An E-CRISPR nucleic acid detection method based on the exonuclease I end protection effect and CRISPR / Cas12a trans-cleavage signal amplification and its application

Abstract

This invention discloses an E-CRISPR nucleic acid detection method and its application based on the exonuclease I end-protection effect and CRISPR / Cas12a trans-cleavage signal amplification. The invention designs a bifunctional single-stranded DNA probe with a target recognition region at its 3' end and a Cas12a activation sequence at its 5' end. When the target RNA is present, it forms an RNA-DNA hybrid structure with the probe's 3' end, protecting the probe from exonuclease I degradation. The 5' end activation sequence of the intact probe triggers the CRISPR / Cas12a trans-cleavage activity, cleaving a fluorescent reporter molecule to generate a signal. In the absence of a target, the probe is degraded by exonuclease I, resulting in no signal output. This invention eliminates the need for nucleic acid amplification and offers advantages such as high sensitivity, strong specificity, simple operation, short detection time, and independence from expensive instruments. It can be used for the detection of low-abundance targets such as long non-coding RNA MIR155HG, providing a new technical means for the detection of perioperative depression-related molecular markers in cervical cancer patients and for studying the antidepressant mechanism of esketamine.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and molecular diagnostics, specifically to an E-CRISPR nucleic acid detection method and its application based on the exonuclease I end-protection effect and CRISPR / Cas12a trans-cleavage signal amplification. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors of the female reproductive system, and surgical treatment remains the primary treatment for early-stage and some locally advanced cervical cancers. With continuous improvements in perioperative management, the overall survival rate of cervical cancer patients has significantly improved; however, postoperative psychological problems are increasingly attracting attention. Studies have shown that patients undergoing radical hysterectomy for cervical cancer often experience significant anxiety and depression due to surgical trauma and bodily stress, postoperative pain, immune dysfunction, and concerns about disease prognosis and fertility, severely impacting postoperative recovery quality and long-term prognosis. Currently, the mechanisms of perioperative depression are not fully understood. Recent studies have found that immune inflammatory responses, neuroendocrine imbalances, and changes in neuroplasticity may jointly participate in the development of depression. Among these, the role of immune-related long non-coding RNAs (lncRNAs) in inflammation regulation and neuropsychiatric disorders is gaining increasing attention. The MIR155 host gene (MIR155HG), as a precursor host gene of miR-155, participates in the regulation of immune inflammatory responses, cytokine release, and neurological function. Studies have shown that the MIR155HG / miR-155 signaling axis is closely related to depressive states and inflammation levels, suggesting that MIR155HG may become an important molecular biomarker for perioperative depression.

[0003] Currently, in clinical and experimental research, the detection of long non-coding RNA mainly employs reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). This technology aims to achieve quantitative analysis of low-abundance RNA molecules. Its technical structure involves: firstly, extracting RNA and reverse transcribing it to generate cDNA; then, amplifying the cDNA using quantitative PCR to detect the target nucleic acid. It offers high sensitivity and good quantitative capabilities, and is therefore widely used in gene expression analysis.

[0004] However, the existing RT-qPCR technology still has the following shortcomings: (1) The detection process is relatively complex, requiring multiple steps such as RNA extraction, reverse transcription and amplification, and the operation time is long; (2) The detection process depends on expensive fluorescent PCR instruments and professional experimental personnel, which is not conducive to the application of rapid detection in primary medical institutions and bedside; (3) lncRNA usually has low expression abundance, and the background of clinical serum samples is complex, which is easily affected by non-specific amplification, RNA degradation and amplification efficiency fluctuations, thus affecting the accuracy of detection; (4) There is a risk of aerosol contamination during the PCR amplification process, which may lead to false positive results; (5) The traditional method still has limited ability to detect low abundance targets rapidly and with high sensitivity, which is difficult to meet the needs of instant detection and portable detection.

[0005] In recent years, CRISPR / Cas nucleic acid detection technology has attracted widespread attention due to its high specificity and high sensitivity. In particular, the CRISPR / Cas12a system, after recognizing specific target DNA, can activate its non-specific trans-cleavage activity against single-stranded DNA, thereby achieving amplified nucleic acid signal detection. A search revealed the following prior art to be closest to this application: Comparative document 1 (Wakaba P, Muramatsu A, Imagawa T, Furuse Y, Saito N, Uno N. CRISPR-Cas12a biosensing via transcription of crRNA from PCR or LAMP products for pathogen detection. Biosens Bioelectron. 2026;310:118860): A nucleic acid detection method based on the trans-cleavage activity of CRISPR / Cas12a. The technical solution is as follows: Objective—To establish a nucleic acid detection method based on the trans-cleavage activity of CRISPR / Cas12a; The method involves guiding Cas12a to recognize specific target DNA via crRNA, activating Cas12a to non-specifically cleave single-stranded DNA fluorescent reporter probes in the presence of the target, thereby releasing a fluorescent signal; Results—Highly specific nucleic acid detection is achieved, laying the foundation for the development of CRISPR molecular diagnostic technology.

[0006] However, the prior art still has the following shortcomings: (1) Most detection systems still rely on pre-amplification steps such as PCR, RPA or LAMP; (2) The detection process is relatively complex and the amplification step is prone to aerosol contamination; (3) Existing technologies are mainly used for pathogen nucleic acid detection and are rarely used in the detection of molecular markers related to depression in the perioperative period of tumors; (4) There are no reports on combining CRISPR / Cas12a technology with MIR155HG detection and the study of the antidepressant mechanism of esketamine.

[0007] Furthermore, previous studies have found that exonuclease I (Exo I) can specifically degrade single-stranded DNA, and RNA-DNA hybrid structures can protect DNA ends. Although some studies have attempted to utilize the end-protective effect of Exo I for nucleic acid detection, existing techniques generally suffer from limited signal amplification efficiency, insufficient detection stability, and difficulties in coupling with CRISPR systems. In particular, no studies have been reported specifically targeting the detection of low-abundance MIR155HG.

[0008] Therefore, there is an urgent need to develop a novel nucleic acid detection method that does not require complex amplification, has high sensitivity and specificity, and is suitable for rapid detection of low-abundance MIR155HG, in order to meet the needs of detecting perioperative depression-related molecular markers in cervical cancer patients and studying the antidepressant mechanism of esketamine. Summary of the Invention

[0009] To address the problems of existing technologies, the purpose of this invention is to provide an E-CRISPR nucleic acid detection method and its application based on the exonuclease I end protection effect and CRISPR / Cas12a trans-cleavage signal amplification.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a nucleic acid detection method based on the exonuclease I end protection effect and the amplification of the CRISPR / Cas12a trans-cleavage signal.

[0011] Secondly, this application provides an E-CRISPR detection kit for implementing the method.

[0012] Thirdly, this application provides the application of a kit in the preparation of a kit for the detection of molecular markers related to perioperative depression in cervical cancer patients or for the study of the antidepressant mechanism of esketamine.

[0013] The first aspect of this application provides a nucleic acid detection method based on the end protection effect of exonuclease I and the amplification of the CRISPR / Cas12a trans-cleavage signal, characterized by the following steps: (1) providing a bifunctional single-stranded DNA probe, wherein the 3' end of the probe is the target nucleic acid recognition region and the 5' end is the CRISPR / Cas12a activation sequence; (2) incubating the sample to be tested, the bifunctional single-stranded DNA probe and exonuclease I together, so that if the target nucleic acid is present, it specifically binds to the 3' end recognition region of the probe to form an RNA-DNA hybrid structure, thereby protecting the 3' end of the probe from degradation by exonuclease I, while the unprotected probe is degraded by exonuclease I; (3) inactivating exonuclease I; (4) adding a CRISPR / Cas12a detection system to the system obtained in step (3), wherein the detection system contains Cas12a protein, crRNA and a fluorescent reporter molecule, wherein the crRNA can specifically bind to the 5' end activation sequence of the bifunctional single-stranded DNA probe; (5) detecting the fluorescence signal, and judging the presence or quantity of the target nucleic acid based on the intensity of the fluorescence signal.

[0014] Further, in step (1), the nucleotide sequence of the bifunctional single-stranded DNA probe is shown in SEQ ID NO.1.

[0015] Further, in step (4), the nucleotide sequence of crRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent reporter molecule is: 6-FAM-TTATTT-BHQ1; the target nucleic acid is long non-coding RNA MIR155HG.

[0016] Further, in step (2), the incubation conditions include: first incubating at 37°C to allow the target and probe to bind, then adding exonuclease I to continue the reaction, and then heating at 80°C to inactivate exonuclease I.

[0017] Furthermore, in step (4), the reaction conditions of the CRISPR / Cas12a detection system include: incubation at 37°C for 10 minutes; the fluorescent reporter molecule is a single-stranded DNA reporter probe with fluorescent groups and quenching groups labeled at both ends respectively.

[0018] Furthermore, in steps (1) to (5), the method does not require a nucleic acid amplification step.

[0019] A second aspect of this application provides an E-CRISPR detection kit for implementing a method. The E-CRISPR detection kit includes: a bifunctional single-stranded DNA probe with the nucleotide sequence shown in SEQ ID NO.1; exonuclease I; Cas12a protein; crRNA with the nucleotide sequence shown in SEQ ID NO.2; a fluorescent reporter molecule with the nucleotide sequence 6-FAM-TTATTT-BHQ1; and instructions for use.

[0020] Furthermore, the target recognition region of the bifunctional single-stranded DNA probe is specifically targeted at the long non-coding RNA MIR155HG.

[0021] The third aspect of this application provides a method or kit for the preparation of kits for the detection of molecular markers related to perioperative depression in patients with cervical cancer or for the study of the antidepressant mechanism of esketamine.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Significantly improved detection sensitivity: This invention utilizes a cascade mechanism of target RNA-induced Exo I end protection effect and CRISPR / Cas12a trans-cleavage signal amplification to achieve highly sensitive detection of low-abundance long non-coding RNA MIR155HG without the need for nucleic acid amplification steps such as PCR. Compared to the traditional RT-qPCR-dependent exponential amplification mode, this invention avoids amplification bias and efficiency fluctuation problems, and has superior detection ability for low-expression lncRNAs.

[0023] (2) Enhanced detection specificity: This invention constructs a dual-specificity recognition system: the first layer is the complementary base pairing between the 3' end of the bifunctional single-stranded DNA probe and the target RNA, forming an RNA-DNA hybrid structure to protect the probe; the second layer is the specific recognition of the crRNA of the CRISPR / Cas12a system with the activation sequence at the 5' end of the probe. The dual-gating mechanism effectively reduces non-specific background signals and false positive results, improving detection accuracy.

[0024] (3) Simplified detection process and shortened detection time: In this invention, the reaction with exonuclease I is carried out at 37°C. In step (4), the CRISPR / Cas12a detection system only needs to be incubated at 37°C for 10 minutes. The entire detection process does not require complex operations such as RNA reverse transcription and PCR thermal cycling. Compared with the detection time of several hours of traditional RT-qPCR, this invention can complete the detection in a shorter time, which significantly improves the detection efficiency.

[0025] (4) Reduced dependence on large instruments and improved accessibility: The entire process of this invention is carried out under constant temperature conditions (37℃, 80℃ inactivation), eliminating the need for high-end thermal cycling equipment such as real-time fluorescence PCR instruments. Only a common constant temperature metal bath and a simple fluorescence detection device (such as an ELISA reader or portable fluorometer) are required to complete the detection. This makes the invention particularly suitable for primary healthcare institutions, point-of-care rapid testing, and molecular diagnostic scenarios in areas with limited resources.

[0026] (5) Reduced false-positive background signal and improved result stability: Under target-free conditions, the bifunctional single-stranded DNA probe is completely degraded by Exo I due to lack of protection, blocking the generation of CRISPR / Cas12a activation signals at the source. Exo I is inactivated by heating at 80℃, avoiding the influence of residual enzymes on the subsequent detection system. This mechanism effectively reduces the background fluorescence of the system, reduces the risk of false positives, and ensures the reliability and repeatability of the detection results.

[0027] (6) Filling the gap in specific application areas: This invention is the first to apply E-CRISPR technology to the detection of MIR155HG, a molecular marker of perioperative depression in cervical cancer patients, and to the study of the antidepressant mechanism of esketamine. It provides a new detection tool that is fast, convenient and sensitive in this field and has good prospects for clinical application and scientific research promotion. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram illustrating the principle of the E-CRISPR detection method of the present invention.

[0030] Figure 2 This is a graph showing the performance analysis of the bifunctional DNA probe of this invention.

[0031] Figure 3 This is a feasibility analysis diagram of the bifunctional DNA probe of the present invention.

[0032] Figure 4 This is a graph showing the application of the method of the present invention to analyze the expression level of MIR155HG in clinical samples.

[0033] Figure 5 This is a sensitivity analysis diagram of the E-CRISPR system for detecting MIR155HG according to the present invention.

[0034] Figure 6This is a selective analysis diagram of the E-CRISPR system of the present invention for detecting MIR155HG. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0037] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention. Reagents not specifically described in detail herein are conventional reagents and are commercially available; methods not specifically described in detail are conventional experimental methods and are known from the prior art.

[0039] The first aspect of this application provides a nucleic acid detection method based on the end protection effect of exonuclease I and the amplification of the CRISPR / Cas12a trans-cutting signal, comprising the following steps: (1) providing a bifunctional single-stranded DNA probe, wherein the 3' end of the probe is the target nucleic acid recognition region and the 5' end is the CRISPR / Cas12a activation sequence; (2) incubating the sample to be tested, the bifunctional single-stranded DNA probe and exonuclease I together, so that if the target nucleic acid is present, it specifically binds to the 3' end recognition region of the probe to form an RNA-DNA hybrid structure, thereby protecting the 3' end of the probe from degradation by exonuclease I, while the unprotected probe is degraded by exonuclease I; (3) inactivating exonuclease I; (4) adding a CRISPR / Cas12a detection system to the system obtained in step (3), wherein the detection system contains Cas12a protein, crRNA and a fluorescent reporter molecule, wherein the crRNA can specifically bind to the 5' end activation sequence of the bifunctional single-stranded DNA probe; (5) detecting the fluorescence signal, and judging the presence or quantity of the target nucleic acid based on the fluorescence signal intensity.

[0040] In some embodiments, in step (1), the nucleotide sequence of the bifunctional single-stranded DNA probe is shown in SEQ ID NO.1.

[0041] In some embodiments, in step (4), the nucleotide sequence of the crRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the fluorescent reporter molecule is 6-FAM-TTATTT-BHQ1, wherein 6-FAM is a fluorescent group and BHQ1 is a quencher group. The target nucleic acid is the long non-coding RNA MIR155HG.

[0042] In some embodiments, in step (2), the incubation conditions include: first incubating at 37°C to allow the target to bind to the probe, then adding exonuclease I to continue the reaction, and then heating at 80°C to inactivate exonuclease I.

[0043] In some embodiments, in step (4), the reaction conditions of the CRISPR / Cas12a detection system include: incubation at 37°C for 10 minutes; the fluorescent reporter molecule is a single-stranded DNA reporter probe with fluorescent groups and quenching groups labeled at both ends.

[0044] In some embodiments, the method does not require a nucleic acid amplification step in steps (1) to (5).

[0045] A second aspect of this application provides an E-CRISPR detection kit for implementing a method. The E-CRISPR detection kit includes: a bifunctional single-stranded DNA probe with the nucleotide sequence shown in SEQ ID NO.1; exonuclease I; Cas12a protein; crRNA with the nucleotide sequence shown in SEQ ID NO.2; a fluorescent reporter molecule with the nucleotide sequence 6-FAM-TTATTT-BHQ1; and instructions for use.

[0046] In some embodiments, the target recognition region of the bifunctional single-stranded DNA probe is specifically targeted at the long non-coding RNA MIR155HG.

[0047] The third aspect of this application provides a method or kit for the preparation of a kit for detecting perioperative depression-related molecular markers in cervical cancer patients or for studying the antidepressant mechanism of esketamine. Example 1

[0048] The present invention provides a nucleic acid detection method based on the exonuclease I end protection effect and CRISPR / Cas12a trans-cleavage signal amplification, comprising the following steps: (1) providing a bifunctional single-stranded DNA probe, wherein the 3' end of the probe is the target nucleic acid recognition region and the 5' end is the CRISPR / Cas12a activation sequence; the nucleotide sequence of the bifunctional single-stranded DNA probe is shown in SEQ ID NO.1; (2) The test sample, bifunctional single-stranded DNA probe and exonuclease I are incubated together so that if the target nucleic acid is present, it will specifically bind to the 3' end recognition region of the probe to form an RNA-DNA hybrid structure, thereby protecting the 3' end of the probe from degradation by exonuclease I, while the unprotected probe is degraded by exonuclease I; the incubation conditions include: first incubating at 37°C to allow the target to bind to the probe, then adding exonuclease I to continue the reaction, and then heating at 80°C to inactivate exonuclease I.

[0049] (3) Inactivate exonuclease I; (4) Add the CRISPR / Cas12a detection system to the system obtained in step (3). The detection system contains Cas12a protein, crRNA, and a fluorescent reporter molecule. The crRNA can specifically bind to the 5' activation sequence of the bifunctional single-stranded DNA probe. The nucleotide sequence of the crRNA is shown in SEQ ID NO.2. The nucleotide sequence of the fluorescent reporter molecule is 6-FAM-TTATTT-BHQ1, where 6-FAM is a fluorescent group and BHQ1 is a quencher group. The target nucleic acid is the long non-coding RNA MIR155HG. The reaction conditions of the CRISPR / Cas12a detection system include: incubation at 37°C for 10 minutes. The fluorescent reporter molecule is a single-stranded DNA reporter probe with fluorescent and quencher groups labeled at both ends.

[0050] (5) Detect the fluorescence signal and determine the presence or quantity of the target nucleic acid based on the fluorescence signal intensity. In steps (1) to (5), the method does not require a nucleic acid amplification step. Verification Experiment

[0051] To verify the feasibility and effectiveness of the E-CRISPR detection method described in this invention, the following control group experiment was set up.

[0052] Control Group 1 (Performance Verification of Bifunctional DNA Probe): The experimental results of Control Group 1 show that the bifunctional DNA probe can produce a significantly enhanced fluorescence signal, while the background signal level of the system without the bifunctional DNA probe is extremely low, verifying the feasibility of E-CRISPR detection technology. The results are as follows: Figure 2 As shown, Figure 2 This is a graph showing the performance analysis of the bifunctional DNA probe of this invention.

[0053] Control Group 2 (Feasibility Verification of Different Target Concentrations): The experimental results of Control Group 2 show that after different concentrations of the target bound to the bifunctional DNA probe, the fluorescence signal gradually increased with increasing target concentration, as shown in the following figures. Figure 3 As shown, Figure 3 This is a feasibility analysis diagram of the bifunctional DNA probe of the present invention. Example 2

[0054] This invention discloses an E-CRISPR detection kit for implementing the method. The E-CRISPR detection kit includes: a bifunctional single-stranded DNA probe with the nucleotide sequence shown in SEQ ID NO.1; exonuclease I; Cas12a protein; crRNA with the nucleotide sequence shown in SEQ ID NO.2; a fluorescent reporter molecule with the nucleotide sequence 6-FAM-TTATTT-BHQ1; wherein 6-FAM is modified with a fluorescent group and BHQ1 is modified with a quencher group; and an instruction manual.

[0055] The target recognition region of the bifunctional single-stranded DNA probe is specifically targeted at the long non-coding RNA MIR155HG. Example 3

[0056] The application of a method or kit of the present invention in the preparation of a kit for detecting molecular markers of perioperative depression in cervical cancer patients or for studying the antidepressant mechanism of esketamine.

[0057] The method or kit described in this invention is used in the preparation of a kit for detecting the expression level of MIR155HG in the serum of cervical cancer patients, wherein the MIR155HG expression level can be used to assist in the assessment of perioperative depression risk or for the study of the antidepressant mechanism of esketamine. Figure 4This image shows an analysis of the MIR155HG expression level in clinical samples using the method described in this invention. The results are as follows: Figure 4 As shown, the expression level of MIR155HG in the serum of cervical cancer patients was significantly higher than that in the healthy control group, and the difference was statistically significant (P<0.05). The results indicate that the E-CRISPR detection method described in this invention can effectively detect the expression level of MIR155HG in clinical serum samples, and can be used for the detection of perioperative depression-related molecular markers in cervical cancer patients and for studying the antidepressant mechanism of esketamine. Example 4

[0058] Methods for detecting MIR155HG using E-CRISPR technology 1. Experimental materials and reagents The DNA and RNA sequences used in this embodiment were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The target RNA was a synthetically produced fragment based on the lncRNA MIR155HG (NR_132106.1) sequence in the GenBank database. The nucleotides X to Y at the 3' end of the bifunctional single-stranded DNA probe (SEQ ID NO.1) are complementary to the nucleotides A to B at the MIR155HG (GenBank accession number NR_132106.1) sequence.

[0059] The main reagents included: EnGen® Lba Cas12a (Cpf1), 10×NEBuffer 2.1, Exonuclease I (Exo I), 10×CutSmart Buffer (New England Biolabs, Beijing, China), RNase-free water and 100 mM MgSO4 solution (Beyotime Biotechnology Co., Ltd., Shanghai, China), and ultrapure water prepared by the Millipore purification system.

[0060] 2. Main instruments and equipment A constant-temperature metal bath (for Exo I and Cas12a reactions), and an M200 PRO multi-functional microplate reader (for fluorescence detection). Target protection response (Exo I response) The specific steps are as follows: Add 1 μL of 10×CutSmart Buffer, 3 μL of DNA probe, 1 μL of target, and 4 μL of LNase-free water. Incubate at 37°C for 15 min to allow the target and probe to bind initially. Add Exo I (0.5 U / μL, 1 μL) and bring the buffer to a final volume of 10 μL. Continue the reaction to complete the degradation of undetermined single-stranded nucleic acids. Then, heat at 80°C for 20 min to inactivate Exo I.

[0061] Construct a CRISPR detection system to a final volume of 50 μL. Add: 5 μL 10×NEBuffer, 2 μL 1 μM crRNA, 2 μL 1 μM Cas12a, fluorescent reporter molecule, and RNase-free water to a final volume of 50 μL. Incubate at 37°C for 10 min to terminate the reaction. Transfer the mixture to a black 96-well plate and detect the fluorescence signal using an M200 PRO microplate reader (Ex: 485 nm, Em: 525 nm).

[0062] The core working principle of CRISPR is as follows: Figure 1 As shown, the method mainly consists of two parts: Exo I and the CRISPR / Cas12a system. First, a bifunctional single-stranded DNA probe was designed, with the 3' end being the target recognition region (pink), which can specifically bind complementary to the target; the 5' end is the CRISPR / Cas12a activation sequence (blue). Ensuring the probe structure is intact allows for activation of CRISPR / Cas12a trans-cleavage activity. When the target is present, the target RNA and the 3' recognition region of the probe undergo base pairing, forming a stable hybrid structure at the probe end, preventing Exo I degradation and providing end protection. Subsequently, the blue region of the probe specifically binds to the CRISPR / Cas12a acrRNA system, activating trans-cleavage activity and enabling the cleavage of fluorescent reporter molecules (FQRepotter), thus generating a significant fluorescent signal. In the absence of a target, the probe capable of activating CRISPR / Cas12a is effectively degraded by Exo I, losing its ability to activate CRISPR / Cas12a trans-cleavage activity and producing only a very weak fluorescent signal.

[0063] Sensitivity analysis of the E-CRISPR detection method: The sensitivity of this method was evaluated using fluorescence signals from targets of different concentrations (see...). Figure 5 ). Figure 5 This is a sensitivity analysis chart of the E-CRISPR system for detecting MIR155HG according to the present invention. Within a certain range, there is a good linear relationship between fluorescence intensity and target concentration, with the linear regression equation being y = 9338.14x + 119.68 (R² = 0.99), where y is fluorescence intensity and x is target concentration. The detection limit was calculated using the 3σ / k method, where σ is the standard deviation of the fluorescence signal in the blank control and k is the slope of the standard curve. The results show that the detection limit of this system is 4.76 pM, and the linear range is 4 pM to 0.4 nM.

[0064] Selectivity analysis of E-CRISPR detection method: The selectivity of this method was evaluated by comparing the fluorescence signal intensity of seven groups of samples, including target RNA, multiple random mRNAs and blank control. Figure 6 This is a selectivity analysis chromatogram of the E-CRISPR system of the present invention for detecting MIR155HG. (See figure) Figure 6 As shown, the fluorescence signal intensity decreased significantly with the increase of the number of base mutations; while the fluorescence signals generated by the three random mRNAs were almost the same as those of the blank control group, indicating that the E-CRISPR detection system can effectively distinguish between target sequences, mutant sequences, and other nucleic acid sequences, and has good selectivity.

[0065] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A nucleic acid detection method based on the exonuclease I terminal protection effect and CRISPR / Cas12a trans-cleavage signal amplification, characterized in that... The steps include: (1) providing a bifunctional single-stranded DNA probe, wherein the 3' end of the probe is the target nucleic acid recognition region and the 5' end is the CRISPR / Cas12a activation sequence; (2) incubating the sample to be tested, the bifunctional single-stranded DNA probe and exonuclease I together, so that if the target nucleic acid is present, it specifically binds to the 3' end recognition region of the probe to form an RNA-DNA hybrid structure, thereby protecting the 3' end of the probe from degradation by exonuclease I, while the unprotected probe is degraded by exonuclease I; (3) inactivating exonuclease I; (4) adding a CRISPR / Cas12a detection system to the system obtained in step (3), wherein the detection system contains Cas12a protein, crRNA and fluorescent reporter molecule, wherein the crRNA can specifically bind to the 5' end activation sequence of the bifunctional single-stranded DNA probe; (5) detecting the fluorescence signal, and judging the presence or quantity of the target nucleic acid based on the fluorescence signal intensity.

2. The nucleic acid detection method according to claim 1, characterized in that: In step (1), the nucleotide sequence of the bifunctional single-stranded DNA probe is shown in SEQ ID NO.

1.

3. The nucleic acid detection method according to claim 1, characterized in that: In step (4), the nucleotide sequence of the crRNA is shown in SEQ ID NO.2, the nucleotide sequence of the fluorescent reporter molecule is 6-FAM-TTATTT-BHQ1, and the target nucleic acid is long non-coding RNA MIR155HG.

4. The nucleic acid detection method according to claim 1, characterized in that: In step (2), the incubation conditions include: first incubating at 37°C to allow the target and probe to bind, then adding exonuclease I to continue the reaction, and then heating at 80°C to inactivate exonuclease I.

5. The nucleic acid detection method according to claim 1, characterized in that: In step (4), the reaction conditions of the CRISPR / Cas12a detection system include: incubation at 37°C for 10 minutes; the fluorescent reporter molecule is a single-stranded DNA reporter probe with fluorescent groups and quenching groups labeled at both ends.

6. The nucleic acid detection method according to claim 1, characterized in that: In steps (1) to (5), the method does not require a nucleic acid amplification step.

7. An E-CRISPR detection kit for implementing the method of claim 1, characterized in that: The E-CRISPR detection kit includes: a bifunctional single-stranded DNA probe with the nucleotide sequence shown in SEQ ID NO.1; exonuclease I; Cas12a protein; crRNA with the nucleotide sequence shown in SEQ ID NO.2; the fluorescent reporter molecule with the nucleotide sequence 6-FAM-TTATTT-BHQ1; and instructions for use.

8. The reagent kit according to claim 7, characterized in that: The target recognition region of the bifunctional single-stranded DNA probe is specifically targeted at the long non-coding RNA MIR155HG.

9. The use of the method of claim 1 or the kit of claim 7 in the preparation of a kit for detecting perioperative depression-related molecular markers in cervical cancer patients or for studying the antidepressant mechanism of esketamine.

Images