A CRISPR and magnetic bead nucleic acid probe-based test strip positive color development detection method and application thereof

Abstract

The application discloses a test strip positive color developing detection method based on CRISPR and magnetic bead nucleic acid probes and application thereof. The test strip positive color developing visual detection method comprises the following steps: mixing a to-be-detected sample with a CRISPR-Cas nucleic acid cutting system and a magnetic bead coupled double-stranded nucleic acid probe, the probe is provided with a single-stranded connecting area which can be recognized by the CRISPR-Cas nucleic acid cutting system and is provided with biotin and hapten markers; if the target nucleic acid exists in the to-be-detected sample, the Cas enzyme side cutting activity is activated, the single-stranded connecting area is cut, and the complete double-stranded nucleic acid probe is released from the magnetic bead; the released double-stranded nucleic acid probe is loaded to a lateral chromatographic test strip, if the detection line and the quality control line develop color at the same time, it is determined that the sample is positive, and if only the quality control line develops color, it is determined that the sample is negative. The application realizes a positive color developing positive interpretation mode, is more in line with the conventional interpretation habit, and improves the detection accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a positive colorimetric detection method for test strips based on CRISPR and magnetic bead nucleic acid probes and its application, which is particularly suitable for point-of-care testing (POCT) and visual interpretation of respiratory pathogens. Background Technology

[0002] Visualized nucleic acid detection technology combining the CRISPR system with lateral chromatography test strips is one of the important development directions for point-of-care testing (POCT). This type of method combines the advantages of nucleic acid recognition specificity and on-site visual readout of test strips, and has good application prospects in respiratory pathogen screening, point-of-care testing at the grassroots level, and portable in vitro diagnostics.

[0003] Currently, one of the mainstream technologies for nucleic acid detection of respiratory pathogens is quantitative real-time PCR. This method has high sensitivity and specificity, but it usually relies on thermal cyclers, fluorescence detection equipment, and professional operators. The detection scenarios are mostly limited to laboratories or institutions with the corresponding instruments, making it difficult to fully meet the needs of rapid on-site detection and immediate interpretation.

[0004] To meet the needs of point-of-care testing (POCT), visual detection methods combining CRISPR nucleic acid recognition with colloidal gold lateral chromatography test strips have been gradually developed in recent years. These methods typically utilize Cas12a or similar nucleases to trigger cleavage activity after recognizing the target nucleic acid, and then achieve band color development through signal conversion between the reporter probe and the test strip capture system.

[0005] The closest existing technology to this invention is a detection scheme based on CRISPR cutting of the reporter probe and combining it with test strips for visual interpretation. Although this type of scheme can achieve on-site visual reading of nucleic acid test results, its common reading mode is mostly a negative colorimetric interpretation method, that is, a visible red band appears on the test line for negative samples, while for strongly positive samples, the test line does not show color or the color is weakened because the reporter probe is cut or the signal is turned off.

[0006] The above-mentioned negative colorimetric reading method has the following defects: (1) The test results do not conform to the conventional interpretation habits of test strips. Users are usually more likely to accept the intuitive logic of "positive results show a test line, negative results do not show a test line" or "positive results have an extra line", but in the negative colorimetric mode, the test line of strong positive samples does not show color, while the test line of negative samples shows a red band, which can easily cause comprehension difficulties. (2) The user experience is poor. The test line is visible in negative samples and weakens or even disappears in positive samples, which is contrary to the conventional test perception and is not conducive to grassroots operators and non-professional users to quickly and accurately judge the results. (3) The accuracy of interpretation is easily affected. For weak positive, boundary samples or complex on-site environments, the change in the strength of the test line in the negative colorimetric mode is not intuitive enough, which can easily lead to misreading, missed reading or misjudgment of positive results.

[0007] Therefore, it is still necessary to provide a CRISPR detection system that conforms to conventional interpretation habits and enables visual reading of positive test strips, so that the negative sample test line does not appear and the positive sample test line shows a clear red band, thereby improving user experience and accurate interpretation of test results. Summary of the Invention

[0008] To address the shortcomings of existing technologies and practical needs, this invention provides a positive colorimetric detection method for test strips based on CRISPR and magnetic bead nucleic acid probes, and its application. By constructing a magnetic bead-coupled nucleic acid probe system, the target nucleic acid recognition, CRISPR cleavage and release, and test strip colorimetric interpretation are structurally coupled, so that the detection line does not appear in the case of negative samples and a clear red band appears in the case of positive samples. This forms a positive colorimetric visualization detection mode that is more in line with normal interpretation habits and improves the accuracy of test strip result interpretation.

[0009] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a positive colorimetric visualization detection method for test strips based on CRISPR and magnetic bead nucleic acid probes. The method includes: mixing the sample to be tested with a CRISPR-Cas nucleic acid cleavage system and a magnetic bead-conjugated double-stranded nucleic acid probe, wherein the magnetic bead-conjugated double-stranded nucleic acid probe has a single-stranded linker region that can be recognized by the CRISPR-Cas nucleic acid cleavage system and is labeled with biotin and hapten; if the target nucleic acid is present in the sample to be tested, the Cas enzyme lateral cleavage activity is activated to cleave the single-stranded linker region, thereby releasing the complete double-stranded nucleic acid probe from the magnetic bead; the released double-stranded nucleic acid probe is loaded onto a lateral chromatography test strip; if both the test line and the control line show color, the sample is determined to be positive; if only the control line shows color, the sample is determined to be negative.

[0010] The detection principle of this invention is as follows: When the target nucleic acid is present in the sample to be tested, the target nucleic acid, crRNA, and Cas protease form a recognition and cleavage system, activating the paracleavage activity of the Cas protease. The activated Cas protease cleaves the single-stranded linker region located in the probe structure on the magnetic bead surface, releasing the complete double-stranded probe originally immobilized on the surface of the carboxyl magnetic bead from the solid-phase support. The released double-stranded probe enters the lateral chromatography test strip, forming a detection line (T line) under the combined action of the anti-hapten antibody gold label and streptavidin; simultaneously, excess anti-hapten antibody gold label is captured by goat anti-mouse secondary antibody at the control line (C line), thus forming a positive colorimetric interpretation result of "positive samples showing colorimetric T and C lines simultaneously, and negative samples showing colorimetric C line only". The schematic diagram of the principle is shown in the figure below. Figure 1 As shown.

[0011] Preferably, the CRISPR-Cas nucleic acid cleavage system contains Cas protease and crRNA that specifically recognizes the target nucleic acid.

[0012] Preferably, the Cas protease includes any one of Cas12a, Cas12b, Cas12c, Cas13a, or Cas14.

[0013] Preferably, the double-stranded nucleic acid probe includes a main strand and a complementary strand, one end of the main strand is connected to a magnetic bead and the other end is labeled with biotin, and the complementary strand is labeled with a hapten.

[0014] Preferably, the main strand sequence of the double-stranded nucleic acid probe includes the sequences shown in SEQ ID NO.1-SEQ ID NO.3, and the complementary strand sequence includes the sequence shown in SEQ ID NO.4; when an RNA linker is used to adapt the Cas13a system, the main strand probe sequence includes the sequence shown in SEQ ID NO.5.

[0015] Preferably, the crRNA sequence in the CRISPR-Cas nucleic acid cleavage system includes sequences as shown in SEQ ID NO.6-SEQ ID NO.9.

[0016] Preferably, the sequence of the target nucleic acid includes the sequences shown in SEQ ID NO.10-SEQ ID NO.13.

[0017] The specific nucleic acid sequences used in this invention are shown in Table 1.

[0018] Table 1 Preferably, the magnetic beads are carboxyl magnetic beads, and the magnetic beads are coupled to double-stranded nucleic acid probes through covalent connection of an amino group at one end of the main chain to the carboxyl magnetic beads.

[0019] Preferably, the hapten includes any one of digoxin, fluorescein, or dinitrophenol.

[0020] Preferably, the single-strand connection region is located on the main strand, on the complementary strand, or between the hybridization regions of the main strand and the complementary strand.

[0021] Preferably, the detection line of the lateral chromatography test strip is coated with streptavidin, the control line is coated with goat anti-mouse secondary antibody, and the gold label recognition component is an anti-hapten antibody gold label.

[0022] Preferably, the cutting conditions are: temperature 35℃-38℃ (e.g., 35℃, 37℃ or 38℃), time 20-40min (e.g., 20 min, 30 min or 40 min).

[0023] Preferably, the sample to be tested includes a respiratory pathogen nucleic acid sample.

[0024] Preferably, the respiratory pathogen includes any one or a combination of at least two of the following: influenza A virus, SARS-CoV-2, respiratory syncytial virus, adenovirus, or rhinovirus.

[0025] Secondly, the present invention provides a positive colorimetric visualization device for test strips based on CRISPR and magnetic bead nucleic acid probes, the positive colorimetric visualization device for test strips comprising: a sample processing module, a cutting and release module, a chromatography colorimetric module, and a result interpretation module; The sample processing module is used to perform the following: mixing the sample to be tested with a CRISPR-Cas nucleic acid cleavage system and a magnetic bead-coupled double-stranded nucleic acid probe; the magnetic bead-coupled double-stranded nucleic acid probe is provided with a single-stranded linker region that can be recognized by the CRISPR-Cas nucleic acid cleavage system, and is labeled with biotin and hapten. The cleavage and release module is used to perform the following: when the target nucleic acid is present in the sample to be tested, activate the Cas enzyme paracleavage activity to cleave the single-stranded linker region, so that the complete double-stranded nucleic acid probe is released from the magnetic bead; The chromatography colorimetric module is used to perform the following: receiving the released double-stranded nucleic acid probe using a lateral chromatography test strip and performing chromatography colorimetric analysis; The result interpretation module is used to perform the following: determine the result based on the color development of the test line and the control line. If both the test line and the control line develop color, the sample is determined to be positive; if only the control line develops color, the sample is determined to be negative.

[0026] Thirdly, the present invention provides the application of the test strip positive colorimetric visualization detection method described in the first aspect or the test strip positive colorimetric visualization device based on CRISPR and magnetic bead nucleic acid probes described in the second aspect in the preparation of products for detecting respiratory pathogens.

[0027] Preferably, the respiratory pathogen includes any one or a combination of at least two of the following: influenza A virus, SARS-CoV-2, respiratory syncytial virus, adenovirus, or rhinovirus.

[0028] The positive colorimetric visualization detection method, device, and application of the test strips described in this invention are not directly used for disease diagnosis, but can be used for the following non-disease diagnostic purposes: (a) Environmental monitoring: used to detect pathogens or target nucleic acids in environmental samples (including but not limited to water sources, soil, air, and surface swabs in public places) to assess the environmental biosafety status; (b) Food safety testing: used to detect pathogenic microorganisms or adulterants in food samples (including but not limited to fresh food, processed food, and drinking water) to ensure food safety; (c) Customs quarantine: Used to detect quarantine pathogens or biological products in inbound and outbound articles (including but not limited to animals and plants and their products, passenger baggage, and postal items) to prevent the cross-border spread of harmful organisms; (d) Scientific research: Nucleic acid detection used in basic laboratory research, including but not limited to scientific research such as pathogen tracing, gene expression analysis, and CRISPR technology optimization; (e) Pathogen screening: Pathogen screening for non-clinical scenarios, including but not limited to disease screening in livestock farms, health monitoring in public places, and initial screening in large-scale population screening. The screening results need to be combined with other information for comprehensive judgment and do not directly lead to a disease diagnosis conclusion.

[0029] In the aforementioned non-disease diagnosis application scenarios, the test results are only intermediate data or auxiliary information and are not directly used to determine the disease state of a human or animal. Those skilled in the art will understand that when using the method or device described in this invention for clinical diagnostic purposes, it is necessary to combine it with other information such as clinical symptoms and imaging examinations for comprehensive judgment. This invention does not involve direct disease diagnosis steps.

[0030] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention pre-immobilizes double-stranded nucleic acid probes on the surface of carboxyl magnetic beads and sets single-stranded linker regions that can be cleaved by Cas protease in the probe structure. This allows Cas protease to release complete double-stranded labeling units that can participate in the capture and color development of the test strip instead of directly shutting down the detection line signal using the traditional negative color development method when the target nucleic acid is present. This results in a positive color development and visualization detection result on the lateral chromatography test strip, thus realizing a continuous coupled signal conversion path of "solid phase immobilization-cleavage release-chromatographic color development".

[0031] (2) Existing visual detection methods for CRISPR-based test strips mostly adopt a negative colorimetric interpretation mode, that is, the T line of a negative sample is colored, while the T line of a positive sample is not colored or weakened. This is contrary to the interpretation habit of conventional immunochromatographic test strips and is prone to misinterpretation. This invention achieves a positive colorimetric interpretation mode through a magnetic bead fixation and cutting release strategy: only the C line of a negative sample is colored (the T line is not colored), while the T line and C line of a positive sample are colored at the same time. This interpretation mode is more in line with the clinical rapid detection and the intuitive understanding of test strips by ordinary users, and significantly improves the accuracy of result interpretation and user experience.

[0032] (3) In this invention, the positive sample detection line shows a clear red band, while the negative sample detection line does not appear, which can improve the intuitiveness and accuracy of result interpretation and reduce the risk of misreading, omission, and difficulty in judging boundary samples.

[0033] (4) The present invention improves the flexibility of probe structure design by using a modular design of the main chain, complementary chain and single chain connection region to separate the magnetic bead fixation function, the cutting and release function and the chromatography capture function.

[0034] (5) The method described in this invention can be adapted to different crRNAs and target sequences, and has the potential to be extended to the nucleic acid detection of other respiratory pathogens, and is suitable for the development of rapid detection products. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the positive colorimetric detection principle of the test strip based on CRISPR and magnetic bead nucleic acid probes of the present invention.

[0036] Figure 2 The figures show the feasibility verification results of positive colorimetric detection under different probe loadings. Figure A shows the colorimetric results of test strips for positive / negative samples in the 0.1 OD group and the 0.2 OD group, and Figure B shows the statistical results of the corresponding T-line grayscale values.

[0037] Figure 3 The results of optimizing the capture conditions of the lateral chromatography test strip are shown in Figure A, where Figure A shows the color development results of the test strip under different combinations of streptavidin streaking amount and gold-labeled antibody spraying amount, and Figure B shows the statistical results of the corresponding T-line gray value.

[0038] Figure 4 The figures show the results of the detection and sensitivity verification of the influenza A virus template concentration gradient. Figure A shows the color development results of the test strips corresponding to different concentrations of IAV-T102 template, and Figure B shows the statistical results of the gray value of the T line and the linear fitting results.

[0039] Figure 5The images show the results of the specificity verification for respiratory pathogen detection. Image A shows the color development results of the test strips for detecting influenza A virus, SARS-CoV-2, and Mycoplasma pneumoniae templates, while Image B shows the statistical results of the corresponding T-line grayscale values.

[0040] Figure 6 The images show the universality verification results for replacing the SARS-CoV-2 template and corresponding crRNA. Image A shows the color development results of the test strips for positive / negative samples, and Image B shows the statistical results of the corresponding T-line grayscale values.

[0041] Figure 7 The figure shows the feasibility verification results of the Cas13a and RNA linker probe system. Figure A shows the color development results of the test strips for positive / negative samples, and Figure B shows the statistical results of the corresponding T-line gray values.

[0042] Figure 8 This is a graph showing the accuracy verification results of the method of the present invention on known qPCR templates. N1-N4 are templates confirmed as negative by qPCR, and P1-P4 are templates confirmed as positive by qPCR. Detailed Implementation

[0043] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] In this embodiment, the R buffer is 100 mM MES buffer, pH 4.5; the W buffer is 1×PBS, pH 7.4; and the S buffer is 1×PBS plus 0.05% ProClin® 300, pH 7.4.

[0046] The R buffer can be the coupling buffer from the Antibody Coupling Kit A buffer kit, which is from Suzhou NanoMicro Technology Co., Ltd.; the PBS / BSA reagents used for the W buffer are from Shenzhen Kejie Industrial Development Co., Ltd.

[0047] In the lateral chromatography test strip, the T line is coated with streptavidin (SA, 1 μg / μL), the C line is coated with goat anti-mouse IgG secondary antibody (1 μg / μL), and the gold label recognition component is anti-digoxin antibody gold label; the goat anti-mouse IgG secondary antibody catalog number is IGGAb-KJ03R, specification 1 mg / vial; the anti-digoxin monoclonal antibody catalog number is 200-002-156, specification 1 mg.

[0048] The reagents used in the above lateral chromatography test strips were sourced as follows: streptavidin was purchased from Bioss (Beijing, China); goat anti-mouse IgG secondary antibody and PBS / BSA related reagents were purchased from Shenzhen Kejie Industrial Development Co., Ltd.; and anti-digoxin monoclonal antibody (IgG fraction) was purchased from Beijing Xinbosheng Biotechnology Co., Ltd.

[0049] The main strand DNA, complementary strand DNA, crRNA, and target template nucleic acid in the following examples were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

[0050] Example 1 This embodiment provides a positive colorimetric detection method for test strips based on CRISPR and magnetic bead nucleic acid probes, specifically including the following steps: (1) Preparation of main strand DNA in nucleic acid probe Amino-modified and biotin-labeled main-strand DNA (SEQ ID NO.3) was dissolved in R buffer (100 mM MES, pH 4.5) to prepare the nucleic acid solution to be coupled. Different probe loadings were set: 0.12 pmol / mg magnetic beads and 0.25 pmol / mg magnetic beads.

[0051] (2) Pretreatment of magnetic beads Take 20 μL of carboxyl magnetic beads (Antibody Coupling Kit A magnetic bead suspension, 50 mg / mL; Suzhou Nanomicro Technology Co., Ltd., product MagneStar MP3-COOH, catalog number MPHCC-300, particle size 3 μm), wash 3 times with R buffer, discard the supernatant after magnetic separation, and retain the magnetic bead precipitate.

[0052] The Antibody Coupling Kit A magnetic bead suspension and its accompanying buffer solution are from Suzhou NanoMicro Technology Co., Ltd.; the magnetic bead product is MagneStar MP3-COOHcarboxylated magnetic beads, catalog number MPHCC-300, with a particle size of 3 μm.

[0053] (3) Main chain coupling in nucleic acid probes To control the probe density on the magnetic bead surface, the dosage of amino-modified main-strand DNA was set to 0.12 and 0.25 pmol / mg of magnetic beads, respectively. Specifically, 12 μL of 10 nM main-strand DNA working solution or 5 μL of 50 nM main-strand DNA working solution was mixed with 1 mg of pretreated carboxyl magnetic beads, and the volume was brought up to 30 μL with R buffer. Pre-coupling was performed at 25°C and 500 rpm for 30 min. Then, 15 μL of freshly prepared EDC solution (100 mg / mL, prepared with ice-cold R buffer) was added, and R buffer was added to bring the total volume to 100 μL. Coupling was performed at 25°C and 500 rpm for 4 h, allowing the amino-modified main-strand DNA to be covalently coupled to the carboxyl magnetic bead surface via EDC-mediated amide bonds. The EDC was purchased from Sigma-Aldrich; the R buffer was from the Antibody Coupling Kit A buffer kit.

[0054] (4) Sealing treatment After coupling, the magnetic beads were washed twice with 2× sodium citrate buffer (SSC) to remove carbodiimide (EDC) and residual coupling solution. Then, salmon sperm DNA blocking solution prepared with 2×SSC was added for blocking. The final concentration of salmon sperm DNA in the blocking solution was 0.1 mg / mL. The blocking conditions were 25℃, 500 rpm, and 20 min to reduce non-specific adsorption.

[0055] (5) Complementary strand hybridization The blocked nucleic acid probe backbone magnetic beads were resuspended in 2×SSC and hybridized with digoxigenin-labeled complementary DNA (SEQ ID NO.4) to form double-stranded probe magnetic beads. The complementary DNA was added at 1.2 times the molar amount of the backbone DNA. The hybridization conditions were 25℃, 400 rpm, and 45 min.

[0056] (6) Washing after hybridization The hybridized magnetic beads were washed three times with 0.2×SSC to remove unhybridized complementary strands and residual free nucleic acids.

[0057] (7) CRISPR-Cas12a cleavage and release Add the Cas12a cleavage system to the reaction tube containing the hybridized magnetic beads. The total volume of the cleavage system in a single tube is 25 μL, including: 1.25 μL of 10×NEBuffer 2.1, 1.25 μL of EnGen® Lba Cas12a (Cpf1, 1 μM), 1.25 μL of crRNA (SEQ ID NO.7, 1 μM), 1 μL of the nucleic acid of the sample to be tested (IAV-T102, SEQ ID NO.10, 10 μM), and 20.25 μL of enzyme-free water. After adding the above system to the magnetic beads, react at 37℃ and 500 rpm for 30 min to allow Cas12a to cleave the single-stranded linker region and release the complete double-stranded probe; the sample to be tested is a synthetic double-stranded DNA fragment containing the target nucleic acid sequence of influenza A virus.

[0058] The EnGen® Lba Cas12a (Cpf1) was purchased from New England Biolabs (USA); the crRNA and template nucleic acid were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; and the enzyme-free water was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0059] (8) Sample loading and color development After cutting, add 55 μL of W buffer (1×PBS, pH 7.4) to bring the total volume of the system to 80 μL. After magnetic separation to remove the magnetic beads, take the supernatant (containing the released double-stranded nucleic acid probe) and use it for sample loading onto the test strip. The colorimetric analysis is completed within about 15 minutes. Simultaneous color development of the T line and C line indicates a positive sample, while color development of only the C line indicates a negative sample.

[0060] The results are as follows Figure 2 As shown in Figures A and B, positive samples in both the 0.12 pmol / mg and 0.25 pmol / mg magnetic bead groups exhibited simultaneous T-line and C-line color development, while negative samples only showed the C-line. Statistical results of the corresponding T-line grayscale values ​​show that the grayscale value of positive samples is significantly higher than that of negative samples, indicating that the magnetic bead nucleic acid probe system constructed in this invention can achieve positive colorimetric interpretation. The 0.25 pmol / mg magnetic bead group showed a stronger positive signal and can be used as the preferred probe loading condition for subsequent experiments.

[0061] Example 2 The only difference between this embodiment and Example 1 is that the nuclease in the CRISPR-Cas nucleic acid cleavage system is Cas13a, the crRNA used is the sequence shown in SEQ ID NO.8, and the short single-stranded linker of the main chain probe is replaced with an RNA linker (SEQ ID NO.5). The remaining steps and conditions are the same as in Example 1. The results are as follows: Figure 7 As shown in Figures A and B, Cas13a can also achieve the positive colorimetric visualization detection of the present invention.

[0062] Example 3 The difference between this embodiment and Embodiment 1 is that the test sample is replaced with a SARS-CoV-2 template (SEQ ID NO. 11), and the crRNA in the system is replaced with the sequence shown in SEQ ID NO. 9, which corresponds to the SARS-CoV-2 target sequence. All other steps and conditions are the same as in Embodiment 1.

[0063] The results are as follows Figure 6 As shown in Figures A and B, positive colorimetric detection can still be achieved after replacing the SARS-CoV-2 template and corresponding crRNA, indicating that the method of the present invention has the feasibility of adapting and extending to different target sequences.

[0064] Example 4 This embodiment optimizes the capture conditions of the lateral chromatography test strip.

[0065] Take the double-stranded nucleic acid probe magnetic beads prepared and hybridized according to Example 1, and release the double-stranded probes according to the cutting conditions described in Example 1; use the released double-stranded probes as chromatographic samples to compare the effects of different T-line streptavidin streaking amounts and gold-labeled anti-digoxigenin antibody spraying amounts on the color development intensity.

[0066] Lateral chromatography test strips were prepared by setting the streptavidin streaking volume to 1.0 μL / cm, 1.5 μL / cm, and 2.0 μL / cm, and the gold-labeled anti-digoxigenin antibody spray volume to 10 μL / cm, 15 μL / cm, and 20 μL / cm. The same volume of release probe sample was added to each test strip. Visual color development results were recorded after 5 minutes, and the T-line gray value was read and statistically analyzed after 15 minutes.

[0067] The results are as follows Figure 3 As shown in Figures A and B, when the amount of streptavidin (SA) applied is 1.5 μL / cm and the amount of gold-labeled anti-digoxigenin antibody sprayed is 20 μL / cm, the gray value of line T is the highest and line C remains clear. This indicates that the combination can improve the positive detection signal while ensuring the effectiveness of the control line. Therefore, it is the preferred condition for the preparation of subsequent lateral chromatography test strips.

[0068] Example 5 Accuracy verification.

[0069] To verify the accuracy of the detection method of the present invention, nucleic acid templates whose results had been pre-confirmed by real-time quantitative PCR (qPCR) were selected as verification objects, including 4 qPCR positive templates and 4 qPCR negative templates. These templates were numbered and used as blind samples, and were tested according to the method described in Example 1. Positive or negative results were determined based on the color development of the test strips.

[0070] The results are as follows Figure 8 As shown, all four qPCR positive templates were determined to be positive using the method of this invention, with a positive sample detection rate of 100% (4 / 4); all four qPCR negative templates were determined to be negative using the method of this invention, with a negative sample concordance rate of 100% (4 / 4). The overall concordance rate between the method of this invention and known qPCR results is 100% (8 / 8).

[0071] The results show that, in the accuracy verification using known qPCR templates as the detection targets, the method of this invention can correctly distinguish between positive and negative samples, demonstrating good accuracy. The detection limit is 5 pM, the linear range is 5-1600 pM, and the number of replicates is 3.

[0072] Example 6 Sensitivity verification.

[0073] The IAV-T102 template was serially diluted from high to low concentrations, and a template-free blank control was set up. The same volume of template working solution was added to each reaction tube, and Cas12a cleavage and release and lateral chromatography were performed as in Example 1. After color development, the test strip photographs were recorded, and the T-line gray value was read. Sensitivity and linearity were analyzed by plotting the template concentration or its logarithm on the x-axis and the T-line gray value on the y-axis.

[0074] The results are as follows Figure 4 As shown in Figures A and B, as the concentration of IAV-T102 template decreases, the T-line of the test strip gradually weakens, and the corresponding gray value of the T-line decreases in a concentration-dependent manner. Within the tested range, the gray value of the T-line has a good linear relationship with the logarithm of the target nucleic acid concentration, indicating that the method of the present invention can be used for concentration gradient detection of target nucleic acids.

[0075] Example 7 Specificity verification.

[0076] Referring to Example 1, the influenza A virus target template IAV-T102 and non-target templates SC2-T72 and MP-T72 were added respectively, and each template was detected at the same final concentration. The remaining cleavage, release, and lateral chromatography colorimetric steps were the same as in Example 1. After colorimetric development, the test strip photograph was recorded, and the gray value of the T line was read to evaluate the ability of the system of the present invention to distinguish between target and non-target sequences.

[0077] The results are as follows Figure 5 As shown in Figures A and B, under the same detection conditions, the influenza A virus target template produces a clear T-line signal, while non-target templates such as SARS-CoV-2 and Mycoplasma pneumoniae do not produce a clear T-line signal or only show background signals, indicating that the system of the present invention has good recognition specificity for target sequences.

[0078] In summary, this invention constructs a magnetic bead-coupled nucleic acid probe system, structurally coupling target nucleic acid recognition, CRISPR cleavage and release, and test strip colorimetric interpretation. This results in no detection line appearing for negative samples and a clear red band appearing for positive samples, thus forming a positive colorimetric visualization detection mode that is more in line with normal interpretation habits and improving the accuracy of test strip result interpretation.

[0079] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for positive colorimetric visualization detection of test strips based on CRISPR and magnetic bead nucleic acid probes, characterized in that, The positive colorimetric visualization detection method for the test strip includes: mixing the sample to be tested with a CRISPR-Cas nucleic acid cleavage system and a magnetic bead-conjugated double-stranded nucleic acid probe. The magnetic bead-conjugated double-stranded nucleic acid probe has a single-stranded linker region that can be recognized by the CRISPR-Cas nucleic acid cleavage system and is labeled with biotin and hapten. If the target nucleic acid is present in the sample to be tested, the Cas enzyme lateral cleavage activity is activated to cleave the single-stranded linker region, releasing the complete double-stranded nucleic acid probe from the magnetic bead. The released double-stranded nucleic acid probe is loaded onto a lateral chromatography test strip. If both the test line and the control line show color, the sample is considered positive; if only the control line shows color, the sample is considered negative.

2. The positive colorimetric visualization detection method for test strips according to claim 1, characterized in that, The CRISPR-Cas nucleic acid cleavage system contains Cas protease and crRNA that specifically recognizes the target nucleic acid; Preferably, the Cas protease includes any one of Cas12a, Cas12b, Cas12c, Cas13a, or Cas14.

3. The positive colorimetric visualization detection method for test strips according to claim 1 or 2, characterized in that, The double-stranded nucleic acid probe includes a main strand and a complementary strand. One end of the main strand is connected to a magnetic bead and the other end is labeled with biotin. The complementary strand is labeled with a hapten. Preferably, the magnetic beads are carboxyl magnetic beads, and the double-stranded nucleic acid probe is covalently linked to the carboxyl magnetic beads through an amino group at one end of the main chain.

4. The positive colorimetric visualization detection method for test strips according to claim 3, characterized in that, The hapten includes any one of digoxigenin, fluorescein, or dinitrophenol; Preferably, the single-strand connection region is located on the main strand, on the complementary strand, or between the hybridization regions of the main strand and the complementary strand.

5. The method for positive colorimetric visualization detection of test strips according to any one of claims 1-4, characterized in that, The detection line of the lateral chromatography test strip is coated with streptavidin, the control line is coated with goat anti-mouse secondary antibody, and the gold label recognition component is an anti-hapten antibody gold label.

6. The method for positive colorimetric visualization detection of test strips according to any one of claims 1-5, characterized in that, The cutting conditions are: temperature 35℃-38℃, time 20-40 min.

7. The method for positive colorimetric visualization detection of test strips according to any one of claims 1-6, characterized in that, The samples to be tested include nucleic acid samples of respiratory pathogens; Preferably, the respiratory pathogen includes any one or a combination of at least two of the following: influenza A virus, SARS-CoV-2, respiratory syncytial virus, adenovirus, or rhinovirus.

8. A positive colorimetric visualization device for test strips based on CRISPR and magnetic bead nucleic acid probes, characterized in that, The positive colorimetric visualization device for the test strip includes: a sample processing module, a cutting and release module, a chromatography colorimetric module, and a result interpretation module; The sample processing module is used to perform the following: mixing the sample to be tested with a CRISPR-Cas nucleic acid cleavage system and a magnetic bead-coupled double-stranded nucleic acid probe; the magnetic bead-coupled double-stranded nucleic acid probe is provided with a single-stranded linker region that can be recognized by the CRISPR-Cas nucleic acid cleavage system, and is labeled with biotin and hapten. The cleavage and release module is used to perform the following: when the target nucleic acid is present in the sample to be tested, activate the Cas enzyme paracleavage activity to cleave the single-stranded linker region, so that the complete double-stranded nucleic acid probe is released from the magnetic bead; The chromatography colorimetric module is used to perform the following: receiving the released double-stranded nucleic acid probe using a lateral chromatography test strip and performing chromatography colorimetric analysis; The result interpretation module is used to perform the following: determine the result based on the color development of the test line and the control line. If both the test line and the control line develop color, the sample is determined to be positive; if only the control line develops color, the sample is determined to be negative.

9. The positive colorimetric visualization detection method for test strips according to any one of claims 1-7 or the positive colorimetric visualization device for test strips based on CRISPR and magnetic bead nucleic acid probes according to claim 8, in the preparation of products for detecting respiratory pathogens.

10. The application according to claim 9, characterized in that, The respiratory pathogens include any one or a combination of at least two of the following: influenza A virus, SARS-CoV-2, respiratory syncytial virus, adenovirus, or rhinovirus.

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