Method for diagnosing cancer using cfdna

Abstract

Provided is a method for detecting a biomarker specifically expressed or overexpressed in cancer cells present in a liquid sample without a PCR or nucleic acid amplification step. Alternatively, methods are provided for detecting mutations (e.g., SNPs) in cancer cell biomarkers without a PCR or nucleic acid amplification step.SOLUTION: The diagnostic method of the present invention relates to a technique of concentrating and isolating small-sized cfDNA from a liquid sample such as urine, cerebrospinal fluid, plasma, blood, pleural effusion, or body fluid, and then detecting a biomarker overexpressed in a specific cancer with ultra-high sensitivity without PCR. Since the detection method according to an embodiment of the present invention does not require a PCR amplification reaction, the time taken to diagnose cancer can be greatly reduced. In addition, it can be used for Point-Of-CareTesting (POCT) that can perform real-time analysis on site and can simultaneously search for a large number of genes in a short period of time.SELECTED DRAWING: Figure 2a

Description

[Technical Field]

[0001] The present invention relates to a cancer diagnostic method using cell-free DNA having a double helix structure, and more specifically Specifically, it amplifies biomarker genes that are specifically expressed or overexpressed in cancer. The present invention relates to a process-free detection method and an apparatus for using the same. [Background technology]

[0002] Recently, the importance of early diagnosis of cancer has increased significantly worldwide. However, until now, the methods for diagnosing cancer have not been widely studied. This has been done through invasive methods such as tissue sampling and endoscopy. The tissue test involves removing a portion of the suspected diseased area and examining it under a microscope. Therefore, to obtain tissue samples, needles, punches, endoscopes, or laparoscopes are used. In order to perform this procedure, the body must be incised, which causes some inconvenience to the patient. It leaves scars and takes a long time to heal.

[0003] As an alternative to invasive diagnostic and testing methods, molecular diagnostic methods using liquid biopsies are gaining attention. Liquid biopsy uses a non-invasive method, In addition, the test results can be quickly confirmed. Unlike tissue samples, liquid biopsies allow for multifaceted analysis of disease. In particular, liquid biopsies are expected to be extremely useful in cancer diagnosis. In particular, it is possible to detect cancer cell-derived cancers present in the blood of various body parts simply by testing body fluids such as blood and urine. It is predicted that DNA analysis will enable detailed observation of cancer development and metastasis. It is measured.

[0004] Molecular diagnostics is a typical in vitro diagnostic technique that uses genetic information from blood, urine, etc. A diagnostic technique that detects changes in DNA or RNA from samples through numerical or image analysis. This has the advantage of being highly accurate and not requiring tissue testing, making it a popular method for genomic analysis. With the rapid development of molecular analysis technology, it is being applied to cancer diagnosis technology based on the advantages of cost reduction. Attempts are being made.

[0005] Cell-free DNA (hereinafter referred to as cfDNA) is a DNA fragment that is extracted from blood. cfDNA refers to DNA derived from cells present in the same tissue. In many cases, cfDNA not only has a nucleotide structure but also a coiled-coil structure. They may be derived from tumor cells. They may also be derived from blood, plasma, or other materials obtained from cancer patients. In bodily fluids such as urine, cfDNA derived from tumor cells can be found.

[0006] cfDNA found in cancer patients is derived from cell necrosis, cell death, or normal cells in the urinary tract and cfDNA is often derived from human and / or cancer cells. Therefore, it is released into the urine, blood, etc., depending on the biological sample, such as blood, plasma, or urine. As technology for isolating and detecting cfDNA in samples develops, liquid biopsies are becoming increasingly available for cancer risk patients. It is anticipated that this will become a more effective and reliable tool for monitoring In particular, urine, cerebrospinal fluid (CSF), and blood Plasma, pleural effusion, ascites, blood, or other body fluids are easily obtained samples, so repeated Through this sampling, large amounts of specimens can be collected in a simple and non-invasive manner.

[0007] However, cfDNA in liquid samples such as blood and urine can be analyzed to identify mutations present in genes. There are many difficulties with the current state of technology in diagnosing cancer early by looking at the Therefore, we are developing methods to easily detect cfDNA, as well as improving detection sensitivity and accuracy. There is a need for technologies for early diagnosis of cancer.

[0008] In addition, Korean Patent No. 10-1751962 discloses a method for detecting cfDNA. A chain polymerization reaction is carried out using a primer, and a probe that can complementarily bind to cfDNA is added. However, it has been reported that cfDNA can be quantified using However, there remains a problem that separate polymerases and experimental equipment are required to carry out the reaction. Not only are there problems with the number of defects, but there is also the problem that on-site diagnosis is not easy.

[0009] In addition, Korean Patent No. 10-1701618 has a patent for a method to effectively separate cfDNA. discloses a nanostructure whose surface properties can be changed by changing an electric field. The nanostructure can bind and release cfDNA through electrical changes, cfDNA can be easily isolated from samples. However, what kind of cfDNA is present? To confirm its existence, a chain polymerization reaction must still be used. There are limits.

[0010] A variety of primer sets are used to amplify cfDNA by chain polymerization. Not only is it necessary, but it is also time-consuming since it involves complex steps. Therefore, it overcomes the limitation of PCR and can extract cfDNA with high accuracy. Research is being continuously conducted to develop methods for analyzing the cfDNA is found at extremely low levels in liquid samples, allowing for high accuracy and fewer samples. Research on developing methods for obtaining and analyzing DNA in small amounts and on efficient analysis methods Research is ongoing. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Korean Patent No. 10-1751962 [Patent Document 2] Korean Patent No. 10-1701618 Summary of the Invention [Problem to be solved by the invention]

[0012] Conventionally, to detect cfDNA, a primer complementary to cfDNA was required to bind to the target. The denaturation process that converts double-stranded cfDNA into single-stranded DNA is essential. To detect fDNA, a process of applying heat is essential, and the process involves reacting with polymerases, etc. A process is essential.

[0013] However, the present inventors have found that the DNA transcription process occurs actively in cancer cells, and that There are many DNA sections in which double-stranded DNA has dissociated into single strands, and from such cancer cells The released cfDNA allows the probe to bind to the cfDNA without the need for a denaturing process. This discovery led to the development of the present invention.

[0014] Therefore, one aspect of the present invention is to detect blood cfDNA using a probe having a sequence complementary to cfDNA. Bacteria that are specifically expressed or overexpressed in cancer cells present in liquid samples such as plasma or urine The present invention provides a method for detecting biomarkers without a PCR or nucleic acid amplification step.

[0015] In another embodiment, mutations (e.g., SNPs) in cancer cell biomarkers are detected by PCR or nucleic acid sequencing. A method of detection without an amplification step is provided. [Means for solving the problem]

[0016] To achieve the above object, one aspect of the present invention is to provide a method for preparing a cell-free DNA (cell-free DNA) containing: Biological samples isolated from individuals containing cfDNA (hereinafter referred to as cfDNA) and positive control samples b) Mixing the charged substances; c) Separating the positively charged substances bound to cfDNA c) adding to the mixture a probe and a marker having a sequence complementary to the cfDNA. d) mixing probes and primers that do not bind to cfDNA sequentially or simultaneously; and e) detecting said marker from said sample. A method for diagnosing cancer by detecting genes derived from cancer cells without amplification, A is a probe derived from a cancer cell and having a sequence complementary to the cfDNA. The gene complementarily binds to genes known to be cancer biomarkers, and is used to diagnose or detect cancer. The present invention provides a method for predicting the prognosis of [Effects of the Invention]

[0017] The cancer diagnosis method of the present invention is carried out by using a liquid such as urine, cerebrospinal fluid, plasma, blood, pleural effusion, or body fluid. After isolating small-sized cfDNA from the sample, cancer-specific or hyperspecific detection can be performed without PCR. The present invention relates to a technique for detecting expressed biomarkers with ultra-high sensitivity. This method does not require PCR amplification, which significantly reduces the time it takes to diagnose cancer. In addition, it allows for immediate analysis on-site, and many genes can be analyzed simultaneously in a short period of time. Point-of-Care Testing (POC) In particular, according to the method of the present invention, the IL-14-16 nucleotides present in normal human blood can be used for CT. It can distinguish between cfDNA and cfDNA present in the blood of cancer patients, allowing for effective treatment of various cancers. can be detected effectively. [Brief explanation of the drawings]

[0018] [Figure 1a] FIG. 1 shows a scanning electron microscope (SEM) image of positively charged nanowires (PEI / Ppy NW). [Figure 1b] FIG. 1 shows a scanning electron microscope image of HRP / streptavidin-conjugated nanoparticles. [Figure 2a] Figure 1 shows a conceptual diagram of the fabrication of a nanostructure (PEI / mPpy NW) with the cationic polymer polyethyleneimine (PEI) attached to its surface, and the method of using it to detect and recover cfDNA. [Figure 2b] This figure shows photographs of the process of detecting and recovering cfDNA using a magnetic nanostructure (PEI / mPpy NW) with the cationic polymer polyethyleneimine (PEI) attached to its surface. [Figure 3] This is a diagram illustrating the process of collecting cfDNA using an Eppendorf tube. [Figure 4]This figure shows the level of PD-L1 DNA expression and PD-L1 mRNA expression measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines. [Figure 5] This figure shows the level of PD-L1 DNA expression and PD-L1 mRNA expression measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines. [Figure 6] This figure shows the level of EpCAM DNA expression and EpCAM mRNA expression measured from cfDNA of EpCAM-positive or EpCAM-negative cancer cell lines. [Figure 7] This figure shows the level of EpCAM DNA expression and EpCAM mRNA expression measured from cfDNA of EpCAM-positive or EpCAM-negative cancer cell lines. [Figure 8] This figure shows the level of FOLR1 DNA expression and FOLR1 mRNA expression measured from cfDNA of FOLR1-positive or FOLR1-negative cancer cell lines. [Figure 9] This figure shows the level of FOLR1 DNA expression and FOLR1 mRNA expression measured from cfDNA of FOLR1-positive or FOLR1-negative cancer cell lines. [Figure 10] FIG. 1 shows the levels of EGFR DNA expression and EGFR mRNA expression measured from cfDNA of EGFR-positive or EGFR-negative cancer cell lines. [Figure 11] FIG. 1 shows the levels of EGFR DNA expression and EGFR mRNA expression measured from cfDNA of EGFR-positive or EGFR-negative cancer cell lines. [Figure 12]FIG. 1 shows the levels of ERBB2 DNA expression and ERBB2 mRNA expression measured from cfDNA of ERBB2-positive or ERBB2-negative cancer cell lines. [Figure 13] FIG. 1 shows the levels of ERBB2 DNA expression and ERBB2 mRNA expression measured from cfDNA of ERBB2-positive or ERBB2-negative cancer cell lines. [Figure 14] FIG. 1 shows the level of OGT DNA expression measured from cfDNA of OGT-positive or OGT-negative cancer cell lines. [Figure 15] FIG. 1 shows the level of OGT DNA expression measured from cfDNA of OGT-positive or OGT-negative cancer cell lines. [Figure 16] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 17] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 18] FIG. 1 shows the level of CEA DNA expression measured from cfDNA of CEA-positive or CEA-negative cancer cell lines. [Figure 19] FIG. 1 shows the level of PSA DNA expression measured from cfDNA of PSA-positive or PSA-negative cancer cell lines. [Figure 20] FIG. 1 shows the level of PSA DNA expression measured from cfDNA of PSA-positive or PSA-negative cancer cell lines. [Figure 21]This is a graph showing the level of CA19-9 DNA expression measured from cfDNA of CA19-9-positive or CA19-9-negative cancer cell lines. [Figure 22] This is a graph showing the level of CA19-9 DNA expression measured from cfDNA of CA19-9-positive or CA19-9-negative cancer cell lines. [Figure 23] This is a graph showing the level of CA125 DNA expression measured from cfDNA of CA125-positive or CA125-negative cancer cell lines. [Figure 24] This is a graph showing the level of CA125 DNA expression measured from cfDNA of CA125-positive or CA125-negative cancer cell lines. [Figure 25] FIG. 1 shows the level of AFP DNA expression measured from cfDNA of AFP-positive or AFP-negative cancer cell lines. [Figure 26] FIG. 1 shows the level of AFP DNA expression measured from cfDNA of AFP-positive or AFP-negative cancer cell lines. [Figure 27] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 28] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 29] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from prostate cancer patients. [Figure 30] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 31]FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 32] FIG. 1 shows the DNA expression levels of PSA, PSMA, PAP, and PAC3 measured using plasma obtained from normal humans. [Figure 33] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from lung cancer patients. [Figure 34] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from lung cancer patients. [Figure 35] FIG. 1 shows the DNA expression levels of NSE, SCC, CEA, Cyfra21-1 and TPA measured using plasma obtained from normal humans. [Figure 36] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 37] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 38] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from thyroid cancer patients. [Figure 39] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from normal humans. [Figure 40] FIG. 1 shows the DNA expression levels of CEA, NSE, TG, and CALCA measured using plasma obtained from normal humans. [Figure 41] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients. [Figure 42]FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients. [Figure 43] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from patients with cystitis. [Figure 44] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from patients with cystitis. [Figure 45] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from normal humans. [Figure 46] FIG. 1 shows the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from normal humans. [Figure 47] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from breast cancer patients. [Figure 48] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from breast cancer patients. [Figure 49] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from normal humans. [Figure 50] FIG. 1 shows the DNA expression levels of CA27-29, CA15-3 and CEA measured using plasma obtained from normal humans. [Figure 51] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using plasma obtained from colon cancer patients. [Figure 52] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using plasma obtained from colon cancer patients. [Figure 53]FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 54] FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 55] FIG. 1 shows the results of measuring the DNA expression levels of CEA and CA19-9 using plasma obtained from normal humans. [Figure 56] FIG. 1 shows the DNA expression levels of CA19-9, CA125, and CEA measured using plasma obtained from bile duct cancer patients. [Figure 57] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 58] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 59] FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from gastric cancer patients. [Figure 60] FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from normal humans. [Figure 61] FIG. 1 shows the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using plasma obtained from normal humans. [Figure 62] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 63] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 64] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 65]FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from pancreatic cancer patients. [Figure 66] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 67] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 68] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 69] FIG. 1 shows the DNA expression levels of CA19-9, CA125 and CEA measured using plasma obtained from normal humans. [Figure 70] FIG. 1 is a graph showing the DNA expression level of CPT1A measured using plasma obtained from lung cancer patients. [Figure 71] FIG. 1 shows the results of measuring the level of CPT1A DNA expression using plasma obtained from normal humans. [Figure 72] FIG. 1 is a graph showing the DNA expression level of CPT1A measured using urine collected from bladder cancer patients. [Figure 73] FIG. 1 shows the results of measuring the DNA expression level of CPT1A using urine collected from normal humans. [Figure 74] FIG. 1 shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive cancer cell lines or PD-L1-negative cancer cell lines not treated with IFN-γ, and the presence or absence of PD-L1 detection measured using a method according to one embodiment of the present invention. [Figure 75] This figure shows the level of IFN-γ DNA expression measured from cfDNA of PD-L1-positive cancer cell lines or PD-L1-negative cancer cell lines that were not treated with IFN-γ. [Figure 76] This figure shows the level of DNA expression of IFNR1 (IFN-γ receptor) measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines that were not treated with IFN-γ. [Figure 77] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 78] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 79] This figure shows the DNA expression levels of PD-L1, IFNG, and IFNR1 measured from cfDNA of PD-L1-positive or PD-L1-negative cancer cell lines treated with IFN-γ. [Figure 80] This figure shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 81] This is a graph showing the level of IFN-γ DNA expression measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines with or without IFN-γ treatment. [Figure 82] This figure shows the level of IFNR1 DNA expression measured from cfDNA of PD-L1-positive cancer cell lines and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 83] This figure shows the level of PD-L1 DNA expression measured from cfDNA of PD-L1-positive and PD-L1-negative cancer cell lines, with or without IFN-γ treatment. [Figure 84a] Figure 84a is a diagram illustrating the detection steps of the present invention. Figure 84a is a diagram illustrating a method for collecting cfDNA from a patient's body fluid using nanowires (PEI / Ppy NW) with polyethyleneimine (PEI) attached to their surface, and then analyzing gene mutations within about 60 minutes through a reaction with a probe and HRP / streptavidin-nanoparticles (HRP / st-tagged NP). [Figure 84b]Figure 84b is a schematic diagram of a method for detecting unstable cfDNA using nanowires, probes, and HRP / streptavidin nanoparticles. [Figure 84c] 84c shows a process for detecting gene mutations using a spin column with nanowires that do not contain magnetic nanoparticles. In one embodiment of the present invention, a step of treating the lysis buffer may be additionally included. [Figure 84d] Figure 84d shows a timeline of a method for detecting unstable cfDNA in samples such as blood, cerebrospinal fluid, or pleural effusion. [Figure 84e] Figure 84e is a timeline showing a method for detecting unstable cfDNA in a sample such as urine. [Fig. 84f] Figure 84f is a diagram illustrating the differences in denaturing conditions depending on the state of cfDNA obtained from blood. [Figure 84g] Figure 84g is a diagram illustrating the differences in denaturing conditions depending on the state of cfDNA obtained from urine, saliva, and sputum. [Figure 85] This figure shows the separation of cfDNA using a spin column with nanowires that do not contain magnetic nanoparticles. The top photo is an SEM image of the spin column before centrifugation, and the bottom photo is an SEM image of the spin column with separated cfDNA after centrifugation. [Figure 86] This is a graph showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion and PIK3CA measured from blood collected from lung cancer patients using a syringe. [Figure 87] This is a diagram showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion and PIK3CA measured from blood collected from lung cancer patients using a lancet. [Figure 88] This is a graph showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion measured from blood collected from normal subjects using a syringe needle. [Figure 89]This is a diagram showing the DNA expression levels of cancer-related biomarkers such as AKL Fusion measured from blood collected from normal subjects using a lancet. [Figure 90] This figure shows the expression level of EML4-ALK confirmed by RT-PCR from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 91] This figure shows the level of EML4-ALK expression confirmed by Western blotting from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 92] This is a graph showing the results of confirming the level of EML4-ALK expression from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines using RT-PCR and Western blotting. [Figure 93] This figure shows the level of DNA expression of EML4-ALK fusion var.1 or EML4-ALK fusion var.3 measured from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 94] This is a graph showing the level of DNA expression of EML4-ALK fusion var.1 or EML4-ALK fusion var.3 measured from cfDNA of EML4-ALK variant 3a / b positive cell (H2228) and EML4-ALK negative cell (A549, H1993, PC9, RT4) cancer cell lines. [Figure 95]This is a graph showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var.3, KRAS, SYP, NCAM1, and NKX2-1, in blood collected from patients with small cell lung cancer. As a result, EML4-ALK fusion was found in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var.3, not var.1, indicating a poor response to the ALK TKI crizotinib. [Figure 96] This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to the ALK TKI crizotinib, resulting in a partial response (PR) for the patient. [Figure 97] This is a diagram measuring the DNA expression level of cancer-related biomarkers such as EML4-ALK fusion var. 3 in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA. Since the ctDNA results showed that the EML4-ALK fusion was var. 3, not var. 1, and the patient did not respond well to the ALK TKI crizotinib, alectinib was prescribed from the start, and the patient's response was awaited. [Figure 98] This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var.3, BRAFV800E, and TP53, in blood collected from cancer patients. As a result, EML4-ALK fusion was found in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var.3, not var.1, indicating a poor response (PD) to the ALK TKI crizotinib. [Figure 99]This is a diagram showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to the ALK TKI crizotinib, resulting in a partial response (PR) for the patient. [Figure 100] This is a graph showing the DNA expression levels of cancer-related biomarkers, such as EML4-ALK fusion var. 1, measured in blood collected from cancer patients. As a result, EML4-ALK fusion was found to be present in both cancer tissue and blood ctDNA, and the ctDNA results showed that the EML4-ALK fusion was var. 1, not var. 3, indicating a good response to ALK TKI and a partial response (PR) in the patient. [Figure 101] This figure shows the level of OGT protein expression confirmed in vitro using Western blotting from cfDNA of each cancer cell line. [Figure 102] This figure shows the level of OGT mRNA expression confirmed in vitro using RT-PCR from cfDNA of each cancer cell line. [Figure 103] This is a graph showing the results of confirming the level of OGT mRNA expression from cfDNA of each cancer cell line in vitro using RT-PCR. [Figure 104] This is a diagram showing the level of OGT DNA expression measured in vitro from cfDNA of each cell line. [Figure 105] This is a graph showing the results of measuring the level of OGT DNA expression from cfDNA of each cell line in vitro. [Figure 106] This figure shows the results of confirming the level of OGT expression in each cell line in vitro through Western blot, RT-PCR, and cfDNA detection. [Figure 107]This is a photograph of OGT cfDNA detected in vitro from each cell line on nanowires that do not contain magnetic nanoparticles. [Figure 108] This is a graph showing the quantification of cfDNA obtained from urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 109] This is a graph showing the analysis of the DNA expression level of OGT from cfDNA obtained from the urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 110] This is a graph showing the analysis of the DNA expression level of OGT from cfDNA obtained from the urine of normal subjects, cystitis patients, and bladder cancer patients. [Figure 111] This is a diagram showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 112] This is a diagram showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 113] This is a diagram showing the analysis of OGT DNA expression levels in cfDNA obtained from urine samples of various cancer patients in a blind test. [Figure 114] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 115] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 116] This is a diagram showing the analysis of the DNA expression levels of BRAF V600E and TERT C250T from cfDNA obtained from the tissues of thyroid cancer patients. [Figure 117] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 118]This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 119] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 120] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 121] This is a graph showing the DNA expression levels of cancer-related biomarkers such as SYP, CgA, NCAM1, and NKX2-1 in blood collected from patients with small cell lung cancer. In particular, the patient in Figure 120 was diagnosed with small cell lung cancer (SCLC) at Korea Atomic Energy Hospital and prescribed crizotinib, but it was found to be ineffective. [Figure 122] FIG. 1 shows the results of measuring the DNA expression levels of SYP, CgA, NCAM1, and NKX2-1 using blood obtained from non-small cell lung cancer patients. [Figure 123] FIG. 1 shows the level of CEA DNA expression in blood collected from lung cancer patients before and after anti-cancer treatment, and the prognosis of the patients. [Figure 124] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 125] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 126]This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 127] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 128] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from lung cancer patients. [Figure 129] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 130] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 131] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 132] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 133] This is a graph showing the DNA expression levels of cancer-related biomarkers such as NSE and CEA measured using blood collected from normal subjects. [Figure 134] This is a graph showing the DNA expression levels of cancer-related biomarkers such as PSA, PSMA, PAP, and PCA3 measured using blood collected from prostate cancer patients. [Figure 135] This is a graph showing the DNA expression levels of cancer-related biomarkers such as PSA, PSMA, PAP, and PCA3 measured using blood obtained from normal subjects. [Figure 136] FIG. 10 shows the results of measuring the DNA expression level of TMPRSS2-ERG fusion using blood collected from prostate cancer patients and normal subjects. [Figure 137] This is a graph showing the DNA expression levels of CEA, NSE, TG (Thyroglobulin), and CALCA measured using blood obtained from thyroid cancer patients. [Figure 138] This is a graph showing the DNA expression levels of CEA, NSE, TG (Thyroglobulin), and CALCA measured using blood obtained from normal humans. [Figure 139] This is a graph showing the DNA expression levels of BRAF mutation (V600E) and TERT promoter mutation (C228T, C250T) measured using blood collected from thyroid cancer patients and normal subjects. [Figure 140] This is a diagram showing the DNA expression levels of OGT, FGFR3, TP53, NMP22, and Cyfra21-1 measured using urine collected from bladder cancer patients, hematuria patients, and normal subjects. [Figure 141] FIG. 1 shows the DNA expression levels of CA27-29 and CEA measured using blood collected from breast cancer patients and normal subjects. [Figure 142] FIG. 1 shows the DNA expression levels of CEA and CA19-9 measured using blood collected from colon cancer patients and normal subjects. [Figure 143] FIG. 1 shows the results of measuring the DNA expression levels of CA 19-9, CEA, and CA123 using blood collected from bile duct cancer patients and normal subjects. [Figure 144] FIG. 1 shows the results of measuring the DNA expression levels of CA 19-9, CEA, and CA123 using blood collected from bile duct cancer patients and normal subjects. [Figure 145] This is a graph showing the DNA expression levels of CEA, CA19-9, CGB and Cyfra21-1 measured using blood collected from gastric cancer patients and normal subjects. [Figure 146] FIG. 1 shows the results of measuring the DNA expression levels of CA125 and CEA using blood collected from ovarian cancer patients and normal subjects. [Figure 147] FIG. 1 shows the results of measuring the DNA expression levels of CEA, CA19-9, and CA125 using blood obtained from pancreatic cancer patients. [Figure 148] FIG. 1 shows the DNA expression levels of CEA, CA19-9 and CA125 measured using blood obtained from normal subjects. [Figure 149] These are the results of early diagnosis conducted by measuring the DNA expression level of cancer-related biomarkers using blood collected from normal individuals (PC: Positive control, PC is a tool to confirm whether the early cancer diagnosis experiment was carried out correctly and has no relation to the early cancer diagnosis results). [Figure 150] These are the results of early diagnosis conducted by measuring the DNA expression level of cancer-related biomarkers using blood collected from normal individuals (PC: Positive control, PC is a tool to confirm whether the early cancer diagnosis experiment was carried out correctly and has no relation to the early cancer diagnosis results). [Figure 151] FIG. 1 is a diagram summarizing biomarkers by cancer type used in one example of the present invention. [Figure 152] FIG. 1 is a diagram summarizing biomarkers by cancer type used in one example of the present invention. [Figure 153] This figure shows the absorbance of cfDNA present in the urine of HPV-positive cervical cancer patients (HPV16(+) and HPV18(+)) and HPV-negative healthy controls (HPV-) to confirm the presence or absence of binding of probes specific to HPV18 or HPV16. [Fig. 154] This figure shows the results of sequentially reacting cfDNA isolated from the urine of cervical cancer patients with probes specific to HPV16, EGFR19 deletion, HPV18, and EGFR21 L858R, and then confirming whether or not each probe bound to the cfDNA. [Figure 155] This table shows the analysis of gene mutations in lung cancer patients using cfDNA obtained from the plasma of 151 lung cancer patients. [Figure 156] cfDNA was collected from the plasma of lung cancer patients without EGFR mutations (wild type), with EGFR exon 19 deletion, and with EGFR exon 21 L858R. A probe specific to EGFR exon 19 Del was then mixed with cfDNA, and the gene mutations in the lung cancer patients were confirmed through analysis of UV spectrum absorbance (ΔOD, 500nm~650nm). [Figure 157] This figure shows the specificity and sensitivity of gene mutations after cfDNA was collected from the plasma of a lung cancer patient with EGFR exon 19 deletion and mixed with a probe specific to EGFR exon 19 Del. [Figure 158] cfDNA was collected from the plasma of lung cancer patients without EGFR mutations (wild type), with EGFR exon 19 deletion, and with EGFR exon 21 L858R. A probe specific to EGFR exon 21 L858R was added, and the patient's gene mutations were confirmed through analysis of UV spectrum absorbance (ΔOD, 500nm~650nm). [Figure 159] This figure shows the specificity and sensitivity of the patient's gene mutation after collecting cfDNA from the plasma of a lung cancer patient with EGFR exon 21 L858R and adding a probe specific to EGFR exon 21 L858R. [Figure 160] This figure shows the CP and DP sequences of the EGFR exon 19 deletion. In this study, we analyzed cfDNA mutations in lung cancer patients using CP_1 and DP. CP is a probe designed to complement the sequence containing or adjacent to the mutation, and DP is a probe designed to complement the sequence distant from the mutation. [Figure 161] Figure 1 shows the CP and DP sequences of EGFR exon 20 T790M. In this study, we analyzed cfDNA gene mutations in lung cancer patients using CP2 and DP. [Figure 162] Figure 1 shows the CP and DP sequences of EGFR exon 21 L858R. In this study, CP2 and DP were used to analyze cfDNA mutations in lung cancer patients. [Figure 163]cfDNA obtained from the plasma of a lung cancer patient with EGFR exon 19 deletion and EGFR exon 20 T790M gene mutation was reacted with probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, and EGFR exon 21 L858R. HRP / streptavidin nanoparticles (containing a large amount of HRP) were then added, and the detection of cfDNA was confirmed by color change and UV absorbance. [Fig. 164] cfDNA collected from the plasma of a lung cancer patient with the same EGFR exon 19 deletion and EGFR exon 20 T790M genetic mutation as in Figure 163 was reacted with probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, and EGFR exon 21 L858R. Then, HRP / streptavidin complex (a 1:1 complex of HRP and streptavidin) was added, and the detection of cfDNA was confirmed by color change and UV absorbance. This figure confirms that the HRP / streptavidin complex generates more noise than HRP / streptavidin nanoparticles. [Figure 165] cfDNA was extracted from the plasma of five lung cancer patients with EGFR exon 19 deletion and exon 20 T790M gene mutations. The results were then reacted with probes specific for EGFR exon 19 Del, EGFR exon 20 T790M, and EGFR exon 21 L858R and HRP / streptavidin nanoparticles (HRP / st-tagged NPs), and with probes specific for EGFR exon 19 Del, EGFR exon 20 T790M, and EGFR exon 21 L858R and HRP / streptavidin complexes (HRP and streptavidin bound in a 1:1 ratio). The results were compared to confirm the concordance between the cancer tissue and genotype. [Figure 166]To detect gene mutations in cfDNA collected from the plasma of a lung cancer patient with EGFR exon 20 T790M and EGFR exon 21 L861Q gene mutations, probes specific for EGFR exon 19 deletion (Del19), EGFR exon 20 T790M, EGFR exon 21 L858R, and EGFR exon L861Q were mixed with HRP / st-tagged NP. As a result, gene mutations were observed only in EGFR exon 20 T790M and EGFR exon 21 L861Q, as confirmed by UV absorbance, just like in the cancer tissue. [Figure 167] To detect gene mutations in cfDNA obtained from the plasma of lung cancer patients with ALK-EML4 fusion and ALK point mutation (I1171N / T) gene mutations, probes specific for ALK-EML4 fusion and ALK point mutation (T1151, L1152P, L1152R, C1156Y, I1171N / T) and HRP / st-tagged NP were mixed together. As a result, it was confirmed that the ALK-EML4 fusion and ALK point mutation (I1171N / T) genotypes were detected in the same way as in the cancer tissue. [Figure 168] To detect gene mutations in cfDNA obtained from the plasma of thyroid cancer patients with the BRAF V600E gene mutation, a BRAF V600E-specific probe and HRP / st-tagged NP were mixed together. As a result, it was confirmed that the BRAF V600E gene mutation was detected in the same manner as the patient's genotype. [Figure 169] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after samples collected from normal human blood were denatured under various temperature conditions. [Figure 170] This figure shows the results of detecting unstable cfDNA depending on the processing conditions after samples collected from patient blood were denatured under various temperature conditions. [Figure 171]This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after denaturing fDNA obtained from mutant cell lines under various temperature conditions. [Figure 172] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 30 minutes. [Fig. 173] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 60 minutes. [Fig. 174] This figure shows the results of detecting unstable cfDNA depending on the treatment conditions after treating fDNA obtained from mutant cell lines with DNase at 37°C for 120 minutes. [Figure 175] This figure shows the results of treating cfDNA with 1 μl or 2 μl of DNase at 24°C for 120 minutes to confirm the difference between unstable cfDNA and stable cfDNA due to DNase activity. [Figure 176] This figure shows the results of treating 1 μl or 2 μl of DNase at 3°C ​​for 120 minutes to confirm the difference between unstable cfDNA and stable cfDNA due to DNase activity. [Figure 177] FIG. 10 is a diagram showing a specific example of a cutoff value when detecting the EML4-ALK fusion gene using cfDNA from the plasma of a lung cancer patient in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] <Terminology> As used herein, the term "cell-free DNA" refers to DNA fragments that are free of DNA. cfDNA is a type of DNA derived from tumor cells in urine from cancer patients. Cancer cell-derived D found in biological samples such as cerebrospinal fluid, plasma, blood, or body fluids Circulating tumor DNA (ctDNA) ) and urine, cerebrospinal fluid, pleural effusion, ascites, plasma, blood, saliva, sputum, or cfDNA can be present in biological samples such as body fluids. , about 80bp to about 10kbp, about 100bp to about 1kbp, about 120bp to about 500bp cfDNA can have a size of about 150 bp to about 200 bp. The size of the fragment can be about 165 bp to about 170 bp. Furthermore, the cfDNA contains small fragments of about 80 bp or less. It may contain cfDNA.

[0020] As used herein, the term "unstable cfDNA" refers to "stable cfDNA." This means that the cfDNA is thermodynamically unstable compared to the denatured cfDNA. The unstable cfDNA can be denatured under slightly harsher conditions than those used for denaturation. The reason why cfDNA is generated is that it has an unstable double helix structure. Specifically, cfDNA derived from genes overexpressed in cancer cells is unstable. This may be a specific example of cfDNA.

[0021] As used herein, the term "cfDNA having an unstable double helix structure" refers to It has a lower Tm value than cfDNA with a stable double helix structure or The method is characterized in that it is denatured under conditions in which cfDNA having a double helix structure is not denatured. Tm stands for melting temperature, and is the melting temperature of double-stranded DNA. The Tm value is the temperature at which 50% of the DNA becomes single-stranded. The nucleotide sequence may vary, but genomic DNA is composed of many nucleotides. Since the tides are hydrogen-bonded, do not heat at about 92°C to about 95°C for more than 5 minutes, or at about 98°C. Heating must be done for at least 2 minutes. Also, genomic DNA is degraded at temperatures below approximately 90°C. Denaturation does not occur easily. In this case, a stable double helix structure is formed. Assuming that cfDNA has an average of approximately 170 bp of nucleotides, the genome DNA It may have a Tm value similar to that of NA.

[0022] However, the "cfDNA having an unstable double helix structure" is a stable double helix. Therefore, it has a stable double helix structure. The cfDNA containing the compound is left under the following conditions: i) at room temperature for about 1 minute to about 120 minutes; ii) at about 90°C iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C to about 75°C for about 30 seconds to about 60 minutes; ) heating at about 25°C to about 40°C for about 10 minutes to about 120 minutes; vi) heating with protease for about vii) Treatment with DNase for 10 seconds to 30 minutes conditions; and viii) chemicals (e.g., sodium hydroxide, DMSO, surfactants, etc.). After denaturation under any one of conditions selected from the group consisting of conditions for treating The probes were about 15-30 mer in length and had a sequence complementary to a partial sequence of the NA. When the reaction is carried out, the cfDNA having a stable double helix structure binds to the probe. In this case, "room temperature" means room temperature, which may be about 18°C ​​to about 25°C. In addition to the above conditions, the method further includes heating at about 40°C to about 65°C for about 5 minutes to about 80 minutes. can be done.

[0023] However, cfDNA, which has an unstable double helix structure, is prone to the following problems i) to viii) After treatment under one of the conditions, the DNA was bound to a probe of about 15mer to about 30mer. When a ligation reaction was carried out, it was confirmed that the probe bound to the ligated protein. , about 15mer to about 30mer or about 20mer to about 25mer, and about 21mer , about 22-mer, about 23-mer, or about 24-mer probes.

[0024] In this case, the cfDNA with the unstable double helix structure is called circulating tumor DNA (circ ctDNA).

[0025] As used herein, the term "probe" refers to a probe that is a target cfDN The probe is designed to detect DNA or RNA that is unstable to cfDNA. It may have a sequence designed to be capable of complementary binding. The term used, "probes with sequences complementary to cfDNA," refers to probes that are It can complementarily bind to the target double-stranded cfDNA that is present in the liquid sample and that you want to detect. It means a probe having a nucleic acid sequence.

[0026] In this case, the probe can be produced in two ways. The first probe (hereinafter referred to as CP) is designed to bind to the target site. One of them is a second probe (hereafter referred to as D) designed to bind to the area around the damaged area. The DP can be approximately 1000 kDa from the target DNA sequence or the region where the damage occurred. Complementary binding to sequences located 10 bp to approximately 100 bp, or approximately 20 bp to approximately 50 bp away It can be designed so that

[0027] Here, complementary binding means that the appropriate hybrid Probe under hybridization conditions can bind to the target cfDNA and form a duplex, At least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary sequences. do.

[0028] Hybridization conditions may vary depending on, for example, the length of the probe, the complementarity of the probe, the hybridization The salt concentration (i.e., ionic strength) in the lysis buffer can be determined experimentally by one skilled in the art. Generally, stringent hybridization conditions are those that allow a polynucleotide to hybridize to its target site. It can bind preferentially to its complementary sequence and with higher affinity than any other region on the target. The condition is that the DNA fragment can bind to the complement of a polynucleotide sequence having 20 bases. Exemplary stringent conditions for hybridization are approximately 50% G+C content, 50 mM salt (Na+ ) and an annealing temperature of 60°C. For longer sequences, higher temperatures may be used. Hybridization can be performed. Generally, stringent conditions are used to ensure that annealing is carried out. The melting temperature of the nucleotide is about 5°C below the melting temperature. The "melting temperature" is the temperature at which the target polynucleotide melts at a given ionic strength, pH, and polynucleotide concentration. The temperature at which 50% of the polynucleotides complementary to a given oligonucleotide can bind complementary to it.

[0029] In the present specification, the first probe and the second probe may be used simultaneously, or the first probe may be used alone or in combination. Damaged cfDNA can be effectively detected using either a primary or secondary probe, respectively. It was also confirmed that the probe contained a substance such as biotin to bind to the marker. Alternatively, the probe may be in the form of a marker directly bound to it or a phosphorylated probe. In this case, the marker may be a nanoparticle, a fluorescent dye, a fluorescent The probe may be a protein or an enzyme. The probe may also be added at the same time as the marker. , may be added sequentially.

[0030] In one embodiment of the present invention, the probe capable of complementary binding to the target cfDNA is The antibody can bind to a region containing a sequence specific to the cancer cells. For example, the antibody can bind to a region containing a sequence specific to ovarian cancer or For breast cancer-specific sequences, BRCA1 exon 7 and BRCA1 exon 10 , BRCA1 exon 11, BRCA1 exon 15. In addition, sequences specific to gastric cancer are present in TP53, and in colon cancer, they are present in MSH2. In the case of a sequence specific to lung cancer, it may be an SNP present in EGFR. In addition, in the case of a sequence specific to liver cancer, the sequence is selected from SNPs present in FGFR3. It is possible.

[0031] Biomarker genes derived from cancer cells and specific to cancer cells are known to those skilled in the art. For example, see the following literature: Circulating Cell-Free DNA in Plasma / Serum of Lung C ancer Patients as a Potential Screening and Prognostic Tool、Pathak et al,Clinica l Chemistry October 2006 vol.52 no.10 18 33-1842;Cell-free Tumor DNA in Blood Pla sma As a Marker for Circulating Tumor Ce lls in Prostate Cancer,Schwarzenbach et al,Clin Cancer Res Feb.1,2009 15;1032;Ce ll-free DNA:measurement in various carci nomas and establishment of normal refere nce range,Wua et al,Clinica Chimica Acta ,Volume 321,Issues 1-2,July 2002,Pages 7 7-87;Detection of Circulating Tumour DNA in the Blood(Plasma / Serum)of Cancer Pat ients,Anker et al,Cancer and Metastasis Reviews 1999,Volume 18,Issue 1,pp 65-73; Cell-free nucleic acids as biomarkers in cancer patients,Schwarzenbach et al,Nat ure Reviews Cancer 11,426-437(June 2011) ;Circulating Tumor-Specific DNA:A Marker for Monitoring Efficacy of Adjuvant The rapy in Cancer Patients,Fiegl et al,Canc er Res Feb.15,2005 65;1141.

[0032] In one embodiment of the present invention, the probe capable of complementary binding to the target cfDNA is It can complementarily bind to the region overexpressed in the above cancer cells. Such a region may be a biomarker for cancer cells. The genes may be, but are not limited to, those shown in Figures 151 and 152. Furthermore, throughout this specification, various biomarkers for specific tumor / cancer cells are mentioned. Exemplary probes that bind complementary to the marker genes and biomarker genes are described in the Examples. The probe further comprises a biotin or avidin-based protein. Specifically, the marker may include avidin, streptavidin, avidin (streptavidin) or a combination thereof Preferably, the probe further comprises any one of the following: It can be in the form of

[0033] As used herein, the term "isolated biological sample" refers to a sample isolated from a human body, such as urine, The term "sample" refers to a sample of saliva, cerebrospinal fluid, pleural effusion, ascites, plasma, blood, sputum or body fluid. The isolated biological sample may be a liquid sample isolated from the human body. It can be obtained from the liquid.

[0034] As used herein, the term "positively charged substance" refers to a material that carries a positive charge. They can be used in the form of nanoparticles, nanowires, net structures or filters. The shape of the positively charged substance is not limited to the above. The nanowire or membrane may have a positively charged surface. The membrane can be manufactured using a conductive polymer. Polyacetylene, polypyrrole role), poly(thiophene), polyparaphenylene Poly(para-phenylene)), poly(3,4-ethylenedioxythiophene) thiophene (poly(3,4-ethylenedioxythiophene)), Poly(phenylene sulfide), poly (poly(para-phenylene vinylene) ne) and polyaniline The length and diameter can be adjusted depending on the fabrication method. In the case of -, the diameter is about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 100 nm to approximately 400 nm, approximately 150 nm to approximately 350 nm, approximately 200 nm to approximately 400 nm, or The diameter can be selected in the range of about 100 nm to about 300 nm, and the length can be selected in the range of several μm to about 100 μm, and the length can be selected in the range of about 1 μm to about 100 μm. 0 μm to about 100 μm, about 15 μm to about 50 μm, about 15 μm to about 40 μm, or about 1 It can be selected from the range of 5 μm to about 30 μm.

[0035] In one embodiment, the nanowire has a diameter of about 200 nm and a length of about 18 μm. The nanowires may also be prepared in a form in which biotin is bound. do.

[0036] The surface of the nanowire or membrane may be modified with a cationic polymer. The cationic polymer is not limited to a specific type. Polyethyleneimine (PEI) or polylysine (polylysine, PLL). Also, cationic branched polymer polyethylene lenimine (cationic branched polymer polyethy The nanowires modified with such cationic polymers can be Or the membrane can have a positively charged surface to capture cfDNA. The surface charge of the nanowire or membrane is approximately 20mV to about 80mV, about 30mV to about 60mV, about 35mV to about 50mV The surface charge may be about 36 mV, about 37 mV, about 38 mV, or about 39 mV. , about 40 mV, about 41 mV, about 42 mV, about 43 mV, or about 44 mV.

[0037] In one embodiment, positively charged nanowires efficiently bind cfDNA even at low concentrations. In particular, the large surface area for binding to target molecules such as DNA can be successfully captured. Nanowire features such as enhanced mobility facilitate interaction with DNA. This allows for effective collection of cfDNA.

[0038] As used herein, the term "marker" refers to a double helix structure derived from a cancer cell. A substance for effectively detecting and / or quantifying cfDNA having , quantum dots, substances that decompose specific substrates to produce color reactions, and irradiation with specific wavelengths Specifically, the marker may be a substance that emits light when exposed to light. P (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), RFP(Red Fluorescent Protein) or CFP (Cyan Fluorescent Protein) ) or the marker can be a fluorescent protein such as alkaline phosphatase (alkaline phosphatase, AP), HRP (Horseradis h peroxidase), or beta-galactosidase (β-galactos The enzyme may be a chromogenic or bioluminescent enzyme such as BGAL.

[0039] The chromogenic enzyme mediates a color- or luminescent reaction by reacting with a substrate. Such substrates include ABTS, OPD, AmplexRed, DAB, AEC, TMB, homovanillic acid, and luminol can be used. In the case of AP, BCI P(5-Bromo-4-Chloro-3-Indolyl Phosphate) / NBT (nitriblue tetrazolium), pNPP (p-Nitrop henyl Phosphate), Fast Red TR / Naphthol AS -MX and CDP-Star (Disodium 2-chloro-5-(4-methyl thoxyspiro[1,2-dioxetane-3,2'-(5-chlorot ricyclo[3.3.1.1 3.7 ]decan])-4-yl]-1-phenyl l phosphate) can be used, and in the case of BGAL, X-gal (5-bromo o-4-chloro-3-indolyl-β-d-galactopyranosi de) or ONPG(ortho-Nitrophenyl-β-galactosi) Any one substrate selected from the group consisting of Luminescent enzymes are derived from the enzymes found in fireflies (Photinus pyralis) and sea pansies (Renil la sp.), the copepod Metridia longa, and the bib Vibrio bacteria or dinoflagellate The luciferase may be a luciferase of the type I or II.

[0040] The marker may additionally include a substance that can bind to the probe. If biotin is bound to the probe, the marker may further comprise an avidin-based protein. Specifically, the marker may include avidin, streptavidin, avidin (streptavidin) or a combination thereof It may further include any one of the following:

[0041] The marker may also include biotin. In such a case, the probe may be avidin. Specifically, the marker may further comprise avidin (avidin). idin, streptavidin, or a combination thereof The compound may further comprise any one selected from the group consisting of:

[0042] One example of such a marker is a marker composed of a conductive polymer and hyaluronic acid. It can be used in the form of nanoparticles bound with streptavidin and HRP. In this case, the conductive polymer is as described above, and preferably polypyrrole. Still another specific example is a conductive polymer and hyaluronic acid. It is used in the form of nanoparticles to which streptavidin and fluorescent proteins are bound. The size of the HRP nanoparticles is about 20 nm to about 150 nm, and the size of the HRP nanoparticles is about 30 nm to about 150 nm. The HRP nanoparticles may have a particle size of about 0 nm to about 120 nm, or about 40 nm to about 100 nm. The diameter of the crystal is about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, or about 80 nm. It can be m.

[0043] In addition, a substrate compatible with the marker can be used to induce a color reaction of the marker. In this case, the substrate can be added simultaneously with the marker, but it is also possible to add the substrate after adding the marker. The marker and the substrate can be added before or after the reaction. As a specific example, when HRP is used as a marker, ABTS(2,2 '-Azinobis[3-ethylbenzothiazoline-6-sulf onic acid]-diammonium salt), OPD(o-Phenyl enediamine dihydrochloride), AmplexRed, DA B(3,3'-diaminobenzidine tetrahydrochlori de), AEC(3-Amino-9-ethylcarbazole), TMB(3, 3',5,5'-Tetramethylbenzidine), homovanillic acid or Luminol can be used as a substrate. When using a specific wavelength of light, the presence of light emitted after irradiation with light of a specific wavelength, rather than the substrate, is important. The marker can be detected by its presence or absence.

[0044] According to one embodiment, the diagnostic method detects target cfDNA with high precision and accuracy. This is effective even when the biological sample contains only trace amounts of target cfDNA. Therefore, it can be useful for detecting cancer cells in the early stages.

[0045] Identifying specific abnormal cells / tissues present in a biological sample, such as specific cancer biomarkers By detecting the presence or absence of cfDNA, the cancer can be diagnosed. sis, prognosis, or metastasis status It is possible to identify resistance / tolerance to conventional treatment methods. It is also possible to predict the tolerance.

[0046] <Prostate cancer> How prostate cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the prostate cancer cells. The method for diagnosing prostate cancer involves detecting genes derived from the cfDNA, and The probes having sequences complementary to genes known to be biomarkers for prostate cancer are The present invention provides a method for diagnosing prostate cancer by detecting a known biomarker for prostate cancer. The selected gene can be a gene that encodes a protein that is overexpressed in prostate cancer.

[0047] Specifically, the probes having a sequence complementary to the cfDNA include KLK3, FOLH1, PCA3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR 2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TM A small number selected from the group consisting of EFF2, TMPRSS2-ERG, and combinations thereof The gene may be one that binds complementarily to at least one of the genes.

[0048] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0049] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0050] In addition, nanowires labeled with avidin-based proteins were placed in a PEI solution containing biotin. The nanowires were mixed with cations via the interaction of biotin-avidin proteins. Addition of cationic branched polyethyleneimine (PEI) As a result, polyethyleneimine, a cationic polymer, is displayed. Nanoparticles are densely packed and irregularly arranged in the surface-bound nanostructure (PEI / mPpy NW). can be embedded in a distributed manner.

[0051] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0052] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of prostate cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in prostate cancer cells. The nucleic acid sequence that is overexpressed in cancer cells exhibits an appropriate expression level in normal cells. refers to a nucleic acid sequence that is overexpressed in certain cancer cells.

[0053] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The degree or standard of the effect can be determined by measuring the absorbance using a marker and then plotting a receiver operating characteristic curve. The OD value determined by the maximum sensitivity and specificity can be used as a standard. The wavelength of light to be irradiated for the measurement of cfD can be appropriately determined by the marker. NA can be double-stranded unwinding of DNA In addition, cfDNA can be determined appropriately depending on the purpose.

[0054] In addition, the cfDNA derived from the prostate cancer cells is i) a double helix derived from normal cells. ii) have a lower Tm value compared to cfDNA with the structure, or ii) are derived from normal cells The compound is characterized by being denatured under conditions in which cfDNA having a double helix structure is not denatured. It is possible.

[0055] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0056] The gene overexpressed in the prostate cancer cells was KLK3 (NCBI Gene ID: 35 4), FOLH1 (NCBI Gene ID:2346), ACPP (NCBI Ge ne ID:55), PCA3(NCBI Gene ID:50652), PDE4D 7(NCBI Gene ID:5144), SFMBT2(NCBI Gene ID:5144) :57713), EFEMP1(NCBI Gene ID:2202), RETN(N CBI Gene ID:56729), ACADL(NCBI Gene ID:33 ), AGR2 (NCBI Gene ID:10551), COL1A1 (NCBI G ene ID:1277), FAM13C(NCBI Gene ID:220965) , GPX8(NCBI Gene ID:493869), GRHL2(NCBI Ge ne ID:79977), HNF1A(NCBI Gene ID:6927), HO XB13 (NCBI Gene ID:10481), KLK2 (NCBI Gene ID:3817), MYBPC1(NCBI Gene ID:4604), NR0B1 (NCBI Gene ID:190), PITX2(NCBI Gene ID:53 08), SFRP4 (NCBI Gene ID:6424), SLCO1B3 (NCB I Gene ID:28234), TMEFF2(NCBI Gene ID:236 71), CPT1A (NCBI Gene ID:1374), IFNG (NCBI G ene ID:3458), CD274(NCBI Gene ID:29126), F OLR1 (NCBI Gene ID:2348), EPCAM (NCBI Gene ID:4072), OGT(NCBI Gene ID:8473), TMPRSS2- It may be any one selected from the group consisting of ERG and combinations thereof.

[0057] As a specific example, genes that are specifically present in prostate cancer include KLK3, FOLH1, and PC A3, PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, COL1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KL K2, MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEF F2, TMPRSS2-ERG gene, and additionally ACPP, CPT1A, IFN G, CD279, CD274, ERBB2, EGFR, FOLR1, and EPCAM and diagnosing prostate cancer by additionally detecting one or more additional marker genes selected from the group consisting of: The additional marker gene may be a prostate cancer specific marker gene. However, when used in combination with the gene specifically present in prostate cancer, This can significantly improve the sensitivity and specificity of cancer diagnostic methods.

[0058] As used herein, the term "KLK3" refers to kallikrein-3, ga mma-seminoprotein or prostate-specific antigen The PSA gene is a gene encoding the prostate specific antigen (PSA). is a proteolytic enzyme synthesized in the epithelial cells of the prostate gland and is rarely found in tissues other than the prostate gland. It is rarely expressed and is a useful tumor marker used in the selection of prostate cancer. PSA is useful not only for screening for prostate cancer but also for determining recurrence after surgery. do.

[0059] As used herein, the term "FOLH1" refers to a prostate-specific cell membrane antigen (proLAP). state-specific membrane antigen (PSMA) PSMA is a gene that is highly expressed in the prostate and is expressed in prostate cancer cells. It is known that PSMA expression increases approximately 8 to 12 times compared to normal prostate cells. PSMA is used as a tumor marker for diagnosing prostate cancer. do.

[0060] As used herein, the term "ACPP" refers to prostatic acid phosphatase (P The gene encoding prostatic acid phosphatase (PAP) The PAP is an enzyme produced in the prostate. Its expression appears to be increased in men with cancer or prostate disease, suggesting that prostate cancer It is also used as an indicator of prostate disease.

[0061] As used herein, the term "PCA3" refers to a non-co-expressing protein in human prostate tissue. The PCA3 gene is expressed in the form of ding RNA. PCA3 is expressed only in the prostate and is highly overexpressed in prostate cancer cells. It is used as a tumor marker for cancer.

[0062] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence such that the sequence is complementary to the cfDNA. The antibody is a nucleic acid molecule that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a biotin (biotin The probe may be a marker bound to a biotin-binding protein. Can be combined with Carr.

[0063] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), BFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0064] The marker may be bound to a biotin-binding protein. The protein is avidin, an avidin-based protein. , streptavidin, traptavidin or neutravidin A protein that can specifically bind to biotin can be neutravidin. Any substance can be used without limitation. It may be leptavidin-linked.

[0065] As used herein, the term "avidin" refers to the protein found in the oviducts of birds, reptiles and amphibians. It is a homotetrameric protein produced in egg white and has a high affinity for biotin. Its function in nature has not yet been elucidated, but it binds to biotin, which is essential for bacterial growth. It is believed that its use is to inhibit bacterial growth by binding to

[0066] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A strain of Streptomyces avidinii with a molecular weight of approximately 60kD a tetrameric biotin-binding protein of the formula a. It has very low homology to the avidin However, its structure is very similar. Like avidin, it has antibacterial activity and acts in response to biotin. It has extremely high binding strength. Unlike avidin, it does not contain carbohydrates and is an acidic isoelectric It has a low solubility (pI=5) and is significantly lower than avidin. It is commercially available. Suitable streptavidin, e.g., Thermo Scientific Pierce Streptavidin is a recombinant form of streptavidin with a molecular weight of approximately 53 kDa. Streptavidin has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI of ribosomal IgG results in a lower level of nonspecific binding (especially These characteristics of streptavidin allow it to have a wide range of detection systems. This makes it an ideal reagent for stem cell selection.

[0067] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0068] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (approximately 60 kDa) compared to IgG, yet maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. The amount of ATP is reduced, lowering the isoelectric point (pI = approximately 6.3) to prevent nonspecific binding to avidin. Effectively eliminates the cause. Lysine residues remain available, so streptavidin It can be easily derivatized and conjugated like biotin. Its combined activity and low nonspecific binding make it an ideal biotin-binding protein. It can be used.

[0069] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of the marker is detected by observing the light emitted after irradiating it with light of a certain wavelength. Moreover, when the marker is luciferase, it is possible to use a substrate such as luciferin. The presence of the marker was confirmed by measuring the bioluminescence that appeared after the addition of the marker using a bioluminometer. It can be detected.

[0070] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures the prostate cancer-derived cfDNA. This can be done so that only the cfDNA that is present can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can be done for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds or In one embodiment, the denaturing step may be performed for about 90 seconds. can be carried out before step c).

[0071] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0072] Prostate cancer diagnostic kit Another aspect of the present invention is a method for producing a prostate cancer-specific gene-specific biotin-binding protein. a binding probe; a positively charged substance; an avidin-based protein-bound marker; and and providing a diagnostic kit for prostate cancer including instructions.

[0073] In this case, the genes specifically expressed in prostate cancer are KLK3, FOLH1, and PCA3. , PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, CO L1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2 , MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2 TMPRSS2-ERG, TMPRSS2-ERG, and combinations thereof. There can be more than one.

[0074] The instructions also state that the kit is configured to treat prostate cancer using the following protocol: The kit may be described as capable of diagnosing: a) from a biological sample isolated from an individual; b) Isolate cfDNA using a positively charged substance contained within the The cfDNA was then subjected to the biotin-conjugated probe and the primers included in the kit. c) probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0075] Also, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, OGT and combinations thereof. A complementary binding biotin-conjugated probe may additionally be included.

[0076] The probe, positively charged substance, and marker are as described above.

[0077] Prostate cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in prostate cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. d) a detection unit for detecting the marker; and e) a detection unit for detecting the marker based on whether or not the marker is detected. If the sample contains cfDNA that has a sequence complementary to the probe and is derived from prostate cancer, and determining a gene derived from a prostate cancer cell from the sample without amplification. The present invention provides a device for detecting and diagnosing prostate cancer.

[0078] In this case, the genes specifically expressed in prostate cancer are KLK3, FOLH1, and PCA3. , PDE4D7, SFMBT2, EFEMP1, RETN, ACADL, AGR2, CO L1A1, FAM13C, GPX8, GRHL2, HNF1A, HOXB13, KLK2 , MYBPC1, NR0B1, PITX2, SFRP4, SLCO1B3, TMEFF2 TMPRSS2-ERG, TMPRSS2-ERG, and combinations thereof. There can be more than one.

[0079] Also, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, OGT and combinations thereof. A complementary binding biotin-conjugated probe may additionally be included.

[0080] <Lung cancer> How lung cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, The method for diagnosing lung cancer comprises detecting a gene of the present invention and using a nucleic acid sequence complementary to the cfDNA. The probe binds to genes known as lung cancer biomarkers, and detects lung cancer. In this case, a gene known as a lung cancer biomarker is used to diagnose lung cancer. The gene may be a gene encoding a protein that is overexpressed in the host.

[0081] Specifically, the probes having a sequence complementary to the cfDNA include ENO2, SART3, KRT19, PLAT, EGFR, ALK, ROS1, RET, ERBB2, PI3K, S100P, MMP11, CDCA7, S100A2, ETV4, TOP2A, UBE2 C and combinations thereof. It may be one that binds complementarily.

[0082] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. In one embodiment, nitrocellulose, which has a strong affinity for nucleic acids, can be used. Nitrocellulose membrane can be used. In addition, as a specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a filter, a mesh structure, or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure. Alternatively, it may be a positively charged membrane.

[0083] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0084] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0085] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0086] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of lung cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in lung cancer cells. The nucleic acid sequence overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but is not expressed at a specific level in normal cells. It means a nucleic acid sequence that is overexpressed in cancer cells. Specifically, the nucleic acid sequence is overexpressed in cancer cells. The degree or cutoff for the absorbance (optical density) using the marker is When the density is measured, the OD value may be about 0.010 or more. Specifically, the degree or standard of overexpression of the nucleic acid sequence in cancer cells is determined using a marker. When measuring absorbance, the OD value is about 0.012 or about 0.015 or more. In this case, the wavelength of light to be irradiated for measuring the absorbance can be appropriately determined depending on the marker. The cfDNA is a DNA in which the double strand is unwound (unwinding o cfDNA can be determined appropriately depending on the purpose.

[0087] In addition, the cfDNA derived from the lung cancer cells has i) a double helix structure derived from normal cells. or ii) have a lower Tm value compared to cfDNA derived from normal cells. It is characterized by being denatured under conditions where cfDNA having a double helix structure does not denature. Possible.

[0088] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C (v) Heating conditions: about 25°C to about 40°C for 10 seconds to about 30 minutes at about 75°C to about 75°C; vi) heating for 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; and and vii) treating with DNase for about 1 minute to about 30 minutes.

[0089] The gene overexpressed in the lung cancer cells was ENO2 (NCBI Gene ID: 2026 ), SART3(NCBI Gene ID:9733), ACPP(NCBI Gen e ID:55), KRT19(NCBI Gene ID:3880), PLAT(N CBI Gene ID:5327), EGFR(NCBI Gene ID:1956) ), KRAS (NCBI Gene ID:3845), ALK (NCBI Gene ID:238), ROS1(NCBI Gene ID:6098), RET(NCBI Gene ID:5979), ERBB2(NCBI Gene ID:2064), PI3K (NCBI Gene ID:5291), S100P (NCBI Gene ID:6286), MMP11(NCBI Gene ID:4320), CDCA7( NCBI Gene ID:83879), S100A2(NCBI Gene ID: 6273), ETV4(NCBI Gene ID:2118), TOP2A(NCBI Gene ID:7153), UBE2C(NCBI Gene ID:11065) , CPT1A(NCBI Gene ID:1374), IFNG(NCBI Gene ID:3458), CD274(NCBI Gene ID:29126), FOLR 1(NCBI Gene ID:2348), EPCAM(NCBI Gene ID: 4072), OGT (NCBI Gene ID: 8473), and combinations thereof It can be any one selected from the group consisting of:

[0090] As a specific example, genes that are specifically present in lung cancer include SART3, PLAT, ALK, ROS1, PI3K, S100P, CDCA7, S100A2, and ETV4 genes. Additionally, ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2 , MMP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD27 4. Detecting ERBB2, EGFR, FOLR1, or EPCAM genes to diagnose lung cancer It is possible.

[0091] As used herein, the term "ENO2" refers to Gamma-enolase or Enolase 2 or NSE (neuron-specific enolase) NSE refers to a gene encoding an enzyme known to cause small cell lung cancer (SLC). single cell lung cancer, neuroblastoma ), tumor markers of medullary thyroid cancer It is used as a car.

[0092] As used herein, the term "SART3" refers to the squamous cell carcinoma antigen (Squamo The gene encoding the HIV-1 cell carcinoma antigen (SCCA) SCCA refers to a gene that causes not only squamous cell carcinoma of the cervix, but also cancer of the vulva and vagina. Many squamous cell carcinoma patients, including those with esophageal, tongue, and pharyngeal cancer, test positive in their blood. It is used as a tumor marker.

[0093] As used herein, the term "KRT19" refers to Cyfra21-1, CK-19 (cytokeratin-19) or K19 (keratin-19) Cyfra21-1 refers to a gene encoding a protein. It is known to be associated with cancers originating from epithelial cells, such as head and neck cancer. yfra21-1 is expressed in higher blood levels in patients with pneumonia or lung disease than in normal individuals. It has been reported that it can be used as a tumor marker.

[0094] As used herein, the term "PLAT" refers to a protein involved in the breakdown of blood mochi. It encodes the Tissue plasminogen activator (TPA). This refers to genes that are present.

[0095] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0096] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0097] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without any restrictions. In one embodiment, the marker is bound to streptavidin. Possible.

[0098] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. It has a pI of approximately 5 and significantly lower solubility than avidin. It is commercially available. Suitable streptavidin, e.g., Thermo Scientific Pierce Streptavidin is a recombinant form of streptavidin with a molecular weight of approximately 53 kDa. It is a ubiquitin and has a near-neutral isoelectric point (pI = about 6.8 to about 7.5). The lack of glycosylation and low pI of avidin result in a lower level of nonspecific binding (especially , lectin binding). These characteristics of streptavidin allow for many detection This makes it an ideal reagent for the system.

[0099] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0100] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI = approximately 6.3) and eliminates the main cause of nonspecific binding to avidin. Lysine residues remain available, effectively removing the causative agent. It can be easily derivatized and conjugated like biotin, and has high biotin binding capacity. Its high potency and low nonspecific binding make it an ideal biotin-binding protein for a wide range of applications. You can be there.

[0101] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of the marker is detected by observing the light emitted after irradiating it with light of a certain wavelength. Moreover, when the marker is luciferase, it is possible to use a substrate such as luciferin. The presence of the marker was confirmed by measuring the bioluminescence that appeared after the addition of the marker using a bioluminometer. It can be detected.

[0102] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures lung cancer-derived cDNA. This can be done so that only fDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one embodiment, the cfDNA can be denatured for 1 second. This can be done before step c).

[0103] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Leave at about 90 to 95°C for about 1 second to 1 minute iii) Heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) Heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes ) heating at about 60°C to about 75°C for about 1 minute to about 30 minutes; v) heating at about 25°C to about 40°C for about Heating for 5 to 60 minutes; vi) Treatment with protease for 1 to 10 minutes and vii) treatment with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: does not denature double-stranded cfDNA derived from normal cells, but denatures cfDNA derived from cancer cells. By selectively denaturing only the NA, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions i) to vii) are used after collecting cfDNA bound to a positively charged substance. In addition, the denaturing conditions i) to vii) can be determined by the temperature, protease, and The DNase treatment time can be adjusted as needed as long as it does not denature stable cfDNA. do.

[0104] Lung cancer diagnostic kit Another aspect of the present invention is a biotin-binding protein that binds complementary to a gene specifically expressed in lung cancer. Probe; positively charged substance; avidin-based protein-bound marker; and description The present invention provides a lung cancer diagnostic kit including a diagnostic kit for lung cancer.

[0105] In this case, the genes specifically expressed in lung cancer are SART3, PLAT, ALK, RO S1, PI3K, S100P, CDCA7, S100A2, ETV4, and combinations thereof The combination may be any one or more selected from the group consisting of:

[0106] The instructions also state that the kit is configured to diagnose lung cancer according to the following protocol: a) from a biological sample isolated from an individual into a kit b) Isolate cfDNA using a positively charged substance; cfDNA was analyzed using the biotin-conjugated probe and marker included in the kit. c) probes that do not bind to cfDNA and removing the marker; and d) detecting a signal of said marker.

[0107] Also ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, M MP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from the group The device may additionally include a

[0108] The probe, positively charged substance, and marker are as described above.

[0109] Lung cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in lung cancer; and and nanoparticles containing streptavidin and a marker are added sequentially or simultaneously. a response unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker by detecting the presence or absence of the marker. The sample contains a sequence complementary to the probe, and it is determined that cfDNA derived from lung cancer is present. The information processing unit detects genes derived from lung cancer cells from the sample without amplification, and determines whether the lung The present invention aims to provide a device for diagnosing cancer.

[0110] In this case, the genes specifically expressed in lung cancer are SART3, PLAT, ALK, RO S1, PI3K, S100P, CDCA7, S100A2, ETV4, and combinations thereof The combination may be any one or more selected from the group consisting of:

[0111] Also ENO2, ACPP, KRT19, EGFR, KRAS, RET, ERBB2, M MP11, TOP2A, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, or EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from the group The device may additionally include a

[0112] <Thyroid cancer> How thyroid cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the marker comprising: This method diagnoses thyroid cancer by detecting genes derived from the cfDNA. The probes with sequences complementary to genes known to be biomarkers for thyroid cancer were used. The present invention provides a method for diagnosing thyroid cancer by detecting a thyroid cancer biomarker known to be a thyroid cancer biomarker. The selected gene may be a gene encoding a protein that is overexpressed in thyroid cancer.

[0113] Specifically, the probes having a sequence complementary to the cfDNA include TG, CALCA, and AP. At least one selected from the group consisting of OC1, HIG2, and combinations thereof It may be one that binds complementary to one gene.

[0114] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0115] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0116] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0117] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0118] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of thyroid cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in thyroid cancer cells. The nucleic acid sequence that is overexpressed in cancer cells exhibits an appropriate expression level in normal cells. refers to a nucleic acid sequence that is overexpressed in specific cancer cells.

[0119] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0120] Furthermore, the cfDNA derived from the thyroid cancer cells is i) a double helix derived from normal cells. ii) have a lower Tm value compared to cfDNA with the structure, or ii) are derived from normal cells The compound is characterized by being denatured under conditions in which cfDNA having a double helix structure is not denatured. It is possible.

[0121] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0122] The gene overexpressed in the thyroid cancer cells was ACPP (NCBI Gene ID: 55 ), ENO2(NCBI Gene ID:2026), TG(NCBI Gene I D:7038), CALCA(NCBI Gene ID:796), APOC1(NC BI Gene ID:341), HIG2(NCBI Gene ID:29923) , TYRO3(NCBI Gene ID:7301), CPT1A(NCBI Gen e ID:1374), IFNG (NCBI Gene ID:3458), CD274 (NCBI Gene ID:29126), FOLR1(NCBI Gene ID: 2348), EPCAM (NCBI Gene ID: 4072), and combinations thereof The compound may be any one selected from the group consisting of:

[0123] As a specific example, genes that are specifically present in thyroid cancer include TG, CALCA, and APOC 1, HIG2 gene, and additionally ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1 or EPCA Thyroid cancer can be diagnosed by detecting the M gene.

[0124] As used herein, the term "TG" refers to thyroglobulin. , Tg) is a gene encoding the thyroglobulin. When thyroid cancer develops or metastasizes, the level of thyroglobulin in the blood increases. The blood thyroglobulin level is used as a marker for thyroid cancer.

[0125] As used herein, the term "CALCA" refers to calcitonin gene-related peptide. (calcitonin gene-related peptide) It means a gene that

[0126] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0127] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0128] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0129] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0130] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0131] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0132] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to use a group such as luciferin. The presence of the marker was confirmed by measuring the bioluminescence that appeared after adding the substrate using a bioluminometer. can be detected.

[0133] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but rather denatures the denatured DNA. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. You can also change the time for about 1 second, 5 seconds, 10 seconds, 30 seconds, 60 seconds, or 90 seconds. In one embodiment, the step of denaturing the cfDNA can be performed by the step c). It can be done in front of the floor.

[0134] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0135] Thyroid cancer diagnostic kit Another aspect of the present invention is a method for producing a thyroid cancer-specific gene-specific biotin-binding protein. a binding probe; a positively charged substance; an avidin-based protein-bound marker; and and providing a thyroid cancer diagnostic kit including instructions.

[0136] In this case, the genes specifically expressed in thyroid cancer are TG, CALCA, APOC1, HIG2, and combinations thereof. .

[0137] The instructions also state that the kit is configured to treat thyroid cancer using the following protocol: The kit may be described as capable of diagnosing: a) from a biological sample isolated from an individual; b) Isolate cfDNA using a positively charged substance contained within the The cfDNA was then subjected to the biotin-conjugated probe and the primers included in the kit. c) probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0138] Also, ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD27 4. ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from the group The device may additionally include a

[0139] The probe, positively charged substance, and marker are as described above.

[0140] Thyroid cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in thyroid cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. d) a detection unit for detecting the marker; and e) a detection unit for detecting the marker based on whether or not the marker is detected. If the sample contains cfDNA that has a sequence complementary to the probe and is derived from thyroid cancer, and determining, from the sample, genes derived from thyroid cancer cells without amplification. The objective of the present invention is to provide a device for detecting and diagnosing thyroid cancer.

[0141] In this case, the genes specifically expressed in thyroid cancer are TG, CALCA, APOC1, HIG2, and combinations thereof. .

[0142] Also, ENO2, ACPP, TYRO3, CPT1A, IFNG, CD279, CD27 4. ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from the group The device may additionally include a

[0143] <Bladder cancer> How Bladder Cancer is Diagnosed

[0144] One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting said marker from said sample without amplification, said marker being derived from bladder cancer cells. A method for diagnosing bladder cancer by detecting a gene encoding the cfDNA, The probe binds to a gene known as a bladder cancer biomarker. A method for diagnosing bladder cancer is provided, in which genes known to be biomarkers for bladder cancer are used. may be a gene encoding a protein that is overexpressed in bladder cancer.

[0145] Specifically, the probes having a sequence complementary to the cfDNA include OGT, FGFR3, T P53, NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC176 24, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE 3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L , HOXA9, ISL1, ALDH1A3, and combinations thereof. The gene may be one that binds complementarily to at least one of the genes.

[0146] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0147] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0148] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0149] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0150] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of bladder cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in bladder cancer cells. The nucleic acid sequence that is overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but It refers to a nucleic acid sequence that is overexpressed in specific cancer cells.

[0151] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0152] Furthermore, the cfDNA derived from the bladder cancer cells has i) a double helix structure derived from normal cells. ii) have a lower Tm value compared to cfDNA with the same structure, or ii) are derived from normal cells It is characterized by being denatured under conditions where cfDNA with a double helix structure does not denature. It's possible.

[0153] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0154] The gene overexpressed in the bladder cancer cells was OGT (NCBI Gene ID: 8473 ), FGFR3(NCBI Gene ID:2261), TP53(NCBI Gen e ID:7157), NUMA1(NCBI Gene ID:4926), KRT1 9(NCBI Gene ID:3880), COCH(NCBI Gene ID:1 690), CELSR3 (NCBI Gene ID:1951), HMOX1 (NCB I Gene ID:3162), KIF1A(NCBI Gene ID:547), MGC17624(NCBI Gene ID:404550), MTAP(NCBI Gene ID:4507), PFKFB4(NCBI Gene ID:5210), S100A8 (NCBI Gene ID:6279), RSPH9 (NCBI Gen e ID:221421), CCNB1(NCBI Gene ID:891), FOX M1(NCBI Gene ID:2305), FANCB(NCBI Gene ID:2305) :2187), FANCC(NCBI Gene ID:2176), FANCD2(N CBI Gene ID:2177), RUSC1-AS1(NCBI Gene ID :284618), CACNA1B(NCBI Gene ID:774), IMP-1 (NCBI Gene ID:10642), PDE3A(NCBI Gene ID: 5139), POU3F4 (NCBI Gene ID:5456), SOX3 (NCB I Gene ID:6658), DMC1(NCBI Gene ID:11144) , PLXDC2 (NCBI Gene ID:84898), ZNF312 (NCBI Gene ID:55079), SYCP2L(NCBI Gene ID:22171) 1), HOXA9 (NCBI Gene ID:3205), ISL1 (NCBI Ge ne ID:3670), ALDH1A3(NCBI Gene ID:220), CP T1A(NCBI Gene ID:1374), IFNG(NCBI Gene ID:1374) :3458), CD274(NCBI Gene ID:29126), FOLR1(N CBI Gene ID:2348), EPCAM(NCBI Gene ID:407 2) and combinations thereof.

[0155] As a specific example, genes that are specifically present in bladder cancer include OGT, FGFR3, and TP53. , NUMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MTAP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FAN CC, FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, POU3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HO XA9, ISL1, ALDH1A3 genes, and additionally KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1 Bladder cancer can be diagnosed by detecting the EPCAM gene.

[0156] As used herein, the term "OGT" refers to O-GlcNAc transfer It refers to the gene that encodes the enzyme.

[0157] As used herein, the term "FGFR1" refers to fibroblast growth factor receptor 1 (fGFR1). It encodes inflammatory cytokines (inflammatory cytokines) and cytokine receptor 1 (IL-1). This refers to genes that are present.

[0158] As used herein, the term "NUMA1" refers to NMP22 (nuclear m The gene encoding NMP2 (atrix protein-22) is 2 has been found in higher than normal levels in the urine of patients with certain types of cancer, including bladder cancer. It is widely used as a bladder cancer marker.

[0159] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0160] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0161] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without any restrictions. In one embodiment, the marker is bound to streptavidin. Possible.

[0162] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding force. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point ( It has a pI of 5 and significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce St Reptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. and has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of affinity and low pI result in lower levels of nonspecific binding (especially with lectins) compared to avidin. Such characteristics of streptavidin make it possible to develop a detection system with many It can be chosen as an ideal reagent.

[0163] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0164] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI = 6.3) and eliminates the main cause of nonspecific binding to avidin. Lysine residues remain available, so streptavidin It can be easily derivatized or conjugated as shown above. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0165] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to use a group such as luciferin. The presence of the marker was confirmed by measuring the bioluminescence that appeared after adding the substrate using a bioluminometer. can be detected.

[0166] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures bladder cancer-derived cfDNA. This can be done so that only cfDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be set to about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 9 seconds. In one embodiment, the cfDNA can be denatured for 0 seconds. This can be done before step c).

[0167] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0168] Bladder cancer diagnostic kit Another aspect of the present invention is a method for producing a bladder cancer-specific gene-specific biotin-binding protein. a positively charged substance; an avidin-based protein-bound marker; and The present invention provides a bladder cancer diagnostic kit including a diagnostic kit for bladder cancer.

[0169] In this case, the genes specifically expressed in bladder cancer are OGT, FGFR3, TP53, N UMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MT AP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC , FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, PO U3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA 9, ISL1, ALDH1A3, and any combination thereof There can be one or more.

[0170] The instructions also state that the kit is configured to diagnose bladder cancer according to the following protocol: a) from a biological sample isolated from an individual in a kit; b) separate the cfDNA using a positively charged substance contained in the The cfDNA was then subjected to the biotin-conjugated probe and markers included in the kit. c) Mix probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0171] Also, KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ER BB2, EGFR, FOLR1, EPCAM, and combinations thereof Add a biotin-conjugated probe that binds complementary to at least one gene that is identified. It can be included essentially.

[0172] The probe, positively charged substance, and marker are as described above.

[0173] Bladder cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in bladder cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. a reaction unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker. The sample contained cfDNA with a sequence complementary to the probe, indicating the presence of bladder cancer-derived cfDNA. and detecting a gene derived from a bladder cancer cell from the sample without amplification, the gene comprising: an information processing unit for detecting a gene derived from a bladder cancer cell from the sample without amplification; The present invention aims to provide a device for diagnosing bladder cancer using a bladder cancer diagnostic system.

[0174] In this case, the genes specifically expressed in bladder cancer are OGT, FGFR3, TP53, N UMA1, COCH, CELSR3, HMOX1, KIF1A, MGC17624, MT AP, PFKFB4, S100A8, RSPH9, FOXM1, FANCB, FANCC , FANCD2, RUSC1-AS1, CACNA1B, IMP-1, PDE3A, PO U3F4, SOX3, DMC1, PLXDC2, ZNF312, SYCP2L, HOXA 9, ISL1, ALDH1A3, and any combination thereof There can be one or more.

[0175] Also, KRT19, CCNB1, CPT1A, IFNG, CD279, CD274, ER BB2, EGFR, FOLR1, EPCAM, and combinations thereof Add a biotin-conjugated probe that binds complementary to at least one gene that is identified. It can be included essentially.

[0176] <Breast cancer> How breast cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting said marker from said sample without amplification, said marker being derived from breast cancer cells. The method for diagnosing breast cancer is to detect a gene of the cfDNA, and to detect a gene of the cfDNA having a sequence complementary to the cfDNA. The probe binds to genes known as breast cancer biomarkers, and detects breast cancer. In this case, genes known as biomarkers for breast cancer are used to diagnose breast cancer. The gene may be a gene encoding a protein that is overexpressed in the host.

[0177] Specifically, the probes having a sequence complementary to the cfDNA include MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6 ST, MGP, TRBC1, MMP11, COL10A1, C10orf64, COL1 1A1, POTEG, FSIP1, HER2, and combinations thereof The gene may be one that binds complementarily to at least one of the genes.

[0178] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0179] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0180] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0181] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0182] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from genes of breast cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in breast cancer cells. The nucleic acid sequence overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but is not expressed at a specific level in normal cells. It refers to a nucleic acid sequence that is overexpressed in cancer cells.

[0183] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0184] In addition, the cfDNA derived from the breast cancer cells has i) a double helix structure derived from normal cells. or ii) have a lower Tm value compared to cfDNA derived from normal cells. It is characterized by being denatured under conditions where cfDNA having a double helix structure does not denature. Possible.

[0185] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0186] The gene overexpressed in the breast cancer cells was MUC1 (NCBI Gene ID: 4582 ), ACPP(NCBI Gene ID:55), MEST(NCBI Gene I D:4232), TYRO3(NCBI Gene ID:7301), NR1D1(N CBI Gene ID:9572), UBE2C(NCBI Gene ID:110) 65), BIRC5 (NCBI Gene ID:332), RACGAP1 (NCBI Gene ID:29127), DHCR7(NCBI Gene ID:1717) , STC2(NCBI Gene ID:8614), AZGP1(NCBI Gene ID:563), RBBP8(NCBI Gene ID:5932), IL6ST( NCBI Gene ID:3572), MGP(NCBI Gene ID:4256) ), TRBC1 (NCBI Gene ID:28639), MMP11 (NCBI G ene ID:4320), COL10A1(NCBI Gene ID:1300), C10orf64 (NCBI Gene ID:57705), COL11A1 (NCB I Gene ID:1301), POTEG(NCBI Gene ID:40478 5), FSIP1 (NCBI Gene ID:161835), HER2 (NCBI Gene ID:2064), CPT1A(NCBI Gene ID:1374), I FNG (NCBI Gene ID:3458), CD274 (NCBI Gene I D:29126), FOLR1(NCBI Gene ID:2348), EPCAM( NCBI Gene ID: 4072) and combinations thereof It can be any one of the following:

[0187] As a specific example, genes that are specifically present in breast cancer include MEST, NR1D1, and BIRC. 5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, M GP, TRBC1, MMP11, COL10A1, C10orf64, COL11A1, POTEG, FSIP1, HER2 genes, and additionally MUC1, ACPP, TY RO3, UBE2C, CPT1A, IFNG, CD279, CD274, ERBB2, E Breast cancer can be diagnosed by detecting the GFR, FOLR1 or EPCAM gene.

[0188] As used herein, the term "MUC1" refers to CA 15-3 (Carcinoma a antigen 15-3) and CA 27-29. CA15-3 is a gene that increases the likelihood of early recurrence of breast cancer. It has been shown to be effective in treating breast cancer and is used as a breast cancer marker.

[0189] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0190] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0191] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without any restrictions. In one embodiment, the marker is bound to streptavidin. Possible.

[0192] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0193] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0194] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3) and eliminates the main cause of nonspecific binding to avidin. Lysine residues remain available, so streptavidin It can be easily derivatized or conjugated as shown above. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0195] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0196] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures breast cancer-derived cDNA. This can be done so that only fDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one embodiment, the cfDNA can be denatured for 2 seconds. c) can be performed before step c.

[0197] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0198] Breast cancer diagnostic kit Another aspect of the present invention is a biotin-binding protein that binds complementary to a gene specifically expressed in breast cancer. Probe; positively charged substance; avidin-based protein-bound marker; and description The present invention provides a breast cancer diagnostic kit including a diagnostic kit for breast cancer.

[0199] In this case, the genes specifically expressed in breast cancer include MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP , TRBC1, MMP11, COL10A1, C10orf64, COL11A1, PO Any of the following selected from the group consisting of TEG, FSIP1, HER2, and combinations thereof There can be one or more.

[0200] The instructions also state that the kit is configured to diagnose breast cancer according to the following protocol: a) from a biological sample isolated from an individual into a kit b) Isolate cfDNA using a positively charged substance; cfDNA was analyzed using the biotin-conjugated probe and marker included in the kit. c) probes that do not bind to cfDNA and removing the marker; and d) detecting a signal of said marker.

[0201] Also, MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD27 9, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin that binds complementary to at least one gene selected from the group consisting of The antibody may additionally comprise a nucleotide-binding probe.

[0202] The probe, positively charged substance, and marker are as described above.

[0203] Breast cancer diagnostic equipment Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in breast cancer; and and nanoparticles containing streptavidin and a marker are added sequentially or simultaneously. a response unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker by detecting the presence or absence of the marker. The sample contains a sequence complementary to the probe, and it is determined that cfDNA derived from breast cancer is present. The information processing unit detects genes derived from breast cancer cells from the sample without amplification, and The present invention aims to provide a device for diagnosing cancer.

[0204] In this case, the genes that are expressed complementarily in breast cancer include MEST, NR1D1, BIRC5, RACGAP1, DHCR7, STC2, AZGP1, RBBP8, IL6ST, MGP , TRBC1, MMP11, COL10A1, C10orf64, COL11A1, PO Any of the following selected from the group consisting of TEG, FSIP1, HER2, and combinations thereof There can be one or more.

[0205] Also, MUC1, ACPP, TYRO3, UBE2C, CPT1A, IFNG, CD27 9, CD274, ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin that binds complementary to at least one gene selected from the group consisting of The antibody may additionally comprise a nucleotide-binding probe.

[0206] <Colon cancer> How colon cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the marker being derived from colon cancer cells. This method detects genes from the cfDNA and diagnoses colorectal cancer. The probes containing the nucleotides bind complementary to genes known to be biomarkers for colon cancer. A method for diagnosing colon cancer is provided, in which genes known to be biomarkers for colon cancer are used. may be a gene encoding a protein that is overexpressed in colon cancer.

[0207] Specifically, the probe having a sequence complementary to the cfDNA is NCKAP1, AUNI P, NOTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD 51AP1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KL K6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6 ST2, EGFL6, CXCL3, CA9, PROX1, SPP1, CST1, CXCL 2, TSTA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPI NB5, TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, P RC1, PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX 38, TTLL2, PRR7, CANP, KIAA010, and their combinations It can be one that complementarily binds to at least one gene selected from the group consisting of .

[0208] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0209] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0210] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0211] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0212] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of colon cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in colorectal cancer cells. The nucleic acid sequence that is overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but It refers to a nucleic acid sequence that is overexpressed in specific cancer cells.

[0213] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0214] Furthermore, the cfDNA derived from the colon cancer cells has i) a double helix structure derived from normal cells. ii) have a lower Tm value compared to cfDNA with the same structure, or ii) are derived from normal cells It is characterized by being denatured under conditions where cfDNA with a double helix structure does not denature. It's possible.

[0215] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0216] The gene overexpressed in the colon cancer cells was ACPP (NCBI Gene ID: 55). , FLU3(NCBI Gene ID:837968), TYRO3(NCBI Ge ne ID:7301), NCKAP1(NCBI Gene ID:10787), A UNIP(NCBI Gene ID:79000), NOTUM(NCBI Gene ID:147111), KRT5(NCBI Gene ID:3852), TUBB (NCBI Gene ID:203068), COL6A1(NCBI Gene I D:1291), JUP (NCBI Gene ID:3728), COTL1 (NCB I Gene ID:23406), CK7(NCBI Gene ID:3855), CK20 (NCBI Gene ID:54474), CDX2 (NCBI Gene ID:1045), MUC2(NCBI Gene ID:4583), MELTF(N CBI Gene ID:4241), SDC2(NCBI Gene ID:6383) ), EFEMP2 (NCBI Gene ID:30008), DEFA5 (NCBI Gene ID:1670), ASB9(NCBI Gene ID:140462), CHEK1 (NCBI Gene ID:1111), MAD2L1 (NCBI Gen e ID:4085), ENC1(NCBI Gene ID:8507), CSE1L (NCBI Gene ID:1434), RAD51AP1(NCBI Gene I D:10635), ERICH3(NCBI Gene ID:127524), SLC 7A11(NCBI Gene ID:23657)、KRT23(NCBI Gene ID:25984)、PLAU(NCBI Gene ID:5328)、CCNB1 (NCBI Gene ID:891)、MELK(NCBI Gene ID:983 3)、CDCA1(NCBI Gene ID:83540)、KLK6(NCBI G ene ID:5653)、CKS2(NCBI Gene ID:1164)、IFI TM1(NCBI Gene ID:8519)、DPEP1(NCBI Gene I D:1800)、CDH3(NCBI Gene ID:1001)、ANLN(NCB I Gene ID:54443)、CXCL1(NCBI Gene ID:2919 )、CTHRC1(NCBI Gene ID:115908)、CEACAM6(NC BI Gene ID:4680)、LCN2(NCBI Gene ID:3934) 、HS6ST2(NCBI Gene ID:90161)、EGFL6(NCBI G ene ID:25975)、CXCL3(NCBI Gene ID:2921)、C A9(NCBI Gene ID:768)、ATAD2(NCBI Gene ID: 29028)、PROX1(NCBI Gene ID:5629)、SPP1(NCB I Gene ID:6696)、CST1(NCBI Gene ID:1469)、 CXCL2(NCBI Gene ID:2920)、TSTA3(NCBI Gene ID:7264)、RRM2(NCBI Gene ID:6241)、MMP3(N CBI Gene ID:4314)、MMP7(NCBI Gene ID:4316 )、MMP10(NCBI Gene ID:4319)、CXCL5(NCBI Ge ne ID:6374), SERPINB5(NCBI Gene ID:5268), TEAD4 (NCBI Gene ID:7004), BUB1 (NCBI Gene ID:699), CDC2(NCBI Gene ID:983), CLDN2(NCB I Gene ID:9075), HSPH1(NCBI Gene ID:10808) ), LY6G6D (NCBI Gene ID:58530), PRC1 (NCBI G ene ID:9055), PUS1(NCBI Gene ID:80324), SQ LE(NCBI Gene ID:6713), TOP2A(NCBI Gene ID:6713) :7153), TTK(NCBI Gene ID:7272), DSCC1(NCBI Gene ID:79075), ECT2(NCBI Gene ID:1894), RNF183 (NCBI Gene ID:138065), FBXO39 (NCBI Gene ID:162517), TEX38(NCBI Gene ID:37497) 3), TTLL2 (NCBI Gene ID:83887), PRR7 (NCBI G ene ID:80758), CANP(NCBI Gene ID:823), KIA A0101(NCBI Gene ID:9768), CPT1A(NCBI Gene ID:1374), IFNG(NCBI Gene ID:3458), CD274( NCBI Gene ID:29126), FOLR1(NCBI Gene ID:2 348), EPCAM (NCBI Gene ID:4072), KRAS (NCBI Gene ID: 3845) and combinations thereof It could be one.

[0217] As a specific example, genes that are specifically present in colorectal cancer include NCKAP1, AUNIP, and N OTUM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFE MP2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51A P1, ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DPEP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2 , EGFL6, CXCL3, CA9, PROX1, SPP1, CST1, CXCL2, T STA3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5 , TEAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1 , PUS1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TTLL2, PRR7, CANP, KIAA010 genes, and additionally ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, SDC2, ASB9 , CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TO P2A, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, E Colon cancer can be diagnosed by detecting the GFR, FOLR1, or EPCAM gene .

[0218] As used herein, the term "FLU3" refers to CA19-9 (Carcinoma This refers to a gene encoding a protein containing the antigen 19-9.

[0219] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0220] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0221] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0222] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0223] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0224] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0225] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0226] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but does denature colon cancer-derived cfDNA. This can be done so that only cfDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be set to about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 9 seconds. In one embodiment, the cfDNA can be denatured for 0 seconds. This can be done before step c).

[0227] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0228] Colon cancer diagnostic kit Another aspect of the present invention is a method for producing a biotin-binding protein that binds complementarily to a gene specifically expressed in colon cancer. a positively charged substance; an avidin-based protein-bound marker; and The present invention aims to provide a diagnostic kit for colorectal cancer, which includes a diagnostic document.

[0229] In this case, the genes specifically expressed in colon cancer are NCKAP1, AUNIP, NOT UM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP 2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1 , ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DP EP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, E GFL6, CXCL3, CA9, PROX1, SPP1, CST1, CXCL2, TST A3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, T EAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, P US1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TT From the group consisting of LL2, PRR7, CANP, KIAA0101 and combinations thereof It may be any one or more selected from the above.

[0230] The instructions also state that the kit is configured to diagnose colon cancer according to the following protocol: a) from a biological sample isolated from an individual in a kit; b) separate the cfDNA using a positively charged substance contained in the The cfDNA was then subjected to the biotin-conjugated probe and markers included in the kit. c) Mix probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0231] In addition, ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, S DC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274 , ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from may additionally include:

[0232] The probe, positively charged substance, and marker are as described above.

[0233] Colon cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in colon cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. a reaction unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker. The sample contained a sequence complementary to the probe, indicating the presence of cfDNA derived from colorectal cancer. and detecting a gene derived from a colon cancer cell from the sample without amplification, the gene comprising: an information processing unit for detecting a gene derived from a colon cancer cell from the sample; The present invention aims to provide a device for diagnosing colorectal cancer using a CT scan.

[0234] In this case, the genes specifically expressed in colon cancer are NCKAP1, AUNIP, NOT UM, KRT5, TUBB, COL6A1, JUP, CDX2, MELTF, EFEMP 2, DEFA5, CHEK1, MAD2L1, ENC1, CSE1L, RAD51AP1 , ERICH3, SLC7A11, KRT23, PLAU, CDCA1, KLK6, DP EP1, CDH3, ANLN, CXCL1, CTHRC1, LCN2, HS6ST2, E GFL6, CXCL3, CA9, PROX1, SPP1, CST1, CXCL2, TST A3, RRM2, MMP3, MMP7, MMP10, CXCL5, SERPINB5, T EAD4, BUB1, CDC2, CLDN2, HSPH1, LY6G6D, PRC1, P US1, SQLE, TTK, ECT2, RNF183, FBXO39, TEX38, TT From the group consisting of LL2, PRR7, CANP, KIAA0101 and combinations thereof It may be any one or more selected from the above.

[0235] In addition, ACPP, FLU3, TYRO3, COTL1, CK7, CK20, MUC2, S DC2, ASB9, CCNB1, MELK, CKS2, IFITM1, CEACAM6, ATAD2, TOP2A, CPT1A, DSCC1, IFNG, CD279, CD274 , ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from may additionally include:

[0236] <Bile duct cancer> How bile duct cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the marker being derived from cholangiocarcinoma cells. This method detects genes from the cfDNA and diagnoses bile duct cancer. The probe binds to a gene known as a biomarker for bile duct cancer. A method for diagnosing bile duct cancer is provided, in which genes known to be biomarkers for bile duct cancer are used. may be a gene encoding a protein that is overexpressed in bile duct cancer.

[0237] Specifically, the probes having sequences complementary to the cfDNA include MUC16, ASH1L, , DOCK70, and combinations thereof. The gene may be complementary to one of the two genes.

[0238] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0239] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0240] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0241] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0242] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of bile duct cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in cholangiocarcinoma cells. The nucleic acid sequence that is overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but It refers to a nucleic acid sequence that is overexpressed in specific cancer cells.

[0243] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0244] Furthermore, the cfDNA derived from the bile duct cancer cells has i) a double helix structure derived from normal cells. ii) have a lower Tm value compared to cfDNA with the same structure, or ii) are derived from normal cells It is characterized by being denatured under conditions where cfDNA with a double helix structure does not denature. It's possible.

[0245] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0246] The gene overexpressed in the cholangiocarcinoma cells is ACPP (NCBI Gene ID: 55). , FLU3(NCBI Gene ID:837968), MUC16(NCBI Ge ne ID:94025), ASH1L(NCBI Gene ID:55870), D OCK7 (NCBI Gene ID:85440), CPT1A (NCBI Gene ID:1374), IFNG(NCBI Gene ID:3458), CD274( NCBI Gene ID:29126), FOLR1(NCBI Gene ID:2 348), EPCAM (NCBI Gene ID:4072), CA125 (NCBI Gene ID:94025), CEACAM5(NCBI Gene ID:104 8) and combinations thereof.

[0247] As a specific example, genes that are specifically present in bile duct cancer include MUC16, ASH1L, and DO CK70 gene, and additionally ACPP, FLU3, CPT1A, DSCC1, IF NG, CD279, CD274, ERBB2, EGFR, FOLR1 or EPCAM genes Cholangiocarcinoma can be diagnosed by detecting the gene.

[0248] As used herein, the term "MUC16" refers to CA-125 (Carcinoma of the CA-125 refers to a gene encoding a soluble antigen (CA-125). , tumor markers or biomarkers that are positive in the blood of some patients with certain types of cancer It is used as a

[0249] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0250] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0251] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0252] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0253] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0254] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0255] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0256] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures cholangiocarcinoma-derived cfDNA. This can be done so that only cfDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be set to about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 9 seconds. In one embodiment, the cfDNA can be denatured for 0 seconds. This can be done before step c).

[0257] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0258] Bile duct cancer diagnostic kit Another aspect of the present invention is a method for producing a biotin-binding protein that binds to a gene that is specifically expressed in bile duct cancer. a positively charged substance; an avidin-based protein-bound marker; and The present invention aims to provide a diagnostic kit for bile duct cancer, which includes a diagnostic certificate.

[0259] In this case, the genes specifically expressed in the bile duct cancer are MUC16, ASH1L, DOCK 7 and combinations thereof.

[0260] The instructions also state that the kit is configured to diagnose bile duct cancer according to the following protocol: a) from a biological sample isolated from an individual in a kit; b) separate the cfDNA using a positively charged substance contained in the The cfDNA was then subjected to the biotin-conjugated probe and markers included in the kit. c) Mix probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0261] Also, ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD27 4. ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that binds complementarily to at least one gene selected from the group The device may additionally include a

[0262] The probe, positively charged substance, and marker are as described above.

[0263] Bile duct cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in bile duct cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. a reaction unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker. The sample contained cfDNA with a sequence complementary to the probe, indicating the presence of cfDNA derived from bile duct cancer. and detecting a gene derived from a bile duct cancer cell from the sample without amplification, the information processing unit including: The objective of the present invention is to provide a device for diagnosing bile duct cancer using a cholangiocarcinoma.

[0264] In this case, the genes specifically expressed in bile duct cancer include ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1, EPCAM and combinations thereof. do.

[0265] Also, ACPP, FLU3, CPT1A, DSCC1, IFNG, CD279, CD27 4. ERBB2, EGFR, FOLR1, EPCAM, and combinations thereof A biotin-binding probe that specifically binds to at least one gene selected from the group The device may additionally include a

[0266] <Stomach cancer> How stomach cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, The method for diagnosing gastric cancer is to detect the gene of the present invention, and to detect the gene of the present invention. The probe binds to genes known as biomarkers for gastric cancer, and In this case, a gene known as a biomarker for gastric cancer is used to diagnose gastric cancer. The gene may be a gene encoding a protein that is overexpressed in the host.

[0267] Specifically, the probes having sequences complementary to the cfDNA include CGB, PARP1, F OXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, M ATN3, CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GR M6, INSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CF L1, NRP2, ANXA10, TFF2, CDCA5, NUSAP1, and their combinations A gene that complementarily binds to at least one gene selected from the group consisting of a combination of It is possible that.

[0268] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0269] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0270] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0271] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0272] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of gastric cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in gastric cancer cells. The nucleic acid sequence overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but is not expressed at a specific level in normal cells. It refers to a nucleic acid sequence that is overexpressed in cancer cells.

[0273] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0274] Furthermore, the cfDNA derived from the gastric cancer cells has i) a double helix structure derived from normal cells. or ii) have a lower Tm value compared to cfDNA derived from normal cells. It is characterized by being denatured under conditions where cfDNA having a double helix structure does not denature. Possible.

[0275] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0276] The genes overexpressed in the gastric cancer cells include ACPP (NCBI Gene ID: 55), FLU3(Gene ID:837968), CGB(NCBI Gene ID:10 82), KRT19 (NCBI Gene ID:3880), PARP1 (NCBI Gene ID:142), FOXO3A(NCBI Gene ID:2309), M ED30 (NCBI Gene ID:90390), ERBB2 (NCBI Gene ID:2064), CCNE1(NCBI Gene ID:898), MYC(NC BI Gene ID:4609), EGFR(NCBI Gene ID:1956) 、KRAS(NCBI Gene ID:3845)、TFF1(NCBI Gene ID:7031)、FABP1(NCBI Gene ID:2168)、CK20(N CBI Gene ID:54474)、MUC2(NCBI Gene ID:458 3)、SDC2(NCBI Gene ID:6383)、LAMP5(NCBI Ge ne ID:24141)、MATN3(NCBI Gene ID:4148)、CL IP4(NCBI Gene ID:79745)、NOX4(NCBI Gene I D:50507)、ADRA2C(NCBI Gene ID:152)、CSK(NC BI Gene ID:1445)、FZD9(NCBI Gene ID:8326) 、GALR1(NCBI Gene ID:2587)、GRM6(NCBI Gene ID:2916)、INSR(NCBI Gene ID:3643)、LPHN1( NCBI Gene ID:22859)、LYN(NCBI Gene ID:406 7)、MRGPRX3(NCBI Gene ID:117195)、ADCY3(NC BI Gene ID:109)、HDAC2(NCBI Gene ID:3066) 、CFL1(NCBI Gene ID:1072)、COTL1(NCBI Gene ID:23406)、NRP2(NCBI Gene ID:8828)、ANXA1 0(NCBI Gene ID:11199)、TFF2(NCBI Gene ID: 7032)、CDCA5(NCBI Gene ID:113130)、ATAD2(N CBI Gene ID:29028)、ASB9(NCBI Gene ID:140 462), MMP1 (NCBI Gene ID:4312), CEACAM6 (NCB I Gene ID:4680), DSCC1(NCBI Gene ID:79075 ), CKS2 (NCBI Gene ID:1164), CST1 (NCBI Gene ID:1469), IFITM1(NCBI Gene ID:8519), NUSA P1(NCBI Gene ID:51203), MELK(NCBI Gene ID:51203) :9833), LGALS3BP(NCBI Gene ID:3959), CPT1A (NCBI Gene ID:1374), IFNG(NCBI Gene ID:34 58), CD274 (NCBI Gene ID:29126), FOLR1 (NCBI Gene ID:2348), EPCAM(NCBI Gene ID:4072), CEACAM5 (NCBI Gene ID: 1048) and combinations of these It can be any one selected from the group consisting of:

[0277] As a specific example, genes that are specifically present in gastric cancer include CGB, PARP1, and FOXO3. A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3 , CLIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, I NSR, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, N RP2, ANXA10, TFF2, CDCA5, NUSAP1 genes, and additionally ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1, CK 20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CEACA M6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOLR1 Gastric cancer can be diagnosed by detecting the EPCAM gene.

[0278] The term "CGB" as used herein refers to human chorionic gonadotropin (hCG). It refers to the gene that codes for the hormone (gonadotropin). may also produce the hormone hCG. Therefore, it is important to measure it when the patient is not pregnant. High levels of the hormone hCG may be a sign of a cancer diagnosis.

[0279] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0280] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0281] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0282] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0283] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0284] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0285] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0286] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures the cDNA derived from gastric cancer. This can be done so that only fDNA can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can also be performed for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one embodiment, the cfDNA can be denatured for 2 seconds. c) can be performed before step c.

[0287] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0288] Stomach cancer diagnostic kit Another aspect of the present invention is a biotin-binding protein that binds complementary to a gene specifically expressed in stomach cancer. Probe; positively charged substance; avidin-based protein-bound marker; and description The present invention provides a gastric cancer diagnostic kit including a test kit.

[0289] In this case, the genes specifically expressed in gastric cancer are CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, C LIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INS R, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP 2, ANXA10, TFF2, CDCA5, NUSAP1, and their combinations It may be any one or more selected from the group consisting of:

[0290] The instructions also state that the kit is configured to diagnose stomach cancer according to the following protocol: a) from a biological sample isolated from an individual into a kit b) Isolate cfDNA using a positively charged substance; cfDNA was analyzed using the biotin-conjugated probe and marker included in the kit. c) probes that do not bind to cfDNA and removing the marker; and d) detecting a signal of said marker.

[0291] Also ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1 , CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CE ACAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3 BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOL At least one selected from the group consisting of R1, EPCAM, and combinations thereof It may additionally contain a biotin-conjugated probe that binds complementarily to one of the genes.

[0292] The probe, positively charged substance, and marker are as described above.

[0293] Gastric cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in gastric cancer; and and nanoparticles containing streptavidin and a marker are added sequentially or simultaneously. a response unit; d) a detection unit for detecting the marker; and e) detecting the presence or absence of the marker by detecting the presence or absence of the marker. The sample contains a sequence complementary to the probe, and it is determined that cfDNA derived from gastric cancer is present. The information processing unit detects genes derived from stomach cancer cells from the sample without amplification, and The present invention aims to provide a device for diagnosing cancer.

[0294] In this case, the genes specifically expressed in gastric cancer are CGB, PARP1, FOXO3A, MED30, CCNE1, MYC, TFF1, FABP1, LAMP5, MATN3, C LIP4, NOX4, ADRA2C, CSK, FZD9, GALR1, GRM6, INS R, LPHN1, LYN, MRGPRX3, ADCY3, HDAC2, CFL1, NRP 2, ANXA10, TFF2, CDCA5, NUSAP1, and their combinations It may be any one or more selected from the group consisting of:

[0295] Also ACPP, FLU3, KRT19, ERBB2, EGFR, KRAS, DSCC1 , CK20, MUC2, SDC2, COTL1, ATAD2, ASB9, MMP1, CE ACAM6, DSCC1, CKS2, CST1, IFITM1, MELK, LGALS3 BP, CPT1A, IFNG, CD279, CD274, ERBB2, EGFR, FOL At least one selected from the group consisting of R1, EPCAM, and combinations thereof It may additionally contain a biotin-conjugated probe that binds complementarily to one of the genes.

[0296] <Pancreatic cancer> How pancreatic cancer is diagnosed One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification. The method for diagnosing pancreatic cancer involves detecting genes derived from the cfDNA, and The probes with sequences complementary to genes known to be biomarkers for pancreatic cancer are The present invention provides a method for diagnosing pancreatic cancer by detecting a pancreatic cancer-related gene that is known to be a biomarker for pancreatic cancer. The selected gene can be a gene that encodes a protein that is overexpressed in pancreatic cancer.

[0297] Specifically, the probes having sequences complementary to the cfDNA include SMAD4, APC, G At least one gene selected from the group consisting of NAS and combinations thereof It may be one that binds complementary to the molecule.

[0298] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0299] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0300] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0301] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0302] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of pancreatic cancer cells. Specifically, cfDNA may contain nucleic acid sequences that are overexpressed in pancreatic cancer cells. The nucleic acid sequence that is overexpressed in cancer cells exhibits an appropriate expression level in normal cells. refers to a nucleic acid sequence that is overexpressed in specific cancer cells.

[0303] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0304] In addition, the cfDNA derived from the pancreatic cancer cells is i) a double helix derived from normal cells. ii) have a lower Tm value compared to cfDNA with the structure, or ii) are derived from normal cells The compound is characterized by being denatured under conditions in which cfDNA having a double helix structure is not denatured. It is possible.

[0305] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0306] The gene overexpressed in the pancreatic cancer cells was KRAS (NCBI Gene ID: 38 45), SMADA4 (NCBI Gene ID:4089), APC (NCBI G ene ID:324), GNAS(NCBI Gene ID:2788), MUC1 (NCBI Gene ID:4582), CEACAM5(NCBI Gene ID:4582) :1048), CEACAM1(NCBI Gene ID:634), MUC16(N CBI Gene ID: 94025) and combinations thereof It can be any one of the following:

[0307] As a specific example, genes that are specifically present in pancreatic cancer include SMAD4, APC, and GNA. S gene, and additionally KRAS, MUC1, MSLN, CEACAM1, CEAC Pancreatic cancer can be diagnosed by detecting the AM5 or MUC16 gene.

[0308] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0309] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0310] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0311] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0312] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0313] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0314] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0315] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but does denature pancreatic cancer-derived cfDNA. This can be done so that only the cfDNA that is present can be selectively denatured. The denaturation step can be carried out at about 50°C to about 100°C for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. It can be done for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds or about In one embodiment, the denaturing step may be performed for 90 seconds. It can be carried out before step c).

[0316] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0317] Pancreatic cancer diagnostic kit Another aspect of the present invention is a method for producing a pancreatic cancer-specific gene-specific biotin-binding protein. a binding probe; a positively charged substance; an avidin-based protein-bound marker; and and providing a diagnostic kit for pancreatic cancer including instructions.

[0318] In this case, the genes specifically expressed in pancreatic cancer include SMAD4, APC, GNAS, and and combinations thereof.

[0319] The instructions also state that the kit is configured to treat pancreatic cancer using the following protocol: The kit may be described as capable of diagnosing: a) from a biological sample isolated from an individual; b) Isolate cfDNA using a positively charged substance contained within the The cfDNA was then subjected to the biotin-conjugated probe and the primers included in the kit. c) probes that do not bind to cfDNA and and d) removing the marker; and d) detecting a signal of said marker.

[0320] Also, KRAS, MUC1, MSLN, CEACAM1, CEACAM5 or MUC1 6 and combinations thereof. A complementary binding biotin-conjugated probe may additionally be included.

[0321] The probe, positively charged substance, and marker are as described above.

[0322] Pancreatic cancer diagnostic device Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in pancreatic cancer; and adding nanoparticles containing streptavidin and a marker sequentially or simultaneously. d) a detection unit for detecting the marker; and e) a detection unit for detecting the marker based on whether or not the marker is detected. If the sample contains cfDNA that has a sequence complementary to the probe and is derived from pancreatic cancer, and extracting a gene derived from a pancreatic cancer cell from the sample without amplification, the gene including an information processing unit for determining The objective of the present invention is to provide a device for detecting and diagnosing pancreatic cancer.

[0323] In this case, the genes specifically expressed in pancreatic cancer include SMAD4, APC, GNAS, and and combinations thereof.

[0324] Also, KRAS, MUC1, MSLN, CEACAM1, CEACAM5 or MUC1 6 and combinations thereof. A complementary binding biotin-conjugated probe may additionally be included.

[0325] <Early diagnosis and prognosis prediction> Cancer diagnosis methods One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the marker being derived from the cancer cell. This is a method for early diagnosis or prognosis prediction of cancer by detecting genes, and is based on the cfDNA. The probes with complementary sequences bind to genes known to be cancer biomarkers. The present invention provides a method for early diagnosis or prognosis prediction of cancer.

[0326] Specifically, the probes having a sequence complementary to the cfDNA include CPT1A, IFNG, IFNGR1, CD279, CD274, and combinations thereof The gene may be one that binds complementarily to at least one of the genes. Alternatively, two or more genes selected from the above group can be combined.

[0327] The cancers include lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, and head and neck cancer. , esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer Bladder cancer, rectal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, lymphoma, acute leukemia, multiple It can be any one selected from the group consisting of myeloma and combinations thereof.

[0328] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0329] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0330] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0331] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0332] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of cancer cells. Specifically, cfDNA can contain nucleic acid sequences that are overexpressed in cancer cells. The nucleic acid sequence that is overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but is overexpressed in specific cancer cells. It refers to a nucleic acid sequence that is overexpressed in a cell.

[0333] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0334] In addition, the cfDNA derived from the cancer cells has i) a double helix structure derived from normal cells. ii) have a lower Tm value compared to cfDNA derived from normal cells; or It is characterized by being denatured under conditions where cfDNA having a helical structure does not denature. Ugh.

[0335] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0336] The genes overexpressed in the cancer cells are FNG (NCBI Gene ID: 3458), IFNGR1 (NCBI Gene ID:3459), CD279 (NCBI Gen e ID:5133) and CD274 (NCBI Gene ID:29126) and combinations thereof. , when the combination of genes overexpressed in the cancer cells is two, IFNG / IFNG R1, IFNG / CD274 or IFNG / CD279, and overexpressed in the cancer cells When the combination of three genes is involved, IFNG / IFNGR1 / CD274, IF NG / CD274 / CD279 or IFNGR1 / CD274 / CD279, When the combination of four genes is overexpressed in cancer cells, it is called INFG / IFNGR. 1 / CD274 / CD279. When analyzing the data, the reliability of the analysis results can be improved.

[0337] As used herein, the term "IFNG" refers to interferon gamma (interferon gamma). The term refers to the gene encoding the rferon gamma.

[0338] As used herein, the term "IFNGR1" refers to an interferon gamma receptor. It encodes Interferon gamma receptor 1 (Interferon gamma receptor 1). It means genes.

[0339] The term "CD274" as used herein refers to PD-L1 (Programme d death-ligand 1) refers to the gene encoding it.

[0340] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0341] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0342] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0343] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0344] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0345] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0346] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0347] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures cancer-derived cfDNA. This can be done so that only DNA can be selectively denatured. The denaturation step can be carried out at about 50° C. to about 100° C. for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. You can also set the time to about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one embodiment, the step of denaturing the cfDNA can be performed by ) step can be performed before.

[0348] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0349] Cancer diagnostic kits Another aspect of the present invention is a method for producing a biotin-binding protein that binds complementary to a gene that is specifically expressed in cancer. a probe; a positively charged substance; an avidin-based protein-bound marker; and instructions. and providing a kit for early diagnosis or prognosis prediction of cancer, comprising:

[0350] In this case, the genes specifically expressed in the cancer include CPT1A, IFNG, IFNGR1, Any one selected from the group consisting of CD279 and CD274 and combinations thereof There can be one or more.

[0351] The instructions also state that the kit is configured to diagnose cancer early using the following protocol: The present invention relates to a method for detecting cancer or cancer prognosis, and the method may be described as: a) isolated from an individual; cfDNA is isolated from the biological sample using a positively charged substance included in the kit. b) The isolated cfDNA is treated with the biotin-conjugated probe and and the markers included in the kit, either sequentially or simultaneously; c) cfDN a) removing probes and markers that do not bind to a) and b) removing probes and markers that do not bind to a) and c ... Detect the signal.

[0352] The probe, positively charged substance, and marker are as described above.

[0353] Cancer diagnostic equipment Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in the cancer; and and nanoparticles containing streptavidin and a marker are added sequentially or simultaneously. d) a detection unit for detecting a marker; and e) detecting the sample based on the presence or absence of the detection of the marker. information that determines the presence of cfDNA derived from cancer, which has a sequence complementary to the probe in the A method for detecting cancer cell-derived genes from the sample, including a processing section, without amplification, to detect cancer at an early stage. The present invention provides a device for diagnosing or predicting the prognosis of cancer.

[0354] In this case, the genes specifically expressed in the cancer include CPT1A, IFNG, IFNGR1, Any one selected from the group consisting of CD279, CD274, and a combination thereof It could be more than that.

[0355] <Cancer> Method for confirming cancer, cancer metastasis, and cancer resistance to drugs One aspect of the present invention is to provide a) cell-free DNA (hereinafter referred to as cfDNA) A biological sample isolated from an individual containing a substance called a 'positively charged substance' is mixed with the sample. b) separating the positively charged substance to which the cfDNA is bound; c) adding the mixture to the positively charged substance; A probe and a marker having a sequence complementary to the cfDNA are mixed sequentially or simultaneously. d) removing probes and markers that do not bind to the cfDNA; and and e) detecting the marker from the sample without amplification, the marker being derived from the cancer cell. Detecting genes to determine whether cancer is present, whether cancer has metastasized, and / or whether it is resistant The method for determining whether or not a cancer cell is a cancer cell-derived cfDNA is a cancer cell-derived cfDNA. The probe with a sequence complementary to A is an indicator of the presence or absence of cancer and cancer metastasis. Genes known to be biomarkers of resistance or resistance The present invention provides a method for determining the metastatic state or the presence or absence of resistance of cancer, by complementarily binding to In this case, genes known as biomarkers for cancer, the presence or absence of cancer metastasis, or resistance, Single nucleotide polymorphisms in cancer hism; snp) (see Examples 6 and 7) .

[0356] Specifically, the probes having a sequence complementary to the cfDNA include CPT1A, IFNG, IFNGR1, CD279, CD274G, and combinations thereof. The gene may be one that binds complementarily to at least one of the genes.

[0357] The probe having a sequence complementary to the cfDNA is used to detect genes overexpressed in cancer cells, genes specifically present in the nucleus, genes associated with metastasis, or genes associated with drug resistance It may be a complementary binding.

[0358] In one embodiment, the genes overexpressed in cancer cells are CPT1A, IFNG, IFNG R1, CD279, CD274, NSE, SCC, CEA, cyfra21-1, TPA , NMP22, OGT, Thyroglobulin(TG), Calcitonin( CALCA), BRAF V600E, TERT C228T / C250T, AFP, β -HCG(CGB), CA19-9, PSA, PSMA, PAP, PCA3, TMPRS S2-ERG, CA125, HIF-1a, VEGF, CA15-3, HER2, SCC (SART3), TOP2A, MCM2, p16INK4a(CDKN2A), Ki-6 7 (MKI167), HE4 (WEDC2), and combinations thereof. It could be any one of them.

[0359] In one specific example, the gene specifically present in cancer is CPT1A, IFNG, IFN GR1, CD279, CD274 genes, and additionally NSE, SCC, CEA, c yfra21-1, TPA, NMP22, OGT, Thyroglobulin (TG) , Calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG(CGB), CA19-9, PSA, PSMA, PA P, PCA3, TMPRSS2-ERG, CA125, HIF-1a, VEGF, CA1 5-3, HER2, SCC(SART3), TOP2A, MCM2, p16INK4a( CDKN2A), Ki-67 (MKI167), or HE4 (WEDC2) genes were detected. It can be used to diagnose the type of cancer.

[0360] In one embodiment, the gene associated with cancer metastasis is a gene encoding a "proliferation "on" and "invasion" related genes, e.g., Ki67, S TK15, Survivin, Cyclin B1, MYBL2, Stromelysi n3, Cathepsin L2.

[0361] In one embodiment, the drug resistance-related genes are determined by the drug and cancer type. As a specific example, the gene associated with acquired resistance to EGFR-TKI is EGF R T790M, PI3K, BRAF, MAPK1, HER2, KRAS, NRAS, R Group consisting of B deletion, p53 deletion, PTEN and NFkB It can be any one selected from:

[0362] The purpose of using the separated biological sample is to detect cfDNA present in the sample. Therefore, cfDNA in samples can be isolated and / or enriched using a variety of methods. As a specific example, nitrocellulose, which has a strong affinity for nucleic acids, can be used. A nitrocellulose membrane can be used. In one specific example, a positively charged antibody is used to capture negatively charged cfDNA. The positively charged substance can be a nanoparticle, a nanowire, or the like. The filter may be a mesh structure or a positively charged filter, but is not limited to this shape. A specific example of the "positively charged substance" is a positively charged nanostructure or Alternatively, it may be a positively charged membrane.

[0363] An example of a nanostructure may include a cationic polymer. There is no limitation on the type of polymer. A specific example of a cationic polymer is polyethylene. Polyethyleneimine (PEI) is a cationic branched polymer Polyethyleneimine (cationic branched polymer po lyethyleneimine).

[0364] In addition, streptavidin-labeled nanowires were mixed with biotin-conjugated PEI solution. Upon mixing, cationic binding was performed on the nanowires through biotin-streptavidin interactions. Branched polyethyleneimine (cationic branched PEI) was added to As a result, the cationic polymer polyethyleneimine is attached to the surface. The nanoparticles are densely and irregularly distributed in the combined nanostructure (PEI / mPpy NW). It can be laid and embedded.

[0365] Such nanowires successfully separated genomic DNA and cfDNA at low concentrations with high efficiency. In particular, the large surface area for binding to target molecules such as DNA, D Nanowire features such as enhanced mobility for enhanced interaction with NAs can effectively It can efficiently and effectively capture target cfDNA.

[0366] In this case, the target cfDNA refers to the cfDNA of interest to be detected. In this case, the cfDNA has a double strand. The cfDNA may be derived from the genes of cancer cells. Specifically, cfDNA can contain nucleic acid sequences that are overexpressed in cancer cells. The nucleic acid sequence that is overexpressed in the cancer cells is expressed at an appropriate level in normal cells, but is overexpressed in specific cancer cells. It refers to a nucleic acid sequence that is overexpressed in a cell.

[0367] Specifically, the degree or cutoff of the nucleic acid sequence overexpressed in cancer cells is When measuring the optical density using a meter, the OD value was 0 More specifically, the nucleic acid sequence may be overexpressed in cancer cells. The standard for the degree of chromatin degradation is when the OD value reaches 0.012 when measuring the absorbance using a marker. In this case, the absorbance may be 0.015 or more. The wavelength can be appropriately determined by a marker. It can also be the unwinding of DNA. The fDNA can be determined appropriately.

[0368] In addition, the cfDNA derived from the cancer cells has i) a double helix structure derived from normal cells. ii) have a lower Tm value compared to cfDNA derived from normal cells; or It is characterized by being denatured under conditions where cfDNA having a helical structure does not denature. Ugh.

[0369] In addition, the cfDNA is complementary to the cfDNA under any one of the following conditions: It can bind to a probe of about 15 mer to about 30 mer: i) at room temperature ii) leaving it for about 1 minute to about 120 minutes; ii) heating at about 90°C to about 95°C for about 1 second to about 3 minutes iii) heating at about 75°C to about 90°C for about 1 second to about 5 minutes; iv) heating at about 60°C ℃ to about 75℃ for about 30 seconds to about 30 minutes; v) about 25℃ to about 40℃ for about 10 minutes vi) heating for about 120 minutes; vi) treating with protease for about 1 minute to about 30 minutes; vii) DNase treatment for about 1 minute to about 30 minutes; and viii) chemicals (e.g., , sodium hydroxide, DMSO, surfactants, etc.).

[0370] As used herein, the term "probe" refers to a probe for detecting cfDNA. The probe refers to a DNA or RNA capable of complementary binding to cfDNA. The probe may have a specific sequence complementary to the cfDNA. is a nucleic acid that can complementarily bind to the target double-stranded cfDNA present in plasma that you want to detect. In this case, the probe has a sequence of biotin. The probe may be a marker bound to a biotin-binding protein. It can be combined with

[0371] As used herein, the term "marker" refers to a probe that detects binding to cfDNA. The markers refer to substances used to detect the presence of a substance. Specifically, the marker may be a quantum dot, HRP, or an enzyme. The fluorescent protein may be any one selected from the group consisting of: The markers are GFP (green fluorescent protein), BF P (blue fluorescent protein), CFP (cyan flu orescent protein), YFP(yellow fluorescent protein) or HRP (horse radish peroxidase) It can be, but is not limited to these.

[0372] The marker may be bound to a biotin-binding protein. The proteins are avidin-based proteins such as streptavidin and trastuzumab. Traptavidin or neutravidin However, any protein that can specifically bind to biotin can be used without restriction. In one embodiment, the marker is a streptavidin-bound marker. It is possible that.

[0373] As used herein, the term "streptavidin" refers to the nucleotide sequence of Streptomyces avium A 60kDa molecular weight isolate from Streptomyces avidinii It is a tetrameric biotin-binding protein of the formula 1. Although it has very low homology with avidin, Its structure is very similar to that of avidin. It has antibacterial activity, similar to avidin, and is anti-biotin. It always has a high binding strength. Unlike avidin, it does not contain carbohydrates and has an acidic isoelectric point. (pI=5) and has significantly lower solubility than avidin. Streptavidin, e.g., Thermo Scientific Pierce S Treptavidin is a recombinant form of streptavidin with a molecular weight of 53 kDa. It has a near-neutral isoelectric point (pI = 6.8 to 7.5). The lack of glycosylation and low pI result in lower levels of nonspecific binding (especially rectification) compared to avidin. This allows for a detection system with many of the properties of streptavidin. This makes it an ideal reagent for

[0374] As used herein, the term "traptavidin" means is a term used to refer to a variant or mutein of streptavidin. It exhibits approximately 10 times slower dissociation rate for biotin, increased mechanical strength, and thermal stability. Traptavidin is a protein with improved affinity for biotin.

[0375] The term "neutravidin" as used herein means "deglycosylated avidin." It is also called avidin, and was created to avoid the major drawbacks of natural avidin and streptavidin. As the name suggests, it is produced by deglycosylating avidin, and It has a reduced molecular weight (60 kDa) compared to IgG, but maintains high biotin binding capacity. Deglycosylation of the avidin reduces lectin binding to undetectable levels. This lowers the isoelectric point (pI=6.3), which is the main cause of nonspecific binding to avidin. Lysine residues remain available, allowing for the efficient removal of streptavidin. It can be easily derivatized or conjugated like a thiol. It also has high biotin-binding capacity. and low non-specific binding, making it an ideal biotin-binding protein for various uses. It is possible.

[0376] As used herein, the term "detecting a marker" refers to the detection of a probe and a biotinylated marker. The step of detecting the bound marker through avidin-avidin reaction is also referred to as the step of detecting the bound marker. Detection can be by color change, change in UV absorbance, presence or absence of bioluminescence, change in fluorescent response, or electrochemical Specifically, the method for detecting the marker can be performed by using The procedure can be performed differently depending on the marker used. For example, when HRP is used as a marker, When used, the marker is detected by observing the color reaction that occurs through the reaction of hydrogen peroxide with a substrate. In addition, when the marker is a fluorescent protein such as GFP, The presence or absence of a marker is detected by observing the light emitted after irradiating it with light of a specific wavelength. Moreover, when the marker is luciferase, it is possible to The presence of the marker is determined by measuring the bioluminescence that appears after adding the substrate with a bioluminometer. The presence of

[0377] In addition, the diagnostic method of the present invention may further include a step of denaturing cfDNA. During this process, the denaturation step does not denature normal double-stranded cfDNA, but denatures cancer-derived cfDNA. This can be done so that only DNA can be selectively denatured. The denaturation step can be carried out at about 50° C. to about 100° C. for about 0.1 seconds to about 5 minutes. A specific example of the denaturation temperature is about 95°C, and the denaturation time is usually about 0.1 seconds to about 8 minutes. You can also set the time to about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, or about 90 seconds. In one embodiment, the step of denaturing the cfDNA can be performed by ) step can be performed before.

[0378] Specifically, before the step c), the sample or the cfDNA bound to the positively charged substance is 1) Leave at room temperature for about 1 to 10 minutes; ii) Heat at about 90 to 95°C for 1 second to 1 minute Heating conditions: iii) heating at about 75°C to about 90°C for about 10 seconds to about 3 minutes; iv) heating at about Heating conditions: 60°C to 75°C for 1 minute to 30 minutes; v) Heating conditions: 25°C to 40°C for 5 minutes vi) Heating for about 60 minutes; vi) Treating with protease for about 1 minute to about 10 minutes; and vii) treating with DNase I for about 1 minute to about 10 minutes. The method may further include a step of denaturing the polymer under any one of the following conditions: Double-stranded cfDNA derived from normal cells is not denatured, but cfDNA derived from cancer cells is. By selectively denaturing only the nucleotides, it is possible to further facilitate binding with the probe. The denaturing conditions i) to vii) can be carried out after the sample is obtained. The denaturing conditions in ) to vii) are performed after the cfDNA bound to the positively charged substance is obtained. In addition, the denaturing conditions i) to vii) can be changed by the temperature, protease and DNA. The ase treatment time can be adjusted as appropriate as long as it does not denature stable cfDNA.

[0379] Cancer diagnostic kits Another aspect of the present invention is a method for producing a biotin-binding protein that binds complementary to a gene that is specifically expressed in cancer. a probe; a positively charged substance; an avidin-based protein-bound marker; and instructions. and providing a cancer diagnostic kit comprising:

[0380] In this case, the genes specifically expressed in the cancer include CPT1A, IFNG, IFNGR1, Any one selected from the group consisting of CD279, CD274G, and a combination thereof There can be more than one.

[0381] The instructions also state that the kit is configured to diagnose cancer using the following protocol: a) from a biological sample isolated from an individual to be contained in the kit; b) Isolate cfDNA using a positively charged substance; Biotin-conjugated probes and markers included in the kit for fDNA c) Mix probes and primers that do not bind to cfDNA. and d) detecting the signal of said marker.

[0382] In addition, NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, T hyroglobulin (TG), calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG(CGB), CA 19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125 , HIF-1a, VEGF, CA15-3, HER2, SCC(SART3), TOP2 A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE 4 (WEDC2) and combinations thereof It may additionally contain a biotin-conjugated probe that binds complementarily to one of the genes.

[0383] The probe, positively charged substance, and marker are as described above.

[0384] Cancer diagnostic equipment Another aspect of the invention is a method for detecting cfDNA from a biological sample isolated from an individual. a) a mixing section that mixes charged substances; b) a section that removes positively charged substances bound to cfDNA; a collection unit for removing the sample; c) a positively charged substance to which the cfDNA is bound; a biotin-conjugated probe capable of binding complementarily to the gene specifically expressed in the cancer; and and nanoparticles containing streptavidin and a marker are added sequentially or simultaneously. d) a detection unit for detecting a marker; and e) detecting the sample based on the presence or absence of the detection of the marker. information that determines the presence of cfDNA derived from cancer, which has a sequence complementary to the probe in the and detecting genes derived from cancer cells from the sample, without amplification, to diagnose cancer. The object of the present invention is to provide an apparatus for

[0385] In this case, the genes specifically expressed in the cancer include CPT1A, IFNG, IFNGR1, Any one selected from the group consisting of CD279 and CD274 and combinations thereof There can be one or more.

[0386] In addition, NSE, SCC, CEA, cyfra21-1, TPA, NMP22, OGT, T hyroglobulin (TG), calcitonin (CALCA), BRAF V600E, TERT C228T / C250T, AFP, β-HCG(CGB), CA 19-9, PSA, PSMA, PAP, PCA3, TMPRSS2-ERG, CA125 , HIF-1a, VEGF, CA15-3, HER2, SCC(SART3), TOP2 A, MCM2, p16INK4a (CDKN2A), Ki-67 (MKI167), HE 4 (WEDC2) and combinations thereof It may additionally contain a biotin-conjugated probe that binds complementarily to one of the genes. [Example]

[0387] The present invention will be described in more detail below with reference to the following examples. These are merely examples and are not intended to limit the scope of the present invention.

[0388] Experimental Method 1: cfDNA Detection Method Derived from Tumor-Specific Genes Step 1: Sample preparation and nanowire addition Immediately after receiving the patient's plasma, urine, saliva, or sputum, the sample is subjected to 3000Xg at 4°C for 10 minutes. The patient's plasma, urine, saliva, or sputum was diluted in DPBS at a certain ratio. In the case of plasma, 1 μl to 30 μl of plasma was mixed with 150 μl of DW and spun. Spin column (Type G or Type Q) and PEI Py nanowires (150 μl) were added and the mixture was heated in a thermomixer. The mixture was mixed at room temperature for 20 minutes at a speed of 1,200 rpm using a mixing machine.

[0389] Stage 2: Vacuum / Washing / Temperature Denaturation After attaching the spin column to a vacuum suction device, suction was applied at 550 mBar. 400 μl of 1x DPBS was added and aspirated again. The same process was repeated once more. Only the nanowire-DNA complexes obtained through the step 2 were scanned. Filter into a pin column. If temperature denaturation is required, use a heat block preheated to 95°C. Place the evacuated spin column into the heating block and heat at 95°C for 1 minute. After incubation, the samples were immediately removed. Puru did not go through this process.

[0390] Nanowire-based spin column without magnetic nanoparticles The upper photo shows the separation of cfDNA using the centrifuge (Figure 85). The bottom image shows the SEM image of the pin column, with the cfDNA separated after centrifugation. This is an SEM image of a pin column.

[0391] Step 3: Adding probes and HRP / STR NPs The probe (200 μl) and HRP / STR NPs solution ( 200 μl) was placed in each spin column. The mixture was mixed at room temperature for 30 minutes at a speed of 850 rpm to 1,000 rpm using a spin mixer. After attaching the column to a vacuum device, suction was applied. 400 μl of 1x DPBS was added, and The same process was repeated once more.

[0392] Step 4: TMB reaction for gene mutation detection After replacing the collection tube with a new one, The rum was diluted with 200 μl sodium acetate buffer (0.2 M, pH 7. 0), 50 μl H2O2 (0.1 M) was added using a syringe pump. After adding the solution to the spin column in order, the solution was incubated for 3 minutes. After incubation, spin the column at 3,500 rpm to 5,000 rpm for 3 minutes. The solution collected in the collection tube was centrifuged for 10 seconds. 200 μl of the mixture was transferred to a 96-well plate and then measured with a UV / VIS spectrophotometer (s absorbance in the wavelength range of 500nm to 850nm using a spectrophotometer was measured.

[0393] Overview of detection methods The detection steps of the present invention are diagrammed in Figures 84a-g. (PEI)-bound polypyrrole nanowires (PEI / Ppy NW) were used. After collecting cfDNA from the patient's body fluid, a probe that complementarily binds to the target cfDNA is used. probe and HRP / streptavidin-nanoparticles (HRP / st-tagged N Schematic diagram of a method for analyzing cancer cell-derived genes within 60 minutes through a reaction with P Nanowires, probes, and HRP / streptavidin nanoparticles were used. Schematic representation of the method for detecting cfDNA from cancer cells (Figure 84b). Using a spin column, genes derived from cancer cells were extracted. FIG. 84c shows the process of detecting the lysis bar. The method may further include a step of treating the buffer, such as blood, cerebrospinal fluid, or pleural effusion. A timeline showing a method for detecting cancer cell-derived cfDNA from various samples. Figure 84d shows the results of a method for detecting cfDNA derived from cancer cells in samples such as urine. The method is shown in a timeline (Figure 84e). This is a diagram illustrating the difference in denaturation conditions depending on the state (Figure 84f). This is a diagram illustrating the differences in denaturation conditions depending on the state of the cfDNA obtained (Figure 84g).

[0394] Production Example 1. Production of nanowires surface-treated with cationic polymers As shown in Figure 1a, the cationic polymer polyethyleneimine (polyeth Nanowires with PEI (polyethyleneimine) attached to their surfaces were fabricated by anodic oxidation. One side of aluminum (anodic aluminum oxide, AAO) is Q1 50T Modular Coating System (Quorum Technologies, U K) to 5 × 10 -3 The gold (Au) layer (approximately 15 mbar and 50 mA) was grown for 600 seconds. All electrochemical experiments were performed on gold (Au)-coated AA O mold, platinum wire counter electrode and Ag / AgCl (3.0M NaCl type) reference electrode Potentiostat / galvanostat with polarity (BioLogic Measurements were performed using a meter (SP-150).

[0395] For the fabrication of nanowires surface-treated with cationic polymers (PEI / Ppy NWs) The pores of the AAO mold were filled with 0.01M poly(4-styrenesulfonic acid) (poly(4-st yrene sulfonic acid) and 1 mg / ml biotin (biot 7 at 1.0 V (vs. Ag / AgCl) with a 0.01 M pyrrole solution containing Apply chronoamperometry for 1 minute to charge Chemical vapor deposition was carried out.

[0396] The resulting AAO template was washed multiple times with distilled water and then dissolved in 2M sodium hydroxide (NaOH) solution. After immersion in the solution for 3 hours, the cells were sonicated using a Bioruptor UCD-200 (D A free-standing polypyrrole doped with biotin molecules was placed in a ion-exchange (iagenode) Free-standing Ppy NWs were obtained. The nanowires were treated with 30 mM N-(3-dimethylaminopropyl) )-N-ethylcarbodiimide hydrochloride(EDC) and 6 mM N-hydroxysuccinimide (NHS) were added to The carboxylic acid (-COOH) groups were activated. After adding the solution, the mixture was reacted at room temperature for 1 hour, and then washed with water to remove polyethyleneimine from the surface. The nanostructure (PEI / Ppy NW) was obtained. EI / Ppy NW) was dispersed in deionized water and stored at room temperature until use.

[0397] By this manufacturing method, the AAO template is selectively dissolved, and then each polypillar is Pyrolyzed (PPy) nanowires were released from the AAO template and attached to the nanowires by biotin-streptidase. Cationic branched polyethyleneimine (CPE) is synthesized through the interaction of putavidin with c branched PEI, 25 kDa) was additionally conjugated.

[0398] Production Example 2: Production of nanowires surface-treated with cationic polymers By substantially the same method as in Preparation Example 1, polylysine was used instead of polyethyleneimine. We obtained nanostructures (PL / Ppy NWs) with PyPy nanoparticles attached to their surfaces.

[0399] Preparation Example 3. Preparation of polypyrrole nanoparticles labeled with HRP and streptavidin For the preparation of HRP- and streptavidin-conjugated nanoparticles, polyvinyl pillowcases were used. Polyvinylpyrrolidone (PVP) 0.5g in 12.5ml of 3 After adding distilled water and stirring for 30 minutes, 65 μl of pyrrole was added. After further stirring for 10 minutes, 0.5 ml of 0.75 g / ml FeCl3 solution was added. After adding the hyaluronic acid solution (400 mg / 20 ml) 20 ml of HCl was added and stirred for 3 hours to obtain polypyrrole-hyaluronic acid nanoparticles ( Ppy-HA-NPs) were prepared.

[0400] Dialyze the mixture against triple distilled water using a membrane with a pore size of MWCO 50,000 for 2 days. Large particle aggregates are collected at 1,200 The 200 μg of the above-prepared PBS was centrifuged at 400 rpm for 3 minutes, removed, and then freeze-dried. Ppy-HA-NPs were placed in 1 ml of triple distilled water and then diluted with 100 mM EDC / 50 mM An NHS solution was added and reacted for 45 minutes to activate the carboxyl groups of hyaluronic acid. Centrifuge at 15,000 rpm for 10 minutes, remove the supernatant, and wash twice. Ppy-HA-NPs were added with 1 mg of HRP and 1 mg of streptavidin. The mixture was then centrifuged at 15,000 rpm for 10 minutes and the supernatant was collected. After removing the HCl, the plate was stored in triple distilled water. HRP and streptavidin were attached to the surface. The morphology of the nanoparticles (HRP / st-tagged NPs) was investigated using a scanning electron microscope. was observed (Fig. 1b).

[0401] Example 4: Probe fabrication A probe was developed to detect cfDNA with an unstable double helix structure. The probe was designed to be different depending on the type of cancer cfDNA that was desired to be detected. In this case, biotin was bound to the probe. The specific nucleic acid sequence information of the probe is as follows: The results are as shown in Tables 1, 5, 9, and 11-28 depending on the type of cancer being diagnosed.

[0402] I. Confirmation of accuracy of gene detection derived from cancer cell lines Example 1. Detecting expressed genes in cancer cell lines Example 1.1. Confirmation of PD-L1 detection accuracy PD-L1-positive cancer cell lines and known MD obtained from ATCC and the Korean Cell Line Bank A-MB-231, HCC827, H1975, PC9, and H460 cancer cell lines and PD -L1-negative cancer cell lines, A549, MDA-MB-468, HeLa, and MC The accuracy of PD-L1 detection was confirmed using the F7 cancer cell line. Cell lines and PD-L1-negative cancer cell lines were identified using CCLE (Cancer Cell Lymphocyte Activation PD-L1 mRNA levels in each cancer cell line provided by the Encyclopedia The classification was based on the mRNA level.

[0403] Specifically, we analyzed the PD-L1-positive and PD-L1-negative cancer cell lines. After extracting genomic DNA, sonication is performed. It was prepared in the form of DNA (fragmented DNA). 50ng / μl of fDNA was used. After placing it in PBS, add the nanowires prepared in Preparation Example 1 and react for 20 minutes. After that, the DNA was denatured at 95°C for 1 minute, Biotinylated PD-L1 probe (biotinylated PD-L1 probe) The probe used was the following: Shown in Table 1.

[0404] [Table 1]

[0405] For colorimetric detection, TMB(3,3' ,5,5'-Tetramethylbenzidine) and H2O2 in sodium acetate The detection results were obtained by processing the solution added to the sodium acetate buffer. Analysis of DNA-based PD-L1 expression (ΔOD) of each cancer cell line revealed that 2 MDA-MB-231, HCC827, H1975, PC9, and H46 in replicates 0 Clear PD-L1 DNA expression (ΔOD; cutoff On the other hand, A549, MDA-MB-461, HeLa, In the MCF7 cancer cell line, two replicate experiments both showed no PD-L1 DNA expression. In addition, the PD-L1 DNA expression results of each cancer cell line and CC were compared (Figures 4 and 5). Each of the following was obtained from the LE (cancer cell line encyclopedia): The results of PD-L1 (CD274) mRNA expression in each cancer cell line were compared. PD-L1 (CD274) mRNA levels are shown in Table 2 below.

[0406] [Table 2]

[0407] As a result, MDA-MB-231, HCC827, H1975, PC9 and H460 On the other hand, high mRNA expression was observed in cancer cell lines A549, MDA-MB-461, and , HeLa and MCF7 cancer cell lines showed almost no mRNA expression.

[0408] Example 1.2. Confirmation of EpCAM detection accuracy EpCAM-positive cancer cell lines obtained from ATCC and the Korean Cell Line Bank and known M DA-MB468, HCC827, MCF7, H1975 and MDA-MB-231 Cancer cell lines and known EpCAM-negative cancer cell lines A549, H460, and HeLa The accuracy of EpCAM detection was confirmed using cell lines. The EpCAM-negative and EpCAM-negative cancer cell lines were identified as CCLE (Cancer Cell Line Engraftment). The EpCAM mRNA levels of each cancer cell line provided by the cyclopedia were used as a standard. and classified them.

[0409] Specifically, genomic DNA from EpCAM-positive and EpCAM-negative cancer cell lines was After extraction, fDNA was prepared by sonication. 50 ng / μl of fDNA was added to PB After adding the nanowires prepared in Preparation Example 1 to S, the reaction was continued for 20 minutes and the nanowires were separated. After that, the mixture was denatured at 95°C for 1 minute, and then the biotin-labeled EpCA was added. Add M probe (biotinylated EpCAM probe) for 20 minutes. The reaction was further carried out using the probes shown in Table 3 below.

[0410] [Table 3]

[0411] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The DNA-based EpCAM expression (ΔOD) of each cancer cell line was analyzed to confirm the detection results. Analysis revealed that PC9, MDA-MB468, HCC827, and MCF7 were cloned in two replicate experiments. , EpCAM distinct in fDNA only in H1975 and MDA-MB-231 cancer cell lines DNA expression (ΔOD; cutoff OD>0.012) was shown. 0 and HeLa cancer cell lines showed no EpCAM DNA expression in two replicate experiments. In addition, the EpCAM DNA expression results of each of the cancer cell lines and C Obtained from CLE (cancer cell line encyclopedia) The EpCAM mRNA expression results of each cancer cell line were compared. The mRNA values ​​are shown in Table 4 below.

[0412] [Table 4]

[0413] As a result, PC9, MDA-MB468, HCC827, MCF7, H1975 and M It showed high mRNA expression in DA-MB-231 cancer cell lines. The IL-1 and HeLa cancer cell lines showed almost no mRNA expression.

[0414] Example 1.3. Confirmation of FOLR1 detection accuracy FOLR1-positive cancer cell lines obtained from ATCC and the Korean Cell Line Bank and known HeLa cells were used. La, MDA-MB-468, HCC827, MCF7 and MDA-MB-231 cancers Cell lines and known FOLR1-negative cancer cell lines: H460, PC9, H1975, and A5 The accuracy of FOLR1 detection was confirmed using 49 cancer cell lines. Cell lines and FOLR1-negative cancer cell lines were identified using CCLE (Cancer Cell Line e Encyclopedia) for the mRNA levels of FOLR1 in each cancer cell line. were classified based on the criteria.

[0415] Specifically, genomic DNA from FOLR1-positive and FOLR1-negative cancer cell lines was After extraction, fDNA was prepared by sonication. 50 ng / μl of fDNA was added to PB After adding the nanowires prepared in Preparation Example 1 to S, the reaction was continued for 20 minutes and the nanowires were separated. After that, the mixture was denatured at 95°C for 1 minute, and then biotin-labeled FOLR was added. Add 1 probe (biotinylated FOLR1 probe) and incubate for 20 minutes. The reaction was further carried out using the probes shown in Table 5 below.

[0416] [Table 5]

[0417] For colorimetric detection, TMB and H2O2 were added to sodium acetate (Sodium acetate). e) The solution added to the buffer was processed to confirm the detection results. Analysis of FOLR1 expression (ΔOD) in HeLa, MDA- fDNA of MB468, HCC827, MCF7 and MDA-MB-231 cancer cell lines Only in the presence of FOLR1 DNA expression (ΔOD; cutoff OD>0.012) was clearly observed. On the other hand, in H460, PC9, H1975, and A549 cancer cells, Repeat experiments did not show FOLR1 DNA expression (Fig. 8 and Fig. 9). FOLR1 DNA expression results of cancer cell lines and CCLE (cancer cell line The FOLR1 mRNA results for each cancer cell line obtained from the encyclopedia The FOLR1 mRNA levels for each cancer cell line are shown in Table 6 below.

[0418] [Table 6]

[0419] As a result, HeLa, MDA-MB-468, HCC827, MCF7, and MDA-M High mRNA expression was observed in B-231 cancer cell lines. The H1975 and A549 cancer cell lines showed little mRNA expression.

[0420] Example 1.4. Confirmation of EGFR detection accuracy EGFR-positive cancer cell lines and known HeL obtained from ATCC and the Korean Cell Line Bank a, PC9, A549, H1975, H460, MDA-MB-468, MCF7 and MDA-MB-231 cancer cell line and MCF7 cancer cell line, known as an EGFR-negative cancer cell line The accuracy of EGFR detection was confirmed using EGFR-positive cancer cell lines and EGFR-positive cancer cell lines. FR-negative cancer cell lines are identified in CCLE (Cancer Cell Line Encyclopedia The EGFR mRNA levels of each cancer cell line provided by pedia were used as the standard for classification. .

[0421] Specifically, genomic DNA was extracted from EGFR-positive and EGFR-negative cancer cell lines. After extraction, the fDNA was prepared by sonication. 50 ng / μl of fDNA was dissolved in PBS. After the addition, the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes before separation. After that, the mixture was denatured at 95°C for 1 minute, and then the biotin-labeled EGFR protein was added. A biotinylated EGFR probe was added and the mixture was further reacted for 20 minutes. The probes used are shown in Table 7 below.

[0422] [Table 7]

[0423] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The DNA-based EGFR expression (ΔOD) of each cancer cell line was analyzed. Analysis showed that HCC827, PC9, MDA-MB468, MCF7, and and MDA-MB-231 cancer cell lines showed clear EGFR DNA expression (Δ On the other hand, in the MCF7 cancer cell line, However, two repeated experiments showed no EGFR DNA expression (Fig. 10 and Fig. 11). In addition, the EGFR DNA expression results of each cancer cell line and CCLE were used to determine the EGF of each cancer cell line. The EGFR mRNA expression results for each cancer cell line were compared with those for the other cancer cell lines. 8.

[0424] [Table 8]

[0425] As a result, HeLa, H460, PC9, H1975, MDA-MB-468, MCF7 and MDA-MB-231 cancer cell lines. The CF7 cancer cell line showed almost no mRNA expression.

[0426] Example 1.5. Confirmation of ERBB2 (HER2) detection accuracy ERBB2-positive cancer cell lines and known MCs obtained from ATCC and the Korean Cell Line Bank F7, PC9, A549, H1975, H460, MDA-MB468, MCF7 and MDA-MB-231 cancer cell line and H460 cancer cell line, which is known to be ERBB2-negative cancer cell line. The accuracy of ERBB2 detection was confirmed using ERBB2-positive cancer cell lines and The ERBB2-negative cancer cell lines were CCLE (Cancer Cell Line Ency The ERBB2 mRNA levels of each cancer cell line provided by the cyclopedia were used as a standard. and classified them as follows.

[0427] Specifically, genomic DNA from ERBB2-positive and ERBB2-negative cancer cell lines was After extraction, fDNA was prepared by sonication. 50 ng / μl of fDNA was added to PB After adding the nanowires prepared in Preparation Example 1 to S, the reaction was continued for 20 minutes and the nanowires were separated. After that, the mixture was denatured at 95°C for 1 minute, and then the biotin-labeled ERBB was added. Add probe 2 (biotinylated ERBB2 probe) and incubate for 20 minutes. Further reaction was carried out using the probes shown in Table 9 below.

[0428] [Table 9]

[0429] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The DNA-based ERBB2 expression (ΔOD) of each cancer cell line was analyzed to confirm the detection results. Analysis revealed that MCF7, PC9, A549, H1975, and MDA-M were isolated from two replicates. ER was clearly observed in B468, MCF7, and MDA-MB-231 cancer cell lines using only fDNA BB2 DNA expression (ΔOD; cutoff OD>0.010) was shown. The H460 cancer cell line showed no ERBB2 DNA expression in two repeated experiments ( 12 and 13). In addition, the ERBB2 DNA expression results of each of the cancer cell lines and CCL E. ERBB2 mRNA expression results of each cancer cell line were compared. R mRNA values ​​are shown in Table 10 below.

[0430] [Table 10]

[0431] As a result, the experiments showed that MCF7, PC9, A549, H1975, MDA-MB468, MC High mRNA expression was observed in F7, H460, and MDA-MB-231 cancer cell lines. Ta.

[0432] Example 1.6. OGT (O-linked β-N-acetylglucosamin e transferase) Confirmation of detection accuracy OGT-positive cancer cell lines and known UMU obtained from ATCC and the Korean Cell Line Bank C3, KU19-19, 253J, J82, T24, MBT2 cancer cell lines and OGT negative RT4, MDCK, HBL EpC, and Jurkat cancer cell lines were used. The accuracy of OGT detection was confirmed by using OGT-positive and OGT-negative cancer cell lines. The cell line is listed in the CCLE (Cancer Cell Line Encyclopedia). The OGT mRNA levels of each cancer cell line provided were used as a criterion for classification.

[0433] Specifically, we analyzed genomes from OGT-positive and OGT-negative cancer cell lines. c) DNA was extracted and sonicated to prepare fDNA. After the NA was placed in PBS, the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes. After that, the biotin-labeled fragments were separated by a thermal denaturation process at 95°C for 1 minute. Add biotinylated OGT probe and incubate for 20 minutes. The probes used are shown in Table 11 below.

[0434] [Table 11]

[0435] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by analyzing the results of the OGT-positive and OGT-negative cancer cell lines. The fDNA was detected using the nanowire produced in Production Example 1, and then DNA-based OGT was detected. Analysis of the expression (ΔOD) showed that UMUC3, KU19-19, and 253J were significantly different in two replicate experiments. , J82, T24, and MBT2 cancer cell lines showed clear OGT DNA expression (Δ On the other hand, the positive bladder cancer cell lines RT4 and normal bladder cancer cell lines MDCK and HBL_EpC, or Jurkat cells In kat T-lymphocyte cells, OGT DNA expression was observed in both experiments. This was not the case (Figures 14 and 15).

[0436] II. Detection of tumor markers derived from cancer cell lines Example 2. Detection of cancer cell line-derived biomarkers Example 2.1. CEA detection LoVo, a known CEA-positive cancer cell line obtained from ATCC and the Korean Cell Line Bank , MKN45, and SW1116 cancer cell lines, and HCT8, a known CEA-negative cancer cell line. CEA was detected using HCT15, HeLa, and MDA-MB-231 cancer cell lines. In this case, CEA-positive and CEA-negative cancer cell lines were treated with CCLE (Cancer Cell Lysis Encephalopathy). CE of each cancer cell line provided by the Cancer Cell Line Encyclopedia Classification was based on the mRNA level of A.

[0437] Specifically, genomic DNA was extracted from CEA-positive and CEA-negative cancer cell lines. After that, it was sonicated to prepare fDNA form. 50ng / μl of fDNA was added to PBS. After that, the nanowires were added and reacted for 20 minutes before separation. After a temperature denaturation process for 1 minute, a biotin-labeled CEA probe (biotinyl The chromatographic CEA probe was added and the reaction was continued for 20 minutes. The probes used are shown in Table 12 below.

[0438] [Table 12]

[0439] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by analyzing the DNA-based CEA expression (ΔOD) of each cancer cell line. The results showed that the fD of LoVo, MKN45, and SW1116 cancer cell lines was significantly higher in three replicate experiments. Only NA showed clear CEA DNA expression (ΔOD; cutoff OD>0.010). On the other hand, HCT8, HCT15, HeLa, and MDA-MB-231 cancer cells The strain showed no CEA DNA expression in all three experiments (FIGS. 16-18).

[0440] Example 2.2. PSA detection PSA-positive cancer cell lines and known LNEs and IFN-γ-glucanase-1 (IFN-γ-glucanase) were obtained from ATCC and the Korean Cell Line Bank. and LNCaP cancer cell lines and known PSA-negative cancer cell lines PC3, DU145, and PSA was detected using the MCF7 cancer cell line. SA-negative cancer cell lines are classified as CCLE (Cancer Cell Line Encyclopedia The PSA mRNA levels of each cancer cell line provided by the National Cancer Institute (NRI) were used as the basis for classification.

[0441] Specifically, genomic DNA was extracted from PSA-positive and PSA-negative cancer cell lines. After that, it was sonicated to prepare fDNA form. 50ng / μl of fDNA was added to PBS. Then, the nanowires prepared in Preparation Example 1 were added and reacted for 20 minutes, followed by separation. After a temperature denaturation process at 95°C for 1 minute, the biotin-labeled PSA probe ( biotinylated PSA probe) was added and the reaction was continued for 20 minutes. The probes used are shown in Table 13 below.

[0442] [Table 13]

[0443] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by analyzing the DNA-based PSA expression (ΔOD) of each cancer cell line. As a result, in two replicate experiments, clear results were obtained using only fDNA from LNE and LNCaP cancer cell lines. PSA DNA expression (ΔOD; cutoff OD>0.010) was shown. In PC3, DU145, and MCF7 cancer cell lines, PSA DNA expression was significantly higher in both experiments. No phenomenon was observed (Figures 19 and 20).

[0444] Example 2.3. CA19-9 Detection Known CA19-9 positive cancer cell lines obtained from ATCC and the Korean Cell Line Bank Capan1, Capn2, and AsPC1 cancer cell lines and CA19-9 negative cancer cell lines CA19-9 was detected using the well-known MIA-PaCa2 and Panc1 cancer cell lines. In this case, CA19-9 positive and CA19-9 negative cancer cell lines were treated with CCL. Cancer Cell Line Encyclopedia (E) provides information on each cancer The cell lines were classified based on the mRNA level of CA19-9.

[0445] Specifically, genomic DNA was analyzed from CA19-9 positive and negative cancer cell lines. The DNA was extracted and sonicated to prepare fDNA. 50 ng / μl of fDNA was used. After placing in PBS, nanowires were added and reacted for 20 minutes before separation. After a thermal denaturation process at ℃ for 1 minute, the biotin-labeled CA19-9 probe ( Add a biotinylated CA19-9 probe and react for 20 minutes. The probes used are shown in Table 14 below.

[0446] [Table 14]

[0447] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by DNA-based CA19-9 expression (ΔOD) of each cancer cell line. Analysis of the results showed that Capan1, Capn2, and AsPC1 cancer cells were Clear CA19-9 DNA expression (ΔOD; cutoff OD>0) was observed in the fDNA of the strain. On the other hand, in the MIA-PaCa2 and Panc1 cancer cell lines, Both experiments showed no CA19-9 DNA expression (FIGS. 21 and 22).

[0448] Example 2.4. CA125 detection A5, a known CA125-positive cancer cell line obtained from ATCC and the Korean Cell Line Bank CA125 was measured using 49 cancer cell lines and the A431 cancer cell line, which is known to be CA125-negative. CA125 was detected in both CA125-positive and CA125-negative cancer cell lines. is provided by CCLE (Cancer Cell Line Encyclopedia) The cancer cell lines were classified based on the CA125 mRNA level.

[0449] Specifically, genomic DNA from CA125-positive and CA125-negative cancer cell lines was After extraction, fDNA was prepared by sonication. 50 ng / μl of fDNA was added to PB After the mixture was placed in S, the nanowires were added and reacted for 20 minutes before separation. After a 1-minute thermal denaturation step at 400°C, a biotin-labeled CA125 probe (bio Tinylated CA125 probe) was added and the reaction was continued for another 20 minutes. The probes used are shown in Table 15 below.

[0450] [Table 15]

[0451] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by analyzing the DNA-based CA125 expression (ΔOD) of each cancer cell line. As a result of the analysis, in two repeated experiments, clear CA125 was detected using only fDNA from the A549 cancer cell line. DNA expression (ΔOD; cutoff OD>0.010) was shown. One cancer cell line showed no CA125 DNA expression in both experiments (Figure 23 and Figure 24).

[0452] Example 2.5. AFP detection Huh7, a known AFP-positive cancer cell line obtained from ATCC and the Korean Cell Line Bank , HepG2, Hep3B, and PLC cancer cell lines, as well as AFP-negative cancer cell lines and known S NU475, SNU387, SNU423, SNU449, SK Hep1 and HeL AFP was detected using cancer cell lines. Cancer cell lines are classified as CCLE (Cancer Cell Line Encyclopedia). The AFP mRNA levels of each cancer cell line provided by ia) were used as the criterion for classification.

[0453] Specifically, genomic DNA was extracted from AFP-positive and AFP-negative cancer cell lines. After that, it was sonicated to prepare fDNA form. 50ng / μl of fDNA was added to PBS. After that, the nanowires were added and reacted for 20 minutes before separation. After a temperature denaturation process for 1 minute, the biotin-labeled AFP probe (biotinyl A fused AFP probe was added and the reaction was continued for 20 minutes. The probes used are shown in Table 16 below.

[0454] [Table 16]

[0455] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The detection results were confirmed by analyzing the DNA-based AFP expression (ΔOD) of each cancer cell line. As a result, Huh7, HepG2, Hep3B, and PLC cancer cell lines were identified in two replicate experiments. Clear AFP DNA expression (ΔOD; cutoff OD>0.010) was observed in only the fDNA of On the other hand, SNU475, SNU387, SNU423, SNU449, S K Hep1 and HeLa cancer cell lines showed AFP DNA expression in both experiments. This was not the case (Figures 25 and 26).

[0456] III. Confirmation of biomarker detection using plasma or urine of cancer patients Example 3. Detection of biomarkers derived from cancer patients Example 3.1. Confirmation of tumor marker detection using plasma from prostate cancer patients PSA, PSMA, PAP, and PCA3 tumor markers for prostate cancer were detected. PSA and PS were used as prostate cancer markers. MA, PAP, and PAC3 are highly expressed in prostate cancer patients and are currently being investigated in cancer tissues and blood. Check the levels of prostate cancer antigens (PSA, PSMA, PAP, PAC3) through a blood test. It is used to differentiate prostate cancer by

[0457] Specifically, plasma was collected from normal humans or prostate cancer patients, and nanowires were added to the plasma. Add the ctDNA (circulating tumor DNA) and let it react for 20 minutes. The ctDNA was isolated. During this process, the ctDNA attached to the nanowire to form a complex. The NA and nanowire complexes were i) used at 27°C without temperature denaturation and ii) The experiment was carried out in two cases: one in which the sample was used after a temperature denaturation process at 95°C for 1 minute, and the other in which the sample was used after a temperature denaturation process at 95°C for 1 minute. Then, each biotinylated probe ) and HRP and streptavidin-labeled polypyrrole nanoparticles (HRP / The NPs were added and reacted for 20 minutes. The probes used are shown in Table 17 below.

[0458] [Table 17]

[0459] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The expression of PSA, PSMA, PAP, and PAC3 was confirmed. P, PAC3 ctDNA expression (ΔOD) was analyzed, and it was found that temperature Without denaturation or after a temperature denaturation process, clear PSA, PSMA, PAP, PAC3 ctDNA expression (ΔOD; cutoff OD>0.015) was demonstrated. On the other hand, in normal humans, both without thermal denaturation and after thermal denaturation, PSA, PSMA, PAP, and PAC3 ctDNA expression (ΔOD; cutoff OD > 0.015) was not observed (Figures 27 to 32).

[0460] Example 3.2. Confirmation of tumor marker detection using plasma from lung cancer patients NSE, SCC, CEA, and Cyfr were detected in plasma obtained from normal subjects or lung cancer patients. a21-1, TPA lung cancer tumor markers were detected. NSE, S CC, CEA, Cyfra21-1, and TPA are highly expressed in lung cancer patients, and currently Lung cancer antigens (NSE, SCC, CEA, Cyfra21) are detected through cancer tissue and blood tests. 1, TPA) values ​​are used to differentiate lung cancer.

[0461] Specifically, plasma was collected from normal humans or lung cancer patients, and nanowires were added to the plasma. The mixture was incubated for 20 minutes to separate ctDNA (circulating tumor DNA). At this time, the ctDNA adheres to the nanowire to form a complex. and nanowire complexes were used at i) 27°C without temperature denaturation and ii) 95°C. The experiment was carried out in two cases: one in which the sample was used after being subjected to a temperature denaturation process at a temperature of ℃ for 1 minute, and the other in which the sample was used after being subjected to a temperature denaturation process at a temperature of ℃ for 1 minute. , biotinylated probes and and polypyrrole nanoparticles labeled with HRP and streptavidin (HRP / st The reaction was continued for 20 minutes. The lobes are shown in Table 18 below.

[0462] [Table 18]

[0463] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The expression of NSE, SCC, CEA, Cyfra21-1, and TPA was confirmed. NA-based NSE, SCC, CEA, Cyfra21-1, TPA ctDNA expression ( As a result of analyzing the ΔOD, it was found that there was no or no thermal denaturation in two lung cancer patients. If the sexual process is followed, clear NSE, SCC, CEA, Cyfra21-1, TPA c tDNA expression (ΔOD; cutoff OD>0.010) was observed. In the case of the sample without temperature denaturation and with temperature denaturation, NSE, SCC, and CE A, Cyfra21-1 and TPA ctDNA expression (ΔOD; cutoff OD > 0. 010) was not shown (Figures 33 to 35).

[0464] Example 3.3. Confirmation of tumor marker detection using plasma from thyroid cancer patients CEA, NSE, TG, CA from plasma obtained from normal subjects or thyroid cancer patients LCA detected tumor markers for thyroid cancer. CEA and NSE were used as thyroid cancer markers. , TG, and CALCA are highly expressed in thyroid cancer patients, and currently, cancer tissue and blood samples Check the levels of thyroid cancer antigens (CEA, NSE, TG, CALCA) through screening. It is used to differentiate thyroid cancer.

[0465] Specifically, plasma was collected from normal humans or thyroid cancer patients, and nanowires were added to the plasma. Add the ctDNA (circulating tumor DNA) and let it react for 20 minutes. The ctDNA was isolated. During this process, the ctDNA attached to the nanowire to form a complex. The NA and nanowire complexes were i) used at 27°C without temperature denaturation and ii) The experiment was carried out in two cases: one in which the sample was used after a temperature denaturation process at 95°C for 1 minute, and the other in which the sample was used after a temperature denaturation process at 95°C for 1 minute. Then, each biotinylated probe ) and HRP and streptavidin-labeled polypyrrole nanoparticles (HRP / The NPs were added and reacted for 20 minutes. The probes used are shown in Table 19 below.

[0466] [Table 19]

[0467] For colorimetric detection, a solution of TMB and H2O2 in sodium acetate buffer was prepared. The expression of CEA, NSE, TG, and CALCA was confirmed by ctDNA-based CEA analysis. Analysis of ctDNA expression (ΔOD) of NSE, TG, and CALCA revealed that thyroid cancer patients In subjects without thermal denaturation or after thermal denaturation, clear CEA and NS E, TG, CALCA ctDNA expression (ΔOD; cutoff OD>0.010) On the other hand, in normal humans, the temperature denaturation process was not observed. Furthermore, CEA, NSE, TG, and CALCA ctDNA expression (ΔOD; cutoff O D>0.010) was not observed (Figures 36 to 40).

[0468] Example 3.4. Confirmation of tumor marker detection using urine from bladder cancer patients OGT, FGFR3, TP53, and NMP were detected in urine collected from normal subjects or bladder cancer patients. 22. Cyfra21-1 was detected as a tumor marker for bladder cancer. OGT, FGFR3, TP53, NMP22, and Cyfra21-1 were detected in the urine of bladder cancer patients. It can be used to differentiate bladder cancer by showing high values ​​in

[0469] Specifically, after collecting urine from normal humans or bladder cancer patients, nanowires were added and then The reaction was allowed to proceed for 10 minutes to separate ctDNA (circulating tumor DNA). At this time, ctDNA adheres to the nanowire to form a complex. The Nowire complex was used at i) 27°C without temperature denaturation and ii) 95°C. The experiment was carried out in two cases: one in which the sample was used after undergoing a temperature denaturation process at room temperature for 1 minute, and the other in which the sample was used after undergoing a temperature denaturation process at room temperature for 1 minute. Each biotinylated probe and Polypyrrole nanoparticles labeled with HRP and streptavidin (HRP / st-t The reaction ...

Claims

[Claim 1] a) mixing a biological sample isolated from an individual containing cell-free DNA (hereinafter referred to as cfDNA) with a positively charged substance; b) separating the positively charged substance to which the cfDNA is bound; c) sequentially or simultaneously mixing a probe having a sequence complementary to the cfDNA and a marker with the mixture; d) removing probes and markers that do not bind to the cfDNA; and e) detecting said marker; a method for diagnosing prostate cancer by detecting a gene derived from a prostate cancer cell from the sample without amplification, the cfDNA is derived from a prostate cancer cell; A method for diagnosing prostate cancer, in which a probe having a sequence complementary to the cfDNA binds complementarily to a gene known to be a biomarker for prostate cancer.

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