A CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip

Abstract

This utility model relates to the field of test strip technology and discloses a CRISPR-based ultra-high sensitivity malaria nucleic acid test strip, including a test box. The test box includes a PVC base plate, and a sample pad, a gold-labeled pad, an NC detection membrane, and an absorbent pad are sequentially connected to the upper surface of the PVC base plate. In use, this CRISPR-based ultra-high sensitivity malaria nucleic acid test strip utilizes the trans-cleavage characteristics after CRISPR-Cas enzyme activation, allowing a single target to trigger a cascade reaction, achieving exponential signal amplification. This improves the detection of Plasmodium nucleic acid in each microliter of blood, effectively identifying asymptomatic carriers. Line C1 uses an anti-FAM antibody to identify uncut probes, monitoring sample pretreatment effects and avoiding interference from impurities. Line C2 uses goat anti-mouse secondary antibody to identify the gold-labeled mouse monoclonal antibody, verifying the test strip's function. The combination of these two greatly enhances the reliability of the detection results, providing accurate evidence for malaria prevention and control, and effectively blocking malaria transmission.

Description

Technical Field

[0001] This utility model relates to the field of test strip technology, and in particular to an ultra-high sensitivity malaria nucleic acid test strip based on CRISPR. Background Technology

[0002] As is well known, malaria is a global infectious disease caused by Plasmodium infection. Timely and accurate detection of Plasmodium nucleic acid is key to the prevention and control of malaria, especially the screening of asymptomatic carriers, which directly affects the effectiveness of blocking the transmission chain. Currently, malaria nucleic acid detection technology uses colloidal gold test strips for detection.

[0003] Traditional colloidal gold test strips rely on direct antibody-antigen binding, and the signal intensity is linearly related to the target concentration. The detection limit is limited by the enrichment efficiency of colloidal gold particles, making it difficult to detect asymptomatic carriers. Utility Model Content

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip. When used, this CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip utilizes the trans-cleavage characteristics of CRISPR-Cas enzyme activation, allowing a single target to trigger a cascade reaction, achieving exponential signal amplification. This improves the detection of Plasmodium nucleic acid in every microliter of blood, effectively identifying asymptomatic carriers. Line C1 uses anti-FAM antibodies to identify uncut probes, monitoring sample pretreatment effects and avoiding interference from impurities. Line C2 uses goat anti-mouse secondary antibody to identify the gold-labeled mouse monoclonal antibody, verifying the test strip's function. The combination of these two greatly enhances the reliability of the detection results, providing accurate evidence for malaria prevention and control, and effectively blocking malaria transmission.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A CRISPR-based ultra-high sensitivity malaria nucleic acid test strip includes a test box. The test box includes a PVC base plate. A sample pad, a gold label pad, an NC detection membrane, and an absorbent pad are sequentially connected to the upper surface of the PVC base plate. The NC detection membrane has three functional lines: a T-line detection line located in the middle of the membrane near the sample end, a C1-line pretreatment control line located on the side of the T-line closer to the sample end, and a C2-line test strip control line located at the absorbent end of the membrane.

[0007] The T-line detection line is pre-coated with anti-digoxigenin antibody to identify digoxigenin labels on the probe; the C1-line pretreatment control line is pre-coated with anti-fluorescein antibody to identify fluorescein labels on uncut probes; the C2-line test strip control line is pre-coated with goat anti-mouse secondary antibody to identify mouse monoclonal antibody in the gold-labeled pad; and the gold-labeled pad is pre-coated with colloidal gold labels, including colloidal gold-streptavidin complex and colloidal gold-mouse monoclonal antibody complex.

[0008] Compared with existing malaria nucleic acid test strips, this malaria nucleic acid test strip only requires three steps to complete: "sample addition - waiting - interpretation". The sample pad is pretreated with a buffer solution to maintain Cas enzyme activity, so the detection does not require additional temperature control equipment and can be carried out efficiently at room temperature. The gold label pad, NC detection membrane and absorbent pad work together to effectively resist inhibitors in complex samples, ensuring stable liquid migration and signal display, and has high practical performance.

[0009] Furthermore, the anti-digoxigenin antibody of the T-line detection line captures the Biotin-DIG fragment released after CRISPR-Cas enzyme cleavage by targeting and binding to the digoxigenin label on the probe. The Biotin-DIG fragment is the product of a nucleic acid probe modified with biotin and digoxigenin at both ends, respectively, after Cas enzyme cleavage.

[0010] The above-described technical solution utilizes the high specificity and amplification effect of CRISPR-Cas enzyme cleavage, enabling the amplification of extremely small amounts of Plasmodium nucleic acid signals. The anti-digoxin antibody in the T-line targets and binds to the Biotin-DIG fragment, capturing a large amount of this cleaved fragment and significantly enhancing the detection signal. This allows for the detection of extremely low concentrations of Plasmodium nucleic acid in the sample, greatly improving detection sensitivity and aiding in the early detection of malaria infection, including asymptomatic carriers.

[0011] Furthermore, the anti-fluorescein antibody of the C1 line pretreatment control line specifically recognizes intact probes that have not been cleaved by Cas enzyme. The two ends of the intact probe are modified with fluorescein and biotin-digoxigenin conjugate, respectively, to monitor the removal effect of FAM antibody magnetic beads on the intact probes during sample pretreatment.

[0012] Using the above technical solution, during sample pretreatment, the role of FAM antibody magnetic beads is to remove intact probes that have not been cleaved by Cas enzyme, thus preventing these intact probes from interfering with subsequent detection results. The anti-fluorescein antibody in the C1 line can specifically recognize these intact probes. If sample pretreatment is insufficient and intact probes remain, the C1 line will develop color. By observing the color development of the C1 line, it is possible to visually determine whether the sample pretreatment has achieved the expected results, promptly identify and correct any potential problems, and ensure the reliability of the detection results.

[0013] Furthermore, the goat anti-mouse secondary antibody in the C2 line control line of the test strip specifically binds to the mouse monoclonal antibody in the gold-labeled pad. The mouse monoclonal antibody is a non-specific control antibody used to verify the release ability of the gold-labeled pad marker in the test strip and the liquid migration function of the detection membrane.

[0014] The above technical solution utilizes the specific binding of the goat anti-mouse secondary antibody at line C2 to the mouse monoclonal antibody in the gold-labeled pad. This process verifies the basic functionality of the test strip. If the gold-labeled pad marker is released normally and the detection membrane exhibits good liquid migration, the mouse monoclonal antibody in the gold-labeled pad will migrate to line C2 and bind with the goat anti-mouse secondary antibody, causing line C2 to develop color. If line C2 does not develop color, it indicates a potential problem with the release of the gold-labeled pad marker or obstructed liquid migration in the detection membrane. In this case, the test result is invalid, and the test strip needs to be replaced for retesting, thus ensuring the validity of the test results.

[0015] Furthermore, the colloidal gold-streptavidin complex of the gold pad captures the Biotin-DIG fragment generated after CRISPR-Cas enzyme cleavage through the high affinity binding of biotin and streptavidin, and the colloidal gold particles have a particle size of 20-40 nm, which is used to achieve colloidal gold enrichment and amplification of the detection signal.

[0016] The above technical solution utilizes a colloidal gold-streptavidin complex on a gold-labeled pad, which binds to streptavidin with high affinity, to efficiently capture the Biotin-DIG fragment generated after CRISPR-Cas enzyme cleavage. The colloidal gold particles, with a diameter of 20-40 nm, possess a large specific surface area and excellent optical properties. Multiple colloidal gold particles can bind to a single Biotin-DIG fragment, enriching and amplifying the detection signal at the T-line. This signal amplification allows the originally weak Plasmodium nucleic acid signal to be detected by the naked eye or simple detection equipment, improving detection sensitivity and visualization.

[0017] Furthermore, the sample pad is made of pretreated glass fiber or nonwoven fabric, the pretreatment including a buffer system to maintain the pH and ionic strength required for the CRISPR-Cas enzyme reaction, ensuring that the Cas enzyme is efficiently activated and cuts the probe at room temperature;

[0018] The above technical solution maintains the pH and ionic strength required for the CRISPR-Cas enzyme reaction through the sample pad pretreatment buffer system, providing a suitable reaction environment for the Cas enzyme. This allows the Cas enzyme to efficiently activate and cleave the probe at room temperature without the need for additional temperature control equipment, simplifying the detection process and reducing detection costs.

[0019] Furthermore, a sample dispensing hole is provided at the front end of the upper surface of the detection box, the sample dispensing hole is located above the sample pad, and a detection groove is provided in the middle of the upper surface of the detection box, the detection groove is located above the three functional lines;

[0020] The above technical solution facilitates sample addition via the sample dispensing port, and the detection cell allows for clear observation of the color development of the T-line, C1-line, and C2-line, enabling operators to quickly and accurately interpret the test results. Simultaneously, the design of the detection cell protects the detection membrane from external environmental interference, ensuring the stability and reliability of the test results.

[0021] This utility model has the following beneficial effects:

[0022] 1. This invention proposes a CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip. Compared with existing malaria nucleic acid detection test strips, this CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip utilizes the trans-cleavage characteristics after CRISPR-Cas enzyme activation. A single target can trigger a cascade reaction, achieving exponential signal amplification. This improves the detection of Plasmodium nucleic acid in every microliter of blood, effectively identifying asymptomatic carriers. Line C1 uses anti-FAM antibody to identify uncut probes, monitoring sample pretreatment effects and avoiding interference from impurities. Line C2 uses goat anti-mouse secondary antibody to identify gold-labeled mouse monoclonal antibody, verifying the test strip's function. The combination of these two greatly enhances the reliability of the detection results, providing accurate evidence for malaria prevention and control, and effectively blocking malaria transmission.

[0023] 2. This utility model proposes a CRISPR-based ultra-high sensitivity malaria nucleic acid test strip. Compared with existing malaria nucleic acid test strips, this malaria nucleic acid test strip only requires three steps to complete: "sample addition - waiting - interpretation". The sample pad is pretreated with a buffer solution to maintain Cas enzyme activity, so the detection does not require additional temperature control equipment and can be carried out efficiently at room temperature. The gold label pad, NC detection membrane and absorbent pad work together to effectively resist inhibitors in complex samples, ensuring stable liquid migration and signal display, and has high practical performance. Attached Figure Description

[0024] Figure 1 An isometric view of a CRISPR-based ultra-high sensitivity malaria nucleic acid test strip proposed in this utility model;

[0025] Figure 2 This is an isometric view of the test strip in the CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip proposed in this utility model;

[0026] Figure 3 for Figure 1 Enlarged view of point A in the middle;

[0027] Figure 4 This is a front view of the test strip of a CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip proposed in this utility model.

[0028] Legend:

[0029] 1. Detection box; 11. Sample dispensing port; 12. Detection slot; 2. PVC base plate; 21. Sample pad; 22. Gold label pad; 23. NC detection membrane; 231. C1 line pretreatment quality control line; 232. T line detection line; 233. C2 line test strip quality control line; 24. Absorbent pad. Detailed Implementation

[0030] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0031] Reference Figure 1-4 The present invention provides an embodiment of a CRISPR-based ultra-high sensitivity malaria nucleic acid test strip, comprising a test box 1, the test box 1 comprising a PVC base plate 2, a sample pad 21, a gold label pad 22, an NC detection membrane 23 and an absorbent pad 24 connected sequentially on the upper surface of the PVC base plate 2, the NC detection membrane 23 having three functional lines, namely a T-line detection line 232 located in the middle of the sample end, a C1-line pretreatment quality control line 231 located on the side of the T-line close to the sample end, and a C2-line test strip quality control line 233 located at the absorbent end of the membrane.

[0032] T-line test line 232 is pre-coated with anti-digoxigenin (DIG) antibody to identify the digoxigenin (DIG) label on the probe. C1-line pretreatment control line 231 is pre-coated with anti-fluorescein (FAM) antibody to identify the fluorescein (FAM) label on the uncut probe. C2-line test strip control line 233 is pre-coated with goat anti-mouse secondary antibody to identify the mouse monoclonal antibody in the gold-labeled pad 22. The gold-labeled pad 22 is pre-coated with colloidal gold labels, including colloidal gold-streptavidin (SA) complex and colloidal gold-mouse monoclonal antibody complex.

[0033] Compared to existing malaria nucleic acid test strips, this malaria nucleic acid test strip only requires three steps to use: "sample addition - waiting - interpretation". The sample pad 21 is pretreated with a buffer solution to maintain Cas enzyme activity, so the detection can be carried out efficiently at room temperature without additional temperature control equipment. The gold label pad 22, NC detection membrane 23 and absorbent pad 24 work together to effectively resist inhibitors in complex samples, ensuring stable liquid migration and signal display, and have high practical performance.

[0034] The anti-digoxin DIG antibody of T-line detection line 232 targets and binds to the digoxin DIG label on the probe, capturing the Biotin-DIG fragment released after CRISPR-Cas enzyme cleavage. The Biotin-DIG fragment is the product of nucleic acid probes modified with biotin and digoxin DIG at both ends respectively after Cas enzyme cleavage.

[0035] The CRISPR-Cas enzyme cleavage reaction exhibits high specificity and amplification, enabling the amplification of even minute amounts of Plasmodium nucleic acid signals. The T-line anti-digoxin DIG antibody targets and binds to the Biotin-DIG fragment, capturing a large amount of this cleaved fragment and significantly enhancing the detection signal. This allows for the detection of extremely low concentrations of Plasmodium nucleic acid in the sample, greatly improving detection sensitivity and aiding in the early detection of malaria infection, including asymptomatic carriers.

[0036] The anti-fluorescein antibody FAM antibody in the C1 line pretreatment quality control line 231 specifically recognizes intact probes that have not been cleaved by Cas enzyme. The two ends of the intact probe are modified with fluorescein FAM and biotin-digoxigenin-DIG conjugate, respectively, to monitor the removal effect of FAM antibody magnetic beads on the intact probes during sample pretreatment.

[0037] During sample pretreatment, the role of FAM antibody magnetic beads is to remove intact probes that have not been cleaved by Cas enzyme, thus preventing these intact probes from interfering with subsequent detection results. The anti-fluorescein FAM antibody in the C1 line can specifically recognize these intact probes. If sample pretreatment is insufficient and intact probes remain, the C1 line will develop color. By observing the color development of the C1 line, one can intuitively determine whether the sample pretreatment has achieved the expected results, promptly identify and correct any potential problems, and ensure the reliability of the detection results.

[0038] The goat anti-mouse secondary antibody in control line 233 of the C2 line test strip specifically binds to the mouse monoclonal antibody in the gold-labeled pad 22. The mouse monoclonal antibody is a non-specific control antibody used to verify the release ability of the gold-labeled pad 22 marker in the test strip and the liquid migration function of the detection membrane.

[0039] The specific binding of the goat anti-mouse secondary antibody at line C2 to the mouse monoclonal antibody in the gold-labeled pad 22 verifies the basic functionality of the test strip. If the gold-labeled pad 22 marker is released normally and the detection membrane exhibits good liquid migration, the mouse monoclonal antibody in the gold-labeled pad 22 will migrate to line C2 and bind with the goat anti-mouse secondary antibody, causing line C2 to develop color. If line C2 does not develop color, it indicates a potential problem with the release of the gold-labeled pad 22 marker or obstructed liquid migration in the detection membrane. In this case, the test result is invalid, and the test strip needs to be replaced for retesting, thus ensuring the validity of the test results.

[0040] The colloidal gold-streptavidin SA complex of the gold pad 22 captures the Biotin-DIG fragment generated after CRISPR-Cas enzyme cleavage by binding biotin to streptavidin SA with high affinity. The colloidal gold particles have a particle size of 20-40 nm and are used to achieve colloidal gold enrichment and amplification of the detection signal.

[0041] The colloidal gold-streptavidin SA complex of the gold-labeled pad 22 binds to streptavidin SA with high affinity, efficiently capturing the Biotin-DIG fragment generated after CRISPR-Cas enzyme cleavage. The colloidal gold particles, with a diameter of 20-40 nm, possess a large specific surface area and excellent optical properties. Multiple colloidal gold particles can bind to a single Biotin-DIG fragment, enriching and amplifying the detection signal at the T-line. This signal amplification allows the originally weak Plasmodium nucleic acid signal to be detected by the naked eye or simple detection devices, improving detection sensitivity and visualization.

[0042] Sample pad 21 is made of pretreated glass fiber or non-woven fabric. The pretreatment includes a buffer system to maintain the pH value of 7.0-8.0 and ionic strength required for the CRISPR-Cas enzyme reaction, ensuring that the Cas enzyme is efficiently activated and cuts the probe at room temperature.

[0043] The pretreatment buffer system in sample pad 21 maintains the pH value (7.0-8.0) and ionic strength required for the CRISPR-Cas enzyme reaction, providing a suitable reaction environment for the Cas enzyme. This allows the Cas enzyme to efficiently activate and cleave the probe at room temperature without the need for additional temperature control equipment, simplifying the detection process and reducing detection costs.

[0044] The front end of the upper surface of the test box 1 is provided with a sample feeding hole 11, which is located above the sample pad 21. The middle part of the upper surface of the test box 1 is provided with a test groove 12, which is located above the three functional lines.

[0045] The sample addition port 11 facilitates sample introduction, while the detection chamber 12 allows for clear observation of the color development of the T-line, C1-line, and C2-line, enabling operators to quickly and accurately interpret the test results. Simultaneously, the design of the detection chamber 12 protects the detection membrane from external environmental interference, ensuring the stability and reliability of the test results.

[0046] Working principle: During use, the sample to be tested is dripped into the sample application hole 11 of the test kit 1. The sample falls onto the sample pad 21, which is made of pretreated glass fiber or non-woven fabric. The buffer solution contained in the sample pad maintains the efficient activation of the CRISPR-Cas enzyme at room temperature under suitable pH (7.0-8.0) and ionic strength. If the sample contains Plasmodium nucleic acid, the Cas enzyme will recognize and bind to the target nucleic acid sequence. After activation, it trans-cleaves the nucleic acid probes modified with fluorescein and biotin-digoxigenin at both ends, respectively, to produce a large number of Biotin-DIG fragments. The sample flows into the gold-labeled pad 22 through the sample pad 21. The colloidal gold-streptavidin (SA) complex in the gold-labeled pad 22 captures the target nucleic acid by utilizing the high affinity between biotin and streptavidin. The Biotin-DIG fragment is obtained, enabling colloidal gold enrichment and amplification of the detection signal. The sample then continues to migrate to the detection membrane. The anti-digoxigenin antibody on the T line targets and binds to the digoxigenin (DIG) label on the Biotin-DIG fragment. If the sample contains Plasmodium nucleic acid, the T line will show color due to colloidal gold aggregation. The anti-fluorescein antibody on the C1 line specifically recognizes intact probes not cleaved by Cas enzyme. If sample pretreatment is insufficient and intact probes remain, the C1 line will show color, indicating the need for optimized pretreatment. The goat anti-mouse secondary antibody on the C2 line specifically binds to the mouse monoclonal antibody in the gold-labeled pad 22. If the test strip functions normally, the C2 line will show color, demonstrating normal release of the gold-labeled pad 22 label and good liquid migration function of the detection membrane.

[0047] Finally, it should be noted that the above description is only a preferred embodiment of the present utility model and is not intended to limit the present utility model. Although the present utility model has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present utility model should be included within the protection scope of the present utility model.

Claims

1. A CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip, comprising a detection kit (1), characterized in that: The detection box (1) includes a PVC base plate (2). The sample pad (21), gold label pad (22), NC detection membrane (23) and absorbent pad (24) are connected sequentially on the upper surface of the PVC base plate (2). The NC detection membrane (23) is provided with three functional lines, namely the T-line detection line (232) located in the middle of the sample end, the C1-line pretreatment quality control line (231) located on the side of the T-line close to the sample end, and the C2-line test strip quality control line (233) located at the end of the membrane in the direction of the absorbent end. The T-line detection line (232) is pre-coated with anti-digoxigenin DIG antibody to identify the digoxigenin DIG label on the probe. The C1-line pretreatment control line (231) is pre-coated with anti-fluorescein FAM antibody to identify the fluorescein FAM label on the uncut probe. The C2-line test strip control line (233) is pre-coated with goat anti-mouse secondary antibody to identify the mouse monoclonal antibody in the gold-labeled pad (22). The gold-labeled pad (22) is pre-coated with colloidal gold labels, including colloidal gold-streptavidin SA complex and colloidal gold-mouse monoclonal antibody complex.

2. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The anti-digoxin DIG antibody of the T-line detection line (232) targets and binds to the digoxin DIG label on the probe, capturing the Biotin-DIG fragment released after CRISPR-Cas enzyme cleavage. The Biotin-DIG fragment is the product of a nucleic acid probe modified with biotin and digoxin DIG at both ends respectively after Cas enzyme cleavage.

3. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The anti-fluorescein antibody FAM of the C1 line pretreatment quality control line (231) specifically recognizes intact probes that have not been cleaved by Cas enzyme. The two ends of the intact probe are modified with fluorescein FAM and biotin-digoxigenin-DIG conjugate, respectively, to monitor the removal effect of FAM antibody magnetic beads on the intact probes during sample pretreatment.

4. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The goat anti-mouse secondary antibody in the control line (233) of the C2 line test strip specifically binds to the mouse monoclonal antibody in the gold-labeled pad (22). The mouse monoclonal antibody is a non-specific control antibody used to verify the release ability of the gold-labeled pad (22) marker in the test strip and the liquid migration function of the detection membrane.

5. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The colloidal gold-streptavidin SA complex of the gold pad (22) binds to streptavidin SA with high affinity through biotin to capture the Biotin-DIG fragment generated after CRISPR-Cas enzyme cleavage, and the colloidal gold particles have a particle size of 20-40 nm, which is used to achieve colloidal gold enrichment and amplification of the detection signal.

6. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The sample pad (21) is made of pretreated glass fiber or nonwoven fabric. The pretreatment includes a buffer system to maintain the pH value of 7.0-8.0 and ionic strength required for the CRISPR-Cas enzyme reaction, ensuring that the Cas enzyme is efficiently activated and cuts the probe at room temperature.

7. The CRISPR-based ultra-high sensitivity malaria nucleic acid detection test strip according to claim 1, characterized in that: The front end of the upper surface of the test box (1) is provided with a sample feeding hole (11), which is located above the sample pad (21). The middle part of the upper surface of the test box (1) is provided with a test groove (12), which is located above the three functional lines.

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