hours after addition of the appropriate concentration of salt in H146, H1975, and H2279 cell lines.

FIG. 3 compares the UV-vis absorption spectra of gold nanoparticles after addition of the appropriate concentration of salt in H146, H1975, and H2279 cell lines.

Figure 4 shows (a) color change of the gold nanoparticle suspension and (b) TEM image when 3 hours after adding the appropriate concentration of salt in eight cancer tissues (E1 to E8) of eight patients with non-small cell lung cancer .

5 is a diagram schematically illustrating the principle of the detection method of the present invention.

Claims (12)

To detect mutations in epidermal growth factor receptor (EGFR) genes, which are important for predicting drug reactivity in lung cancer patients, 1) extracting nucleic acids from cells or tissues of lung cancer patients, 2) causing the hybridization reaction by treating the oligonucleotide probe complementary to an EGFR (epidermal growth factor receptor) mutant gene without amplifying the extracted nucleic acid, 3) treating metal nanoparticles to the hybridization reactant, 4) treating a salt solution containing 0.01 to 0.3 M Na +, which is a suitable concentration for causing the metal nanoparticles to aggregate, and 5) including checking the agglomeration of the metal nanoparticles, Wherein the sequence of the probe is 5'-TCGCTATCAARACATCTCCG-3 '(R means A or G) or 5'-ATTTTGGGCGGGCCAAACTG-3'. The method of claim 1, wherein the lung cancer is non-small cell lung cancer. The method of claim 1, wherein the metal nanoparticles are gold nanoparticles, silver nanoparticles, platinum nanoparticles, or a combination thereof. The method of claim 1, wherein the mutation of the EGFR gene is a deletion mutation of E746-A750 in exon 19 of EGFR or L858R point mutation of exon 21. The method of claim 1, wherein the drug is a tyrosine kinase inhibitor (TKI). The method of claim 5, wherein the drug is gefitinib or erlotinib. The method of claim 1, wherein the nucleic acid is DNA or RNA. delete delete The method of claim 1, wherein step 3) and step 4) are performed sequentially or simultaneously. The method of claim 1, wherein step 5) is to visually confirm the aggregation of the metal nanoparticles through the color change of the solution. The method of claim 1, wherein step 5) comprises UV-vis absorption spectroscopy, transmission eletron microscopy (TEM) or quasi-elastic light-scattering (QELS). How to check using.

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