Nucleic acid detection method, test strip, kit and application based on CRISPR / Cas12 cis-cleavage

Abstract

The present invention provides a kind of gene detection test paper strip, including a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad that are sequentially arranged on the base plate and connected to each other; a detection line and a control line are provided on the nitrocellulose membrane, the detection line is provided near the conjugate pad, and the control line is provided near the water-absorbing pad; the detection line is coated with a capture group for binding to the modifying group carried by the primer, and the control line is coated with a quality control probe for binding to the labeled probe, wherein the quality control probe coated with the control line is obtained by spraying a mixture of a streptavidin solution and a quality control probe onto the control line position. The present invention generates a sticky end to be hybridized and combines with a signal probe through a specific cleavage reaction between the Cas protein / crRNA complex and the target sequence, and detects the signal of the signal probe at the detection site, thereby improving the specificity and accuracy of the detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a nucleic acid detection method, test strip, kit and application based on CRISPR / Cas12 cis-cleavage. Background Art

[0002] In recent years, nucleic acid test strips for genetic testing have continued to develop. This technology is simple and easy to use, allowing direct visual detection of test results, making it widely used for rapid on-site nucleic acid testing. Nucleic acid test strips are based on traditional immunoassay strips. Their principle is based on a sandwich nucleic acid hybridization reaction, combined with gold nanoparticles or other nanoparticles and enzyme-linked immunosorbent assays (ELISAs) to achieve colorimetric detection on lateral flow test strips. Current typical nucleic acid test strip technologies involve PCR amplification or isothermal amplification using primers bearing two different tags, resulting in a labeled amplification product. This labeled amplification product can then bind to an affinity ligand on the test strip and be captured on the test strip. For example, single-stranded DNA or RNA is labeled and amplified using asymmetric PCR or isothermal amplification. The product then hybridizes specifically with pre-designed T- and C-line probes embedded on the test strip, and is visualized using a substrate to achieve detection. However, existing nucleic acid amplification methods for nucleic acid detection lack spectral properties, resulting in poor amplification efficiency and poor detection results. Furthermore, the complex amplification design is prone to false positives and low specificity.

[0003] In addition, there are lateral flow assays based on the CRISPR-Cas system. The CRISPR-Cas system consists of the Cas protein and a guide RNA that binds to the Cas9 protein. The guide RNA recognizes the PAM sequence on the gene, and the Cas protein cleaves the DNA at a specific site within the PAM sequence, causing DNA fragmentation. Currently, the most typical lateral flow assays based on the CRISPR-Cas system are DETECTR (DNA endonuclease-targeted CRISPR trans reporter) and SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking), developed based on the trans-cleavage mechanism of Cas12a and Cas13a. However, the detection steps are cumbersome. For negative samples, if the C-line does not completely intercept the signal probe, false positive results can occur, and multiplexed detection cannot be achieved in a single reaction. Furthermore, in technologies based on Cas9 opening the double-stranded DNA for hybridization, the sgRNA in the Cas9 / sgRNA system has low specificity for hybridization with the target, resulting in poor specificity and affecting detection specificity. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art and provide a nucleic acid detection method based on CRISPR / Cas12 cis-cleavage. Through the specific cleavage reaction between the Cas protein / crRNA complex and the target sequence, the target sequence is cis-cleaved by CRISPR / Cas12 to produce a dropped sticky end to be hybridized, and the sticky end to be hybridized is combined with a signal probe. By detecting the signal of the signal probe at the detection site, the presence of the target sequence is detected, thereby improving the specificity and accuracy of the detection.

[0005] The present invention is achieved through the following technical solutions:

[0006] A CRISPR-based nucleic acid detection method comprises the following steps: designing primers according to a target sequence, and amplifying the sequence using the genomic DNA or RNA of the sequence to be detected as a template to obtain an amplified fragment sequence of the sequence to be detected; preparing a signal probe, wherein the signal probe comprises a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule, and the 3' end of the label probe is a complementary pairing sequence, wherein the complementary pairing sequence is used to complementarily pair with a sticky end to be hybridized, wherein the sticky end to be hybridized is a PAM distal sequence fragment with a sticky end that falls off after the amplified fragment sequence of the sequence to be detected is cut by the Cas protein; preparing a test system and performing sample detection: mixing the Cas protein and crRNA to form a Cas protein / crRNA complex, and mixing the Cas protein / crRNA complex with the amplified fragment sequence of the sequence to be detected; wherein the Cas protein can cis-cleave the target sequence to generate the sticky end to be hybridized; if the target sequence exists in the sequence to be detected, the Cas protein / crRNA complex can undergo a specific cleavage reaction with the amplified fragment sequence of the sequence to be detected, and combine with the amplified fragment sequence of the sequence to be detected to form a Cas protein / crRNA / sequence to be detected complex, The RNA / sequence to be detected complex system is used as a test system, wherein the Cas protein / crRNA / sequence to be detected complex system includes a Cas protein / crRNA / sequence to be detected complex and a sticky end to be hybridized, and the sticky end to be hybridized is a PAM distal sequence fragment with a sticky end that falls off after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein; if the target sequence is not present in the sequence to be detected, the Cas protein / crRNA does not react with the amplified fragment sequence of the sequence to be detected, and the test system includes Cas protein / crRNA and an uncut amplified fragment sequence of the sequence to be detected; the test system is mixed with a signal probe, and if the target sequence is present in the sequence to be detected, the signal probe can bind to the sticky end to be hybridized in the Cas protein / crRNA / sequence to be detected complex system as a test system; if the target sequence is not present in the sequence to be detected, the signal probe does not react with the test system; detection is performed in a detection carrier, and a detection site for capturing the sticky end to be hybridized is provided on the detection carrier, and whether the signal of the signal probe is obtained is detected at the detection site. If the signal of the signal probe is detected, the sequence to be detected contains the target sequence, otherwise the sequence to be detected does not contain the target sequence.

[0007] The present invention provides a CRISPR-based nucleic acid detection method. The Cas protein / crRNA complex has cis-cleavage activity. In the step of preparing a test system, after the Cas protein / crRNA complex is mixed with an amplified fragment sequence of a sequence to be detected, if a target sequence is present on the amplified fragment sequence of the sequence to be detected, based on the cis-cleavage activity of the CRISPR-Cas12 protein, the Cas protein / crRNA complex searches for and identifies a PAM sequence on the amplified fragment sequence of the sequence to be detected. The crRNA complementarily pairs with a targeting region (a sequence following the PAM site) of the target sequence and cuts at the targeting region. After the amplified fragment sequence of the sequence to be detected is cut, a PAM distal sequence fragment with a sticky end is dropped. That is, the cis-cleavage reaction of the Cas protein / crRNA complex is highly specific. The sticky end to be hybridized is generated by the specific recognition and cleavage of the target gene by the Cas protein / crRNA complex. Because the labeled probe in the signal probe has a complementary pairing sequence complementary to the sticky end to be hybridized, the signal probe binds to the dropped sticky end to be hybridized. When entering the detection carrier, the sticky end to be hybridized is captured at the test site of the detection carrier, and the signal of the signal probe can be obtained at the capture position, thereby detecting that the target sequence is contained in the sequence to be detected. The present invention generates the sticky end to be hybridized through the specific cleavage reaction between the Cas protein / crRNA complex and the target sequence, and allows the sticky end to be hybridized to bind to the signal probe. By detecting the signal of the signal probe at the detection site, the presence of the target sequence is detected, thereby improving the specificity and accuracy of the detection.

[0008] Furthermore, in the step of designing primers based on the target sequence and amplifying using the genomic DNA or RNA of the test object as a template to obtain an amplified fragment sequence of the sequence to be detected, the primer carries a modifying group, and the modifying group is used to modify one end distal to the PAM sequence with a sticky end. Amplification is performed using the genomic DNA or RNA of the test object as a template. If the target sequence is present in the sequence to be detected, an amplified fragment sequence of the sequence to be detected with the modifying group is obtained, wherein the modifying group is modified at the distal end of the PAM sequence with a sticky end. The detection carrier is coated with a quality control site and at least one detection site. The quality control site is coated with a quality control probe that binds to the labeled probe, and the detection site is coated with a substance that binds to the modifying group. The labeled probe is captured at the quality control site to verify whether the detection process is proceeding normally. The detection site is coated with a substance that binds to the modifying group to capture the PAM distal sequence fragment with a sticky end modified with the modifying group, that is, to capture the sticky end to be hybridized, so that the signal of the signal probe is displayed at this site.

[0009] Furthermore, the detection carrier is at least one of a test strip, a microarray chip, a graphene chip, a microfluidic chip, and a fluorescence detector, and the middle section of the labeled probe is complementary to the quality control probe at the quality control site. When designing the labeled probe, to avoid interfering with the pairing of the labeled probe with the sticky end fragment, it is necessary to avoid designing both ends of the labeled probe as sites that bind to the quality control site. Therefore, the middle section of the labeled probe is designed to bind to the quality control site.

[0010] Furthermore, the 5' end of the labeled probe is modified with a spacer sequence and an affinity group, wherein the affinity group has affinity for the label. The affinity group is provided to connect the labeled probe and the label to form a signal probe. The 5' end of the labeled probe is modified with a spacer sequence. The steric hindrance of the label interferes with the binding of the label probe's mid-section sequence to the quality control probe. The spacer sequence separates the label from the label probe's mid-section sequence, improving the reaction efficiency between the sticky end and the quality control probe and reducing detection errors.

[0011] Furthermore, the marker is one of a fluorescent probe, an absorption probe, a chemiluminescent probe, a bioluminescent probe, a catalytic enzyme, and a color-developing nanoparticle; the catalytic enzyme includes at least one of horseradish peroxidase, alkaline phosphatase, and a nanozyme; the color-developing nanoparticles include at least one of gold nanoparticles, nanorods, quantum dots, graphene, latex microspheres, upconversion nanoparticles, and iron oxide; and the detection carrier is one of a test strip, a microarray chip, a graphene chip, a microfluidic chip, and an electrochemical sensor.

[0012] Furthermore, in the step of preparing the test system, the Cas12 protein and crRNA are incubated at room temperature in a reaction buffer at a concentration ratio of 1: 1 to form a Cas protein / crRNA complex, and then the amplified fragment sequence of the sequence to be detected with a modified group is added and mixed, and the reaction is carried out for 10-15 minutes. The ratio of Cas12 protein and crRNA is controlled to form a Cas protein / crRNA complex, which recognizes and cuts the amplified fragment sequence of the sequence to be detected with the target sequence.

[0013] The present invention also provides a genetic testing test strip, comprising a base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially arranged and connected to the base plate; a detection line and a control line are arranged on the nitrocellulose membrane, the detection line is arranged close to the conjugation pad, and the control line is arranged close to the water-absorbing pad; the detection line is coated with a capture group for binding to a modifying group, and the control line is coated with a quality control probe for binding to a labeled probe.

[0014] Furthermore, there are several types of target sequences, and primers for different types of target sequences carry different modification groups. Amplification is performed using the genomic DNA or RNA of the sequence to be detected as a template. If different types of target sequences exist in the sequence to be detected, amplified fragment sequences of the sequence to be detected with different modification groups are obtained. There are several detection lines, and several detection lines are arranged in parallel between the binding pad and the control line. Each detection line is coated with a capture group that binds to one of the modification groups. When amplifying the genomes of different analytes, different genomes bind to different modification groups and can be specifically captured by different detection lines when detected on a genetic test strip, thereby realizing the detection of multiple target sequences using one genetic test strip.

[0015] The present invention also provides a gene detection kit, which includes amplification primers of the target sequence, an amplification reagent and the above-mentioned gene detection test strip.

[0016] The present invention also provides application of the above gene detection kit in gene detection.

[0017] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the CRISPR-based nucleic acid detection method in Example 1.

[0019] Figure 2 This is a schematic diagram of the nucleic acid test strip for gene detection in Example 7.

[0020] Figure 3 This is a graph showing the results of detecting African swine fever virus using the CRISPR-based nucleic acid detection method in Example 7.

[0021] Figure 4 1 is a diagram showing the detection principle of the genetic test strip with two test lines in Example 8.

[0022] Figure 5 1 is a diagram showing the detection principle of the gene detection test strip with three test lines in Example 8.

[0023] Figure 6 This is the detection result of the CRISPR-based nucleic acid detection method after a single-base mutation of the African swine fever virus target in Example 11.

[0024] Figure 7 This is the detection result of the CRISPR-based nucleic acid detection method after the African swine fever virus target was mutated by 2bp in Example 11. DETAILED DESCRIPTION

[0025] The following is a further detailed description of the embodiments of the present invention in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the embodiments of the present invention and are not intended to limit the embodiments of the present invention. It should also be noted that, for ease of description, the accompanying drawings only illustrate portions of the embodiments of the present invention, rather than all structures.

[0026] Furthermore, the terms "first," "second," "third," etc., in the specification and claims are used solely for descriptive purposes to distinguish between identical technical features. They are not to be construed as indicating or implying relative importance, or as implicitly specifying the number of technical features, nor do they necessarily describe a sequential or chronological order. The terms are interchangeable where appropriate. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one of those features.

[0027] Similarly, the terms "fixed" and "connected" used in the specification and claims should not be construed as limited to direct connections. Thus, the expression "device A is connected to device B" should not be limited to devices or systems in which device A is directly connected to device B. Rather, it means that a path exists between device A and device B, which may include other devices or tools.

[0028] Example 1

[0029] This embodiment provides a CRISPR-based nucleic acid detection method, comprising the following steps:

[0030] Step S1: Design primers based on the target sequence, wherein the primers carry a modifying group, which is used to modify one end of the sequence fragment distal to the PAM sequence with a sticky end, and amplify the genomic DNA or RNA of the target object as a template to obtain the amplified fragment sequence of the target sequence with the modifying group. The specific operation is as follows:

[0031] Two primers are designed according to the target sequence, namely the first primer and the second primer, wherein the 5' end of the second primer is modified with biotin;

[0032] Genomic DNA or RNA of the analyte is extracted and amplified using PCR using the analyte's genomic DNA or RNA as a template to obtain an amplified fragment of the sequence to be detected. If the target sequence is present in the sequence to be detected, the resulting amplified fragment of the sequence to be detected is a biotinylated amplified fragment, where the biotin is modified at one end of the sequence distal to the PAM sequence.

[0033] The genomic DNA or RNA of the analyte can also be amplified using amplification methods such as RPA and LMAP.

[0034] Step S2: Prepare a signal probe, which includes a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is the PAM distal sequence fragment with a sticky end that is dropped after the amplified fragment sequence of the sequence to be detected is cut by the Cas protein. The specific operation is as follows:

[0035] Nano-gold was selected as the marker and prepared by citrate reduction method with HAuCl4 as raw material. The prepared nano-gold particle size was 13nm.

[0036] A labeled probe is prepared, wherein the 5' end of the labeled probe is modified with a spacer sequence and a thiol group, the middle section is complementary to the C-line probe, and the 3' end is a complementary pairing sequence. The complementary pairing sequence can be combined with the PAM distal sequence fragment with a sticky end that falls off after being cut by the Cas12 protein; the thiol group has an affinity for gold nanoparticles, and the labeled probe is connected to the gold nanoparticles through the thiol group at the 5' end to obtain a gold nanoparticle probe as a signal probe. The spacer sequence includes but is not limited to poly A or poly T. The steric hindrance of the label will interfere with the binding of the middle section sequence of the labeled probe to the quality control probe. The label is separated from the middle section sequence of the labeled probe by the spacer sequence, thereby improving the reaction efficiency of the sticky end and the quality control probe and reducing detection errors.

[0037] In another embodiment, the marker may be one of a fluorescent group, an absorption probe, a chemiluminescent group, a bioluminescent group, a catalytic enzyme, and a color-developing nanoparticle.

[0038] The catalytic enzyme can be an enzyme with catalytic activity such as horseradish peroxidase, alkaline phosphatase, nanozyme, etc., and the color-developing nanoparticles include at least one of nanogold, nanorods, quantum dots, graphene, latex microspheres, upconversion nanoparticles, iron oxide, etc.

[0039] The labeled probe sequence in this example is shown in Sequence Listing Seq1, specifically:

[0040] 5'-SH-AAAAATTACAGCCAGAACGTCGAGAACTCTCACAA -3'

[0041] Step S3: Prepare a test system and perform sample testing:

[0042] The Cas12 protein and crRNA are mixed, and the amplified fragment sequence of the sequence to be detected is added. The Cas12 protein and crRNA form a Cas protein / crRNA complex; wherein the Cas12 protein can cis-cleave the target sequence to produce a sticky end to be hybridized, including but not limited to one of LbaCas12a protein, FnCas12a protein, AsCas12a protein, BrCas12b protein, and AaCas12b protein.

[0043] If the target sequence exists in the sequence to be detected, the Cas protein / crRNA complex can undergo a specific cleavage reaction with the amplified fragment sequence of the sequence to be detected, and combine with the amplified fragment sequence of the sequence to be detected to form a Cas protein / crRNA / sequence to be detected complex. The Cas protein / crRNA / sequence to be detected complex system is used as a test system, wherein the Cas protein / crRNA / sequence to be detected complex system includes a Cas protein / crRNA / sequence to be detected complex and a sticky end, and the sticky end is a PAM distal sequence fragment with a sticky end that falls off after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein. The specific operation steps are as follows:

[0044] The Cas12 protein and crRNA are mixed in a reaction buffer at a concentration ratio of 1:1 at room temperature, and the amplified fragment sequence of the sequence to be detected obtained by amplification in step S1 and the signal probe in step S2 are added at the same time, and incubated for 10 minutes to form a Cas protein / crRNA complex. The reaction conditions are 37-45 ° C for 10-15 minutes; if the target sequence is present in the sequence to be detected, the Cas protein / crRNA complex searches and recognizes the PAM site on the PAM sequence-4 bases TTTN, and the crRNA binds to the base sequence after the PAM site to react to obtain a Cas protein / crRNA / sequence to be detected complex, and the Cas protein cuts there. After cutting, the sticky end to be hybridized at the distal end of the PAM sequence automatically detaches. Therefore, the Cas protein / crRNA / sequence to be detected complex system as a test system includes the Cas protein / crRNA / sequence to be detected complex and the dropped sticky end to be hybridized, the sticky end to be hybridized is modified with biotin, and the signal probe binds to the biotin on the sticky end to be hybridized.

[0045] If the target sequence does not exist in the sequence to be detected, the Cas protein / crRNA cannot recognize the target sequence and does not react specifically with the amplified fragment sequence of the sequence to be detected. The test system includes the Cas protein / crRNA and the amplified fragment sequence of the sequence to be detected that has not been cut, and the signal probe exists in a free state.

[0046] Detection is performed in a detection carrier, on which a detection site for capturing the sticky end to be hybridized is provided. Whether a signal from a signal probe is obtained is detected at the detection site. If a signal from the signal probe is detected at the detection site, the sequence to be detected contains the target sequence; otherwise, the sequence to be detected does not contain the target sequence.

[0047] The detection carrier can be a test strip, a sensor detection platform, a microarray chip, a graphene chip, a microfluidic chip or a paper chip.

[0048] In this embodiment, the detection carrier is a genetic test strip, which is provided with a detection line and a control line. The detection line is coated with streptavidin bound to biotin, and the control line is coated with streptavidin and a quality control probe bound to a labeled probe. The specific operation of performing detection using the genetic test strip is as follows:

[0049] Add the test solution to the genetic test strip, followed by 20 μl of drive buffer. Read the results within 5 minutes. If both the test and control lines turn red, the test is normal and the target sequence is present in the test sample. The drive buffer consists of: 4× SSC, 0.05% Tween-20 (volume percentage), 1× PBS, and 1% BSA (by weight percentage). If only the control line turns red, the test is normal and the target sequence is not present.

[0050] Figure 1 This is a schematic diagram of the CRISPR-based nucleic acid detection method in Example 1. Figure 1 The specific principle is:

[0051] If the target sequence is present in the analyte, in step S1, an amplified fragment sequence of the sequence to be detected modified with biotin is amplified to obtain a fragment sequence, wherein the biotin is modified at one end of the PAM-distal sequence fragment with a sticky end. In step S3, based on the cis-cleavage activity of the CRISPR-Cas12 protein, the Cas protein / crRNA complex searches for the PAM sequence on the amplified fragment sequence of the sequence to be detected, and then searches for and identifies the target sequence on the amplified fragment sequence of the sequence to be detected. The crRNA complementarily pairs with the target region (the sequence following the PAM site) of the target sequence and cuts at the target region. After the amplified fragment sequence of the sequence to be detected is cut, a PAM-distal sequence fragment with a sticky end is dropped. That is, the cis-cleavage reaction of the Cas protein / crRNA complex is highly specific. The sticky end to be hybridized is generated by the specific recognition and cleavage of the target gene by the Cas protein / crRNA complex. When the sticky end to be hybridized is mixed with the signal probe, the complementary pairing sequence at the 3' end of the signal probe hybridizes and binds to the sticky end to be hybridized.

[0052] In the detection carrier, the biotin modified on the sticky end to be hybridized can be captured at the detection line, causing a certain concentration of signal probes to aggregate at the detection line. In this embodiment, nanogold aggregates at the detection line, causing the detection line to turn red; the remaining signal probes and sticky end fragments continue to flow to the control line and are captured, and the control line turns red.

[0053] If the amplified fragment sequence of the sequence to be detected does not contain the target sequence, then in step S1, no amplified product of the target sequence is obtained. In step S3, the Cas protein / crRNA complex does not react with the amplified fragment sequence of the sequence to be detected. The prepared test system includes the Cas protein / crRNA complex and the uncut amplified fragment sequence of the sequence to be detected, which does not react when mixed with the signal probe. The amplified fragment sequence of the sequence to be detected cannot be captured at the detection line in the detection carrier, and only the signal probe can be captured at the control line and turns red, indicating that the detection process is normal.

[0054] Example 2

[0055] This embodiment provides a CRISPR-based nucleic acid detection method, the main steps of which are similar to those of Example 1, and the main differences from Example 1 are: Step S2: preparing a signal probe, the signal probe including a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is a sequence fragment with a sticky end that falls off the distal end of the PAM sequence after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein. The specific operation is as follows:

[0056] Selecting one of a fluorescent group, a chemiluminescent group, and a bioluminescent group as a marker;

[0057] Prepare a labeled probe, wherein the 5' end of the labeled probe is modified with poly A and an affinity group, the middle section is complementary to the C-line probe, and the 3' end is a complementary pairing sequence, wherein the affinity group has affinity for a fluorescent group, a chemiluminescent group, or a bioluminescent group, and the complementary pairing sequence can be complementary to the sticky end on the distal fragment of the PAM sequence that falls off after being cut by the Cas12 protein.

[0058] The Cas protein / crRNA / sequence to be detected complex system is mixed with the signal probe and introduced into the detection carrier. The detection carrier is a test strip. The detection site of the detection carrier is embedded with a substance that can bind to the modification group. If a fluorescent signal or a luminescent signal carried by the labeled probe is observed at the detection site, it means that the labeled probe and the amplified fragment sequence of the sequence to be detected with the modification group are captured at the detection site, and the sequence to be detected contains the target sequence. Otherwise, the sequence to be detected does not contain the target sequence.

[0059] Alternatively, in step S4, the detection carrier is a fluorescence detector, a detection site is provided in the fluorescence detector, and a substance that can bind to the modification group is provided at the detection site. If a fluorescent signal or a luminescent signal carried by the labeled probe is observed at the detection site, it means that the labeled probe and the sticky end to be hybridized with the modification group are captured at the detection site, and the sequence to be detected contains the target sequence; otherwise, the sequence to be detected does not contain the target sequence.

[0060] Example 3

[0061] This embodiment provides a CRISPR-based nucleic acid detection method. The main steps are similar to those of Example 1. The main differences from Example 1 are:

[0062] In step S2, a signal probe is prepared. The signal probe includes a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is the PAM distal sequence fragment with a sticky end dropped after the amplified fragment sequence of the sequence to be detected is cleaved by the Cas protein. The specific operation is as follows:

[0063] In step S2, a signal probe is prepared. The signal probe includes a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, and a catalytic enzyme is selected as the label;

[0064] Step S3, prepare the test system, and during the sample detection, the Cas protein / crRNA / sequence to be detected complex system and the signal probe are synchronously introduced or step-by-step introduced into the detection carrier, wherein the detection carrier is a test strip or a test tube, and the detection site of the detection carrier is embedded with a substance that can bind to the modification group and a solution that reacts with the catalytic enzyme. If color is observed at the detection site, it means that the labeled probe and the sticky end to be hybridized with the modification group are captured at the detection site, and the catalytic enzyme reacts with the reaction solution at the detection site to develop color, so that color can be observed at the detection site, and the sequence to be detected contains the target sequence, otherwise the sequence to be detected does not contain the target sequence.

[0065] Example 4

[0066] This embodiment provides a CRISPR-based nucleic acid detection method. The main steps are similar to those of Example 1. The main differences from Example 1 are:

[0067] In step S2, gold nanoparticles were selected as the marker and prepared using the citrate reduction method with chloroauric acid HAuCl4 as the raw material. The prepared gold nanoparticles had a particle size of 13 nm.

[0068] A labeled probe is prepared, wherein the 5' end of the labeled probe is modified with poly A and a thiol group, the middle section is complementary to the C-line probe, and the 3' end is a complementary pairing sequence, which can complementarily pair with the sticky end to be hybridized, and the nanogold probe is resuspended in an embedding buffer to obtain a nanogold probe for embedding in a test strip; wherein the embedding buffer includes 20mMNa3PO4, 0.25% volume percentage Tween-20, 20% mass percentage sucrose, and 5% mass percentage bovine serum albumin (BSA).

[0069] In step S3, the detection carrier is a test strip, and a sufficient amount of the labeled probe for embedding is sprayed on the test strip, dried at 37°C for 2 hours, and stored in a dry state at 4°C to embed the labeled probe in the test strip.

[0070] Example 5

[0071] This embodiment provides a CRISPR-based nucleic acid detection method, comprising the following steps:

[0072] Step S1: Design primers based on the target sequence and amplify the target sequence using the genomic DNA or RNA of the target as a template to obtain the amplified fragment sequence of the target sequence. The specific operation is as follows:

[0073] Two primers are designed according to the target sequence, namely the first primer and the second primer; the genomic DNA or RNA of the test object is extracted, and the genomic DNA or RNA of the test object is used as a template to amplify the PCR amplification method to obtain the amplified fragment sequence of the sequence to be detected.

[0074] Step S2, preparing a signal probe, the signal probe comprising a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is the PAM distal sequence fragment with the sticky end dropped after the amplified fragment sequence of the sequence to be detected is cleaved by the Cas protein;

[0075] Step S3, preparing a test system and performing detection in a detection carrier:

[0076] The Cas12 protein and crRNA are mixed to form a Cas protein / crRNA complex, and the Cas protein / crRNA complex is mixed with the amplified fragment sequence of the sequence to be detected. If the target sequence is present in the sequence to be detected, the Cas protein / crRNA complex specifically cuts the amplified fragment sequence of the sequence to be detected, and a Cas protein / crRNA / sequence to be detected complex system is obtained as a test system. The Cas protein / crRNA / sequence to be detected complex system includes a Cas protein / crRNA / sequence to be detected complex and a sticky end to be hybridized, and the sticky end to be hybridized is a PAM distal sequence fragment with a sticky end dropped after the amplified fragment sequence of the sequence to be detected is cut;

[0077] The test system is introduced into a detection carrier, such as an electrochemical sensor or microfluidic chip, pre-embedded with a signal probe. If the target sequence is present in the sequence to be detected, the hybridized sticky ends react with the signal probe, causing a current change in the electrochemical sensor or microfluidic chip. Otherwise, the target sequence is not present.

[0078] Example 6

[0079] This embodiment provides a CRISPR-based nucleic acid detection method for detecting African swine fever virus, comprising the following steps:

[0080] Step S1: Design primers based on the target sequence, wherein the primers carry a modification group, and amplify the genomic DNA or RNA of the target object as a template to obtain an amplified fragment sequence of the target sequence with the modification group. The specific operation is as follows:

[0081] African swine fever virus genomic DNA was extracted using the TIANamp Virus DNA kit;

[0082] A pair of PCR amplification primers were designed based on the p72 gene region of the conserved gene of African swine fever, namely the first primer and the second primer, wherein the second primer was modified with biotin, and a crRNA sequence was designed;

[0083] 2×TaqMix (purchased from Takara) was used for DNA amplification. The PCR amplification system (50µL) for African swine fever virus DNA is shown in Table 1:

[0084] Table 1 PCR amplification system (50µL) for African swine fever virus DNA

[0085]

[0086] Step S2: Prepare a signal probe, which includes a label probe and a label connected to the label probe, wherein the label probe is a nucleic acid molecule; the 3' end of the label probe is a complementary pairing sequence, and the complementary pairing sequence is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is the PAM distal sequence fragment with the sticky end dropped after the amplified fragment sequence of the sequence to be detected is cut by the Cas protein. The specific operation is as follows:

[0087] Nanogold was used as a marker. 100 mL of 1 nM HAuCl₄ was brought to a boil. 10 mL of 38.8 mM sodium citrate solution was quickly added under rapid magnetic stirring. Within 2 minutes, the solution color changed from golden yellow to gray to wine red to translucent red. Stirring was continued for 20 minutes, and the solution was cooled to room temperature to obtain a nanogold colloidal solution. Ultraviolet absorption spectroscopy showed an absorption peak at 520 nm, and the nanogold particle size was 13 nm.

[0088] Prepare a labeled probe, wherein the 5' end of the labeled probe is modified with poly A and thiol, the middle section is complementary to the C-line probe, and the 3' end is a complementary pairing sequence, which can complement the sticky end dropped at the distal end of the PAM sequence after cleavage by the Cas12 protein;

[0089] Dissolve 1OD of labeled probe in 500µL of nanogold colloidal solution, freeze at -40℃ for 3h, centrifuge at 12000rpm and 4℃ for 30min, and repeat three times.

[0090] Step S3: Prepare a test system and perform sample testing:

[0091] The Cas12 protein and crRNA are mixed, and the amplified fragment sequence of the sequence to be detected in step S1 and the signal probe obtained in step S2 are added. The Cas12 protein and crRNA form a Cas protein / crRNA complex to obtain a Cas protein / crRNA / sequence to be detected complex system as a test system. Since there is a target sequence in the amplified fragment sequence of the sequence to be detected in this embodiment, the Cas protein / crRNA / sequence to be detected complex system includes a Cas protein / crRNA / sequence to be detected complex and a sticky end to be hybridized. The sticky end to be hybridized is a PAM distal sequence fragment with a sticky end dropped after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein. The specific operation steps are as follows:

[0092] The Cas12 protein and crRNA are mixed in a reaction buffer at a concentration ratio of 1:1 at room temperature, and the amplified fragment sequence of the sequence to be detected and the signal probe are added. The mixture is incubated at 37°C for 10 minutes. The reaction is completed to obtain a Cas protein / crRNA / sequence to be detected complex system. The Cas protein / crRNA / sequence to be detected complex system includes a Cas protein / crRNA / sequence to be detected complex and a sticky end to be hybridized.

[0093] The specific operations of sample testing are as follows:

[0094] ① Preparation of nucleic acid test strips for gene detection:

[0095] Figure 2 This is a schematic diagram of a nucleic acid test strip for genetic testing. Please refer to Figure 2 The gene detection nucleic acid test strip includes a bottom plate and a sample pad 1, a binding pad 2, a nitrocellulose membrane 3 and a water absorbent pad 4 which are sequentially arranged on the bottom plate and connected to each other.

[0096] Saturate sample pad 1 with sample pad treatment solution (0.25% by volume Triton X-100, 0.05M Tris-HCl, 0.15M NaCl, pH 8.0), dry at 37°C for 2 hours, and store in a desiccator at room temperature. Spray conjugate pad 2 with sufficient conjugate pad treatment buffer, dry at 37°C for 2 hours, and store in a desiccator at 4°C.

[0097] A test line and a control line are set on the nitrocellulose membrane 3. Use a membrane sprayer to spray 6 µL of streptavidin solution onto the test line (T line), and spray 6 µL of a mixture of streptavidin solution and quality control probe onto the control line (C line). Dry at room temperature for 1 h and store at 4°C. The concentration of the streptavidin solution is 1 mg / mL, and the concentration of the quality control probe mixture is 100 µM.

[0098] Assemble a genetic testing nucleic acid test strip. The sample pad 1, conjugation pad 2, nitrocellulose membrane 3, and absorbent pad 4 are sequentially arranged on a base plate and connected. The nitrocellulose membrane 3 is affixed to the center of the base plate. The conjugation pad 2 is placed on one side of the nitrocellulose membrane 3 and overlaps it by 2 mm. The sample pad 1 is located on the side of the conjugation pad 2 away from the nitrocellulose membrane 3 and overlaps it by 2 mm. The absorbent pad 4 is located on the side of the nitrocellulose membrane 3 away from the sample pad 1 and overlaps it by 2 mm. The strip is cut into 4 mm wide test strips using a strip cutter. The detection line on the nitrocellulose membrane 3 is set near the conjugation pad 2, with a width of 2 mm and a distance of 6 mm from the conjugation pad 2. The control line on the nitrocellulose membrane 3 is set near the absorbent pad 4, with a width of 2 mm and a distance of 12 mm from the conjugation pad 2.

[0099] ② Add the test system to the nucleic acid test strip, then add the driving buffer, and read the results within 5 minutes. The driving buffer contains: 4× SSC solution, 0.05% Tween-20 by volume, 1× PBS, and 1% BSA (bovine serum albumin) by weight.

[0100] The base sequences of the first primer, the second primer, crRNA, the signal probe, and the quality control probe are shown in sequence table Seq2-6, and also in Table 2:

[0101] Table 2 Base sequences of the first primer, second primer, crRNA, signal probe, and quality control probe

[0102]

[0103] Figure 3 This is the result of detecting African swine fever virus using the CRISPR-based nucleic acid detection method in Example 6. The detection results are as follows: Figure 3 As shown:

[0104] A positive genetic testing nucleic acid test strip forms a red line at both the C line and the T line, while a negative genetic testing nucleic acid test strip only forms a red line at the C line, indicating that the genetic testing nucleic acid test strip can correctly detect whether it contains the target sequence fragment.

[0105] Example 7

[0106] This embodiment provides a genetic testing test strip, comprising a base plate and a sample pad 1, a conjugation pad 2, a nitrocellulose membrane 3, and a water-absorbing pad 4 sequentially arranged and connected to the base plate; a detection line and a control line are arranged on the nitrocellulose membrane 3, the detection line is arranged near the conjugation pad 2, and the control line is arranged near the water-absorbing pad 4; the detection line is coated with a capture group that binds to the modifying group, and the control line is coated with a quality control probe for binding to the labeled probe.

[0107] Example 8

[0108] This embodiment provides a genetic testing test strip, comprising a base plate and a sample pad 1, a conjugation pad 2, a nitrocellulose membrane 3, and a water-absorbing pad 4 sequentially disposed on and connected to the base plate; a detection line and a control line are disposed on the nitrocellulose membrane 3, the detection line being disposed near the conjugation pad 2, and the control line being disposed near the water-absorbing pad 4;

[0109] There are several detection lines, which are arranged in parallel between the binding pad 2 and the control line. Each detection line is coated with a different capture group; the control line is coated with a quality control probe for binding to the labeled probe.

[0110] Figure 4This is a diagram showing the principle of a genetic test strip with two test lines. Figure 5 This is a diagram of the detection principle of a genetic test strip with three test lines. Please refer to Figure 4-5 When amplifying the genomes of different analytes, there are several target sequences. The primers for different target sequences carry different modification groups. Amplification is performed using the genomic DNA or RNA of different analytes as templates. When different target sequences exist in the sequence to be detected, amplified fragment sequences with different modification groups can be obtained. Different genomes are bound to different modification groups. When tested on a genetic test strip, different genomes can be specifically captured by capture groups on different test lines, thereby enabling the detection of multiple target sequences using a single genetic test strip. In a specific implementation, test lines corresponding to the number of target sequences that need to be detected can be set on the genetic test strip.

[0111] Example 9

[0112] This embodiment provides a method for using the genetic testing test strip described in Example 8. The main steps are similar to those in Example 1, with the main differences being:

[0113] Step S1: Two different pairs of primers are designed based on the target sequence, one for amplifying the Orflab gene and the other for amplifying the N gene. The 5' end of the second primer for the Orflab gene is modified with biotin, and the 5' end of the second primer for the N gene is modified with a C3 spacer group and a hybridization sequence. The hybridization sequence is used for hybridization with the probe coated at position T2 of the test strip.

[0114] In step S3, a test system is prepared and a sample is tested. The test carrier is a genetic test strip having two test lines T1 and T2. The test line T1 is coated with a first capture probe bound to biotin, and the test line T2 is coated with a second capture probe bound to a C3 spacer group and a hybridization sequence.

[0115] Example 10

[0116] This embodiment provides a method for using the genetic testing test strip described in Example 8. The main steps are similar to those in Example 1, with the main differences being:

[0117] Step S1: Three different pairs of primers are designed according to the target sequence, which are used to amplify the IAV gene, IBV gene and N gene respectively. The 5' end of the second primer of the IBV gene is modified with biotin, the 5' end of the second primer of the IAV gene is modified with a C3spacer group and a first hybridization sequence, and the 5' end of the second primer of the N gene is modified with a C3spacer group and a one-end sequence.

[0118] Step S3: Prepare a test system and perform sample detection. The detection carrier is a genetic detection test strip with three detection lines, namely detection line T1, detection line T2 and detection line T3, wherein detection line T1 is coated with a first capture probe bound to biotin, and detection line T2 is coated with a second capture probe bound to a C3spacer group and a one-end sequence. Detection line T2 is coated with a second capture probe bound to a C3spacer group and a one-end sequence.

[0119] Example 11

[0120] To test whether the CRISPR-based nucleic acid detection method can distinguish wild-type targets from mutant targets, the African swine fever virus target was subjected to single-base mutation and 2bp mutation, and CRISPR-based nucleic acid detection was performed respectively, and the operation steps were similar to Example 6. Figure 6 is the detection result of the CRISPR-based nucleic acid detection method after single-base mutation of the African swine fever virus target in Example 11, Figure 7 The detection results of the CRISPR-based nucleic acid detection method after the African swine fever virus target was mutated by 2bp in Example 11 are as follows: Figure 6-7 shown.

[0121] The target sequence length of the African swine fever virus recognized by the CRISPR / Cas12 system is 20bp. After single-base mutations were performed at various sites of the African swine fever virus, the test results were all positive, and the measured concentration was lower than the concentration of the control test group without mutation. This shows that the African swine fever virus can be detected by the CRISPR-based nucleic acid detection method after a single-base mutation occurs, but there are differences with the results of the control test group without mutation. Whether a single-base mutation occurs can be determined by observing the test strip, thereby determining whether it is a wild-type target or a mutant target.

[0122] After a 2bp mutation was made at each site on the African swine fever virus target, the test results for the 2bp mutation at sites 1 / 2 / 6 / 9 / 10 were negative, indicating that the African swine fever virus cannot be detected by the CRISPR-based nucleic acid detection method after a 2bp mutation occurred at a specific site. The occurrence of a 2bp mutation at a specific site can be determined by observing the test strip, thereby distinguishing the wild-type target from the mutant target with a 2bp mutation at a specific site.

[0123] The present invention is not limited to the above-mentioned embodiments. If various changes or modifications of the present invention do not depart from the spirit and scope of the present invention, and if these changes and modifications fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A method for nucleic acid detection based on CRISPR / Cas12 protein, wherein the method is for non-diagnostic purposes, characterized in that: The following steps are involved: Primers are designed according to the target sequence, and genomic DNA of the sequence to be detected is used as a template for amplification to obtain an amplified fragment sequence of the sequence to be detected; the primers are composed of a first primer and a second primer, the base sequence of the first primer is 5'-ATGGATACCGAGGGAATAGC-3'; the base sequence of the second primer is 5'-biotin-CTTACCGATGAAAATGATAC-3'; Preparation of signal probe: The signal probe includes a label probe and gold nanoparticles connected to the label probe, wherein the base sequence of the label probe is 5'-SH-AAAAATTACAGCCAGAACGTCGAGAACTCTCACAA -3', and the 3' end of the label probe is a complementary pairing sequence, which is used to complementarily pair with the sticky end to be hybridized, wherein the sticky end to be hybridized is a PAM distal sequence fragment with a sticky end that falls off after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein; Prepare the test system and conduct sample testing: The Cas12 protein and crRNA are mixed to form a Cas12 protein / crRNA complex, and the Cas12 protein / crRNA complex is mixed with the amplified fragment sequence of the sequence to be detected; wherein the Cas12 protein can cis-cleave the target sequence to produce a sticky end to be hybridized; the sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUCCUGCUGUUUGGAUAUUGUG-3', If the target sequence is present in the sequence to be detected, the Cas12 protein / crRNA complex undergoes a specific cleavage reaction with the amplified fragment sequence of the sequence to be detected, and combines with the amplified fragment sequence of the sequence to be detected to form a Cas12 protein / crRNA / sequence to be detected complex, and the Cas12 protein / crRNA / sequence to be detected complex system is used as a test system, wherein the Cas12 protein / crRNA / sequence to be detected complex system includes Cas12 protein / crRNA / sequence to be detected complex and sticky ends to be hybridized, and the sticky ends to be hybridized are PAM distal sequence fragments with sticky ends that fall after the amplified fragment sequence of the sequence to be detected is cut by the Cas12 protein; If the target sequence is not present in the sequence to be detected, the Cas12 protein / crRNA does not undergo a specific cleavage reaction with the amplified fragment sequence of the sequence to be detected, and the test system includes the Cas12 protein / crRNA and the amplified fragment sequence of the sequence to be detected that is not cut; The test system is mixed with the signal probe. If the target sequence exists in the sequence to be detected, the signal probe binds to the sticky end to be hybridized in the Cas12 protein / crRNA / sequence to be detected complex system as the test system; if the target sequence does not exist in the sequence to be detected, the signal probe does not react with the test system; Detection is performed in a detection carrier, which is provided with a detection site for capturing the sticky end to be hybridized, and whether a signal from the signal probe is obtained is detected at the detection site. If the signal from the signal probe is detected, the sequence to be detected contains the target sequence; otherwise, the sequence to be detected does not contain the target sequence; The detection carrier is coated with a quality control site and at least one detection site, the quality control site is coated with a quality control probe that binds to the labeled probe, and the detection site is coated with a substance for binding to the modifying group. The middle section of the labeled probe is complementary to the quality control probe on the quality control site, and the detection carrier is a test strip.

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