Naked eye visual detection method based on CRISPR / Cas reaction system nucleic acid detection and kit thereof

Abstract

The invention provides a naked eye visual detection method based on CRISPR / Cas reaction system nucleic acid detection and a kit thereof, in the method, a solution of a CRISPR / Cas reaction system is prepared, the solution contains a Cas enzyme with trans-cleavage ability, crRNA and an SSA capture probe, the SSA capture probe is constructed as a recognition element by combining a CRISPR / Cas signal amplification system, and the SSA capture probe is used for detecting nucleic acid in the CRISPR / Cas reaction system. An artificially programmed CG probe with a color developing function, which specifically responds to an SSA probe, is used as a signal transduction element, the SSA probe is used for effectively capturing the CG probe, target nucleic acid is used for activating the activity of CRISPR / Cas trans-cleavage, and the SSA and the CG probe are cleaved, so that colloidal gold is in a free state in a solution, and after magnetic separation is observed by naked eyes, the CG probe and the SSA probe are separated by magnetic force. When the detected sample is positive, the supernatant color is pink, the supernatant of the negative sample and the supernatant of the blank sample are in a colorless state, and the target nucleic acid is detected by distinguishing the colors.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a naked eye visualization detection method and a kit thereof based on nucleic acid detection of a CRISPR / Cas reaction system. Background Art

[0002] The CRISPR / Cas system (Clustered regularly interspaced short palindromicrepeats) was discovered by researchers in bacteria. Since its discovery, it has made great achievements in the field of life sciences. In 2015, ZETSCHE and other scholars discovered that the CRISPR / Cas12a (Cpf1) system has a nuclease that can recognize double-stranded DNA. The CRISPR / Cas12 system can target DNA and activate its ability to target and non-target DNA cutting. Due to its unique trans-cutting activity, CRISPR provides a new direction in the field of nucleic acid detection.

[0003] At present, most of the nucleic acid detection systems established based on the CRISPR method are based on PCR, loop-mediated isothermal amplification, and recombinase polymerase isothermal amplification, and the concentration of target nucleic acids is amplified in vitro to improve the sensitivity of detection; however, this process requires opening the lid, and the operation steps are complicated. At the same time, the operation of opening the lid will increase the risk of contamination. In addition, the interpretation of the results of the above-mentioned CRISPR detection methods requires the use of large instruments and equipment. Traditional fluorescence-based CRISPR detection requires fluorescence quantitative instruments; CRISPR detection based on electrochemical sensors requires the characterization of results through electrical signals, with the help of precision instruments such as potentiometers; CRISPR detection based on Raman scattering spectral sensors requires expensive optical system precision instruments, etc.; two-step amplification and the way to interpret the results are also new and huge challenges for current applications. With the increasing maturity of traditional molecular diagnostic technology, the sensitivity, throughput, detection speed, stability, and reliability of its diagnostic methods are constantly improving, but because its diagnosis requires large instruments and professional operators, it is impossible to make new breakthroughs in scenarios outside the laboratory. In recent years, home self-testing has entered the public eye. You can complete the test at home by following the simple instructions. Currently, home self-testing mainly relies on antibody testing, using chromatography test strips and colloidal gold to determine the results. However, the interpretation is subjective and can easily lead to false results.

[0004] In order to solve the above-mentioned problems and further solve the problems of poor sensitivity and specificity of on-site home testing and dependence on interpretation instruments, it is urgently necessary to invent a detection method that can improve the sensitivity and specificity of on-site testing and accurately interpret the results without the use of instruments. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a naked eye visualization detection method based on CRISPR / Cas reaction system nucleic acid detection, the method comprising the following steps:

[0006] Step 1: Prepare a solution of a CRISPR / Cas reaction system, wherein the solution contains a Cas enzyme with trans-cutting ability, crRNA and an SSA capture probe, wherein the crRNA is composed of a structural sequence and a guide sequence, wherein the crRNA structural sequence forms a complex with the Cas enzyme, and the guide sequence is complementary to a sequence in the target nucleic acid sequence to be tested; the complex scans the entire nucleic acid sample to be tested, and identifies whether there is a complementary sequence to the guide sequence in the nucleic acid sample to be tested; if the complementary sequence exists, the guide sequence forms a double-stranded structure with the complementary sequence, and guides the Cas enzyme to cut the double-stranded structure, while activating the trans-cutting activity of the Cas enzyme to non-specifically cut the single-stranded structure of the nucleic acid in the reaction system;

[0007] The SSA capture probe is composed of a nucleic acid single-strand P and magnetic beads, the magnetic beads are modified with a first conjugate, the single-strand P is composed of two parts, A and B, wherein the A part is a single-strand containing A / T bases, which is a single-strand that can be recognized and cut by the Cas enzyme, and the 5' end of the A part is modified with a second conjugate, and the B part is a single-strand that is less likely to be recognized and cut by the Cas enzyme than the A part; the second conjugate modified at the 5' end of the A part of the single-strand P is paired / coupled with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe;

[0008] Step 2: Add the nucleic acid sample to be tested into a reaction tube containing the CRISPR / Cas reaction system. If the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, the guide sequence of the crRNA forms a double-stranded structure with it, and guides the Cas enzyme to effectively cut the double strand, while activating the trans-cutting activity of the Cas enzyme to non-specifically cut the single strand P of the SSA capture probe in the reaction system, and cut the SSA capture probe into "magnetic bead-A" and "free single strand B". If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the SSA capture probe will not be cut;

[0009] Step 3: After the reaction in step 2 is completed, a CG signal transduction probe is added to the reaction system, wherein the CG signal transduction probe is composed of an H chain and colloidal gold, and the colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain is composed of two parts, C and D, wherein the C part is a single chain containing A / T bases, which is a single chain recognized and cut by the Cas enzyme, and the D part is a single chain that is less likely to be recognized and cut by the Cas enzyme than the C part, and is complementary to the B part, and the end of the C part away from the D part is labeled with colloidal gold; if the nucleic acid sample to be tested contains The target nucleic acid sequence to be detected, the trans-nuclease activity of the Cas enzyme protein is activated, and the single-stranded part of the CG signal transduction probe is also cut, and the CG signal transduction probe is cut into "colloidal gold-C" and "free single-stranded D"; wherein, "free single-stranded D" and the "free single-stranded B" generated by the reaction in step 2 form a "double-stranded BD"; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of three free components: "magnetic beads-A", "double-stranded BD", and "colloidal gold-C";

[0010] If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the trans-nuclease activity of the Cas enzyme will not be activated, and the CG signal transduction probe will not be cut; wherein, the B chain of the complete SSA capture probe and the D chain of the complete CG signal transduction probe will still form a BD complementary double strand, and finally form a "magnetic bead-PH-colloidal gold" complex in which the two probes are combined together, wherein PH is combined together by BD complementarity; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of a "magnetic bead-PH-colloidal gold" complex;

[0011] Step 4: After the reaction in step 3 is completed, the reaction tube is placed on a magnetic stand for magnetic separation, and then the visual result is interpreted by naked eyes; if the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-A" will be adsorbed to the tube wall, while the two components "double-stranded BD" and "colloidal gold-C" will still be free in the solution, so the solution presents the color of colloidal gold; if the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-PH-colloidal gold" complex will be adsorbed on the tube wall, and there is no free colloidal gold in the solution, so the solution is colorless.

[0012] In one embodiment, the single-stranded A portion is 6-12 nt in length, and the A / T content is not less than 70%.

[0013] In one embodiment, the A / T content in the single-stranded A portion is not less than 80%.

[0014] In one embodiment, the single-stranded C portion is longer than 10 nt and has an A / T content of not less than 70%.

[0015] In one embodiment, the A / T content in the single-stranded C portion is not less than 80%.

[0016] In one embodiment, the CRISPR / Cas reaction system is a CRISPR / Cas12a reaction system, and the nucleic acid sample to be tested is a DNA sample.

[0017] In one embodiment, the CRISPR / Cas reaction system is a CRISPR / Cas13a reaction system, and the nucleic acid sample to be tested is an RNA sample.

[0018] In one embodiment, the first conjugate and the second conjugate are a molecular pairing coupling system, which is any one of a streptavidin-biotin pairing coupling system, a biotin-avidin pairing coupling system, a receptor-ligand pair coupling system, an engineered covalent binding pair coupling system, a nucleic acid aptamer and target molecule coupling system, an antigen-antibody coupling system, or a metal chelate coupling system.

[0019] In one embodiment, a naked eye visualization detection kit for nucleic acid detection based on a CRISPR / Cas reaction system is provided, the kit comprising: a Cas enzyme with trans-cutting ability, crRNA, an SSA capture probe and a CG signal transduction probe, the crRNA is composed of a structural sequence and a guide sequence, the crRNA structural sequence forms a complex with the Cas enzyme, and the guide sequence is complementary to a sequence in a target nucleic acid sequence to be detected; the complex scans the entire nucleic acid sample to be detected, and identifies whether there is a complementary sequence to the guide sequence in the nucleic acid sample to be detected, if the complementary sequence exists, the guide sequence forms a double-stranded structure with it, and guides the Cas enzyme to cut the double strand, while activating the trans-cutting activity of the Cas enzyme, which is used to non-specifically cut the single-stranded structure of the nucleic acid in the reaction system;

[0020] The SSA capture probe is composed of a nucleic acid single-strand P and magnetic beads, the magnetic beads are modified with a first conjugate, the single-strand P is composed of two parts, A and B, wherein the A part is a single-strand containing A / T bases, which is a single-strand that can be recognized and cut by the Cas enzyme, and the 5' end of the A part is modified with a second conjugate, and the B part is a single-strand that is less likely to be recognized and cut by the Cas enzyme than the A part; the second conjugate modified at the 5' end of the A part of the single-strand P is paired / coupled with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe;

[0021] The CG signal transduction probe is composed of an H chain and colloidal gold, and the colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain is composed of two parts, C and D, wherein the C part is a single chain containing A / T bases, which is a single chain recognized and cut by the Cas enzyme, and the D part is a single chain that is less likely to be recognized and cut by the Cas enzyme than the C part, and is complementary to the B part, and the end of the C part away from the D part is labeled with colloidal gold; if the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, the trans-nuclease activity of the Cas enzyme protein is activated, and the single-chain part in the CG signal transduction probe will also be cut, and the CG signal transduction probe will be cut into "colloidal gold-C" and "free single-chain D".

[0022] In one embodiment, the single-stranded A portion is 6-12 nt in length, and the A / T content is not less than 70%.

[0023] In one embodiment, the single-stranded C portion is longer than 10 nt and has an A / T content of not less than 70%.

[0024] In the method of the present invention, a Cas protein, crRNA, a programmable SSA capture probe, and an artificially programmed CG probe with a color development function that specifically responds to the SSA capture probe are used as signal transduction elements; in the CRISPR / Cas reaction system, the SSA capture probe can effectively identify and capture the CG probe, and a DNA hybridization reaction occurs. When the target nucleic acid is present, the Cas protein activity is activated, and the artificially programmed single-stranded portion in the SSA and CG probes can be effectively cut, so that the colloidal gold is released in the solution, and after magnetic separation, the supernatant presents a colloidal gold (pink) color visible to the naked eye; when no target nucleic acid is present, the Cas protein activity is not activated, and the SSA probe can effectively capture the CG probe during magnetic separation, the colloidal gold is not released, and the supernatant of the solution is colorless; in the method of the present invention, the SSA capture probe and the CG probe do not need to be adjusted or changed according to different detection targets, and are a pair of universal capture color development systems.

[0025] The CG probe is effectively captured by the SSA capture probe, and the target nucleic acid activates the activity of CRISPR / Cas trans-cutting, cuts the SSA and CG probes, and realizes the free state of colloidal gold in the solution. After magnetic separation, it can be observed with the naked eye that when the test sample is positive, the supernatant is pink, and the supernatant of negative and blank samples is colorless. The detection of the target nucleic acid is achieved by distinguishing the colors. The present invention adopts the above-mentioned simple detection method for visual detection of target genes, without the need for instrumentation to determine the results. The present invention can accurately, quickly and portablely detect fully matched target substances.

[0026] The detection method of the present invention uses single-stranded labeled colloidal gold as a signal transduction element for interpretation, and observes the color of the supernatant through magnetic separation, with clear color distinction, and can get rid of the dependence on instruments in the result interpretation stage; at the same time, the use of nucleic acid detection can solve the current problems of poor antigen sensitivity and stability; further, different detection targets can use a set of interpretation systems and complete the interpretation of the results within 30 minutes, saving costs and energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a schematic diagram of the naked eye visualization detection method for nucleic acid detection based on the CRISPR / Cas reaction system of the present invention;

[0029] Figure 2 It is the fluorescence value trend diagram of the recognition and cleavage ability of Cas enzyme to SSA probes of different lengths and base types;

[0030] Figure 3 The results of chromatography after different base lengths in the H chain were connected to colloidal gold;

[0031] Figure 4 The results of chromatography after different base types in the H chain were connected with colloidal gold;

[0032] Figure 5 It is the fluorescence result after the single strands of different base types in the H chain are cut by the Cas enzyme;

[0033] Figure 6 This is the result of visualizing CRISPR / Cas12 when the initial sample is a DNA sample. Figure 6A is the result of a DNA-positive sample; Figure 6B is the result of a DNA-negative nucleic acid sample, where "1" is 10. 5 Copies / μL, "2" is 10 4 Copies / μL, "3" is 10 3 Copies / μL, "4" is 10 2 copies / μL, “5” is 10 copies / μL, “6” is negative control, “7” is negative control, and “8” is blank. DETAILED DESCRIPTION

[0034] In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the embodiments. Obviously, the embodiments are only examples and cannot limit the present invention. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work should belong to the scope of protection of this application. The experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0035] Example 1 Basic principle of the method of the present invention

[0036] like Figure 1 As shown, the present invention is to establish a method for naked eye visualization detection based on nucleic acid combined with CRISPR / Cas. This method innovates the signal reading system based on CRISPR / Cas, and visually reads positive and negative through the naked eye, without the need for direct judgment by instruments. The specific principle of the method of the present invention is shown in Figure 1 , the method shown comprises the following steps:

[0037] Step 1: Prepare a solution of a CRISPR / Cas reaction system, the solution comprising a Cas enzyme with trans-cutting ability, crRNA and an SSA capture probe, the crRNA is composed of a structural sequence and a guide sequence, the crRNA structural sequence forms a complex with the Cas enzyme, and the guide sequence is complementary to a sequence in the target nucleic acid sequence to be tested; the complex scans the entire nucleic acid sample to be tested, and identifies whether there is a complementary sequence of the guide sequence in the nucleic acid sample to be tested, if there is the complementary sequence, the guide sequence forms a double-stranded structure with it, and guides the Cas enzyme to cut the double strand, while activating the trans-cutting activity of the Cas enzyme, which is used to non-specifically cut the single-stranded structure of the nucleic acid in the reaction system;

[0038] The SSA capture probe is composed of a nucleic acid single-strand P and magnetic beads, the magnetic beads are modified with a first conjugate, the single-strand P is composed of two parts, A and B, wherein the A part is a single-strand containing A / T bases, which is a single-strand that can be recognized and cut by the Cas enzyme, and the 5' end of the A part is modified with a second conjugate, and the B part is a single-strand that is less likely to be recognized and cut by the Cas enzyme than the A part; the second conjugate modified at the 5' end of the A part of the single-strand P is paired / coupled with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe;

[0039] Step 2: Add the nucleic acid sample to be tested into a reaction tube containing the CRISPR / Cas reaction system. If the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, the guide sequence of the crRNA forms a double-stranded structure with it, and guides the Cas enzyme to effectively cut the double strand, while activating the trans-cutting activity of the Cas enzyme to non-specifically cut the single strand P of the SSA capture probe in the reaction system, and cut the SSA capture probe into "magnetic bead-A" and "free single strand B". If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the SSA capture probe will not be cut;

[0040] Step 3: After the reaction in step 2 is completed, a CG signal transduction probe is added to the reaction system, wherein the CG signal transduction probe is composed of an H chain and colloidal gold, and the colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain is composed of two parts, C and D, wherein the C part is a single chain containing A / T bases, which is a single chain recognized and cut by the Cas enzyme, and the D part is a single chain that is less likely to be recognized and cut by the Cas enzyme than the C part, and is complementary to the B part, and the end of the C part away from the D part is labeled with colloidal gold; if the nucleic acid sample to be tested contains The target nucleic acid sequence to be detected, the trans-nuclease activity of the Cas enzyme protein is activated, and the single-stranded part of the CG signal transduction probe is also cut, and the CG signal transduction probe is cut into "colloidal gold-C" and "free single-stranded D"; wherein, "free single-stranded D" and the "free single-stranded B" generated by the reaction in step 2 form a "double-stranded BD"; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of three free components: "magnetic beads-A", "double-stranded BD", and "colloidal gold-C";

[0041] If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the trans-nuclease activity of the Cas enzyme will not be activated, and the CG signal transduction probe will not be cut; wherein, the B chain of the complete SSA capture probe and the D chain of the complete CG signal transduction probe will still form a BD complementary double strand, and finally form a "magnetic bead-PH-colloidal gold" complex in which the two probes are combined together, wherein PH is combined together by BD complementarity; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of a "magnetic bead-PH-colloidal gold" complex;

[0042] Step 4: After the reaction in step 3 is completed, the reaction tube is placed on a magnetic stand for magnetic separation, and then the visual result is interpreted by naked eyes; if the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-A" will be adsorbed to the tube wall, while the two components "double-stranded BD" and "colloidal gold-C" will still be free in the solution, so the solution presents the color of colloidal gold; if the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-PH-colloidal gold" complex will be adsorbed on the tube wall, and there is no free colloidal gold in the solution, so the solution is colorless.

[0043] In the present invention, in the CRISPR / Cas reaction system, the Cas enzyme can be Cas12, Cas13, etc.

[0044] In the present invention, the first conjugate and the second conjugate are high-affinity, high-specificity molecular pairing coupling systems, for example, they can be streptavidin (Streptavidin) - biotin (Biotin) pairing coupling system, biotin - avidin (Biotin-Avidin) pairing coupling system, receptor - ligand pair coupling system, engineered covalent binding pair coupling system such as SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, nucleic acid aptamer and target molecule coupling system, antigen - antibody coupling system, metal chelate coupling system; commonly used are streptavidin (Streptavidin) - biotin (Biotin) pairing coupling system, biotin - avidin (Biotin-Avidin) pairing coupling system.

[0045] In the method of the present invention, the experimental results can be interpreted through the above-mentioned simple color difference visible to the naked eye.

[0046] Example 2 Optimization of SSA capture probe in the visualization detection system of the present invention

[0047] The SSA capture probe is composed of a single-stranded nucleic acid P and magnetic beads. The magnetic beads are modified with a first conjugate. The single-stranded P consists of two parts, A and B, wherein the A part is a single-stranded containing A / T bases, and B is an editable single-stranded; the second conjugate modified at the 5' end of the A part of the single-stranded P is paired / combined with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe. In order to verify the effectiveness of the preparation of the SSA capture probe, the A part of the single-stranded P is verified with different lengths and base types, wherein the first conjugate is selected as streptavidin, and more specifically, the A part is modified with biotin at the 5' end and the FAM fluorescent group at the 3' end of the A part, and under the action of the BW buffer, the biotin at the 5' end is coupled with the streptavidin magnetic beads to obtain the SSA probe. The preparation and effectiveness test of the probe include the following specific steps:

[0048] 1. Take 100 μL of magnetic beads and wash them twice with BW Buffer (10 mM Tris-HCl, 1 mM EDTA, 2.0 M NaCl, pH 7.5), then disperse the magnetic beads in 100 μL of BW buffer at a concentration of 10 mg / mL;

[0049] 2. Take 100 μL (10 μM) P and mix it with the magnetic bead dispersion, and incubate it at room temperature for 1-2 hours with rotation to ensure that the first conjugate streptavidin is fully combined with the second conjugate biotin end;

[0050] 3. Separate the magnetic beads by magnetic attraction and wash them 2-3 times with BW Buffer. Transfer the washed magnetic beads to the buffer system and store them at 4°C as SSA capture probes for future use.

[0051] 4. 20μL CRISPR / Cas reaction system: 10×Cas 12a Reaction Buffer, 2μL crRNA (1μM), 1μL 1×Cas 12a Protein, 5μL SSA capture reporter probe, 2μL positive quality control, and the rest is made up to 20μL with water) and mixed into the reaction tube. Specifically, the CRISPR program is: react at 37°C for 10 minutes; 5. After the reaction, magnetic separation is performed, and the solution is taken to observe the fluorescence value results on the fluorescent PCR instrument to determine the cutting efficiency of the Cas enzyme and select the single-stranded P that meets the requirements;

[0052] The specific sequence information and results are shown in Table 1. Figure 2 As shown, when the length and base of the A chain in P change, the recognition ability of the Cas protein is also different. Under the same input amount and amplification time, the results are analyzed by analyzing the fluorescence value in the solution, as follows.

[0053] (1) Through the fluorescence value data analysis of P1-P7 in Table 1, when the sample addition amount is the same, when the length of the A part in the P chain is 4 bases (P1), the fluorescence value in the supernatant is 212.84, and its cutting efficiency is low; Figure 2 As shown in A, when the length of the A part in the P chain is 6-10 (PA2-PA4) bases, its fluorescence value is above 400 and the fluorescence value change trend is that as the number of bases increases, the fluorescence value shows an upward trend. When the number of bases increases to 12 (P5), the fluorescence value is 352.99, and the fluorescence value shows a downward trend. When the number of bases increases to 16 (P7), the fluorescence value is 226.24. At this time, the Cas enzyme has a weaker ability to recognize and cut the A part. Therefore, it can be concluded that when the length of the A part in the P chain is 6-12 bases, the Cas enzyme can more efficiently recognize the A part and exert a better cutting efficiency.

[0054] (2) Furthermore, the base types were changed when the optimal length of PA was 10 bases. By comparing the fluorescence value data (P8-P12) in Table 1 and Figure 2 Analysis of the fluorescence value change curve in B (right) shows that as the number of C / G bases in the PA part increases, its fluorescence value shows a downward trend. When the length of the PA part is 10 bases, of which the number of C / G bases is 0 or 1, its fluorescence value is above 400. When the number of G / C bases is increased to 2, there is a more obvious change in the fluorescence value. When the number of C / G bases increases to 4, the fluorescence value is 249.10, which affects the cutting efficiency of the Cas enzyme. Therefore, it can be concluded that when the A / T content in the single chain is not less than 70%, the Cas enzyme can better recognize the single chain and exert its cutting function. More preferably, when the A / T content is not less than 80%, the Cas enzyme can exert better cutting efficiency on the single chain.

[0055] In summary, when the length of the A part of the P chain in the SSA capture probe is 6-12 nt and the A / T content is not less than 70%, preferably when the A / T content is not less than 80%, it is easier to be recognized by the Cas enzyme and has a higher cutting efficiency.

[0056] Table 1

[0057]

[0058] Implementation 3 Design optimization of CG signal transduction probes in the visualization detection system of the present invention

[0059] Design and preparation of CG signal transduction probe, which consists of an H chain and colloidal gold, wherein the colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain consists of two parts, C and D, wherein the C part is a single chain containing A / T bases, and D is complementary to the B part of the P chain in the above-mentioned SSA probe, and the end of the C part away from the D part is labeled with colloidal gold. Design optimization of CG signal transduction probe: The C part of the H chain is modified with SH at the 5' end and biotin at the 3' end, and the synthesized HC is connected to colloidal gold; the chromatography test strip is used to verify whether the single-chain-colloidal gold connection is effectively completed. When the C line is colored, the T line is colored, which means that the single chain is successfully connected to the colloidal gold, and the T line is not colored, which means the connection fails; when the single chain is effectively connected to the colloidal gold, the C chain part of the H is 5'FAM and 3' quenched, and the synthesized single-chain H is added to the CRISPR system as a reporter probe, and the recognition and cutting efficiency of the Cas protein on its single chain is determined by detecting its fluorescence value; the preparation and testing of the probe includes the following specific steps:

[0060] 1. For the ligation reaction, take 1 mL of colloidal gold, add single-stranded (H) with a final concentration of 4 μM, incubate at 50°C for 24 h, add 100 μL of nucleic acid labeled colloidal gold blocking solution, and incubate at 50°C for 10 h;

[0061] 2. Purify the reaction solution after incubation, centrifuge at 12000rpm for 20min, remove the supernatant, add 1mL of nucleic acid labeled colloidal gold washing solution, shake and mix, centrifuge at 10000rpm for 10min, remove the supernatant. Add 1mL of resuspension buffer to resuspend, and store at 2-8℃ as a CG probe for future use;

[0062] 3. Take 50 μL of the prepared CG probe and add it to the reaction well of the chromatography test strip, and observe the chromatography results;

[0063] 4. 20μL reaction system of CRISPR / Cas: 10×Cas 12a Reaction Buffer, 2μL crRNA (1μM), 1μL 1×Cas 12a Protrinr, 5μL CG capture reporter probe, 2μL positive quality control product, and the rest is supplemented with water to 20μL) and mixed into the reaction tube. Specifically, the CRISPR procedure is: use a fluorescent PCR amplification instrument to react at 37°C for 10 minutes; by observing the changes in its fluorescence value, judge the cutting efficiency of the Cas enzyme and make a selection.

[0064] According to the following result chart, when the length of the C part in the H chain changes, the connection between the colloidal gold and its single chain is different. When the base type of the C part in the H chain is further changed, the recognition ability of the Cas protein is also different. Under the same input amount and amplification time, the chromatography results and the fluorescence value in the solution are analyzed, specifically:

[0065] (1) According to Table 2 and Figure 3 The results of the middle chromatography showed that when the number of bases in the C part was too small (C1-C3), the chromatography results showed that only the C line was colored, and the T line was not colored. The added solution did not contain biotin, indicating that the connection between the single chain and the colloidal gold was unstable, and the resuspended solution did not contain a single chain structure; when the number of bases increased by more than 10 (C4-C8), there was a clear T line on the chromatography test strip, which further indicated that C4-C8 could be successfully connected to colloidal gold;

[0066] (2) Furthermore, the base sequence of C4 was changed from A / T to C / G, and the chromatography verification and Cas enzyme cleavage efficiency test were performed again. The chromatography results ( Figure 4 ) It can be seen that when the base type is changed, the C / T line in the chromatographic strip results has a significant color development, and when the GC content in the sequence is increased, there is no effect on the connection between the single strand and the colloidal gold; through the data in Table 3 and Figure 5 (Fluorescence graph) Analysis of the changes in fluorescence curve and fluorescence value. When the length of the C part in the H chain is consistent, it can be found through the results that when the A / T content in the single chain is 80-100%, its fluorescence value is above 400 without a significant downward trend, and its fluorescence curve has no obvious change; as the proportion of A / T bases in the sequence decreases to 70%, its fluorescence value is lower than 400, and the fluorescence value of the single chain without G / C bases decreases by more than 100, and the fluorescence curve becomes a flatter curve to form a significant distinction; combined with the above results analysis, the change of the base type in the C part of the H chain does not affect the connection with colloidal gold, but has an impact on the recognition and cutting function of the Cas enzyme;

[0067] In summary, when the non-specific chain HC length is more than 10 nt, when the A / T base accounts for no less than 70% of it, preferably, when the A / T base accounts for no less than 80%, it can be well connected with colloidal gold and can be efficiently recognized and cut by the Cas enzyme.

[0068] Table 2

[0069]

[0070] Table 3

[0071]

[0072] Example 4 Using the method of the present invention to detect DNA samples

[0073] Based on the monkeypox detection system (B7R target gene), the applicability was verified using DNA nucleic acid samples combined with the CRISPR / Cas12a visual detection system. The nucleic acid to be tested was the product of isothermal pre-amplification of the standard plasmid. Specific primer information is shown in Table 5.

[0074] Table 5

[0075]

[0076] For the nucleic acid to be tested, the product after RPA reaction was tested using monkeypox standard plasmid (B7R), wherein the RPA reaction system was (50μL RPA reaction system): B7R-F 2μL (10μM), B7R-R 2μL (10μM), 25μL ABuffer, 2.5μL B Buffer, 17.5μL enzyme-free water, 1μL template (standard plasmid or negative quality control product at various concentrations of B7R), mixed and added to the bottom of the reaction tube containing enzyme lyophilized powder, after shaking and mixing, placed in a heated metal bath and set the RPA amplification program to amplification temperature of 39°C and amplification time of 10 minutes. The product after the reaction was used as the nucleic acid sample to be tested for subsequent experimental verification.

[0077] Step 1: Prepare a solution of the CRISPR / Cas reaction system, which includes 2 μL 10×Cas 12aReaction Buffer, 2 μL crRNA (1 μM), 1 μL 1×Cas 12a Protein, 5 μL SSA capture reporter probe, and 8 μL enzyme-free water and mix the reaction tube.

[0078] Step 2: Add the nucleic acid sample to be tested into a reaction tube containing the CRISPR / Cas12a reaction system. When the nucleic acid sample to be tested is a product of positive standard plasmid amplification (positive sample), its concentration is 10 copies / μL, 10 2 copies / μL, 10 3 copies / μL, 10 4 copies / μL, 10 5 Copies / μL; when the nucleic acid sample to be tested is a product of isothermal amplification of a negative control, it is specifically a product of amplification after adding negative extracted nucleic acid and enzyme-free water; the reaction tube after adding the sample to be tested is placed in a constant temperature metal bath, and the program is set to 37°C for 10 minutes;

[0079] Step 3: After the reaction in step 2 is completed, add 20 μL of CG signal transduction probe to all the above reaction tubes respectively; after oscillation and mixing, place in a constant temperature metal bath and set the program to 56°C for 10 min;

[0080] Step 4: After the reaction in step 3 is completed, the reaction tube is placed on a magnetic stand for magnetic separation. For easy observation, the solution is sucked out into eight-row reaction tubes. Specifically, positions 1-5 in the eight-row tube are solutions after magnetic separation of the products of positive standard plasmid amplification for the nucleic acid sample to be tested, and positions 6-8 are solutions after magnetic separation of the products of negative quality control products for the nucleic acid sample to be tested; the eight-row tube is placed on a white background for visual interpretation of the results with the naked eye;

[0081] The results are as follows Figure 6 It can be seen that when different forms of nucleic acid are used as test samples, it can be clearly observed that the supernatant of the positive reaction tube is pink, and the negative and blank products are colorless after magnetic separation. Figure 6 A is a product of isothermal amplification of standard plasmids with different concentrations as the test sample (DNA positive sample). The results show that when the standard plasmid concentration is 10 copies / μL or above, the solution appears pink after magnetic separation; Figure 6 B is an amplification product that does not contain the B monkeypox standard plasmid as the nucleic acid sample to be tested (DNA negative sample). After the above steps, the test is carried out. After magnetic separation, the solution is separated and observed, and the solution can be clearly presented as colorless; further illustrating that when the sample to be tested is DNA, the color development system can make a good distinction between positive and negative based on the CRISPR system based on Cas12a protein.

[0082] Example 5 Detection of RNA samples using the method of the present invention

[0083] Based on the COVID-19 detection system (Orf1a target gene), RNA nucleic acid samples were used in combination with the CRISPR / Cas13a visualization detection system for applicability verification. The nucleic acid to be tested was the RNA nucleic acid sample extracted from the COVID-19 pseudovirus standard. See Table 6 for specific primer information.

[0084] Table 6

[0085]

[0086] Step 1: Prepare the CRISPR / Cas13a reaction system: 2μL Cleavage Buffer (10X), 4μL TransMix (5X), 0.5μL T7 RNA Polymerase (40X), 1μL crRNA (1μM), 1μL Cas13aProtein (2μM), 2μL SSA capture reporter probe, 7.5μL enzyme-free water, mix well and add to the reaction tube.

[0087] Step 2: Add the nucleic acid sample to be tested into a reaction tube containing the CRISPR / Cas13a reaction system. When the nucleic acid sample to be tested is RNA nucleic acid extracted from the new coronavirus pseudovirus standard (positive sample), the specific pseudovirus concentration before extraction is 10 7 copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 2×10 3 When the nucleic acid sample to be tested is a negative control or blank, RNA extracted from the negative sample and enzyme-free water are specifically added; the reaction tube after adding the sample to be tested is placed in a constant temperature metal bath, and the program is set to 42°C for 10 min;

[0088] Step 3: After the reaction in step 2 is completed, add 20 μL of CG signal transduction probe to all the above reaction tubes respectively; after oscillation and mixing, place in a constant temperature metal bath and set the program to 56°C for 10 min;

[0089] Step 4: After the reaction in step 3 is completed, the reaction tube is placed on a magnetic stand for magnetic separation. For easy observation, the solution is sucked out into eight-tube strips. Specifically, positions 1-5 in the eight-tube strip are solutions after magnetic separation when the nucleic acid sample to be tested is an RNA sample extracted from a novel coronavirus pseudovirus standard, and positions 6-8 are solutions after magnetic separation after the above steps when the nucleic acid sample to be tested is a negative nucleic acid sample and enzyme-free water; the eight-tube strip is placed on a white background for visual interpretation of the results with the naked eye;

[0090] The results showed that the RNA nucleic acid extracted from the new coronavirus pseudovirus standard was used as the target nucleic acid to be tested. After magnetic separation, the solution was separated and observed. The results showed that the pseudovirus concentration was 2×10 3 For RNA nucleic acid samples extracted at 30 copies / mL and above, the solution can clearly appear pink; when the sample to be tested is a negative control or a blank RNA negative sample, after magnetic separation, the solution is separated and observed, and the solution can clearly appear colorless; it is further explained that when the sample to be tested is RNA, the color development system can make a good distinction between positive and negative based on the CRISPR system based on Cas13a protein.

[0091] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0092] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A naked eye visualization detection method for nucleic acid detection based on the CRISPR / Cas reaction system, the method comprising the following steps: Step 1: preparing a solution of a CRISPR / Cas reaction system, wherein the solution contains a Cas enzyme with trans-cleavage ability, crRNA and an SSA capture probe, wherein the crRNA is composed of a structural sequence and a guide sequence, wherein the crRNA structural sequence forms a complex with the Cas enzyme, and the guide sequence is complementary to a sequence in the target nucleic acid sequence to be detected; The complex scans the entire nucleic acid sample to be tested, and identifies whether there is a complementary sequence to the guide sequence in the nucleic acid sample to be tested. If the complementary sequence exists, the guide sequence forms a double-stranded structure with the complementary sequence, and guides the Cas enzyme to cut the double strand, while activating the trans-cutting activity of the Cas enzyme to non-specifically cut the single-stranded structure of the nucleic acid in the reaction system; The SSA capture probe is composed of a nucleic acid single-strand P and magnetic beads, the magnetic beads are modified with a first conjugate, the single-strand P is composed of two parts, A and B, wherein the A part is a single-strand containing A / T bases, which is a single-strand that can be recognized and cut by the Cas enzyme, and the 5' end of the A part is modified with a second conjugate, and the B part is a single-strand that is less likely to be recognized and cut by the Cas enzyme than the A part; the second conjugate modified at the 5' end of the A part of the single-strand P is paired / coupled with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe; Step 2: Add the nucleic acid sample to be tested into a reaction tube containing the CRISPR / Cas reaction system. If the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, the guide sequence of the crRNA forms a double-stranded structure with it, and guides the Cas enzyme to effectively cut the double strand, while activating the trans-cutting activity of the Cas enzyme to non-specifically cut the single strand P of the SSA capture probe in the reaction system, and cut the SSA capture probe into "magnetic bead-A" and "free single strand B". If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the SSA capture probe will not be cut; Step 3: After the reaction in step 2 is completed, a CG signal transduction probe is added to the reaction system. The CG signal transduction probe is composed of an H chain and colloidal gold. The colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain is composed of two parts, C and D, wherein the C part is a single chain containing A / T bases, which is a single chain recognized and cut by the Cas enzyme, and the D part is a single chain that is less likely to be recognized and cut by the Cas enzyme than the C part, and is complementary to the B part. The end of the C part away from the D part is labeled with colloidal gold; if the nucleic acid sample to be tested contains The target nucleic acid sequence to be detected, the trans-nuclease activity of the Cas enzyme protein is activated, and the single-stranded part of the CG signal transduction probe is also cut, and the CG signal transduction probe is cut into "colloidal gold-C" and "free single-stranded D"; wherein, "free single-stranded D" and the "free single-stranded B" generated by the reaction in step 2 form a "double-stranded BD"; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of: "magnetic beads-A", "double-stranded BD", "colloidal gold-C" these three free components; If the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, the trans-nuclease activity of the Cas enzyme will not be activated, and the CG signal transduction probe will not be cut; wherein, the B chain of the complete SSA capture probe and the D chain of the complete CG signal transduction probe will still form a BD complementary double strand, and finally form a "magnetic bead-PH-colloidal gold" complex in which the two probes are combined together, wherein PH is combined together by BD complementarity; at this time, in the reaction system, the SSA capture probe and the CG signal transduction probe exist in the reaction system in the form of: "magnetic bead-PH-colloidal gold" complex; Step 4: After the reaction in step 3 is completed, the reaction tube is placed on a magnetic stand for magnetic separation, and then the visual result is interpreted by naked eyes; if the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-A" will be adsorbed to the tube wall, while the two components "double-stranded BD" and "colloidal gold-C" will still be free in the solution, so the solution presents the color of colloidal gold; if the nucleic acid sample to be tested does not contain the target nucleic acid sequence to be tested, after magnetic separation, the "magnetic beads-PH-colloidal gold" complex will be adsorbed on the tube wall, and there is no free colloidal gold in the solution, so the solution is colorless.

2. The method according to claim 1, characterized in that The single-stranded A portion is 6-12 nt in length, and the A / T content is not less than 70%.

3. The method according to claim 2, characterized in that The A / T content in the single-stranded A portion is not less than 80%.

4. The method according to claim 1, characterized in that: The single-stranded C portion is more than 10 nt in length, and the A / T content is not less than 70%.

5. The method according to claim 4, characterized in that The A / T content in the single-stranded C portion is not less than 80%.

6. The method according to claim 1, characterized in that The CRISPR / Cas reaction system is a CRISPR / Cas12a reaction system, and the nucleic acid sample to be tested is a DNA sample.

7. The method according to claim 1, characterized in that The CRISPR / Cas reaction system is a CRISPR / Cas13a reaction system, and the nucleic acid sample to be tested is an RNA sample.

8. The method according to claim 1, characterized in that The first conjugate and the second conjugate are a molecular pairing coupling system, which is any one of a streptavidin-biotin pairing coupling system, a biotin-avidin pairing coupling system, a receptor-ligand pair coupling system, an engineered covalent binding pair coupling system, a nucleic acid aptamer and target molecule coupling system, an antigen-antibody coupling system, or a metal chelate coupling system.

9. A naked eye visualization detection kit for nucleic acid detection based on CRISPR / Cas reaction system, characterized in that: The kit comprises: a Cas enzyme with trans-cutting ability, crRNA, an SSA capture probe and a CG signal transduction probe, wherein the crRNA is composed of a structural sequence and a guide sequence, the crRNA structural sequence forms a complex with the Cas enzyme, and the guide sequence is complementary to a sequence in a target nucleic acid sequence to be tested; the complex scans the entire nucleic acid sample to be tested, and identifies whether there is a complementary sequence of the guide sequence in the nucleic acid sample to be tested, and if the complementary sequence exists, the guide sequence forms a double-stranded structure with the complementary sequence, and guides the Cas enzyme to cut the double strand, while activating the trans-cutting activity of the Cas enzyme, which is used to non-specifically cut the single-stranded structure of the nucleic acid in the reaction system; The SSA capture probe is composed of a nucleic acid single-strand P and magnetic beads, the magnetic beads are modified with a first conjugate, the single-strand P is composed of two parts, A and B, wherein the A part is a single-strand containing A / T bases, which is a single-strand that can be recognized and cut by the Cas enzyme, and the 5' end of the A part is modified with a second conjugate, and the B part is a single-strand that is less likely to be recognized and cut by the Cas enzyme than the A part; the second conjugate modified at the 5' end of the A part of the single-strand P is paired / coupled with the magnetic beads modified with the first conjugate through the first conjugate and the second conjugate to obtain the SSA capture probe; The CG signal transduction probe is composed of an H chain and colloidal gold, wherein the colloidal gold labels the H chain to form a "colloidal gold-H" complex; wherein the H chain is composed of two parts, C and D, wherein the C part is a single chain containing A / T bases, which is a single chain recognized and cut by the Cas enzyme, and the D part is a single chain that is less likely to be recognized and cut by the Cas enzyme than the C part, and is complementary to the B part, and the end of the C part away from the D part is labeled with colloidal gold; if the nucleic acid sample to be tested contains the target nucleic acid sequence to be tested, the trans-nuclease activity of the Cas enzyme protein is activated, and the single-chain part in the CG signal transduction probe will also be cut, and the CG signal transduction probe will be cut into "colloidal gold-C" and "free single-chain D".

10. The kit according to claim 9, characterized in that The single-stranded A portion is 6-12 nt in length, and the A / T content is not less than 70%.

11. The method according to claim 1, characterized in that: The single-stranded C portion is more than 10 nt in length, and the A / T content is not less than 70%.

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