A detection device with complete probe retention effect display

Abstract

This invention relates to the field of nucleic acid detection technology, and more particularly to a detection device, detection method, and application for probe cleavage detection. The detection device is based on a solid-phase carrier and sequentially includes a sample inflow area, a complete probe retention display area, and a probe cleavage product detection area along the sample flow path. The complete probe retention display area is equipped with a retention ligand that specifically binds to the target probe label, used to capture and indicate uncleaved complete probes in the test liquid; the detection area is equipped with a capture ligand that specifically binds to the probe cleavage products, used to detect the products generated by the probe cleavage reaction. By setting the complete probe retention display area before the detection area, a visual indication of whether a complete probe has entered the detection area can be provided, thereby judging the reliability of the detection results and reducing false positives or background signal interference caused by residual complete probes. The detection device may further include a pre-retention structure for complete probes for preliminary capture. This invention is applicable to various probe cleavage detection systems such as CRISPR lateral cleavage detection, RNase H enzyme digestion detection, and T7 endonuclease detection, and has the advantages of simple structure, compatibility with existing lateral chromatography processes, and high reliability of detection results.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to a capture solid phase, detection method and its application for probe cleavage detection, which is particularly suitable for CRISPR side-cleavage effect and enzyme-cleaved probe detection scenarios, solving the problems of false positives and background signal interference caused by intact probe residue, and indicating probe overload. Background Technology

[0002] In nucleic acid probe cleavage detection scenarios, such as test strip detection based on CRISPR side-cutting effect and detection of probe cleavage by enzymes such as T7 or RNase H, there are obvious technical challenges.

[0003] In the detection of CRISPR paracutting effects, such as Figure 1 As shown, the C line of the test strip is used to capture the complete probe, and the T line is used to detect the short chain after cleavage. When there is an excess of probe, causing the C line to fail to completely capture the complete probe, a false positive will occur, and the test strip will not provide any indication. In particular, when both the C line and the T line show color, it is impossible to determine whether the result is positive, due to probe excess, or other reasons that prevented the C line from completely capturing the complete probe.

[0004] In T7 or RNase H enzyme digestion probe detection, the existing design assumes that the probe can only be completely cleaved or not cleaved at all. However, in actual applications, some probes may not bind to the target region or may not be treated by the enzyme after binding, which will generate background signals and interfere with the reliability of the detection.

[0005] Currently, there are no effective solutions in existing technologies to address the aforementioned pain points. In particular, there is no design that uses "complete probe retention + color indication" to determine whether a complete probe remains and enters the detection process. This makes it impossible to avoid false positives and background signal interference at the source, resulting in insufficient detection accuracy and reliability. Summary of the Invention

[0006] To address the technical problems in existing probe cutting detection, such as false positives and background signal interference caused by intact probe residue, and the lack of effective indication methods, this invention provides a method for capturing solid phase and detection, and its application, which realizes the interception and residual indication of intact probe, improves the accuracy and reliability of detection, and fills the gap in the existing technology.

[0007] The core technical solution of this invention is as follows: In the captured solid phase (such as test paper) used for probe cutting detection, at least one complete probe display area is provided. This area can indicate whether a complete probe is present in the test liquid. Simultaneously, in conjunction with the detection area, the probe cutting products are detected, forming a complete "retention-indication-detection" process. This achieves effective retention of complete probes, visual indication of residual status, and simultaneous detection of probe cutting products. The specific strategy is as follows: After the test liquid enters the detection device from the sample inlet, it first undergoes preliminary interception through the intact probe interception system (if there is no pre-interception, it directly enters the interception display area), where most of the intact probes are intercepted. The remaining liquid flows into the intact probe interception display area, where the ligands in the area specifically bind to the remaining intact probes, and the color development indicates whether intact probes remain. After confirming that no intact probes remain in the interception display area, the liquid flows into the detection area, where the captured ligands bind to the probe cleavage products, and the color development indicates the probe cleavage status. Finally, by combining the color development results from the interception display area and the detection area, a reliable detection conclusion is output, fundamentally avoiding false positives and background signal interference caused by intact probe residues.

[0008] The detailed technical solution is as follows: The detection device of this invention uses a captured solid phase as its core carrier. The captured solid phase is selected from one of the following: test strips, microfluidic chips, and solid-phase extraction columns, with test strips being preferred (as they are compatible with existing production processes and facilitate widespread adoption). The detection device as a whole includes a sample inflow end, a complete probe retention system, a complete probe retention display area, a detection area, and a water absorption end. These parts are arranged sequentially along the sample flow direction, wherein: (1) Sample inlet end: used to carry the test liquid containing the target probe. The probe can specifically bind to the test nucleic acid region and can be cleaved by the corresponding cleavage reagent (CRISPR-related protein, T7 endonuclease, RNase H enzyme, etc.). The probe is marked with a specific label, which is selected from one or more of biotin, FAM, and FIFC, and is used for the subsequent retention of the intact probe and the detection of the cleavage product. (2) Complete probe interception system: used to initially intercept the complete probe in the liquid to be tested. According to the different structures of the captured solid phase, it is divided into three implementation forms to adapt to different detection scenarios: ① Form 1 (no pre-withdrawal, corresponding to) Figure 2 -A Structure): No independent pre-retention component is set up. The retention of the complete probe is achieved only through the subsequent complete probe retention display area. It is suitable for scenarios where the initial content of the probe is low and no preliminary enrichment and retention is required. At least two complete probe retention display areas are set up and arranged sequentially along the sample flow direction to achieve multi-level retention and improve the retention effect. ② Form Two (Pre-embedded layer interception, corresponding to...) Figure 2 -B Structure): A complete probe retention pad is set up between the sample inflow end and the complete probe retention display area. The pad contains ligands that specifically bind to the probe label (e.g., avidin is fixed in the pad when the probe is labeled as biotin). This is used to initially retain most of the complete probe in the test liquid, reduce the load on the subsequent retention display area, and improve the thoroughness of retention. ③ Form 3 (Decoupling and interception, corresponding to...) Figure 2 -C structure): An external retention device is set up to be decoupled from the captured solid phase. The external retention device is selected from intact probe capture magnetic beads and solid phase extraction column. The external retention device has a ligand that specifically binds to the probe label fixed inside. The liquid to be tested is first initially retained by the external retention device and then passed into the sample inlet of the captured solid phase. It is suitable for scenarios where there are many impurities in the liquid to be tested and the content of intact probe fluctuates greatly. (3) Complete probe interception display area: Located after the complete probe interception system and before the detection area, it is used to intercept the remaining complete probe after the initial interception and to visually indicate whether the complete probe remains through the color development status. This area is fixed with ligands that specifically bind to the probe label (matching the probe label, such as biotin-avidin, antigen-antibody binding pair). The status is displayed by a preset color development method, which is selected from colloidal gold color development, fluorescence color development, and chemiluminescence color development. Colloidal gold color development is preferred (no special detection equipment is required and the visualization effect is good). There are two types of color display logic for the intercepted display area, which can be selected according to the detection requirements: ① Non-competitive method: The ligand in the interception display area binds directly to the label of the intact probe. If the area is colored, it indicates that there is an intact probe in the test liquid that has not been completely intercepted, and the test result is unreliable (false positives may occur); if the area is not colored, it indicates that the intact probe has been completely intercepted, and the subsequent detection steps can be carried out. ② Competitive method: The interception display area is pre-fixed with a "ligand-chromogenic agent" complex (such as avidin-colloidal gold complex). The intact probe competes with the chromogenic agent to bind to the ligand. If the area remains colored, it indicates that there is no residual intact probe in the test liquid, and the detection result is reliable; if the area disappears from the color, it indicates that there is an intact probe that has not been completely intercepted, and the detection result is unreliable. (4) Detection area: Used to capture the product after probe cleavage and determine the probe cleavage status. The detection area is fixed with capture ligands that specifically bind to the probe cleavage product. The same color development method as the interception display area is used. If the detection area is colored, it is judged as positive (the probe has been successfully cleaved); if the detection area is not colored, it is judged as negative (the probe has not been cleaved). (5) Absorbent end: Located after the detection area, it is used to provide the driving force for sample flow and accelerate the diffusion of the test liquid on the captured solid phase. It is made of materials with excellent water absorption properties (such as absorbent paper or absorbent cotton).

[0009] This device is widely adaptable to various probe cutting and detection scenarios, including but not limited to: (1) CRISPR paracleavage effect detection: Applicable to the detection of paracleavage effect of various CRISPR-related proteins (such as Cas12a and Cas13a), which can effectively avoid false positives caused by excessive probe and improve the reliability of test strip detection; (2) Enzyme-digested probe detection: It is suitable for detection of probes by various nucleases such as T7 endonuclease and RNase H enzyme, and solves the problem of background signal interference caused by some probes not being cleaved; (3) Other probe cutting scenarios: The trapping ligand and the capture ligand of the detection area can be adjusted according to the probe labeling type and the type of cutting reagent to adapt to different nucleic acid detection needs.

[0010] Beneficial effects: ① Addresses existing technical pain points: Effectively retains the complete probe, preventing it from entering subsequent detection stages and causing false positives and background signal interference. At the same time, through color indication, it allows for intuitive judgment of the reliability of the test results, solving the problem that existing test strips cannot indicate the presence of complete probe residue; ② High practicality: It can be achieved based on existing solid-phase capture (such as test strip) production processes, without the need for special equipment, with low difficulty in industrial implementation and repeatable verification; ③ Wide range of applications: It can be adapted to various probe cleavage detection scenarios such as CRISPR side-cleavage effect, T7 enzyme digestion, and RNase H enzyme digestion, and has strong versatility; ④ Simple structure / steps: The design is simple and easy to operate, requiring no additional complicated steps, making it easy to promote and apply. Attached Figure Description

[0012] Figure 1 In current examples of CRISPR test strip usage, both the C-line and T-line show color in certain positive results.

[0013] Figure 2 The diagram illustrates the structure of the test strip of this invention. Structure A includes a sample inlet, at least two complete probe retention display areas, and at least one detection area. Structure B includes a sample inlet, at least one complete probe retention pad, at least one complete probe retention display area, and at least one detection area. Structure C includes a sample inlet, at least one complete probe retention display area, and at least one detection area (this structure requires a complete probe retention device or component decoupled from the test strip). The C-line, NC membrane substrate, absorbent pad, and other classic test strip components are not shown in the diagram.

[0014] Figure 3 : A schematic diagram of the detection method of the present invention. This process is applicable to all captured solid structures of the present invention. Figure 2 -A、 Figure 2 -B、 Figure 2 -C), the specific process steps are as follows: Step 1 (Sample Preparation): Obtain the test liquid containing the target probe. The probe can bind to the nucleic acid region to be tested and can be cleaved by the corresponding cleavage reagent (CRISPR-related protein, T7 endonuclease, RNase H enzyme, etc.). The probe is marked with specific labels (such as biotin, FAM, FIFC, etc.) for subsequent interception and detection.

[0015] Step 2 (Pre-treatment, optional): Select the appropriate retention method based on the structure of the captured solid phase: If it is Figure 2 -B structure (including complete probe absorption pad): the liquid to be tested enters the pad layer directly, and the complete probe is initially retained by the complete probe absorption pad; if it is... Figure 2 -C structure (with decoupled trapping device): The liquid to be tested is first passed through an external trapping device (such as a complete probe capture bead) to initially trap the complete probe; if it is Figure 2 If the -A structure (containing multiple complete probe interception display areas) is used, it will directly proceed to the subsequent interception display stage.

[0016] Step 3 (Complete Probe Retention and Colorimetric Determination): The test liquid, after pre-retention (or without pre-retention), is passed into the complete probe retention display area. This area retains the remaining complete probe through specific binding (such as biotin-avidin binding), and the state is displayed through a preset colorimetric method (colloidal gold colorimetry, etc.). — Scenario 1: Non-competitive method (adapted to Example 1 and Example 3): If the interception display area is colored, it indicates that there is an intact probe that has not been completely intercepted in the test liquid, and the test result is deemed unreliable (false positive may exist), and the sample needs to be reprocessed; if the interception display area is not colored, it indicates that the intact probe has been completely intercepted, and the subsequent detection process can proceed.

[0017] — Scenario 2: Competition method (adapted to Example 2): If the interception display area remains colored, it indicates that there are no residual complete probes in the test liquid, and the test result is reliable, proceeding to the subsequent test stage; if the color of the interception display area disappears, it indicates that there are complete probes that have not been completely intercepted, and the test result is unreliable.

[0018] Step 4 (Detection of probe-cut products): The test liquid that has passed through the interception display area is introduced into the detection area. The detection area captures the products cut by the probe. The probe cutting status is judged by the color development: if the detection area shows color, it is judged as positive (the probe has been successfully cut); if the detection area does not show color, it is judged as negative (the probe has not been cut).

[0019] Step 5 (Result Output): Combining the colorimetric judgment results from Step 3 and the colorimetric results of the detection area from Step 4, output the final detection conclusion to ensure the accuracy and reliability of the detection results.

[0020] Note: In this flowchart, the dashed box marking "pre-retention treatment" indicates an optional step, which depends on the specific structure of the captured solid phase. Figure 2 -A, B, C) can be flexibly selected; the specific criteria for colorimetric judgment can be adjusted according to the probe marking method and the interception principle (competitive method / non-competitive method), all of which fall within the protection scope of this invention.

[0021] Figure 4 :based on Figure 2 -B structure design test paper and results of its application in CRISPR side-cut detection.

[0022] Figure 5 :based on Figure 2 -B structure design test strip and Rnase H cleavage effect detection, result map of complete probe content display area based on competitive method.

[0023] Figure 6 :based on Figure 2 -C structure design and application strategy test strips and results of application to T7 endonuclease paracleavage detection.

[0024] Example 1: Detection of CRISPR sidecut effect based on test paper (capture solid phase) 1. Test strip structure: Reference Figure 2 -B, using an NC membrane as a substrate, with the sample end located at one end. The test strip pad area contains a complete probe absorption pad to absorb the complete probe. A complete probe content display area is located between the sample end and the detection area; colloidal gold colorimetric method is used. The probe is set to 5' biotin-nucleotide chain-FAM-3'. Both the complete probe content display area and the complete probe absorption pad contain biotinylate.

[0025] 2. Detection steps: (1) Prepare the test liquid containing CRISPR probe. After the probe binds to the nucleic acid to be tested, it can be cleaved by CRISPR-related proteins. (2) Drop the test liquid onto the sample end of the test strip. The liquid diffuses along the NC membrane, passes through the padding area, and then passes through the complete probe content display area. (3) Observe the color development status of the complete probe content display area: If the color develops, it means that there are complete probes that have not been completely intercepted in the test liquid. The test result is unreliable and the sample needs to be reprocessed. If the color does not develop, it means that there are no complete probe residues. (4) If the previous step shows that there are no complete probe residues, continue to observe the detection area: Color development means that the probe has been cleaved (positive), and no color development means that the probe has not been cleaved (negative).

[0026] 3. Results show: Figure 4The results show that the area displaying the complete probe content on test strip A is colored, indicating the presence of incompletely trapped probes in the test liquid. Subsequent test areas may show complete probes, making the result unreliable and potentially a false positive. Test strips B and C, on the other hand, do not show color in the area displaying the complete probe content, indicating the absence of completely trapped probes in the test liquid. These results are reliable and are positive.

[0027] Example 2: Detection of the RNase H cleavage effect based on test paper (captured solid phase), displaying the complete probe content area using a competitive method.

[0028] 1. Test strip structure: Reference Figure 2 -B, using an NC membrane as a substrate, with the sample end positioned at one end. The test strip pad area contains a complete probe absorption pad to absorb the complete probe. A complete probe content display area is located between the sample end and the detection area; a colloidal gold colorimetric method is used. The probe is set to 5' biotin-DNA-RNA-DNA-FAM-3'. The complete probe content display area contains biotinavitin. Each complete probe absorption pad itself contains a colloidal gold combination of biotinavitin and a low affinity ligand.

[0029] 2. Detection steps: (1) Prepare the test liquid containing the RNase H probe. After the probe binds to the nucleic acid to be tested, it can be cleaved by the RNase H protein; (2) Add the test liquid to the sample end of the test strip. The liquid diffuses along the NC membrane, passes through the padding area, and then passes through the complete probe content display area; (3) Observe the color development status of the complete probe content display area: if the color development is maintained, it means that there is no complete probe in the subsequent test liquid in this area, and the detection result is reliable; if the color development disappears, it means that the complete probe remains and will flow into the subsequent test area, and the detection result is unreliable; (4) If the color development is maintained in the previous step, continue to observe the test area: color development means that the probe has been cleaved (positive), no color development means that the probe has not been cleaved (negative).

[0030] 3. Results show: Figure 5 The results show that the area displaying the complete probe content on test strip A remains colored, indicating that the subsequent areas of the test liquid do not contain complete probes, and the test result is reliable and positive. The area displaying the complete probe content on test strip B does not show color, indicating that the subsequent areas of the test liquid may contain complete probes, and the test result is unreliable and may be a false positive.

[0031] Example 3: Detection of T7 endonuclease cleavage effect based on test strip (capture solid phase), displaying the intact probe content region using a competitive method.

[0032] 1. Test strip structure: Reference Figure 2-C, using an NC membrane as a substrate, with the sample end positioned at one end, and a complete probe content display area set between the sample end and the detection area; colloidal gold colorimetric method is used. The probe is set to 5' biotin-DNA-FIFC-3'. Biotin-avidin is included in the complete probe content display area.

[0033] 2. Detection steps: (1) Prepare the test liquid containing the T7 endonuclease probe. After the probe binds to the partially complementary test nucleic acid, it can be cleaved by the T7 endonuclease. (2) Capture the test liquid through the intact probe capture magnetic beads. The magnetic beads are coated with avidin. (3) Drop the test liquid onto the sample end of the test paper. The liquid diffuses along the NC membrane, passes through the padding area, and then passes through the intact probe content display area. (4) Observe the color development status of the intact probe content display area: If color development occurs, it indicates that there are intact probes that have not been completely intercepted in the test liquid. The test result is unreliable and the sample needs to be reprocessed. If no color development occurs, it indicates that there are no intact probes remaining and the result is reliable.

[0034] 3. Results show: Figure 6 The results show that the areas displaying the complete probe content on test strips A, E, and F are colored, indicating the presence of incompletely trapped probes in the test liquid. Subsequent test areas may show complete probes, making the results unreliable and potentially false positives. Test strips B, C, and D, on the other hand, do not display color in the areas displaying the complete probe content, indicating the absence of completely trapped probes in the test liquid. These results are reliable and positive.

Claims

1. A detection device for detecting probe cutting reactions, characterized in that, Includes a solid-phase capture support, wherein the following are sequentially arranged on the solid-phase capture support along the sample flow direction: Sample inflow area; Complete probe interception display area; The probe cuts through the product detection area; Wherein: the intact probe interception display area is fixed with an interception ligand that specifically binds to the first marker of the target probe, used to capture and indicate the intact probe that has not been cut in the test liquid; The probe cleavage product detection area is fixed with a capture ligand that specifically binds to the probe cleavage product, used to detect the product generated by the cleavage reaction of the target probe; The target probe is a nucleic acid probe with at least two labels, which can form a hybrid structure with the nucleic acid sequence to be tested and produce a cleavage product that can be recognized by the detection region under the action of the cleavage reagent; The complete probe interception display area indicates whether the complete probe has entered the detection area through color development or signal changes, thereby determining the reliability of the detection results.

2. The detection device according to claim 1, wherein: The capture solid support is a lateral chromatography test paper, and its substrate is a nitrocellulose membrane (NC membrane).

3. The detection device according to claim 1, wherein: The complete probe retention display area is set to at least two and arranged sequentially along the sample flow direction to form a multi-level complete probe retention structure.

4. The detection device according to claim 1, wherein: A pre-entry trapping structure for the complete probe is provided between the sample inflow area and the complete probe trapping display area.

5. The detection device according to claim 4, wherein: The pre-retention structure is a probe capture pad, which has a ligand that specifically binds to the first marker of the target probe.

6. The detection device according to claim 4, wherein: The pre-retention structure is an external retention device decoupled from the captured solid phase carrier.

7. The detection device according to claim 6, wherein: The external interception device is one of the following: Magnetic bead trapping device Solid phase extraction column solid phase adsorption materials Furthermore, the surface of the external interception device is fixed with a ligand that specifically binds to the first marker of the target probe.

8. The detection device according to claim 1, wherein: The complete probe interception display area adopts a non-competitive color rendering mode, characterized in that: When the complete probe is present, the region produces a colorimetric signal; When the complete probe is absent, the region does not produce a colorimetric signal.

9. The detection device according to claim 1, wherein: The complete probe interception display area adopts a competitive color rendering mode, characterized in that: When the complete probe is absent, the region retains a preset colorimetric signal; When the intact probe is present, the colorimetric signal is weakened or disappears.

10. The detection device according to claim 1, wherein: The first mark is selected from one of the following: Biotin, FAM, FITC, DIG, DNP.

11. The detection device according to claim 1, wherein: The second mark is selected from one of the following: FAM, FITC, DIG, DNP, fluorescent dyes, colloidal gold-bonded tags.

12. The detection device according to claim 1, wherein: The cleavage reagent is an enzyme or protein capable of cleaving nucleic acid probes.

13. The detection device according to claim 12, wherein: The cutting reagent is selected from one of the following: CRISPR-related proteins, RNase H, T7 endonuclease, and restriction endonucleases.

14. The detection device according to claim 1, wherein: The signal detection method for the complete probe interception display area and detection area is selected from: Colloidal gold colorimetric detection, fluorescence detection, chemiluminescence detection, and enzyme-catalyzed colorimetric detection.

15. The application of the detection device according to any one of claims 1-14 in nucleic acid detection.

16. The application according to claim 15, wherein: The detection method is selected from one of the following: CRISPR side-cut detection, RNase H probe cleavage detection.

17. A nucleic acid detection method utilizing intact probe residue as an indicator, characterized in that, include: Provide a reaction system containing labeled probes; The probe is hybridized with the nucleic acid to be tested and then cleaved. The reaction solution is introduced into the solid-phase capture and detection device; Determine whether there are any uncut complete probes within the complete probe interception display area; The probe cuts the product in the detection area; The detection result is determined based on the signal combination from steps 4 and 5.

18. A nucleic acid detection kit, characterized in that, include: The detection device according to any one of claims 1-15; Nucleic acid probes with dual labels; Nucleases or CRISPR proteins used to cleave the probe; Reaction buffer system.

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