A CEA detection kit, a detection method and application thereof

Abstract

The application discloses a CEA detection kit, a detection method and application, and belongs to the technical field of biological medicines. The technical problems to be solved are to provide a CEA detection kit to solve the technical problems of dependence on complex equipment, cumbersome operation, insufficient specificity and sensitivity, and non-ideal signal output mode in the prior art. The technical solution points are a CEA detection kit, which comprises: a specific DNA aptamer of CEA; a biotin-labeled forward primer which is complementary to the specific DNA aptamer; a reverse primer which is used for amplifying a DNA template together with the forward primer; the DNA template comprises a sequence complementary to the forward primer and a sequence complementary to the reverse primer; and a CRISPR / Cas protein / crRNA complex, wherein the crRNA is complementary to the sequence of the amplification product of the DNA template.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of medical testing technology, and relates to a CEA detection kit, detection method, and application. Background Technology

[0002] For understanding the technical content of this invention: Early and accurate detection of tumor markers is crucial for cancer screening, prognostic assessment, and monitoring of treatment efficacy. Carcinoembryonic antigen (CEA), as a broad-spectrum tumor marker, is often significantly elevated in the serum of various malignant tumors, including colorectal cancer, gastric cancer, and lung cancer. Currently, the gold standard for CEA detection in clinical laboratories mainly relies on immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). While these methods offer high specificity and accuracy, they generally suffer from the following limitations: reliance on large, sophisticated instruments, cumbersome procedures, long testing cycles, and high costs, making it difficult to implement point-of-care testing (POCT) in primary healthcare institutions, homes, or resource-limited areas.

[0003] In recent years, nucleic acid detection methods based on molecular diagnostic technologies, particularly the CRISPR / Cas system, have developed rapidly due to their advantages of high specificity, high sensitivity, and programmed operation. CRISPR / Cas12a, after recognizing and activating target DNA, exhibits non-specific "trans-cleavage" activity of single-stranded DNA (ssDNA), a characteristic that has been widely used to construct highly sensitive biosensors. To detect non-nucleic acid targets (such as proteins), it is usually necessary to convert the target recognition event into the generation or release of a nucleic acid signal, thereby activating the CRISPR / Cas system. Nucleic acid aptamers, due to their high affinity and specificity similar to antibodies, and their ease of synthesis and modification, are often used as "converters" to convert protein signals into nucleic acid signals.

[0004] Relevant patent documents retrieved: The country of origin is China, publication number CN120539412A, publication date 2025.08.26, entitled "Paper-based Reagent Kit for Detection of Extracellular Vesicle Surface Proteins and Its Application", which discloses a method to improve target specificity by synergistically capturing sEVs with dual aptamers, amplifying the signal by cascading superbranched rolling circle amplification (HRCA) and CRISPR / Cas12a system, and achieving visual detection by detecting the separation of FAM-biotin probes before and after cutting through a transverse flow test strip.

[0005] Relevant non-patent literature retrieved: The document, titled "Research on the Detection of microRNA-21 and Carcinoembryonic Antigen Using a Photoelectrochemical Sensor," authored by Liu Minghui at Qingdao University of Science and Technology and published on April 6, 2023, discloses a method for detecting CEA based on a self-powered photoelectrochemical (PEC) aptamer sensor. The technical solution involves capturing CEA using a dual-aptamer sandwich method, where a Hemin / G4 DNAzyme is attached to the secondary antibody aptamer. This enzyme catalyzes the formation of an insoluble precipitate of the substrate on the electrode surface, leading to a decrease in the photocurrent signal and thus enabling detection.

[0006] The document, titled "CRISPR / Cas System-Mediated Urease Colorimetric Method and the Role of Nanomaterial (ZIF-8) in the Diagnosis and Treatment of Gastric Cancer," by Jin Xin, Shandong University, and published on June 30, 2025, discloses a CRISPR / Cas system-mediated urease colorimetric method for the immediate detection of tumor markers in serum samples. Through feasibility studies, condition optimization, standard curve establishment, sensitivity and specificity investigations, and real-sample testing, the method was demonstrated to accurately and effectively detect tumor markers in serum, providing precise guidance for the diagnosis of gastric cancer (GC) and post-GC surgery. Furthermore, a ZIF-8 nanomaterial loaded with xanthine oxidoreductase (XOR) was designed and synthesized. Material characterization and in vivo and in vitro experiments confirmed its specific aggregation and XOR release at the GC site. Subsequently, it generates ROS and promotes macrophage polarization to achieve a therapeutic effect on GC, aiming to provide a new treatment method for GC.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) Reliance on complex equipment or complex operating procedures: Existing tumor marker detection methods (such as ELISA and chemiluminescence) rely on large instruments and specialized operations, making it difficult to achieve rapid point-of-care testing; traditional nucleic acid detection methods (such as PCR) require thermal cycling equipment, which is complex and time-consuming. For example, the non-patent literature "Research on the detection of microRNA-21 and carcinoembryonic antigen using photoelectrochemical sensors" requires the preparation of complex photoelectrodes and dedicated electrochemical workstations, making it difficult to achieve true portability and POCT. The HRCA amplification used in CN120539412A requires a ligation and circularization step, which is relatively cumbersome. Existing CRISPR detection usually requires a separate pre-amplification step, which increases the complexity of operation and the risk of aerosol contamination.

[0008] (2) The signal output method is not ideal: Although patent document CN120539412A achieves visual detection, it relies on the interpretation of the strips on the test strip, and the results are easily affected by subjective factors, resulting in limited accurate quantitative ability. Non-patent document "CRISPR / Cas system-mediated urease colorimetric method and the role and research of nanomaterial (ZIF-8) in the diagnosis and treatment of gastric cancer" adopts colorimetric output, which has limited sensitivity and is affected by the background color of the sample.

[0009] (3) The design of the aptamer-CRISPR linkage mechanism is complex, resulting in low signal conversion efficiency and insufficient specificity and sensitivity: In order to achieve CRISPR / Cas detection of non-nucleic acid targets such as proteins, it is usually necessary to design a complex aptamer signal conversion and amplification mechanism. For example, in patent CN120539412A, the two aptamers need to recognize two different proteins on the surface of extracellular vesicles, and undergo complex complementary pairing and looping reactions through their respective extended sequences, linker sequences, and circular templates, followed by HRCA amplification, and finally activation of Cas12a to cleave the LFA probe. In addition, in complex biological samples (such as serum and plasma), if there are free single target proteins or non-specific nucleic acid fragments, non-specific amplification or cleavage may be triggered, leading to false positive signals, thereby reducing the specificity and reliability of the detection. Summary of the Invention

[0010] The purpose of this invention is to provide: A CEA detection kit, detection method, and application, and related technologies, to solve the technical problems and combinations thereof in the existing technology, such as reliance on complex equipment, cumbersome operation, insufficient specificity and sensitivity, and unsatisfactory signal output.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Definitions of standard terms can be found in the references “Molecular Cloning: A Laboratory Manual (3rd Edition), Science Press, authors: J. Sambrook and DW Russell, 2002.11”, “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng and Guo Hongwei, 2019-06-19”, and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as centrifugation, incubation, ELISA, chemiluminescence, isothermal amplification (LAMP), NEAR amplification, etc., shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] The term "CRISPR / Cas protein" used in this article refers to the "CRISPR / Cas system," a system of clustered, regularly spaced short palindromic repeats and related proteins, a gene editing and detection tool derived from prokaryotes. For example, CRISPR / Cas12a, guided by crRNA, recognizes a specific DNA sequence and activates its trans-cleavage activity, non-specifically cleaving single-stranded DNA.

[0018] The term "Cas12a" as used in this article refers to CRISPR-associated protein 12a, which belongs to the type V CRISPR-Cas system (a type 2 effector protein) and recognizes and cleaves DNA under the guidance of crRNA. It has dual activities of cis-cleavage (cutting target DNA) and trans-cleavage (non-specific cleavage of single-stranded DNA).

[0019] The term "Cas14" used in this article refers to CRISPR-associated protein 14, belonging to the type V CRISPR-Cas system, and is one of the smallest known Cas proteins (approximately 400-700 amino acids). It can recognize and cleave single-stranded DNA (ssDNA), but has weaker activity towards double-stranded DNA. It is extremely sensitive to mismatches between crRNA and target sequences, exhibiting very high specificity. It does not require a PAM sequence, making recognition more flexible and applicable to highly specific single-stranded DNA detection and aptamer sensing.

[0020] The term "aptamer" as used in this article refers to a single-stranded DNA or RNA molecule obtained through SELEX screening that can bind to target molecules (such as proteins, small molecules, etc.) with high specificity and high affinity. This protocol uses CEA-specific DNA aptamers as recognition elements.

[0021] The term "SELEX" used in this article refers to Systematic Evolution of Ligands by Exponential Enrichment, an in vitro screening method used to screen oligonucleotide sequences, i.e., aptamers, from a large, randomly generated library of artificially synthesized single-stranded nucleic acid (DNA or RNA) sequences through multiple cycles of binding, separation, and amplification.

[0022] The term "fluorescent reporter probe" as used in this article refers to a single-stranded DNA segment labeled with a fluorescent group (such as FAM) and a quencher group (such as BHQ1). When intact, the fluorescence is quenched; after being cleaved by the Cas protein, the fluorescent group and the quencher group are separated, generating a fluorescent signal.

[0023] The term "carcinoembryonic antigen (CEA)" used in this article refers to a broad-spectrum tumor marker that is often abnormally overexpressed in various malignant tumors such as colorectal cancer, gastric cancer, and lung cancer, with a normal serum value generally <5 ng / mL.

[0024] The term "RPA" used in this article refers to Recombinase Polymerase Amplification, which involves the binding of primers to template DNA using a recombinase at a constant temperature (37-42°C). Single-strand binding proteins stabilize the displaced DNA strand, and the strand-displacement DNA polymerase extends the DNA. The reaction is rapid (20-30 minutes) and does not require a thermal cycler.

[0025] The term "liquid phase hybridization capture reagent" as used in this article refers to the process of incubating a single strand of nucleic acid to be captured with a liquid phase hybridization capture reagent (probe) in solution through liquid phase hybridization to form a hybridization complex, thereby capturing the forward primer that has not bound to the specific DNA aptamer.

[0026] The term "signal reporter probe" as used in this article refers to a probe containing single-stranded DNA and labeled with a signal molecule. Based on the labeled signal molecule, it can be divided into fluorescent reporter probes or electrochemical reporter probes. "Fluorescent reporter probes" have both a fluorescent group and a quenching group to generate a fluorescent signal; "electrochemical reporter probes" have electrochemically active molecules to generate an electrochemical signal. For example, single-stranded DNA with one end modified with an electrochemically active molecule (such as ferrocene) will cause a change in electrochemical signal upon cleavage.

[0027] In a first aspect, the present invention provides: a CEA detection kit, comprising: CEA-specific DNA aptamers; A biotin-labeled forward primer, wherein the forward primer is complementary to the specific DNA aptamer; A reverse primer, which, together with the forward primer, is used to amplify the DNA template; The DNA template includes a sequence complementary to the forward primer and a sequence complementary to the reverse primer; A CRISPR / Cas protein / crRNA complex, wherein the crRNA in the CRISPR / Cas protein / crRNA complex is complementary to the amplification product sequence of the DNA template.

[0028] For example, the nucleotide sequence of the specific DNA aptamer is shown in SEQ ID NO:1. It is understood that any aptamer capable of highly specific binding to CEA is acceptable, and the nucleotide sequence of the specific DNA aptamer includes, but is not limited to, SEQ ID NO:1.

[0029] For example, the nucleotide sequence of the forward primer is shown in SEQ ID NO:2. It will be understood that any forward primer capable of complementary pairing with the specific DNA aptamer is acceptable, and the nucleotide sequence of the forward primer includes, but is not limited to, SEQ ID NO:2.

[0030] For example, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3, and the nucleotide sequence of the DNA template is shown in SEQ ID NO:4 and SEQ ID NO:5. It is understood that the reverse primer and the forward primer only need to be able to amplify the DNA template, and the reverse primer and the DNA template include, but are not limited to, the sequences described above.

[0031] According to some embodiments of the present invention, the nucleotide sequence of the crRNA is as shown in SEQ ID NO:6. It is understood that the crRNA need only be complementary to the amplification product sequence of the DNA template, and the nucleotide sequence of the crRNA includes, but is not limited to, SEQ ID NO:6.

[0032] According to some embodiments of the present invention, the Cas protein in the CRISPR / Cas protein / crRNA complex is selected from at least one of Cas12a and Cas14. The Cas protein is preferably Cas12a.

[0033] According to some embodiments of the present invention, the CEA detection kit further includes a solid-phase separation medium for capturing the biotin-labeled forward primer and a complex containing the biotin-labeled forward primer and the specific DNA aptamer.

[0034] Furthermore, the solid-phase separation medium is selected from at least one of streptavidin-modified magnetic beads, filter membranes, and liquid-phase hybridization capture reagents, preferably streptavidin-modified magnetic beads.

[0035] According to some embodiments of the present invention, the CEA detection kit further includes isothermal amplification reaction reagents.

[0036] Furthermore, the isothermal amplification reaction reagent includes recombinase polymerase amplification reaction (RPA) reagent. Preferably, the isothermal amplification reaction reagent is a recombinase polymerase amplification reaction reagent.

[0037] Specifically, the reagents for recombinase polymerase amplification reactions may include recombinase, single-strand binding proteins, strand displacement DNA polymerase, ATP, dNTPs, and reaction buffer. The reaction buffer may contain Tris-HCl, K... + Mg 2+ Ingredients such as...

[0038] Based on the reagent kit disclosed in this invention, those skilled in the art can make conventional settings, optimizations, or adjustments to the isothermal amplification reaction reagents, the volume of the isothermal amplification reaction system, and the types, concentrations, and volumes of components in the reaction system according to actual detection needs. These obvious modifications are all within the scope of this invention.

[0039] According to some embodiments of the present invention, the CEA detection kit further includes a signal reporter probe containing single-stranded DNA.

[0040] Furthermore, the signal reporter probe is selected from fluorescent reporter probes or electrochemical reporter probes. The signal reporter probe is preferably a fluorescent reporter probe, which is beneficial for further improving the detection sensitivity. The fluorescent reporter probe has fluorescent groups and quenching groups at both ends of the single-stranded DNA.

[0041] For example, the CEA detection kit includes: a CEA-specific DNA aptamer; a biotin-labeled forward primer, which is complementary to the specific DNA aptamer; a reverse primer, which, together with the forward primer, is used to amplify a DNA template; the DNA template, which contains sequences complementary to both the forward and reverse primers; a Cas12a / crRNA complex, in which the crRNA is complementary to the amplification product sequence of the DNA template; streptavidin-modified magnetic beads for capturing forward primers that have not bound to the specific DNA aptamer; a recombinase polymerase amplification reaction reagent for amplifying the DNA template with the forward and reverse primers, wherein the amplification product activates the trans-cleavage activity of the Cas12a / crRNA complex; and a fluorescent reporter probe for being cleaved by Cas12a to release a fluorescent signal.

[0042] Please see Figure 1 Taking the CEA detection kit mentioned above as an example, the working principle of this kit includes: The specific DNA aptamer first hybridizes with a biotin-labeled forward primer to form a biotin-forward primer-aptamer complex. Then, the sample to be tested is added and incubated.

[0043] When CEA is present in the test sample: CEA specifically binds to the specific DNA aptamer, causing a conformational change in the aptamer. The aptamer detaches from the biotin-forward primer-aptamer complex and reacts with CEA in the test sample to form an aptamer-CEA complex. Simultaneously, due to the detachment of the aptamer, a free biotin-containing forward primer is formed. After separation, a single DNA strand forward primer (i.e., a free biotin-containing forward primer) is obtained that has not paired with the specific DNA aptamer. Subsequently, a DNA template, reverse primer (primer 2), and isothermal amplification reagents are added to the reaction system for isothermal amplification. The amplification product acts as an activator, activating the trans-cleavage activity of the CRISPR / Cas protein / crRNA complex, which can cleave the signal reporter probe containing single-stranded DNA, releasing a signal.

[0044] When CEA is absent in the sample to be tested: after separation, only the biotin-forward primer-aptamer complex is captured, there is no free forward primer in the system, isothermal amplification cannot be initiated, no amplification product activates the trans-cleavage activity of the CRISPR / Cas protein / crRNA complex, the signal reporter probe containing single-stranded DNA remains intact, and no fluorescence signal is generated.

[0045] Based on the same inventive concept, in a second aspect, the present invention provides: a method for detecting CEA, comprising the following steps: S1, the biotin-labeled forward primer is mixed with the CEA-specific DNA aptamer, and the biotin-labeled forward primer and the specific DNA aptamer are complementary to form a biotin-forward primer-aptamer complex. S2, add the sample to be tested to the biotin-forward primer-aptamer complex and incubate to obtain an incubation solution (when the sample to be tested contains CEA, the specific DNA aptamer detaches from the biotin-forward primer-aptamer complex and binds to CEA in the sample to form an aptamer-CEA complex; at the same time, since the specific DNA aptamer detaches from the biotin-forward primer-aptamer complex, a free biotin-labeled forward primer is formed). S3, capture the biotin-labeled forward primer in the incubation solution to obtain the reaction solution (capture the biotin-labeled forward primer, remove the aptamer-CEA complex in the incubation solution, and retain the solution containing the biotin-labeled forward primer as the reaction solution). S4, add reverse primers, DNA template and isothermal amplification reaction reagent to the reaction solution, perform isothermal amplification, and obtain amplification product; S5, add the CRISPR / Cas protein / crRNA complex and signal reporter probe to the amplification product, perform the cleavage reaction, and read the signal.

[0046] According to some embodiments of the present invention, the sample to be tested can be serum or plasma, preferably serum.

[0047] According to some embodiments of the present invention, before step S1, the detection method may further include centrifuging the sample to be tested to remove impurity particles from the sample.

[0048] According to some embodiments of the present invention, the amount of specific DNA aptamer added is 1 nM to 200 nM, based on the volume of the sample to be tested. Exemplarily, it can be 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, or any value within the range of any two of the above values, preferably 50 nM.

[0049] According to some embodiments of the present invention, in step S1, the concentration of the biotin-labeled forward primer added to the incubation solution is 1 nM-500 nM. Exemplarily, it can be 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, or any value within the range of any two of the above values, preferably 50 nM.

[0050] In step S2, the incubation time can be 5-20 min, and can be, for example, 5 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or any value within the range of any two of the above values, preferably 10 min.

[0051] According to some embodiments of the present invention, in step S4, the isothermal amplification package is selected from recombinase polymerase amplification.

[0052] When isothermal amplification is performed as recombinase polymerase amplification, any commercially available recombinase polymerase amplification reaction reagent can be added to the supernatant.

[0053] According to some embodiments of the present invention, the amplified signal can be read by fluorescence detection or electrochemical detection depending on the different signal reporting probes (fluorescent reporter probe or electrochemical reporter probe).

[0054] Furthermore, when fluorescence detection is selected, a fluorescence microplate reader or a portable fluorescence meter can be used to read the signal intensity.

[0055] For example, a method for detecting CEA includes the following steps: mixing a biotin-labeled forward primer with a CEA-specific DNA aptamer, wherein the biotin-labeled forward primer and the specific DNA aptamer complementarily pair to form a biotin-forward primer-aptamer complex; adding the sample to be tested to the mixture and incubating it; after incubation, adding streptavidin magnetic beads and using magnetic separation technology to capture and separate the biotin-labeled forward primer; then adding a reverse primer, DNA template, and recombinase polymerase amplification reaction reagent to the captured biotin-labeled forward primer and performing isothermal amplification to obtain an amplification product; adding a CRISPR / Cas12a / crRNA complex and a fluorescent reporter probe to the amplification product, performing a cleavage reaction, reading the signal, and calculating the CEA content in the sample to be tested.

[0056] Thirdly, the present invention provides the application of the above-described CEA kit or the above-described detection method in the preparation of products for detecting CEA-related tumor markers, wherein the application is for non-disease diagnosis and treatment purposes.

[0057] The non-disease diagnosis and treatment purposes include at least one of the following: scientific research, drug screening, or industrial applications (manufacturing, assembly, or quality control of reagent kits).

[0058] CEA-related tumors include colorectal cancer, stomach cancer, and lung cancer.

[0059] In addition to CEA, this technology platform can be extended to other tumor markers (AFP, PSA, CA72-4, etc.) by simply changing the corresponding aptamer and crRNA sequence. It is universal and suitable for POCT product development.

[0060] Examples 1-5 of this invention at least support the protection scope of the "CEA detection kit".

[0061] The term "CEA test kit" is derived from the foregoing explanation and / or the corresponding kits in Examples 1-5. Therefore, those skilled in the art can reasonably presume that "CEA test kit," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "CEA test kit."

[0062] In this invention, Examples 1-5 at least support the protection scope of "CEA detection method".

[0063] The term "CEA detection method" is derived from the foregoing explanation and / or the corresponding detection methods in Examples 1-5. Therefore, those skilled in the art can reasonably presume that "CEA detection method," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "CEA detection method."

[0064] Examples 1-5 of this invention at least support the protection scope of "the application of CEA detection kits or detection methods in the preparation of products for detecting CEA-related tumor markers".

[0065] The phrase "the application of CEA detection kits or methods in the preparation of products for detecting CEA-related tumor biomarkers" is derived from the foregoing explanation and / or the use of the corresponding kits and detection methods in Examples 1-5. Therefore, those skilled in the art can reasonably presume that "the application in the preparation of products for detecting CEA-related tumor biomarkers," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "the application in the preparation of products for detecting CEA-related tumor biomarkers."

[0066] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides a CEA detection kit, detection method, and application, which have better technical effects, specifically reflected in the following aspects: 1. High specificity and low background: This invention designs a molecular switch of "biotin-forward primer-aptamer complex" and physically removes unbound primers through a separation step, effectively avoiding non-specific amplification and ensuring low background and high signal-to-noise ratio for signal reading. Compared with existing technologies that rely on dual-target proximity linkages to detect vesicles, this invention provides a novel strategy for directly detecting free CEA protein.

[0067] 2. High sensitivity and strong signal amplification: This invention combines DNA template amplification (first-stage amplification) and CRISPR / Cas protein trans-cleavage (second-stage amplification) to achieve ultrasensitive signal detection of the target protein CEA. Experimental tests show that the detection limit (LOD) for CEA using this invention is as low as 0.42 ng / mL, which is superior to traditional ELISA methods and some existing technologies.

[0068] 3. Constant temperature throughout and simple operation: Compared with PCR method which requires thermal cycling, the isothermal amplification and CRISPR reaction of this invention can be carried out at a constant temperature of 37 ℃-42 ℃, without the need for a complicated thermal cycler, which significantly reduces the equipment threshold, makes the operation simple, and is more suitable for POCT scenarios.

[0069] 4. Accurate and easy-to-quantify results: Compared with colorimetric or test strip methods, the present invention further optimizes the output of fluorescence signals, resulting in stable and objective results, and is easy to perform highly sensitive and accurate quantification using a portable fluorescence instrument.

[0070] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for diagnosing CEA-related tumors, the method comprising performing CEA testing on a subject using the above-described kit or method.

[0071] CEA-related tumors include colorectal cancer, gastric cancer, and lung cancer.

[0072] The subjects mentioned include humans. Attached Figure Description

[0073] Figure 1 This is a schematic diagram illustrating the CEA fluorescence detection principle based on RPA-CRISPR / Cas12a of the present invention.

[0074] Figure 2 This is a graph showing the detection results of the condition optimization experiment in Embodiment 2 of the present invention, wherein, Figure 2 Figure A shows the detection results of the specific DNA aptamer addition amount of CEA. Figure 2 Figure B shows the detection results of the amount of biotin-labeled forward primer added. Figure 2 Figure C shows the results of detecting the amount of reagent added in the recombinase polymerase amplification reaction. Figure 2 Figure D shows the results of detecting the amount of CRISPR / Cas12a / crRNA complex added. Figure 2 Figure E shows the detection results of the amount of fluorescent reporter probe added. Figure 2 The F-plot shows the results of the cutting reaction time detection.

[0075] Figure 3 The results and standard curves for the detection of CEA standards at different concentrations are shown in Example 3 of this invention.

[0076] Figure 4 This is a diagram showing the specific detection results of the method in Embodiment 4 of the present invention.

[0077] Figure 5 This is a graph showing the results of detecting clinical serum samples in Example 5 of the present invention, wherein... Figure 5 Figure A shows the detection results using electrochemiluminescence immunoassay. Figure 5 Figure B shows the detection results using the CEA detection kit and detection method described in Example 1 of this invention. Detailed Implementation

[0078] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0080] Data analysis and statistical analysis were performed using professional data processing software, and significance analysis was conducted using one-way ANOVA. P <0.05 indicates a significant difference.

[0081] The main instrument used in the following examples is a fluorescence spectrometer (Hitachi, Ltd.).

[0082] Example 1: CEA Detection Kit and Detection Method I. The CEA detection kit contains the following components: (1) CEA-specific DNA aptamer, the sequence of which is shown in SEQ ID NO:1; (2) A biotin-labeled forward primer, the sequence of which is shown in SEQ ID NO:2, which is able to pair complementaryly with a CEA-specific DNA aptamer; (3) The reverse primer, the sequence of which is shown in SEQ ID NO:3; (4) DNA template, formed by annealing SEQ ID NO:4 and SEQ ID NO:5; (5) Streptokinin magnetic beads (Beaver Nanotechnology (Suzhou) Co., Ltd., item number 22308-10); (6) Recombinase polymerase amplification (RPA) reaction reagent (Amp Future (Changzhou) Biotechnology Co., Ltd., WLB8201KIT); (7) CRISPR / Cas protein / crRNA complex; wherein the Cas protein is Cas12a and the crRNA sequence is shown in SEQ ID NO:6; (8) A fluorescent reporter probe, the sequence of which is shown in SEQ ID NO:7, with FAM labeled at 5' and BHQ1 labeled at 3'.

[0083] The above nucleotide sequences were synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the sequence information is shown in Table 1.

[0084] Table 1

[0085] II. Detection Method: The CEA detection kit described above is used, and the following steps are included: Step S1: Mix the 50 nM biotin-labeled forward primer with the 50 nM CEA-specific DNA aptamer, wherein the biotin-labeled forward primer and the specific DNA aptamer complement each other to form a biotin-forward primer-aptamer complex. Step S2: Take 40 μL of serum, centrifuge to remove particulate matter, and obtain the sample to be tested. Mix the sample to be tested with the above biotin-forward primer-aptamer complex and 10 μL of streptavidin magnetic beads, and incubate at room temperature for 10 min to obtain the incubation solution. The specific DNA aptamer in the biotin-forward primer-aptamer complex binds to CEA in the sample to be tested, thereby detaching from the biotin-forward primer-aptamer complex to form a free biotin-labeled forward primer. Step S3: Separate and capture the biotin-labeled forward primer using magnetic beads, remove the aptamer-CEA complex, and retain the reaction solution containing the free forward primer; Step S4: Add 50 nM reverse primer, 50 nM DNA template and 0.2 μL recombinase polymerase amplification reaction reagent to the reaction solution, and perform amplification at 37℃ for 20 min to obtain the amplification product. Step S5: Add 10 nM CRISPR / Cas12a / crRNA complex and 1 μM fluorescent reporter probe to the amplification product, perform a room temperature cleavage reaction for 45 min, and then read the signal intensity using a fluorescent microplate reader (Ex / Em: 485 / 520 nm).

[0086] Example 2: Condition Optimization Experiment 1. Specific DNA aptamer addition amount of CEA The detection was performed according to the CEA detection kit and method in Example 1, with the difference that the amount of CEA-specific DNA aptamer added in step S1 was 0, 1 nM, 10 nM, 50 nM, 100 nM, 150 nM, and 200 nM, respectively. The detection results are as follows: Figure 2 As shown in Figure A, based on the sample volume, the addition of specific DNA aptamers ranging from 1 nM to 200 nM can effectively detect CEA, with the best results achieved at an addition of 50 nM.

[0087] 2. Amount of biotin-labeled forward primer added The CEA detection kit and method described in Example 1 were used for detection, with the difference that the amount of biotin-labeled forward primer added in step S1 was 0, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, and 500 nM, respectively. The detection results are as follows: Figure 2 As shown in Figure B, based on the sample volume, biotin-labeled forward primers ranging from 1 nM to 500 nM can effectively detect CEA, with the best results achieved at a concentration of 50 nM.

[0088] 3. Amount of reagent added for recombinase polymerase amplification reaction The CEA detection kit and method described in Example 1 were used for detection, with the difference being that the amount of recombinase polymerase amplification reaction reagent added in step S4 was 0, 0.1 μL, 0.2 μL, 0.3 μL, 0.5 μL, 1 μL, and 2 μL, respectively. The detection results are as follows: Figure 2 As shown in Figure C, 0.1 μL-2 μL of reagent can effectively carry out the amplification reaction, with 0.2 μL being the preferred amount.

[0089] 4. Amount of CRISPR / Cas12a / crRNA complex added The CEA detection kit and method described in Example 1 were used for detection, with the difference being that the amount of CRISPR / Cas12a / crRNA complex (RNP) added in step S5 was 0, 1 nM, 5 nM, 10 nM, 20 nM, 50 nM, and 100 nM, respectively. The detection results are as follows: Figure 2 As shown in Figure D, based on the sample volume, the addition amount of CRISPR / Cas12a / crRNA complex from 1 nM to 100 nM can effectively detect CEA, with the best results achieved when the addition amount is 10 nM.

[0090] 5. Fluorescent reporter probe dosage The CEA detection kit and method described in Example 1 were used for detection, with the difference that the amount of fluorescent reporter probe added in step S5 was 0, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 100 μM, respectively. The detection results are as follows: Figure 2 As shown in Figure E, based on the sample volume, the addition amount of CRISPR / Cas12a / crRNA complex from 0.1 μM to 100 μM can effectively detect CEA, with the best results achieved at an addition amount of 1 μM.

[0091] 6. Cutting reaction time The CEA detection kit and method described in Example 1 were used for detection, with the difference that the cutting reaction time in step S5 was 0, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. The detection results are as follows: Figure 2 As shown in the F-plot, CEA can be effectively detected in reactions ranging from 15 min to 90 min, with the best results observed at 45 min.

[0092] Example 3 Sensitivity Verification CEA standards (Wuxi OriGene Biotechnology Co., Ltd. (i.e., OriGene China), DP0032-P1) were added to a 40 μL reaction system at final concentrations of 0, 1, 1.5, 2, 2.5, 3, 4, 4.5, 5, 10, 15, 20, 25, and 50 ng / mL, respectively, to prepare test samples. The CEA detection kit and method described in Example 1 were then used for detection. The results are as follows: Figure 3 As shown: This confirms that the protocol exhibits a linear relationship (R0) at CEA concentrations ranging from 1.5 to 5.0 ng / mL. 2 =0.992). The detection limit obtained from the 3σ / slope slope is 0.42 ng / mL. It is worth noting that the normal range of CEA in humans is 0-5.0 ng / mL, therefore this protocol is highly sensitive.

[0093] Example 4: Selective Validation CEA group: CEA standard with a final concentration of 5 ng / mL was added to a 40 μL reaction system; Negative control: PBS buffer (pH 7.4); Interfering substance group: Trypsin: 50 ng / mL trypsin (Trypsin, Shanghai Yuanye Biotechnology Co., Ltd., catalog number V87526); AMY: 50 ng / mL amylase (AMY, Shanghai Yuanye Biotechnology Co., Ltd., catalog number 9000-90-2); Lipase: 50 ng / mL lipase (Shanghai Yuanye Biotechnology Co., Ltd., catalog number 9001-62-1); AFP: 50 ng / mL Alpha-fetoprotein (Hangzhou Feixue Gene Technology Co., Ltd., catalog number 11029-61-1); ALP: 50 ng / mL alkaline phosphatase (Shanghai Yuanye Biotechnology Co., Ltd., catalog number 9001-78-9); Pepsin: 50 ng / mL pepsin (Shanghai Yuanye Biotechnology Co., Ltd., catalog number 9001-75-6); PSA: 50 ng / mL PSA (Hangzhou Feixue Gene Technology Co., Ltd.); CA199: 50 ng / mL CA199 (Hangzhou Feixue Gene Technology Co., Ltd.); CA724: 50 ng / mL CA724 (Hangzhou Feixue Gene Technology Co., Ltd.); CA125: 50 ng / mL CA125 (Hangzhou Feixue Gene Technology Co., Ltd.).

[0094] Mixed group (MIX): Add 50 ng / mL of each of the above-mentioned trypsin, amylase, lipase, alpha-fetoprotein, alkaline phosphatase, pepsin, PSA, CA199, CA724, and CA125 to a 5 ng / mL CEA sample.

[0095] The CEA detection kit and method described in Example 1 were used for testing. The results are as follows: Figure 4 As shown: Only the CEA group showed a significant signal increase; the signals of the other groups were not significantly different from the negative control, with a cross-reactivity rate of <3%. Specifically, the cross-reactivity rate = net signal of the interfering substance group / net signal of the CEA group × 100%. A cross-reactivity rate of <3% indicates excellent specificity; the interfering substance produces almost no signal. A cross-reactivity rate range of 3% - 5% (inclusive) indicates good specificity with minimal impact on actual detection. A cross-reactivity rate range of 5% - 10% (exclusive to 10%) indicates general specificity. A cross-reactivity rate of 10% indicates poor specificity.

[0096] Example 5 Clinical Sample Testing Fifteen clinical serum samples were collected (the clinical study has been approved by the Ethics Committee of Shandong Qianfoshan Hospital (Approval No.: YXLL-KY-2022(068)), the serum samples were legally sourced and compliant, and the patients had signed informed consent forms). The CEA detection kit and method described in Example 1 of this invention were used for detection (actual values ​​were recorded). Simultaneously, the samples were also detected using electrochemiluminescence immunoassay (using the Roche carcinoembryonic antigen (CEA) kit, Elecsys CEA, Roche, 11731629322), according to the kit instructions. The results were compared with those obtained using electrochemiluminescence immunoassay (theoretical values). Figure 5 As shown in Table 2, the correlation R between the detection results of this method and the reference method is... 2 =0.991, the average recovery rate was 98.5%, and the RSD was <8%. The recovery rate (%) was calculated as (measured value / theoretical value) × 100%, and the average recovery rate was the average of the recovery rates of 15 samples. , Where n is 15, and Xi is the measured value for each case. This represents the average of the measured values.

[0097] Table 2

[0098] Example 6 Performance Comparison Experiment (1) Comparative Example 1: ELISA method, using kit number E-EL-H6047 purchased from Wuhan Elabscience Biotechnology Co., Ltd., and the test method is described in the kit instructions.

[0099] Using the CEA detection kit and detection method in Example 1 of this invention, and the test method in Comparative Example 1, four samples were tested in parallel. Each sample was tested three times using both Example 1 and Comparative Example 1. CEA standard (Wuxi OriGene Biotechnology Co., Ltd. (i.e., OriGene China), DP0032-P1) was added to a 40 μL reaction system at final concentrations of 2, 3, 4, and 5 ng / mL, respectively, to prepare the test samples. The C1 values ​​of Example 1 and Comparative Example 1 were calculated. found The relative standard deviation (RSD) and the average recovery rate, where C found The average value of three tests is given. The relative standard deviation (RSD) and average recovery rate were tested using the method described in Example 5, and the test results are shown in Table 3.

[0100] Table 3

[0101] The above results demonstrate that the CEA detection kit and detection method of the present invention have high specificity, sensitivity and accuracy.

[0102] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A CEA detection kit, characterized in that, The CEA detection kit includes: CEA-specific DNA aptamers; A biotin-labeled forward primer, wherein the forward primer is complementary to the specific DNA aptamer; A reverse primer, which, together with the forward primer, is used to amplify the DNA template; The DNA template includes a sequence complementary to the forward primer and a sequence complementary to the reverse primer; A CRISPR / Cas protein / crRNA complex, wherein the crRNA in the CRISPR / Cas protein / crRNA complex is complementary to the amplification product sequence of the DNA template.

2. The CEA detection kit according to claim 1, characterized in that, The Cas protein in the CRISPR / Cas protein / crRNA complex is selected from at least one of Cas12a and Cas14a.

3. The CEA detection kit according to claim 1, characterized in that, The CEA detection kit further includes a solid-phase separation medium for capturing the biotin-labeled forward primer and / or a complex containing the biotin-labeled forward primer and the specific DNA aptamer.

4. The CEA detection kit according to claim 3, characterized in that, The solid-phase separation medium is selected from at least one of streptavidin-modified magnetic beads, filter membranes, and liquid-phase hybridization capture reagents.

5. The CEA detection kit according to claim 1, characterized in that, The CEA detection kit also includes isothermal amplification reaction reagents, which include recombinase polymerase amplification reaction reagents.

6. The CEA detection kit according to claim 1, characterized in that, The CEA detection kit also includes a signal reporter probe containing single-stranded DNA.

7. The CEA detection kit according to claim 6, characterized in that, The signal reporting probe is selected from fluorescent reporter probes or electrochemical reporter probes.

8. The CEA detection kit according to claim 1, characterized in that, The nucleotide sequence of the specific DNA aptamer is shown in SEQ ID NO:1; The nucleotide sequence of the forward primer is shown in SEQ ID NO:2; The nucleotide sequence of the reverse primer is shown in SEQ ID NO:3; The nucleotide sequence of the DNA template is shown in SEQ ID NO:4 and SEQ ID NO:5; The nucleotide sequence of the crRNA is shown in SEQ ID NO:

6.

9. A method for detecting CEA, characterized in that, Includes the following steps: S1, the biotin-labeled forward primer is mixed with the CEA-specific DNA aptamer, and the biotin-labeled forward primer and the specific DNA aptamer are complementary to form a biotin-forward primer-aptamer complex. S2, add the sample to be tested to the biotin-forward primer-aptamer complex and incubate to obtain an incubation solution; S3, capture the biotin-labeled forward primer in the incubation solution to obtain the reaction solution; S4, add reverse primers, DNA template and isothermal amplification reaction reagent to the reaction solution, perform isothermal amplification, and obtain amplification product; S5, add the CRISPR / Cas protein / crRNA complex and signal reporter probe to the amplification product, perform the cleavage reaction, and read the signal.

10. The use of the CEA detection kit of any one of claims 1-8 or the detection method of claim 9 in the preparation of a product for detecting biomarkers of CEA-related tumors, wherein the use is for non-disease diagnosis and treatment purposes.

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