A nucleic acid dual-index rapid detection test strip based on CRISPR system and working method

Abstract

The present invention relates to the field of molecular detection technology, in particular to a nucleic acid dual-index rapid detection test strip and working method based on the CRISPR system, the test strip comprising a dual-labeled conjugate pad and a nitrocellulose membrane provided with a quality control line, a first detection line and a second detection line, and a mouse antibody-labeled carrier conjugate and a rabbit antibody-labeled carrier conjugate are provided on the dual-labeled conjugate pad. The present invention adopts the form of combining CRISPR with lateral flow test paper, which is convenient to operate and has a fast detection speed, and visual detection of nucleic acid targets to be detected can be achieved in 5 to 15 minutes. The present invention realizes the simultaneous detection of dual nucleic acid indicators by setting dual-labeled probes, dual-labeled conjugate pads and quality control lines, first detection lines and second detection lines, greatly improving the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and specifically relates to a nucleic acid dual-index rapid detection test strip. Background Art

[0002] CRISPR molecular detection is a new type of molecular detection technology first developed by Feng Zhang's team at the Broad Institute in 2017. The main principle is to use the characteristics of Cas protein that after contacting and binding to the target fragment, its indiscriminate trans-cleavage function is activated and it will cleave all nearby nucleic acid sequences. The result is judged by detecting the cleavage of the probe sequence (Gootenberg, Jonathan, S, et al. Nucleic acid detection with CRISPR-Cas13a / C2c2. [J]. Science, 2017.).

[0003] In 2019, the research team combined the CRISPR method with lateral flow test strips to launch the SHERLOCK technology (Kellner MJ, Koob JG, Gootenberg JS, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases [J]. Nature Protocols, 2019, 14 (10)). This method introduced probe sequences labeled with biotin and fluorescein isothiocyanate (FITC) at both ends into the system. The lateral flow test strip used can detect sequences containing both biotin and FITC: the proximal strip is coated with streptavidin (SA) that can bind to biotin, and the colloidal gold is labeled with anti-FITC antibodies. In addition, there is another strip at the distal end, which is coated with a secondary antibody that can bind to the anti-FITC antibody. This is a sandwich test strip. When there are sequences containing both Biotin and FITC in the sample, a SA-Biotin-probe-FITC-anti-FITC antibody-colloidal gold complex will be formed at the proximal end of the strip, resulting in color development.

[0004] The research team used the sandwich test strip in a competitive manner. When the sample is negative, the CRISPR system is not activated, the probe sequence modified with Biotin and FITC will not be cut, the probe remains intact, and the colloidal gold is completely captured by the SA strip at the proximal end of the test strip through the sandwich method and will not reach the line at the distal end of the test strip.

[0005] When the sample is positive, the probe sequence labeled with Biotin and FITC at both ends is cut off, so that part of the colloidal gold passes through the proximal band and reaches the distal band, presenting a positive result.

[0006] However, the design of the test strip does not allow for the combined detection of multiple indicators. Summary of the invention

[0007] In view of the technical problems existing in the above-mentioned prior art, the present invention provides a nucleic acid dual-index rapid detection test strip and working method based on the CRISPR system with fast detection speed and convenient operation.

[0008] At present, CRISPR nucleic acid detection technology is in the development stage. The requirements for nucleic acid detection in Europe, the United States and China are all dual targets, one as an internal reference indicator and the other as a detection indicator. However, since CRISPR technology is a signal amplification system, after the Cas12 enzyme is activated, it is an indiscriminate cut for ssDNA; after Cas13 is activated, it is an indiscriminate cut for ssRNA. In this way, when designing dual targets, only the strategy of Cas12 and Cas13 dual systems can be adopted. The research and development of the present invention is precisely to cooperate with the research and development of CRISPR dual system color development. At present, there are no nucleic acid test strips that cooperate with CRISPR dual system color development at home and abroad.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A nucleic acid dual-index rapid detection test strip based on the CRISPR system, the test strip comprising a double-labeled binding pad and a nitrocellulose membrane provided with a quality control line, a first detection line and a second detection line.

[0011] Furthermore, the double-labeled conjugate pad is provided with a mouse antibody-labeled carrier conjugate and a rabbit antibody-labeled carrier conjugate.

[0012] Furthermore, the mouse antibody-labeled carrier conjugate includes a mouse anti-FITC antibody-labeled carrier conjugate.

[0013] Furthermore, the rabbit antibody-labeled carrier conjugate includes a rabbit anti-digoxigenin (DIG) antibody-labeled carrier conjugate.

[0014] Furthermore, the labeling carrier is at least one of nano-gold, nano-silver, nano-carbon, colored microspheres and fluorescent microspheres.

[0015] Further, the quality control line, the second detection line and the first detection line are fixed on the nitrocellulose membrane in sequence, the quality control line is arranged at one end close to the conjugate pad, the quality control line is coated with SA, the first detection line is coated with goat anti-mouse secondary antibody, and the second detection line is coated with alpaca anti-rabbit anti-DIG nano monoclonal antibody. After a large number of experimental studies, in order to increase specificity, the inventor's research team used alpaca immunized rabbit anti-DIG primary antibody, which is hereinafter referred to as alpaca anti-rabbit anti-DIG nano monoclonal antibody.

[0016] The working principle of the nucleic acid dual-index rapid detection test strip based on the CRISPR system of the present invention is specifically as follows:

[0017] The CRISPR system is provided with two Cas proteins (Cas12 protein and Cas13 protein), two marker probes, and the two Cas proteins are respectively provided with different guide sequences that can target the nucleic acid molecules to be detected; the two marker probes include a first marker probe and a second marker probe, and the marker probe is formed by coupling a nucleic acid probe and a marker, and when the first marker probe contacts one of the activated Cas proteins, the first marker probe is cut, and when the second marker probe contacts another activated Cas protein, the second marker probe is cut. The first marker probe is formed by coupling the first nucleic acid probe with Biotin and FITC, Biotin is coupled to one end of the first nucleic acid probe, and FITC is coupled to the other end of the first nucleic acid probe. The second marker probe is formed by coupling the second nucleic acid probe with Biotin and DIG, Biotin is coupled to one end of the second nucleic acid probe, and DIG is coupled to the other end of the second nucleic acid probe.

[0018] When the molecule to be detected does not exist in the test sample, the Cas12 protein and the Cas13 protein cannot be activated by the two substances to be detected, the dual-labeled probe cannot be cut, and the mouse anti-FITC antibody-labeled carrier conjugate and the rabbit anti-DIG antibody-labeled carrier conjugate on the dual-labeled binding pad can respectively recognize the FITC and DIG on the dual-labeled probe and combine to form a complete probe-labeled carrier complex, and run on the nitrocellulose membrane through chromatography. When passing the quality control line, the complete probe-labeled carrier complex is captured by the SA coated on the quality control line, and cannot continue to chromatograph to the first detection line and the second detection line, and cannot form a visual signal in the detection line area, indicating that the corresponding molecule to be detected does not exist in the sample.

[0019] When there are two molecules to be detected in the sample to be detected, Cas12 protein and Cas13 protein can be activated by the two substances to be detected, thereby realizing the cutting of the dual-labeled probe. The mouse anti-FITC antibody-labeled carrier conjugate and the rabbit anti-DIG antibody-labeled carrier conjugate on the dual-labeled binding pad can respectively recognize FITC and DIG on the dual-labeled probe and combine to form a cutting probe-labeled carrier complex, and run on the nitrocellulose membrane through chromatography. When passing through the first detection line and the second detection line, the cutting probe-labeled carrier complex is captured by the sheep anti-mouse secondary antibody and the alpaca anti-rabbit anti-DIG nano-monoclonal antibody coated on the first detection line and the second detection line, respectively, and gradually enriched to form a visible signal, indicating the presence of the corresponding molecules to be detected in the sample.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention combines CRISPR with lateral flow test paper, which is easy to operate and has a fast detection speed. Visual detection of the molecules to be detected can be achieved in 5 to 15 minutes.

[0022] (2) The present invention realizes the simultaneous detection of two nucleic acid indicators by setting up a dual-labeled probe, a dual-labeled binding pad and a quality control line, a first detection line and a second detection line, thereby greatly improving the detection efficiency.

[0023] (3) The colloidal gold part of the test strip of the present invention, the first colloidal gold latex microsphere is coupled with a rabbit anti-DIG monoclonal antibody, and the second colloidal gold latex microsphere is coupled with a mouse anti-FITC monoclonal antibody. Under normal design, the two detection lines are respectively embedded with anti-mouse and anti-rabbit 2 antibodies. However, due to the complexity of different samples, it is very easy to cause antibody cross-linking, resulting in false positive non-specific binding. In order to solve the specificity and false positive, the research team of the present invention produced a nano-monoclonal antibody of alpaca anti-rabbit anti-DIG nano-monoclonal antibody, thereby fundamentally solving the problem of non-specific binding and avoiding the occurrence of false positive phenomenon.

[0024] (4) The test strip of the present invention also increases the load of the quality control line, uses more probes, and better reflects the signal amplification effect of CRISPR.

[0025] (5) Since the first colloidal gold latex microspheres and the second colloidal gold latex microspheres are used at the same time, the two microspheres are trapped on the quality control line at the same time, and the natural color presented is purple. When the second colloidal gold latex microspheres coupled with FITC-Biotin are cut, the first detection line is red, and the quality control line will gradually change from purple to blue. When the first colloidal gold latex microspheres coupled with DIG-Biotin are cut, the second detection line is blue, and the quality control line will gradually change from purple to red. When both probes are cut, the purple color of the quality control line will gradually fade or disappear. The present invention also provides two mutually corrected interpretation modes to increase the accuracy and convenience of interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the color development results of the nucleic acid dual-index rapid detection test strip based on the CRISPR system of the present invention.

[0027] Figure 2 The negative control is displayed by different colloidal gold coating, first detection line and second detection line coating antibody labeling and combination methods.

[0028] Figure 3 This is a schematic diagram of the structure of a nucleic acid dual-index rapid detection test strip based on the CRISPR system of the present invention.

[0029] Among them, 1 is the sample pad, 2 is the double-labeled binding pad, 3 is the nitrocellulose membrane, 4 is the absorption pad, 5 is the quality control line, 6 is the first detection line, 7 is the second detection line, 8 is Biotin, 9 is FITC, 10 is DIG, 11 is SA, 12 is the first colloidal gold latex microspheres, 13 is the second colloidal gold latex microspheres, 14 is single-stranded nucleotide, 15 is mouse anti-FITC monoclonal antibody, 16 is rabbit anti-DIG monoclonal antibody, 17 is alpaca anti-rabbit anti-DIG nano monoclonal antibody, and 18 is goat anti-mouse secondary antibody.

[0030] Figure 4 After using the test strip of the present invention to perform RT RAA amplification reaction on the supernatant of pseudovirus lysate with different copy numbers of SARS-CoV-2 ORF1ab gene and E gene, the products are respectively subjected to CRISPR / Cas12a enzyme digestion detection results.

[0031] In the figure, from left to right, test strips 1 to 7 correspond to the initial amplification system, in which the pseudovirus lysis solution added contains 1.4×10 6 Copy, 1.4×10 5 Copy, 1.4×10 4 Copy, 1.4×10 3 Copy, 1.4×10 2 Copy, 1.4×10 1 Copy, 1.4×10 0-1 is the CRISPR / Cas12-RT RAA test strip test results of the copied SARS-CoV-2 ORF1ab gene and E gene; -1 is the CRISPR / Cas12-RT RAA test result of the initial amplification system with an equal volume of lysate supernatant from a healthy human nasal swab without pseudovirus added; -2 is the CRISPR / Cas12-RT RAA test result of the initial amplification system with an equal volume of purified water added.

[0032] Figure 5 The test strip developed by the present invention is used to detect the CRISPR / Cas13a+Cas12-RT RAA results of the supernatant of pseudovirus lysates with different copy numbers of SARS-CoV-2 ORF1ab gene and N gene.

[0033] In the figure, from left to right, test strips 1 to 7 correspond to the pseudovirus lysis solution added to the amplification system containing 6.8×10 6 Copy, 6.8×10 5 Copy, 6.8×10 4 Copy, 6.8×10 3 Copy, 6.8×10 2 Copy, 6.8×10 1 Copy, 6.8×10 0 -1 is the CRISPR / Cas13a+Cas12-RT RAA test result of the initial amplification system with an equal volume of lysate supernatant from a healthy human nasal swab without pseudovirus added; -2 is the CRISPR / Cas13a+Cas12-RT RAA test result of the initial amplification system with an equal volume of purified water added. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] The nucleic acid dual-index rapid detection test strip based on the CRISPR system of the present invention comprises a dual-labeled binding pad and a nitrocellulose membrane provided with a quality control line, a first detection line and a second detection line.

[0037] The inventors have designed a variety of antibody coating methods to screen the best labeling and combination methods of colloidal gold coated antibodies, first detection line coated antibodies and second detection line coated antibodies.

[0038] Among them, the first method is that a mouse antibody-label carrier conjugate and a rabbit antibody-label carrier conjugate are arranged on the double-label binding pad. The mouse antibody-label carrier conjugate is a mouse anti-FITC antibody-label carrier conjugate. The rabbit antibody-label carrier conjugate is a rabbit anti-DIG antibody-label carrier conjugate. The label carrier is nanogold. The quality control line, the second detection line and the first detection line are fixed on the nitrocellulose membrane in sequence, the quality control line is arranged at one end close to the double-label binding pad, the quality control line is coated with SA, the first detection line is coated with goat anti-mouse secondary antibody, and the second detection line is coated with goat anti-rabbit secondary antibody.

[0039] Among them, the second method is that a mouse antibody-label carrier conjugate and a rabbit antibody-label carrier conjugate are arranged on the double-label binding pad. The mouse antibody-label carrier conjugate is a mouse anti-FITC antibody-label carrier conjugate. The rabbit antibody-label carrier conjugate is a rabbit anti-DIG monoclonal antibody-label carrier conjugate. The label carrier is nanogold. The quality control line, the second detection line and the first detection line are fixed on the nitrocellulose membrane in sequence, the quality control line is arranged at one end close to the double-label binding pad, the quality control line is coated with SA, the first detection line is coated with goat anti-mouse secondary antibody, and the second detection line is coated with goat anti-rabbit secondary antibody.

[0040] Among them, the third method is that a mouse antibody-label carrier conjugate and a rabbit antibody-label carrier conjugate are arranged on the double-label binding pad. The mouse antibody-label carrier conjugate is a mouse anti-FITC antibody-label carrier conjugate. The rabbit antibody-label carrier conjugate is a rabbit anti-DIG monoclonal antibody-label carrier conjugate. The label carrier is nanogold. The quality control line, the second detection line and the first detection line are fixed on the nitrocellulose membrane in sequence, and the quality control line is arranged at one end close to the double-label binding pad. The quality control line is coated with SA, the first detection line is coated with sheep anti-mouse secondary antibody, and the second detection line is coated with alpaca anti-rabbit anti-DIG nano monoclonal antibody.

[0041] Add 0.8 μL of 10 μM first labeled probe and 10 μM second labeled probe to 50 μL ultrapure water to prepare negative control. Prepare different test strip samples and use the samples to test the negative control. The test results are expected to be presented as follows Figure 1 The actual test results of negative control are shown in Figure 2 As shown, only the detection situation of method three is consistent with the design idea and performs best. Therefore, the labeling and combination method of the colloidal gold-coated antibody, the first detection line and the second detection line-coated antibody of the nucleic acid dual-index rapid detection test strip based on the CRISPR system is determined to be method three.

[0042] The nucleic acid dual-index rapid detection test strip based on the CRISPR system of the present invention is as follows Figure 3 As shown, it includes a double-labeled conjugate pad 2 and a nitrocellulose membrane 3 provided with a quality control line 5, a first detection line 6 and a second detection line 7.

[0043] The double-labeled conjugate pad 2 is provided with a mouse antibody-labeled carrier conjugate and a rabbit antibody-labeled carrier conjugate. The mouse antibody-labeled carrier conjugate is a mouse anti-FITC antibody-labeled carrier conjugate. The rabbit antibody-labeled carrier conjugate is a rabbit anti-DIG monoclonal antibody-labeled carrier conjugate.

[0044] The label carrier is nano-gold. In other beneficial embodiments, nano-silver, nano-carbon, colored microspheres, fluorescent microspheres, etc. can also be used.

[0045] The quality control line 5, the second detection line 7 and the first detection line 6 are fixed on the nitrocellulose membrane 3 in sequence. The quality control line 5 is arranged at one end close to the double-label binding pad 2. The quality control line 5 is coated with SA 11, the first detection line 6 is coated with goat anti-mouse secondary antibody 18, and the second detection line 7 is coated with alpaca anti-rabbit anti-DIG nano monoclonal antibody 17.

[0046] The CRISPR system is provided with Cas12 protein and Cas13 protein, two kinds of labeling probes, and the two Cas proteins are respectively provided with different guide sequences that can target the molecules to be detected; the two kinds of labeling probes include a first labeling probe and a second labeling probe, and the labeling probes are formed by coupling a nucleic acid probe and a label, and when the first labeling probe contacts one of the activated Cas proteins, the first labeling probe is cut, and when the second labeling probe contacts another activated Cas protein, the second labeling probe is cut. The first labeling probe is formed by coupling a first nucleic acid probe with Biotin 8 and FITC 9, Biotin 8 is coupled to one end of the first nucleic acid probe, and FITC 9 is coupled to the other end of the first nucleic acid probe. The second labeling probe is formed by coupling a second nucleic acid probe with Biotin 8 and DIG 10, Biotin 8 is coupled to one end of the second nucleic acid probe, and DIG 10 is coupled to the other end of the second nucleic acid probe.

[0047] Example 2

[0048] The nucleic acid dual-index rapid test strip based on the CRISPR system was produced using the third method in Example 1. Among them, the test strips (test strips 1 to 6) detect whether the sample contains SARS-CoV-2 ORF1ab gene or N gene, and the negative quality control test strips (test strips 7 to 9) are used as quality control to determine whether the operation process is contaminated and false positive.

[0049] The SARS-CoV-2 virus CRISPR / Cas12a-RT RAA detection primers published by Yong Chen et al. (Yong Chen, Nan Zong, Feidi Ye, et al. Dual-CRISPR / Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection on Automated Centrifugal Microfluidics. Anal. Chem. 2022, 94, 27, 9603–9609.) were used to detect healthy human nasal swab samples mixed with pseudoviruses of different copies of SARS-CoV-2 ORF1ab gene and E gene, healthy human nasal swab samples and negative control samples.

[0050] The specific method of use is: dilute the pseudovirus containing SARS-CoV-2 ORF1ab gene and E gene of Shanghai Fubaiao Biotechnology to 2×10 6 copies / μL, and then serially diluted 10-fold to obtain 2×10 0 copies / μL, 2×10 1 copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 copies / μL and 2×10 5 The pseudovirus dilution with 100 copies / μL is used as the lysis template for subsequent nucleic acid extraction. The nasal swab samples of healthy people were added with different copies of pseudoviruses carrying SARS-CoV-2 ORF1ab gene and E gene of Shanghai Fubaiao Biotechnology. Cool Flash Nucleic Acid Releaser (BT0068) was used for extraction-free lysis, and 34.2 μL of lysis supernatant was drawn, corresponding to 1.4×10 0 Copy, 1.4×10 1 Copy, 1.4×10 2 Copy, 1.4×10 3 Copy, 1.4×10 4 Copy and 1.4×10 5 The copied pseudovirus lysate is used as the amplification template for the subsequent RT RAA. In a single eppendorf tube, the SARS-CoV-2ORF1ab gene (test line 1) or the E gene (test line 2) is amplified by RT RAA, and a negative control with the same volume of water as the template is set as a negative quality control. Among them, the test strip can determine whether the sample contains the SARS-CoV-2ORF1ab gene or the E gene. The negative quality control test strip is used as a quality control to determine that the operation process is free of contamination and false positives.

[0051] The crRNA and signal molecules used in the system are artificially synthesized, wherein the primer sequences are shown in SEQ ID No: 1-8:

[0052] The orf1ab gene RT-RAA forward primer 1 is:

[0053] 5'-GCAATAACAGTTACACCGGAAGCCAATATG-3';

[0054] orf1ab gene RT-RAA reverse primer 1 is:

[0055] 5'-ATCACAACTACAGCCATAACCTTTCCACAT-3';

[0056] orf1ab gene crRNA forward primer:

[0057] 5'-UAAUUUCUACUAAGUGUAGAUGUGGUGCAUCGUGUUGUCUGUAC-3'

[0058] orf1ab gene crRNA reverse primer:

[0059] 5'-UAAUUUCUACUAAGUGUAGAUUACAUACUUACCUUUUAAGUCAC-3'

[0060] Signal molecule Tag ssDNA1: 5'-6-FITC-TTTATT-Biotin-3'

[0061] The forward primer for RT-RAA of E gene is:

[0062] 5'-CGGAAGAGACAGGTACGTTAATAGTTAATAGC-3';

[0063] The reverse primer for RT-RAA of E gene is:

[0064] 5'-AGACCAGAAGATCAGGAACTCTAGAAGAAT-3';

[0065] E gene crRNA forward primer:

[0066] 5'-UAAUUUCUACUAAGUGUAGAUGUGGUAUUCUUGCUAGUUACACU-3'

[0067] E gene crRNA reverse primer:

[0068] 5'-UAAUUUCUACUAAGUGUAGAUCAAGACUCACGUUAACAAUAUUG-3'

[0069] Signal molecule Tag ssDNA2: 5'-DIG-TTTATT-Biotin-3'

[0070] RT-RAA amplification using RT RAA Nucleic Acid Amplification Kit (Basic Version) (JY0203) was used. The RT RAA amplification reaction system for a single sample is as follows:

[0071] Components Dosage (μL) Basal buffer 58.8 ORF1ab or E gene RT-RAA forward primer (40 μM) 1 ORF1ab or E gene RT-RAA reverse primer (40 μM) 1 Sample lysate supernatant or ultrapure water 34.2 Make up to volume with water 95

[0072] Open the reaction unit, add 5 μL of 280 mM MgAc2 to each 0.2 mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed by vortexing. Place the reaction tube at 39°C for 20 min.

[0073] After the reaction is completed, open the reaction tube and aspirate 4 μL of the product, add it to a new 0.2 mL eppendorf tube. The CRISPR / Cas12a-RT RAA cleavage reaction system for a single sample is as follows:

[0074] Components Dosage (μL) Buffer (10X) 2 ORF1ab or E gene RT-RAA forward crRNA (10 μM) 0.2 ORF1ab or E gene RT-RAA reverse crRNA (10 μM) 0.2 RT RAA amplification product 4 Cas12a nuclease 0.4 Signal molecule Tag ssDNA1 or Signal molecule Tag ssDNA2 0.8 Make up to volume with water 20

[0075] Mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken by a vortex shaker. Place the reaction tube at 39°C for 20 minutes.

[0076] After the enzyme cleavage reaction is completed and the cleavage products of different signal molecules are mixed, water is added to the mixed cleavage products to make up to 50μL to determine the test results. The test result of the negative quality control test strip must be negative, which is used to determine whether there is contamination in the operation process. The test result of the nasal swab sample amplification product test strip may be negative or positive, which is used to determine whether the sample contains the ORF1ab gene and / or E gene of SARS-CoV-2. When the number of SARS-CoV-2 copies in the sample is low, it may appear that after the lysate amplification, it may only show positive on test line 1 or test line 2. At this time, the test can be repeated to ensure the accuracy of the results.

[0077] The test results are as follows Figure 4As shown, the invented CRISPR system-based nucleic acid dual-indicator rapid test strip can accurately detect 400 copies / mL of pseudoviruses after amplification of the supernatant of lysate of healthy human nasal swab samples containing pseudoviruses of different copy numbers of SARS-CoV-2 ORF1ab gene and E gene, and CRISPR / Cas12a enzyme cleavage reaction of different signal molecules.

[0078] Example 3

[0079] In this embodiment, a pseudovirus with SARS-CoV-2orf1ab gene and N gene from Shanghai Fubai'ao Biotechnology was used, and SARS-CoV-2N gene Cas12 detection primers provided by Yangyang Sun et al. (2021) (Yangyang Sun, Lei Yu, Chengdu Liu, et al. One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR / Cas12a. J Transl Med. 2021Feb 16; 19(1): 74.) and SARS-CoV-2orf1ab gene Cas13a detection primers and systems provided by Arizti-Sanz J et al. (2020) (Jon Arizti-Sanz, Catherine A Freije, Alexandra C Stanton, et al. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun. 2020Nov 20; 11(1): 5921.) were used for amplification and cutting.

[0080] The crRNA and signal molecules used in the system are artificially synthesized, wherein the primer sequences are shown in SEQ ID No: 9-15:

[0081] orf1ab gene RT-RAA forward primer 2 was: 5′-GAAATTAATACGACTCACTATAGGGCCAAGGTAAACCTTTGGAATTTGGTGCCAC-3′;

[0082] orf1ab gene RT-RAA reverse primer 2 was: 5′-ACTATCATCATCTAACCAATCTTCTTCTTG-3′;

[0083] The crRNA of Cas13a of orf1ab gene is: 5′-CUCUUCUUCAGGUUGAAGAGCAGCAGAA-3′;

[0084] orf1ab FITC Reporter is: 5'-6-FITC-UUUUUUUUUUUUU-Biotin-3';

[0085] The forward primer for RT-RAA of the N gene was: 5′-CAGCAGTAGGGGAACTTCTCCTGCTAGAATGG-3′;

[0086] The reverse primer for RT-RAA of the N gene was: 5′-TGGCCTTTACCAGACATTTTGCTCTCAAGCTG-3′;

[0087] The Cas12 crRNA of the N gene is: 5′-GGGAAUUUCUACUGUUGUAGAUCUGCUGCUUGACAGAUUGA-3′;

[0088] N DIG Reporter is 5'-DIG-TTATTATT-Biotin-3';

[0089] RT-RAA amplification using RT RAA Nucleic Acid Amplification Kit (Basic Version) (JY0203) is used for testing. The test product is tested using this test strip. The reaction system for a single sample is as follows:

[0090] Components Dosage (μL) Basal buffer 58.8 ORF1ab RT-RAA forward primer (20 μM) 0.6 ORF1ab RT-RAA reverse primer (20 μM) 0.6 Cas13a enzyme premix 3 ORF1ab FITC Reporter (10μM) 1.6 RNase Inhibitor (40U / μL) 2 RNase H (5U / μL) 2 T7 RNA Polymerase (50U / μL) 2 ORF1ab Cas13a crRNA (2μM) 0.8 N RT-RAA forward primer (10 μM) 0.6 N RT-RAA reverse primer (10 μM) 0.6 N DIG Reporter (10μM) 1.6 Cas12 enzyme premix 3 N Cas12 crRNA (2 μM) 0.8 Sample lysate supernatant or ultrapure water 17 Make up to volume with water 95

[0091] Dilute the pseudovirus containing SARS-CoV-2 ORF1ab gene and E gene to 2×10 6 copies / μL, and then serially diluted 10-fold to obtain 2×10 0 copies / μL, 2×10 1 copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 copies / μL and 2×10 5 The pseudovirus dilution with 100 copies / μL is used as the lysis template for subsequent nucleic acid extraction. The nasal swab samples of healthy people were added with different copies of pseudoviruses carrying SARS-CoV-2 ORF1ab gene and E gene of Shanghai Fubaiao Biotechnology. Cool Flash Nucleic Acid Releaser (BT0068) was used for extraction-free lysis, and 17 μL of lysate supernatant was drawn, corresponding to 6.8×10 0 Copy, 6.8×101 Copy, 6.8×10 2 Copy, 6.8×10 3 Copy, 6.8×10 4 Copy and 6.8×10 5 The copied pseudovirus lysate is used as the amplification template for the subsequent RT RAA. In a single eppendorf tube, RT RAA amplification and Cas12 and Cas13a cleavage reactions are performed simultaneously on the SARS-CoV-2ORF1ab gene (test line 1) or the N gene (test line 2), and a negative control in which the template is replaced with the same volume of water is set as a negative quality control. Among them, the test strip can determine whether the sample contains the SARS-CoV-2ORF1ab gene or the N gene, and the negative quality control test strip is used as a quality control to determine whether the operation process is contaminant-free and false positive.

[0092] The order of adding samples is negative quality control sample (the supernatant of sample lysate is replaced with the same volume of ultrapure water), nasal swab sample lysate supernatant. After each sample is added, the tube cap must be fastened immediately to avoid aerosol contamination. Mix the above reaction system, add the basic reaction unit, and fully dissolve the lyophilized powder. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator. Open the reaction unit, add 5μL 280mM MgAc2 to each 0.2mL eppendorf tube, mix thoroughly and collect by centrifugation. Note that this step cannot be vigorously shaken and mixed using a vortex oscillator.

[0093] The reaction tube was placed at 39°C for 60 minutes. After the reaction was completed, the eppendorf tube was opened, 5 μL of the amplified product was transferred to a new eppendorf tube, marked, and diluted 15 times before detection.

[0094] The test results are as follows Figure 5 As shown in the figure, through the test results of nasal swabs with different concentrations and without added SARS-CoV-2 pseudovirus, this test strip can accurately detect 400 copies / mL of pseudovirus.

[0095] It can be seen from the above examples that the scheme of the present invention is a lateral flow test strip that can simultaneously detect two different Cas enzyme cleavage products. The test strip can effectively perform dual-target visual detection based on the CRISPR system.

[0096] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0097] The invention shown and described herein can be realized in the absence of any reagents, restrictions specifically disclosed herein. The terms and expressions used are used as illustrative terms rather than restrictions, and it is not desired to exclude any equivalents of the features shown and described or parts thereof in the use of these terms and expressions, and it should be recognized that various modifications are feasible within the scope of the present invention. It should therefore be understood that, although the present invention is specifically disclosed by various embodiments and optional features, modifications and variations of the concepts described herein can be adopted by those of ordinary skill in the art, and it is believed that these modifications and variations fall within the scope of the present invention as defined by the appended claims.

[0098] The contents of the articles, patents, patent applications, and all other documents and electronically available information described or recorded herein are incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such article, patent, patent application, or other document.

Claims

1. A nucleic acid dual-index rapid test strip based on the CRISPR system, characterized in that: The test strip comprises a double-labeled conjugate pad and a nitrocellulose membrane provided with a quality control line, a first detection line and a second detection line; a mouse antibody-labeled carrier conjugate and a rabbit antibody-labeled carrier conjugate are provided on the double-labeled conjugate pad; the mouse antibody-labeled carrier conjugate comprises a mouse anti-FITC antibody-labeled carrier conjugate; the rabbit antibody-labeled carrier conjugate comprises a rabbit anti-DIG antibody-labeled carrier conjugate; The CRISPR system is provided with two Cas proteins and two labeling probes; the two labeling probes are a first labeling probe and a second labeling probe, and the labeling probes are formed by coupling a nucleic acid probe and a label; the first labeling probe is formed by coupling a first nucleic acid probe with Biotin and FITC, wherein Biotin is coupled to one end of the first nucleic acid probe and FITC is coupled to the other end of the first nucleic acid probe; the second labeling probe is formed by coupling a second nucleic acid probe with Biotin and DIG, wherein Biotin is coupled to one end of the second nucleic acid probe and DIG is coupled to the other end of the second nucleic acid probe; The labeling carrier is at least one of nano-gold, nano-silver, nano-carbon, colored microspheres and fluorescent microspheres; The quality control line, the second detection line and the first detection line are fixed on the nitrocellulose membrane in sequence, the quality control line is arranged at one end close to the binding pad, the quality control line is coated with SA, the first detection line is coated with goat anti-mouse secondary antibody, and the second detection line is coated with alpaca anti-rabbit anti-DIG nano monoclonal antibody.

2. The nucleic acid dual-index rapid test strip based on the CRISPR system according to claim 1, characterized in that: The two Cas proteins are respectively provided with different guide sequences that can target the molecules to be detected.

3. The working method of the nucleic acid dual-index rapid detection test strip according to claim 1 or 2, characterized in that: When the molecule to be detected does not exist in the test sample, the two Cas proteins cannot be activated by the two substances to be detected, the dual-labeled probe cannot be cut, and the mouse anti-FITC antibody-labeled carrier conjugate and the rabbit anti-DIG antibody-labeled carrier conjugate on the dual-labeled binding pad can respectively recognize FITC and DIG on the dual-labeled probe and combine to form a complete probe-labeled carrier complex, and run on the nitrocellulose membrane through chromatography. When passing the quality control line, the complete probe-labeled carrier complex is captured by the SA coated on the quality control line and cannot continue to chromatograph to the first detection line and the second detection line, and cannot form a visual signal in the detection line area, indicating that the corresponding molecule to be detected does not exist in the sample; When there are two molecules to be detected in the sample to be detected, the two Cas proteins can be activated by the two substances to be detected, thereby realizing the cutting of the dual-labeled probe. The mouse anti-FITC antibody-labeled carrier conjugate and the rabbit anti-DIG antibody-labeled carrier conjugate on the dual-labeled binding pad can respectively recognize FITC and DIG on the dual-labeled probe and combine to form a cutting probe-labeled carrier complex, and run on the nitrocellulose membrane through chromatography. When passing through the first detection line and the second detection line, the cutting probe-labeled carrier complex is captured by the sheep anti-mouse secondary antibody and the alpaca anti-rabbit anti-DIG nano-monoclonal antibody coated on the first detection line and the second detection line, respectively, and is gradually enriched to form a visible signal, indicating the presence of the corresponding molecules to be detected in the sample.

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