Method for detecting colorectal cancer DNA using lateral flow assay
Patent Information
- Application Number
- PCT/KR2025/004790
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-27
- Filing Date
- 2025-04-09
- Publication Date
- 2026-10-01
Smart Images
Figure KR2025004790_01102026_PF_FP_ABST
Abstract
Description
Detection method of colorectal cancer DNA using lateral flow analysis
[0001] The present invention relates to a method for the early detection of colorectal cancer DNA using a Lateral Flow Assay (LFA), and more specifically, to a method for the early detection of colorectal cancer DNA using a Lateral Flow Assay and a detection kit capable of rapidly detecting circulating tumor DNA (ctDNA) with specific genetic mutations in blood samples by utilizing loop-mediated isothermal amplification (LAMP) technology.
[0002] Colorectal cancer (CRC) remains a major public health issue in the United States, ranking as the second leading cause of cancer-related deaths. Despite advancements in medical technology, the incidence of colorectal cancer is increasing among young adults, highlighting the need for innovative screening and diagnostic methods. Current screening methods, such as fecal immunohistochemistry (FIT) and colonoscopy, are invasive and can lead to delayed diagnosis, particularly in the younger population. Therefore, there is a critical need for accessible and effective diagnostic tools capable of early detection of colorectal cancer.
[0003] The emergence of liquid biopsy technology has enabled a non-invasive alternative to traditional tissue biopsies for detecting and monitoring genetic diseases, particularly cancer, thereby allowing for early detection, real-time monitoring, and personalized treatment. By detecting and analyzing circulating tumor DNA (ctDNA) in blood samples, liquid biopsy can determine the genetic status of a disease in real time. However, the widespread adoption of liquid biopsy faces challenges due to the high cost, complexity, and time-consuming nature of existing methods.
[0004] Furthermore, although LFA has the potential to democratize access to genetic testing, particularly in resource-constrained environments, due to its simplicity, rapid speed, and suitability for point-of-care testing, its utilization has been limited by issues with sensitivity and specificity.
[0005] Prior art literature
[0006] Patent documents
[0007] 1. Republic of Korea Patent Publication No. 10-2020-0089864 (Method for predicting lung cancer based on circulating cancer DNA and circulating cancer cells in the blood),
[0008] 2. Republic of Korea Patent Publication No. 10-2023-0172174 (Method for cancer diagnosis and cancer type prediction using single nucleotide mutations in cell-free nucleic acids).
[0009] Accordingly, the present invention has been devised to solve the aforementioned problems. The objective of the present invention is to improve the accessibility of liquid biopsy by developing a paper-based lateral flow analysis (LFA) method, and to provide a method for the early detection of colorectal cancer DNA using lateral flow analysis that can rapidly detect circulating tumor DNA (ctDNA) with specific genetic mutations in blood samples with a design that is efficient and economical.
[0010] In addition, the invention provides a method for the early detection of colorectal cancer DNA and a detection kit utilizing a lateral flow analysis method that enhances sensitivity and specificity by employing loop-mediated isothermal amplification (LAMP) technology, enabling precise early detection of low-frequency DNA mutations associated with rare genetic diseases, cancer, Alzheimer's disease, etc.
[0011] However, the technical problems to be solved by the present invention are not limited to those mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art to which the present invention belongs from the description below.
[0012] To achieve the above technical objectives, a method for detecting colorectal cancer DNA using a lateral flow analysis method is provided, comprising the steps of: extracting a blood sample (S100); loading the blood sample into a lateral flow analysis (LFA) kit (S110); amplifying a specific target circulating tumor DNA (ctDNA) fragment on the kit using Single Nucleotide Polymorphism (SNP) typing based on a loop-mediated isothermal amplification (LAMP) device (S130); moving the amplified SNP sequence along a nitrocellulose membrane and binding with a gold nanoparticle probe to form a DNA-AuNP complex (S140); and a first probe developing color during the complex formation process so as to be visually detectable (S150); thereby enabling detection of whether colorectal cancer mutant DNA is present in the blood sample based on color development.
[0013] In addition, the specific gene may be at least one of the KRAS, NRAS, PIK3CA, BRAF, and EGFR genes and their related genes, and the mutant gene to be investigated is at least one of BRAF V600E, KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G13D, KRAS A146T, PIK3CA E542K, PIK3CA E545K, and PIK3CA H1047R.
[0014] In addition, the amplification step may include a step in which the 5' end of the FIP (Forward Inner Primer) is extended to the allele of the SNP and the 5' end of the BIP (Backward Inner Primer) is extended to the complementary allele of the SNP.
[0015] Additionally, the method may further include a step (S160) of moving an extra blood sample to a control line to indicate whether the second probe is functioning normally by developing color.
[0016] Additionally, between the loading step (S110) and the amplification step (S130), a step (S120) may be further included in which the multilayer filter of the kit removes impurities, such as red blood cells, from the blood sample and allows only serum to pass through.
[0017] In addition, at least one of the LAMP and the multilayer filter operates on a battery.
[0018] In addition, the loop-mediated isothermal amplifier (LAMP) device amplifies at an isothermal temperature of 60 to 65°C.
[0019] The objective of the present invention as described above is, in another category, to comprise: a loading pad on which a blood sample is loaded; a multilayer filter located below the loading pad that removes impurities such as red blood cells from the blood sample and allows only serum to pass through; a loop-mediated isothermal amplification (LAMP) device located below the multilayer filter that amplifies a specific target circulating tumor DNA (ctDNA) fragment among colorectal cancer mutant DNA using Single Nucleotide Polymorphism (SNP) typing; a battery that supplies power to at least one of the LAMP device and the multilayer filter; a nitrocellulose membrane located below the LAMP device through which the amplified SNP sequence travels; and a first probe of gold nanoparticles that binds to the SNP sequence traveling along the nitrocellulose membrane to form a DNA-AuNP complex. This can also be achieved by a detection kit for colorectal cancer DNA using a lateral flow analysis method, characterized by including a second probe that indicates whether it is operating normally by moving an extra blood sample to a control line and developing color; and enabling detection of whether colorectal cancer mutant DNA is present in a blood sample based on the color development of the first and second probes.
[0020] According to one embodiment of the present invention, Point-of-Care Testing (POCT) can be performed quickly and affordably compared to conventional invasive, costly, and time-consuming diagnostic methods.
[0021] Furthermore, the LFA used in the present invention enables early detection of colorectal cancer by utilizing a loop-mediated isothermal amplification (LAMP) method to detect specific circulating tumor DNA (ctDNA) and genetic mutations in the blood. This LAMP does not require a conventional thermal circulator and can amplify target sequences at an isothermal temperature of 60 to 65°C using an inexpensive battery-powered heater. As a result, it offers the advantages of convenient portability and easy maintenance.
[0022] In addition, the kit of the present invention is battery-powered, enabling the LAMP and multilayer filter to rapidly detect genetic mutations at a low cost.
[0023] However, the effects obtainable from the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.
[0024] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the detailed description of the invention provided below; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings.
[0025] FIG. 1 is a graph showing 10 mutant DNAs identified in 18 colorectal cancer (CRC) studies according to cBioPortal, which can be used for the early detection of colorectal cancer DNA using the lateral flow analysis method of the present invention.
[0026] FIG. 2 is a schematic perspective view of a proposed lateral flow analysis (LFA) kit for ctDNA detection according to the present invention.
[0027] FIG. 3 is a schematic diagram of LAMP-based SNP typing according to the present invention.
[0028] Figure 4 is an example of a 601 bp long ctDNA sequence containing a central SNP allele (the SNP from T to A is shown in bold),
[0029] Figure 5 is a capture of the output screen of PrimerExplorer,
[0030] Figure 6 is an example of a modified LAMP primer sequence to amplify ctDNA containing an SNP allele,
[0031] Figure 7 is an example of a final LAMP primer sequence for amplifying a BRAF_V600E ctDNA fragment.
[0032]
[0033] Below, with reference to the attached drawings, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, since the description of the present invention is merely an example for structural or functional explanation, the scope of the present invention should not be interpreted as being limited by the embodiments described in the text. That is, since the embodiments are subject to various modifications and may take various forms, the scope of the present invention should be understood to include equivalents capable of realizing the technical concept. Furthermore, the objectives or effects presented in the present invention do not imply that a specific embodiment must include all of them or only such effects; therefore, the scope of the present invention should not be understood as being limited by them.
[0034] The meaning of the terms described in this invention should be understood as follows.
[0035] Terms such as "first" and "second" are intended to distinguish one component from another, and the scope of rights shall not be limited by these terms. For example, the first component may be named the second component, and similarly, the second component may be named the first component. When a component is referred to as being "connected" to another component, it should be understood that it may be directly connected to that other component, or that there may be other components in between. Conversely, when a component is referred to as being "directly connected" to another component, it should be understood that there are no other components in between. Meanwhile, other expressions describing the relationship between components, such as "between" and "exactly between," or "adjacent to" and "directly adjacent to," shall be interpreted in the same manner.
[0036] A singular expression should be understood to include a plural expression unless the context clearly indicates otherwise, and terms such as "include" or "have" are intended to specify the existence of the set-up features, numbers, steps, actions, components, parts, or combinations thereof, and should be understood not to preclude the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.
[0037] Unless otherwise defined, all terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having meanings consistent with the context of the relevant technology and should not be interpreted as having an ideal or overly formal meaning unless explicitly defined in this invention.
[0038] Examples
[0039] Hereinafter, the configuration of a preferred embodiment will be described in detail with reference to the attached drawings. First, lateral flow analysis (LFA) can increase the accessibility of genetic testing in environments with limited surrounding conditions (e.g., the field) due to its simplicity, speed, and suitability for point-of-care testing. However, its application has been limited due to issues with the sensitivity and specificity of LFA.
[0040] Colorectal cancer biomarker identification and profiling
[0041] First, 74 mutations in five genes—KRAS, NRAS, PIK3CA, BRAF, and EGFR—were targeted, which is consistent with Agena Bioscience's UltraSEEK® Colon Pane. In the initial analysis, 10 somatic mutations in the BRAF, KRAS, and PIK3CA genes were identified as being associated with colorectal cancer. These three genes are particularly significant in colorectal cancer. Figure 1 is a graph showing the frequency of each of the 10 mutations associated with colorectal and colon adenocarcinoma in 18 studies published on cBioPortal. This study included 7,420 samples collected from a total of 7,244 patients.
[0042] Specifically, one mutation in the BRAF gene, six mutations in the KRAS gene, and three mutations in the PIK3CA gene were identified. The 10 selected mutations—BRAF V600E, KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G13D, KRAS A146T, PIK3CA E542K, PIK3CA E545K, and PIK3CA H1047R—are known to significantly impact the progression of colorectal cancer and patient response to various treatments. The inclusion of these major mutations offers a good opportunity for early detection and personalized treatment strategies.
[0043] BRAF - V600E
[0044] The BRAF gene encodes serine / threonine protein kinases and plays a crucial role in regulating the mitogen-activated protein kinase (MAPK) pathway. BRAF mutations persistently activate these kinases, promoting cell proliferation and the evasion of apoptosis. In particular, the V600E mutation induces abnormal overactivation in the MAPK pathway independently of the RAS. The V600E mutation is located in exon 15 and results in the substitution of valine (V) with glutamate (E) at position 600. Approximately 10% of patients with colorectal cancer (CRC) exhibit BRAF mutations, and V600E is the major variant, accounting for 95% of all BRAF mutations. In addition, about 30% of BRAF mutations correspond to the CpG island methylator phenotype (CIMP) and high levels of microsatellite instability (MSI) resulting from methylation of the MLH1 promoter. These molecular characteristics form specific subtypes of colorectal cancer (CRC) with unique clinical and pathological features.
[0045] The BRAF V600E mutation is associated with resistance to anti-EGFR (anti-Epidermal Growth Factor Receptor) monoclonal antibodies and an overall poor prognosis. This mutation induces aggressive tumor behavior and is associated with lower survival rates compared to wild-type BRAF CRC. Patients with metastatic colorectal cancer (CRC) often develop resistance to EGFR and BRAF inhibitors. Currently, FDA-approved treatments for patients with the BRAF V600E mutation in colorectal cancer (CRC) include combination therapy with the RAF targeted inhibitor encorafenib and the anti-EGFR antibody cetuximab. While this combination therapy has demonstrated improved efficacy compared to monotherapy, managing BRAF-mutated colorectal cancer (CRC) remains challenging due to the development of resistance and tumor heterogeneity.
[0046] Despite these advancements, the clinical management of BRAF-mutated colorectal cancer (CRC) remains challenging. Personalized therapeutic approaches guided by molecular profiling are increasingly recognized as crucial for improving outcomes in this patient population. Ongoing research focuses on developing novel therapeutic strategies, particularly combination therapies, that target multiple pathways and resistance mechanisms. The integration of precision medicine into clinical practice offers hope for more effective treatment responses by providing therapies tailored to the genetic and molecular characteristics of each patient's tumor.
[0047] KRAS - G12V, G12C, G12A, G13D, A146T
[0048] The KRAS gene encodes the GTP / guanosine diphosphate (GDP) binding protein and plays a critical role in regulating the rapid progression fibrosarcoma (RAF)-mitogen-activated protein kinase (MAPK), MEK-extracellular signal-regulating kinase (ERK), and phosphokinositol 3-kinase (PI3K)-protein kinase B (AKT)-rapamycin mechanism target (mTOR) pathways. KRAS mutations have a profound impact on treatment response. In particular, these mutations affect the efficacy of anti-epidermal growth factor receptor (EGFR) therapy, and patients with the wild-type KRAS gene exhibit a better treatment response. Detection of KRAS mutations is considered essential before initiating anti-EGFR antibody therapy. Furthermore, when BRAF mutations co-occur with KRAS mutations, the response to anti-EGFR therapy can become more complex, particularly after first-line treatment.
[0049] Mutations in the RAS protein family deserve particular attention as they are found in approximately half of metastatic colorectal cancer (CRC) cases. Mutations in both KRAS and NRAS have similar effects on downstream growth factor signaling, and harmful mutations in these proteins induce persistent growth signals in cells even in the absence of upstream cell surface signals induced by EGFR or TGFα. Common mutations in KRAS and NRAS occur at codons 12 and 13; these mutations affect the protein's ability to hydrolyze and inactivate bound GTP, resulting in a state of permanently activated RAS protein that does not respond to changes in upstream signaling induced by anti-EGFR therapy. According to a comprehensive study of the COSMIC database, KRAS was identified with mutations at codons 12 and 13 in 18,376 out of 53,978 colorectal cancer samples (34%). Within codon 12, KRAS G12D is the most common, while G12V is the second most common and is associated with a higher risk of mortality. NRAS mutations are less common; although mutations occurred at codons 12 and 13 in 150 out of 8,669 colorectal cancer samples (2%), their mechanisms are similar to those of KRAS mutations. Studies suggest that KRAS and NRAS mutations are likely mutually exclusive at these locations. Furthermore, positive results for KRAS mutations were found to be associated with the severity of metastasis. The likelihood of KRAS mutations increases with each stage of the Dukes' staging system; approximately two-thirds of severely metastatic (stage D) cases carry the mutation, whereas only one-quarter of localized (stage A) cases do. This suggests that liquid biopsy diagnoses can serve as a broader indicator of disease severity.
[0050] PIK3CA - E542K, E545K, H1047R
[0051] The PIK3CA gene encodes the catalytic subunit of PI3-kinase, a critical enzyme involved in the PI3K / AKT signaling pathway that regulates various cellular processes such as cell growth, proliferation, and survival. PIK3CA mutations lead to sustained activation of the PI3K pathway, inducing cell growth and tumor progression. These mutations occur in 10–20% of colorectal cancer (CRC) patients, mostly in exons 9 and 20.
[0052] The E542K and E545K variants are located in exon 9 and involve a mutation in which glutamate (E) is substituted for lysine (K) in the protein's helical domain. These mutations result in the loss of p85-mediated inhibition of catalytic activity, leading to sustained activation of PIK3CA. The H1047R variant is located in exon 20 and involves a mutation in which histidine (H) is substituted for arginine (R) in the protein's kinase domain. This mutation exposes the catalytic loop to the cell membrane, sustainably activating the catalytic subunit of PI3K. This exposure further amplifies the catalytic activity of PI3K, promoting oncogenic signaling. All three variants—E542K, E545K, and H1047R—are considered to be frequently occurring hotspot mutations in colorectal cancer. These mutations are associated not only with promoting tumorigenesis but also with treatment resistance. In particular, these mutations can confer resistance to treatments such as fulvestrant, an estrogen receptor antagonist used in cancer hormone therapy.
[0053] In addition, the presence of PIK3CA mutations can affect the clinical management of colorectal cancer patients and may provide information for combination therapies to overcome resistance and improve treatment efficacy.
[0054] LFA analysis method
[0055] The paper-based lateral flow analysis (LFA) according to the present invention is designed to detect DNA fragments with higher sensitivity and specificity than conventional analysis methods. This LFA utilizes the loop-mediated isothermal amplification (LAMP) method to enhance the ability to detect specific circulating tumor DNA (ctDNA) and genetic mutations in blood. While DNA amplification is crucial for increasing the sensitivity and specificity of the analysis method, conventional thermal circulators were not practical for field use due to the need for expensive laboratory equipment. LAMP overcomes this limitation by enabling isothermal amplification of target sequences at 60 to 65°C. This reaction can be performed using a simple and inexpensive battery-powered heater.
[0056] In addition, the present invention optimizes ctDNA extraction by innovating the blood sample collection procedure. By purifying this process, low-frequency DNA mutations that aid in the diagnosis and monitoring of genetic diseases, including cancer and Alzheimer's disease, can be detected more effectively. The LFA includes a multilayer filter that removes unnecessary components, such as red blood cells, and separates serum containing ctDNA.
[0057] FIG. 2 is a schematic perspective view of a proposed paper-based lateral flow analysis (LFA) kit for ctDNA detection according to the present invention. As shown in FIG. 2, after loading a blood sample, a multilayer filter on the sample loading pad removes impurities and leaves only serum for ctDNA detection. Specific target ctDNA fragments are amplified using LAMP-based single nucleotide polymorphism (SNP) typing. The 5' ends of the Forward Inner Primer (FIP) and Backward Inner Primer (BIP) are designed to extend to the SNP allele and its complementary allele, respectively.
[0058] In an embodiment of the present invention, a LAMP-based signal amplification method is used to increase the sensitivity and specificity of the analysis method. After this optimization work is completed, a clinical evaluation of the performance of the analysis method is performed in the subsequent verification step.
[0059] Objective 1 - Optimization of Paper-Based Lateral Flow Analysis for Enhanced ctDNA Detection:
[0060] Objective 1 is to develop a kit prototype of a paper-based lateral flow analysis (LFA) capable of detecting circulating tumor DNA (ctDNA) and specific DNA mutations in blood with increased sensitivity and specificity using LAMP technology.
[0061] Primers for SNP-specific LAMP amplification
[0062] The goal here is to amplify ctDNA fragments containing specific SNPs that are not wild-type. This is called LAMP-based SNP typing. The key is to ensure that the 5' end of the Forward Inner Primer (FIP) extends to the SNP allele and the 5' end of the Backward Inner Primer (BIP) extends to the SNP's complementary allele.
[0063] FIG. 3 is a schematic diagram of LAMP-based SNP typing according to the present invention, wherein the MEVB vaccine strain in the first figure is a schematic diagram of a DNA fragment that amplifies only SNP alleles, and the wild-type MEV strain in the second figure is not amplified with wild-type alleles.
[0064] Subsequently, the amplified SNP sequence travels along the nitrocellulose membrane and binds to a gold nanoparticle probe to form a DNA-AuNP complex. These nanoparticles emit strong red fluorescence upon hybridization, which is visible to the naked eye and ideal for point-of-care diagnosis. If the target DNA is present, it hybridizes with the complementary sequence on the first probe of the test line to generate a red signal. The excess blood sample continues to the control line, causing the second probe to emit a second red signal. This confirms that the analysis is functioning correctly.
[0065] For example, it shows the technical details of the LAMP primer sequence design for amplifying a ctDNA fragment mutated from T to A in the BRAF gene. This mutation causes valine (V) at the 600th amino acid to be converted to glutamic acid (E) (NM_004333.6(BRAF):c.1799T>A (p.Val600Glu)).
[0066] Objective 1 - Step 1: Obtain the sequence of a 601 bp long ctDNA fragment with a central SNP sequence.
[0067] The ClinVar entry for this variant indicates the coordinates in the reference genome GRCh38 as chr7:140753336. Therefore, chr7:140753036-140753636 represents the 601 bp segment where the SNP is centered. Since the gene is located in the reverse chain, use the UCSC Genome Browser to select the reverse complement option and obtain a FASTA file containing the wild-type allele in the center. Then, manually edit 'T' to 'A' to obtain a ctDNA fragment FASTA file containing the SNP allele.
[0068] Figure 4 is an example of a 601 bp long ctDNA sequence containing a central SNP allele (the SNP from T to A is shown in bold),
[0069] Goal 1 - Step 2: Design LAMP Primers, Run PrimerExplorer on the Website
[0070] Next, PrimerExplorer version 5, a website for generating LAMP primers, was used. The above FASTA file was used as input, and a set of parameters set to automatic determination was used to generate four sets of candidate LAMP primers. Figure 5 is a capture of the PrimerExplorer output screen.
[0071] In Figure 5, the first row is the input FASTA sequence with the SNP allele 'A' marked with an asterisk. The second row is the inverse complementary sequence. The last row, marked with a box, is the candidate sequence aligned with the input FASTA.
[0072] Objective 1 - Step 3: Modify primer sequences obtained from PrimerExplorer to amplify non-wild type SNP alleles
[0073] To amplify only ctDNA containing non-wild type SNP alleles, the reverse of F1c must end at the SNP allele and B1c must start at the SNP allele. The sequence is manually modified for SNP amplification as shown in Fig. 5. Fig. 6 is an example of a modified LAMP primer sequence to amplify ctDNA containing SNP alleles. As shown in Fig. 6, the final four LAMP primer sequences are illustrated to amplify only the ctDNA fragments with the T-to-A mutation of the BRAF gene.
[0074] In Figure 6, the middle removal line indicates the deleted base (C), and the base in bold font (GA) is the inserted base. The base in bold font (T) indicates the SNP allele. The 5'-terminus of FIP is the SNP allele, and the 5'-terminus of BIP is the SNP complementary allele.
[0075] Figure 7 is an example of a final LAMP primer sequence for amplifying a BRAF_V600E ctDNA fragment.
[0076] Objective 2 - Systematic analysis of the sensitivity and specificity of lateral flow analysis methods
[0077] Objective 2 aims to comprehensively evaluate the sensitivity and specificity of lateral flow analysis (LFA) for the detection of circulating tumor DNA (ctDNA). The sensitivity, specificity, and the area under the receiver operation characteristic (ROC) curve (AUC) are calculated, and the criteria are based on these values being 90% or higher.
[0078] The embodiments of the present invention do not use human samples or actual human data; instead, artificially synthesized DNA fragments having the sequences mentioned above are injected at controlled concentrations, and sensitivity and specificity are measured.
[0079] After fabricating the prototype, sensitivity (True Positive Rate, TRP) and specificity analyses were performed to evaluate the performance of the LFA. Each criterion demonstrated an effectiveness of over 90%, and the Area Under the Curve (AUC) value of the Receiver Operating Curve (ROC) was 0.9 or higher.
[0080] As described above, the detailed description of the preferred embodiments of the present invention disclosed is provided to enable those skilled in the art to implement and practice the present invention. Although the present invention has been described with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the invention. For example, those skilled in the art may utilize each configuration described in the embodiments described above in combination with one another. Accordingly, the present invention is not intended to be limited to the embodiments shown herein, but to be given the broadest scope consistent with the principles and novel features disclosed herein.
[0081] The present invention may be embodied in other specific forms without departing from the spirit and essential features of the invention. Accordingly, the above detailed description should not be interpreted restrictively in all respects but should be considered exemplary. The scope of the invention shall be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the invention are included within the scope of the invention. The invention is not intended to be limited to the embodiments shown herein, but to be given the broadest possible scope consistent with the principles and novel features disclosed herein. Furthermore, embodiments may be constructed by combining claims that are not explicitly related in the claims, or by including them as new claims through amendments made after filing.
[0082] This invention was made possible through government support under project number RS-2024-00511676, provided by the Ministry of SMEs and Startups through the Korea Technology Information Promotion Agency for SMEs. This research was conducted as part of the Technology Innovation Program for Startups (TIPS), and the project title is "Development of a POCT device for early diagnosis, recurrence, and drug resistance response monitoring of hematological cancer using paper-based LAMP-LFA technology for DNA biomarker detection, and a hematological cancer-specific DNA-based Human Digital Twin AI integrated platform." The government contribution rate was 100%, and the lead organization for this project was Predictive AI Co., Ltd.
Claims
1. In a method for detecting colorectal cancer DNA, Step of extracting a blood sample (S100); A step (S110) of loading the above blood sample into a lateral flow analysis (LFA) kit; A step (S130) in which a specific target circulating tumor DNA (ctDNA) fragment is amplified on the above kit using Single Nucleotide Polymorphism (SNP) typing based on a Loop-mediated isothermal amplification (LAMP) device; and A step (S140) in which the amplified SNP sequence moves along a nitrocellulose membrane and binds to a gold nanoparticle probe to form a DNA-AuNP complex; and A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized by including a step (S150) in which a first probe develops color so as to be visually detectable during the complex formation process, thereby enabling detection of whether colorectal cancer mutant DNA is present in the blood sample based on the color development.
2. In Paragraph 1, A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized in that the specific gene is at least one of the KRAS, NRAS, PIK3CA, BRAF, and EGFR genes and a related gene.
3. In Paragraph 2, A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized in that the specific gene is at least one of the KRAS, PIK3CA, and BRAF genes and a related gene.
4. In Paragraph 2, A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized in that the above-mentioned mutant gene is at least one of BRAF V600E, KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G13D, KRAS A146T, PIK3CA E542K, PIK3CA E545K, and PIK3CA H1047R.
5. In Paragraph 1, A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized in that the amplification step includes a step in which the 5' end of the FIP (Forward Inner Primer) is extended to the allele of the SNP and the 5' end of the BIP (Backward Inner Primer) is extended to the complementary allele of the SNP.
6. In Paragraph 1, A method for detecting colorectal cancer DNA using a lateral flow analysis method, characterized by further including the step (S160) of moving the extra blood sample to a control line and indicating whether the second probe is operating normally by color development.
7. In Paragraph 1, Between the loading step (S110) and the amplification step (S130), A method for early detection of colorectal cancer DNA using a lateral flow analysis method, characterized by further including a step (S120) in which a multilayer filter of the above kit removes impurities such as red blood cells from the blood sample and allows only serum to pass through.
8. In Paragraph 7, A method for early detection of colorectal cancer DNA using a lateral flow analysis method, characterized in that at least one of the above LAMP and the above multilayer filter operates on a battery.
9. In Paragraph 1, A method for early detection of colorectal cancer DNA using a lateral flow analysis method, characterized in that the loop-mediated isothermal amplification (LAMP) device amplifies at an isothermal temperature of 60 to 65°C.