CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of H1N1 influenza virus and mycoplasma pneumoniae and detection method thereof

Abstract

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of H1N1 influenza virus and mycoplasma pneumoniae and a detection method thereof. The kit and the detection method provided by the invention can be used for timely, sensitively and specifically detecting a nucleic acid target in a sample. The invention provides a lateral flow analysis (LFA) detection platform of a three-stage amplification system integrating recombinase polymerase amplification (RPA), CRISPR / Cas12a and catalytic hairpin assembly (CHA) circulation, the detection platform is used for sensitively and accurately detecting mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1RNA (H1N1RNA), and compared with other researches based on an LFA-CRISPR / Cas12a system, the detection platform has the advantages that the detection efficiency is greatly improved, and the detection cost is reduced. According to the kit and the detection method provided by the invention, nucleic acids of two pathogens can be visually detected on a single LFA at the same time, and the sensitivity is high.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a CRISPR / Cas-based isothermal rapid visual detection kit for H1N1 influenza virus and Mycoplasma pneumoniae and a detection method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Respiratory pathogens such as influenza virus and mycoplasma mostly cause infection symptoms such as fever, cough, swelling and pain, and headache. Their clinical manifestations are similar to those caused by viral and bacterial infections, with acute onset, rapid progression, and atypical symptoms and signs. Therefore, differential diagnosis of respiratory pathogens can quickly identify the pathogens causing respiratory symptoms, allowing doctors to develop targeted treatment plans and health management measures, cure the disease as soon as possible, and avoid excessive use of antibiotics.

[0004] Currently widely used immunological and serological detection methods have problems such as low sensitivity and long detection windows, making them unsuitable for early detection of respiratory pathogens. While nucleic acid detection based on real-time quantitative polymerase chain reaction (qPCR) has excellent sensitivity and specificity, its requirement for specialized equipment, thermal cycling, and trained personnel limits its application in on-site analysis. Therefore, there is an urgent need to develop an alternative detection method for on-site nucleic acid detection of respiratory pathogens with high sensitivity and rapidity to promote medication guidance and large-scale screening to prevent disease transmission.

[0005] Recombinase polymerase amplification (RPA) is a technique for isothermal amplification of target genes, using recombinase and polymerase as core components and single-stranded binding proteins as auxiliary components. It can rapidly amplify target fragments in a short period of time at a constant temperature (37-42°C). The target bands are then separated and detected by conventional gel electrophoresis. It can also be combined with lateral flow chromatography strips and fluorescent probes for detection. The CRISPR / Cas system, composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), can recognize and cleave specific nucleic acid sequences, further increasing the specificity and sensitivity of RPA. The CRISPR / Cas system has been developed as a rapid, portable, low-cost, and highly sensitive molecular detection tool, achieving significant breakthroughs in pathogen detection. Since the optimal temperature for Cas to perform its nuclease function is close to the reaction temperature of RPA, the two can be used in combination, that is, RPA is responsible for isothermal amplification of the target sequence to increase the copy number of the target sequence, and Cas is responsible for specific identification of the target sequence and relies on fluorescent reporter probes to visualize the detection results. However, although the above two methods can complete nucleic acid amplification under constant temperature conditions, in order to achieve highly sensitive nucleic acid detection and analysis, they still require the use of instruments such as fluorimeter and electrochemical workstation. At present, there is still a lack of easy-to-operate and highly sensitive visual detection and analysis tools. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a CRISPR / Cas-based isothermal rapid visual detection kit for H1N1 influenza virus and Mycoplasma pneumoniae and a detection method thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a CRISPR / Cas-based isothermal rapid visualization composition for detecting H1N1 influenza virus and Mycoplasma pneumoniae, comprising:

[0009] Compositions for visually detecting H1N1 influenza virus and compositions for visually detecting Mycoplasma pneumoniae;

[0010] Among them, the composition for visual detection of H1N1 influenza virus includes:

[0011] The first RPA front primer, the sequence of which is shown in SEQ ID NO.1;

[0012] The first post-RPA primer, the sequence of which is shown in SEQ ID NO. 2;

[0013] The first CRISPR RNA, the sequence of which is shown in SEQ ID NO. 3;

[0014] The first blocking strand for blocking hairpin assembly, the sequence of which is shown in SEQ ID NO.4;

[0015] The first initiator strand that catalyzes hairpin assembly; the sequence is shown in SEQ ID NO.5;

[0016] The first probe of the hairpin assembly had the sequence FAM-AAACTCAGCTTGTCAAATATGGTGTGATTTGACAAGCT;

[0017] The second probe was assembled by hairpin assembly, and the sequence was Biotin-ACTTGTCAAATCACACCATATTTGACAAGCATGGTGTGATTT;

[0018] The composition for visually detecting Mycoplasma pneumoniae comprises:

[0019] The second RPA front primer, the sequence of which is shown in SEQ ID NO.6;

[0020] The second post-RPA primer, the sequence of which is shown in SEQ ID NO.7;

[0021] A second CRISPR RNA, the sequence of which is shown in SEQ ID NO. 8;

[0022] A second blocking strand for blocking hairpin assembly, the sequence of which is shown in SEQ ID NO.9;

[0023] A second priming strand that catalyzes hairpin assembly, the sequence of which is shown in SEQ ID NO. 10;

[0024] The third probe for hairpin assembly had the sequence TAMRA-AAAAAAATACAATAGCAAACACCATAGGTGTGAGTGGTGTTTGC;

[0025] The fourth probe was assembled by hairpin assembly, and the sequence was Dig-AAAAAAACACCACTCACACCTATGGTGTTTGGGTGTGAGTG.

[0026] The second aspect of the present invention provides the use of the CRISPR / Cas-based isothermal rapid visual detection composition of H1N1 influenza virus and Mycoplasma pneumoniae according to the first aspect in any of the following applications:

[0027] A1) Detection of H1N1 influenza virus and Mycoplasma pneumoniae;

[0028] A2) Prepare products for detecting H1N1 influenza virus and Mycoplasma pneumoniae.

[0029] In one or more embodiments, the article of manufacture comprises a kit.

[0030] A third aspect of the present invention provides a kit for visually detecting H1N1 influenza virus and Mycoplasma pneumoniae, wherein the kit comprises the CRISPR / Cas-based isothermal rapid visual detection composition for H1N1 influenza virus and Mycoplasma pneumoniae according to the first aspect.

[0031] In one or more embodiments, the kit further comprises a lateral flow test paper, which is provided with a conjugation pad, a T1 line, a T2 line, a quality control C line and an absorption pad in sequence along the transmission direction, wherein the conjugation pad comprises a first specific antibody and a second specific antibody, the first specific antibody is colloidal gold-labeled streptavidin; the second specific antibody is a colloidal gold-labeled anti-TAMRA antibody; the T1 line is coated with an anti-FAM antibody; and the T2 line is coated with an anti-Dig antibody.

[0032] In one or more embodiments, the kit further comprises reagents for recombinase polymerase amplification and Cas12a nuclease;

[0033] Preferably, the reagents for recombinase polymerase amplification include reaction buffer, magnesium acetate, water and lyophilized powder.

[0034] A fourth aspect of the present invention provides a method for visually detecting H1N1 influenza virus and Mycoplasma pneumoniae, comprising visually detecting H1N1 influenza virus and visually detecting Mycoplasma pneumoniae;

[0035] Among them, the method of visually detecting H1N1 influenza virus includes:

[0036] Amplifying the target RNA using a first RPA front primer and a first RPA rear primer to obtain a first amplification product;

[0037] The first amplified product is sequentially incubated with the first CRISPR RNA and the first blocking strand for blocking hairpin assembly for a first time, incubated with the first priming strand for catalyzing hairpin assembly for a second time, and incubated with the first hairpin assembly probe and the second hairpin assembly probe for a third time to obtain an incubated product;

[0038] Immerse the conjugate pad of the lateral flow test paper in the incubated product and read the result after 4 to 6 minutes;

[0039] If the T1 line is red and the quality control C line is blue, it is a positive result; if only the quality control C line is blue, it is a negative result; if the quality control C line does not show color, it indicates that the experiment has failed;

[0040] Visual methods for detecting Mycoplasma pneumoniae include:

[0041] Amplifying the target DNA using a second RPA front primer and a second RPA rear primer to obtain a second amplification product;

[0042] The second amplified product is sequentially incubated with the second CRISPR RNA and the second blocking strand for blocking hairpin assembly for a first time, incubated with the second priming strand for catalyzing hairpin assembly for a second time, and incubated with the third hairpin assembly probe and the fourth hairpin assembly probe for a third time to obtain an incubated product;

[0043] Immerse the conjugate pad of the lateral flow test paper in the incubated product and read the result after 4 to 6 minutes;

[0044] If the T2 line is red and the quality control C line is blue, it is a positive result; if only the quality control C line is blue, it is a negative result; if the quality control C line does not show color, it indicates that the experiment has failed.

[0045] In one or more embodiments, in the method for visually detecting H1N1 influenza virus,

[0046] The first incubation condition is 36-40°C for 28-40 min, preferably 37°C for 30 min.

[0047] In one or more embodiments, in the method for visually detecting H1N1 influenza virus,

[0048] The second incubation condition is 36-40° C. for 8-12 min, preferably 37° C. for 10 min.

[0049] In one or more embodiments, in the method for visually detecting H1N1 influenza virus,

[0050] The third incubation condition is 36-40° C. for 4-6 min, preferably 37° C. for 5 min.

[0051] In one or more embodiments, in the method for visually detecting Mycoplasma pneumoniae,

[0052] The first incubation condition is 36-40°C for 28-40 min, preferably 37°C for 30 min.

[0053] In one or more embodiments, in the method for visually detecting Mycoplasma pneumoniae,

[0054] The second incubation condition is 36-40° C. for 8-12 min, preferably 37° C. for 10 min.

[0055] In one or more embodiments, in the method for visually detecting Mycoplasma pneumoniae,

[0056] The third incubation condition is 36-40° C. for 4-6 min, preferably 37° C. for 5 min.

[0057] The beneficial effects of the present invention are:

[0058] (1) The present invention provides a CRISPR / Cas-based isothermal rapid visual detection kit for H1N1 influenza virus and Mycoplasma pneumoniae and a detection method thereof. The kit and detection method provided by the present invention can detect nucleic acid targets in samples in a timely, sensitive and specific manner. The present invention provides a lateral flow analysis (LFA) detection platform that integrates a three-stage amplification system of recombinase polymerase amplification (RPA), CRISPR / Cas12a and catalytic hairpin assembly (CHA) cycles for sensitive and accurate detection of Mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1 RNA (H1N1RNA). Compared with other studies based on the LFA-CRISPR / Cas12a system, the kit and detection method provided by the present invention can simultaneously visually detect two pathogen nucleic acids on a single LFA with high sensitivity.

[0059] (2) Mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1 RNA (H1N1RNA) were amplified by RPA to produce a large number of amplified products; the amplified products can activate the trans-cleavage activity of CRISPR / Cas12a, and the activated Cas12a can cut the blocking strand (BS) that blocks the hairpin assembly, blocking the hybridization of the blocking strand (BS) and the initiating strand (IS) that catalyzes the hairpin assembly. Under the catalysis of the initiating strand (IS) that catalyzes the hairpin assembly, the hairpin assembly probes combine to form a hybrid duplex; in the detection of influenza A virus subtype H1N1 RNA (H1N1 When detecting RNA), the free initiator chain IS-1 catalyzes the cross-hybridization of the hairpin assembly first probe FAM-H1 and the hairpin assembly second probe Bio-H2 through the CHA-1 cycle, producing a large number of hybrid duplexes FAM-H1 / Bio-H2. These hybrid duplexes will bind to the colloidal gold-labeled streptavidin in the binding pad of the lateral flow test paper and are further captured by the anti-FAM antibody coated on the T1 line, producing a visible red T1 line band; when detecting Mycoplasma pneumoniae DNA (MPDNA), the free initiator chain IS-2 catalyzes the hairpin assembly third probe TAMRA-H3 and the hairpin assembly fourth probe Dig-H4 through the CHA-2 cycle. Cross-hybridization produces a large number of hybrid duplexes TAMRA-H3 / Dig-H4, which bind to the colloidal gold-labeled anti-TAMRA antibody in the binding pad of the lateral flow test paper and are further captured by the anti-Dig antibody coated on the T2 line, producing a visible red T2 line band; on the contrary, if the target is lacking, RPA amplification will not occur, inhibiting the trans-cleavage activity of CRISPR / Cas12a, thereby maintaining the integrity of the blocking strand (BS) that blocks hairpin assembly. The hybridization of the blocking strand (BS) with the initiating strand (IS) that catalyzes hairpin assembly prevents the CHA cycle from generating a signal, and only a blue quality control C line can be observed on the lateral flow test paper. The detection method provided by the present invention successfully detects H1N1 RNA and MP DNA on a single lateral flow strip through multiple isothermal amplifications, with high accuracy and specificity, providing convenience for on-site deployable pathogen detection.

[0060] (3) The detection method provided by the present invention does not require the use of any large-scale instruments or equipment in the entire process from nucleic acid amplification to signal detection, and the signal can be visualized and analyzed by the naked eye. At the same time, the detection method is easy to operate and time-saving, which is helpful for the early detection and monitoring of pathogens and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0062] Figure 1 To visualize the process of detecting H1N1 influenza virus and Mycoplasma pneumoniae;

[0063] Figure 2 This is a gel image of nucleic acid target RPA amplification, where channel 1 is the H1N1 RNA amplification product and channel 2 is the MPDNA amplification product;

[0064] Figure 3 The fluorescence results of CRISPR cleavage activity activated by amplification products are shown in Figure 5. A is the fluorescence curve of the CRISPR / Cas12a-1 system corresponding to H1N1 RNA amplicon activation, B is the quantification of A, C is the fluorescence curve of the CRISPR / Cas12a-2 system corresponding to MP DNA amplicon activation, and D is the quantification of C. The vertical axes in A, B, C, and D are all normalized fluorescence intensities.

[0065] Figure 4 Gel electrophoresis diagram of target activation of the CRISPR system and subsequent CHA cycle initiation, where A and B are the gel electrophoresis results of H1N1 RNA amplicon and MP DNA activation of subsequent CRISPR and CHA amplification, respectively;

[0066] Figure 5 The feasibility results of the lateral flow test strip in detecting target nucleic acid through the detection system are shown in Figures a to d, respectively, for the results of no target, MP DNA alone, H1N1 RNA alone, and a mixture of MP DNA and H1N1 RNA;

[0067] Figure 6 A diagram showing the test strip detecting target nucleic acids at different concentrations;

[0068] Figure 7 The results of quantification of T1 and T2 strips on lateral flow test strips are shown in Figure 1, where A is the linear relationship between T1 signal and H1N1 RNA concentration, and B is the linear relationship between T2 signal and MP DNA concentration.

[0069] Figure 8 are the results of the saliva anti-interference test, where A is the feasibility of the lateral flow test paper to detect the target nucleic acid in saliva; B is the result of the lateral flow test paper detecting different concentrations of targets in saliva. DETAILED DESCRIPTION

[0070] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0071] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0072] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0073] Materials: CRISPR RNA (crRNA) and DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co., Ltd.

[0074] RNA isothermal rapid amplification kit was purchased from Amp Future, catalog number: WLRB8207KIT;

[0075] DNA isothermal rapid amplification kit was purchased from Amp Future, catalog number: WLB8201KIT;

[0076] Cas12a nuclease was purchased from Suzhou Jinan Protein Technology Co., Ltd., catalog number: E373-YH01;

[0077] Aqueous solutions for other experiments were prepared using deionized water (18.2 MΩ·cm);

[0078] Double-labeled nucleic acid test strips were purchased from Tiosbio, catalog number JY0209.

[0079] Figure 1To visualize the process of detecting H1N1 influenza virus and Mycoplasma pneumoniae, Mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1 RNA (H1N1 RNA) were amplified by RPA to produce a large number of amplified products; the amplified products can activate the trans-cleavage activity of CRISPR / Cas12a, and the activated Cas12a can cut the blocking strand (BS) that blocks hairpin assembly, blocking the hybridization of the blocking strand (BS) and the initiating strand (IS) that catalyzes hairpin assembly. Under the catalysis of the initiating strand (IS) that catalyzes hairpin assembly, the hairpin assembly probes bind to form a hybrid duplex; in the detection of influenza A virus subtype H1N1 RNA (H1N1 When RNA is detected, the free initiator chain IS-1 catalyzes the cross-hybridization of the hairpin assembly probe FAM-H1 labeled H1 (FAM-H1) and the hairpin assembly probe biotin labeled H2 (Bio-H2) through the CHA-1 cycle, generating a large number of hybrid duplexes (FAM-H1 / Bio-H2). These hybrid duplexes will bind to the colloidal gold-labeled streptavidin in the binding pad of the lateral flow test paper and are further captured by the anti-FAM antibody coated on the T1 line, generating a visible red T1 line band; in the detection of Mycoplasma pneumoniae DNA (MP When the target is present, the free priming strand IS-2 catalyzes the cross-hybridization of the hairpin assembly probe TAMRA-labeled H3 (TAMRA-H3) and the hairpin assembly probe digoxigenin-labeled H4 (Dig-H4) through the CHA-2 cycle, producing a large number of hybrid duplexes (TAMRA-H3 / Dig-H4). These hybrid duplexes will bind to the colloidal gold-labeled anti-TAMRA antibody in the binding pad of the lateral flow test strip and be further captured by the anti-Dig antibody coated on the T2 line, producing a visible red T2 line band; on the contrary, if the target is absent, RPA amplification will not occur, inhibiting the trans-cleavage activity of CRISPR-Cas12a, thereby maintaining the integrity of the blocking strand (BS) that blocks hairpin assembly. The hybridization of the blocking strand (BS) and the priming strand (IS) that catalyzes hairpin assembly prevents the CHA cycle from generating signals, and only the blue quality control C line can be observed on the lateral flow test strip.

[0080] In Examples 1 and 2, appropriate composition sequences were designed based on the target nucleic acid partial sequences of influenza A virus subtype H1N1 RNA (H1N1 RNA) and Mycoplasma pneumoniae DNA (MP DNA).

[0081] Example 1

[0082] To verify the feasibility of detecting influenza A virus subtype H1N1 RNA (H1N1 RNA):

[0083] (1) RPA amplification: 29.4 μL of buffer A was added to a centrifuge tube containing lyophilized reagents, and the first RPA front primer (2 μL, 10 μM), the first RPA back primer (2 μL, 10 μM), the H1N1 RNA target (nucleic acid sequence as shown in SEQ ID NO. 11), and ddH2O (9.1 μL) were added to the above centrifuge tube; then, buffer B (magnesium acetate: 2.5 μL, 280 mM) was added to initiate the reaction; the reaction mixture was incubated at 37°C for 30 min, and the first amplification product was stored at -20°C until use.

[0084] Figure 2 This is a gel imaging image of nucleic acid target RPA amplification, where channel 1 is the H1N1 RNA amplification product, proving that RPA amplification was successful.

[0085] (2) Confirm that the amplified product can activate CRISPR / Cas12a activity:

[0086] Fluorescence experiment of CRISPR / Cas12a-1 activation after H1N1 RNA amplification: 1 μL of the first CRISPR RNA was pre-mixed with 1 μL of Cas12a nuclease; then 14 μL of Tris-HCl buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 20 mM MgCl2, 0.5% Tween 20) was added; then the first amplification product and the fluorescent probe FQ (2 μL, 2 μM, single-stranded DNA modified with FAM at one end and a quencher at the other end, sequence 5-FAM-TTATT-BHQ-3) were added, mixed and incubated at 37°C for 30 minutes, and then 200 μL of ddH2O was added for fluorescence detection.

[0087] Figure 3 The fluorescence curve of the CRISPR / Cas12a-1 system activated by the amplified product is shown in Figure 2, where A is the fluorescence curve of the CRISPR / Cas12a-1 system activated by the H1N1 RNA amplicon, and B is the quantification of A. Figure 2 As can be seen from A and B, the amplified product can activate CRISPR / Cas12a-1 activity.

[0088] (3) Gel electrophoresis experiment of blocking chain inhibiting CHA cycle:

[0089] For the detection of H1N1 RNA, the first blocking strand BS-1 (1 μL, 1 μM) that blocks hairpin assembly was incubated with the first initiating strand IS-1 (1 μL, 1 μM) that catalyzes hairpin assembly at room temperature for 10 minutes; then the first probe FAM-H1 (2 μL, 2 μL) that catalyzes hairpin assembly in a CHA-1 cycle and the second probe Biotin-H2 (2 μL, 2 μM) that catalyzes hairpin assembly were added to the above solution, reacted for 5 minutes, and finally the solution was analyzed by electrophoresis.

[0090] The results are as follows Figure 4 As shown in A,

[0091] Channel 1: FAM-H1 and Biotin-H2. At this point, the two hairpins remain stable, with no hybridization bands. Channel 2: FAM-H1, Biotin-H2, and IS-1. IS-1 can catalyze the CHA-1 cycle, producing a hybridization band between FAM-H1 and Biotin-H2 (a new band can be seen on the gel electrophoresis). Channel 3: BS-1 is added. BS-1 and IS-1 hybridize, but there is no free IS-1 to catalyze the hybridization between FAM-H1 and Biotin-H2. Therefore, a hybridization band between BS-1 and IS-1 is produced, but no FAM-H1 and Biotin-H2 hybridization band is produced. Channel 4: The corresponding CRISPR system is then introduced. At this point, the CRISPR system is inactive and cannot cleave BS-1. BS-1 and IS-1 hybridize, producing a hybridization band between them, but no FAM-H1 and Biotin-H2 hybridization band is produced. Channel 5: The target is introduced, activating CRISPR, but without the priming strand IS-1, FAM-H1 and Biotin-H2 hybridization bands are still unavailable. Channel 6: Validation of the entire process: When the target is present, the CRISPR system's cleavage activity is activated, cleaving BS-1. Adding IS-1 at this point activates FAM-H1 and Biotin-H2 hybridization, generating hybridization bands. In short: the blocking strand BS-1 successfully blocks IS-1. Furthermore, when the target is present, CRISPR is activated, cleaving BS-1, causing BS-1 blocking to fail. Therefore, free IS-1 can trigger the CHA-1 cycle, generating hybridization bands for FAM-H1 and Biotin-H2.

[0092] (4) Feasibility of lateral flow test strips for detecting target nucleic acids through detection systems

[0093] The RPA product of H1N1 RNA (2 μL) was mixed with the corresponding CRISPR / Cas12a-1 system (2 μL) and BS-1 (1 μM, 1 μL) and incubated at 37°C for 30 minutes. The corresponding initiator chain (IS-1) was then added to the above solution and reacted for 10 minutes. Subsequently, hairpin DNA capable of CHA cycling (FAM-H1 (2 μL, 2 μM) and Biotin-H2 (2 μL, 2 μM) were added and reacted for 5 minutes. After the reaction, the two were mixed and 1× PBS buffer (60 μL) was added. The binding pad of the lateral flow test paper was immersed in the incubated solution for 5 minutes, and the test results could be read visually.

[0094] The results are as follows Figure 5 As shown, Figure 1 a to d are the results of no target, MP DNA alone, H1N1 RNA alone, and a mixture of MP DNA and H1N1 RNA, respectively.

[0095] From the experimental results in this example, it can be seen that the lateral flow analysis (LFA) detection platform designed in this example, which integrates a three-stage amplification system of recombinase polymerase amplification (RPA), CRISPR / Cas12a, and catalytic hairpin assembly (CHA) cycles, can be used to detect influenza A virus subtype H1N1 RNA (H1N1 RNA).

[0096] Example 2

[0097] Verify the feasibility of detecting Mycoplasma pneumoniae DNA (MP DNA):

[0098] (1) RPA amplification: 29.4 μL of buffer A was added to a centrifuge tube containing lyophilized reagents, and the second RPA front primer (2 μL, 10 μM), the second RPA back primer (2 μL, 10 μM), the MP DNA target double strand (the nucleic acid sequence is shown in SEQ ID NO.12, and the nucleic acid sequence of the complementary strand is shown in SEQ ID NO.13) and ddH2O (9.1 μL) were added to the above centrifuge tube; then, buffer B (magnesium acetate: 2.5 μL, 280 mM) was added to initiate the reaction; the reaction mixture was incubated at 37°C for 30 min, and the second amplification product was stored at -20°C for later use.

[0099] Figure 2 This is a gel imaging image of RPA amplification of nucleic acid targets, where channel 2 is the MP DNA amplification product, proving that RPA amplification was successful.

[0100] (2) Confirm that the amplified product can activate CRISPR / Cas12a activity:

[0101] Fluorescence experiment of CRISPR / Cas12a-1 activation after MP DNA amplification: 1 μL of the second CRISPR RNA was pre-mixed with 1 μL of Cas12a nuclease; then 14 μL of Tris-HCl buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 20 mM MgCl2, 0.5% Tween 20) was added; then the second amplification product and the fluorescent probe FQ (2 μL, 2 μM, single-stranded DNA modified with FAM at one end and a quencher at the other end, sequence 5-FAM-TTATT-BHQ-3) were added, mixed and incubated at 37 ° C for 30 min, and then 200 μL of ddH2O was added for fluorescence detection.

[0102] Figure 3 The fluorescence curve of the CRISPR / Cas12a-2 system activated by the MP DNA amplicon is shown in Figure 2, and D is the quantification of C. Figure 2 As shown in C and D, the amplified product can activate CRISPR / Cas12a-2 activity.

[0103] (3) Gel electrophoresis experiment of blocking chain inhibiting CHA cycle:

[0104] For the detection of MP DNA, the second blocking chain BS-2 (1 μL, 1 μM) that blocks hairpin assembly was incubated with the second initiating chain IS-2 (1 μL, 1 μM) that catalyzes hairpin assembly at room temperature for 10 minutes; then the third probe TAMRA-H3 (2 μL, 2 μL) that catalyzes hairpin assembly in a cycle of CHA-2 and the fourth probe Dig-H4 (2 μL, 2 μM) that catalyzes hairpin assembly were added to the above solution, reacted for 5 minutes, and finally the solution was analyzed by electrophoresis.

[0105] The results are as follows Figure 4As shown in Figure B, channel 1 shows TAMRA-H3 and Dig-H4. At this point, the two hairpins remain stable, with no hybridization bands. Channel 2 shows TAMRA-H3, Dig-H4, and IS-2. IS-2 can catalyze the CHA-2 cycle, producing a hybridization band between TAMRA-H3 and Dig-H4 (a new band appears on the gel electrophoresis). Channel 3 shows the addition of BS-2. BS-2 and IS-2 hybridize, but there is no free IS-2 to catalyze the hybridization between TAMRA-H3 and Dig-H4. Therefore, a hybridization band between BS-2 and IS-2 is produced, with no TAMRA-H3 and Dig-H4 hybridization bands. Channel 4 shows the introduction of the corresponding CRISPR system. At this point, the CRISPR system is inactive, so it cannot cleave BS-2. BS-2 and IS-2 hybridize, producing a hybridization band between them, with no TAMRA-H3 and Dig-H4 hybridization bands. Channel 5: The target is introduced, activating CRISPR, but without the triggering strand IS-2, TAMRA-H3 and Dig-H4 hybridization bands are still unavailable. Channel 6: Validation of the entire process: When the target is present, the CRISPR system's cleavage activity is activated, cleaving BS-2. At this point, IS-2 is added, and IS-2 activates TAMRA-H3 and Dig-H4 hybridization, generating hybridization bands. In short: the blocking strand BS-2 successfully blocks IS-2, and when the target is present, CRISPR is activated, which cleaves BS-2, causing BS-2 blocking to fail. Therefore, free IS-2 can trigger the CHA-2 cycle, generating hybridization bands for TAMRA-H3 and Dig-H4.

[0106] (4) Feasibility of lateral flow test strips for detecting target nucleic acids

[0107] The RPA product of MP DNA (2 μL) was mixed with the corresponding CRISPR / Cas12a-2 system (2 μL) and BS-2 (1 μM, 1 μL) and incubated at 37°C for 30 min. The corresponding initiator chain (IS-2) was then added to the above solution and reacted for 10 min. Subsequently, hairpin DNAs (TAMRA-H3 (2 μL, 2 μM) and Dig-H4 (2 μL, 2 μM) capable of CHA cycling were added and reacted for 5 min. After the reaction, the two were mixed and 1× PBS buffer (60 μL) was added. The binding pad of the lateral flow test paper was immersed in the incubated solution for 5 min, and the test results were visually read.

[0108] The results are as follows Figure 5 As shown, Figure 1 a to d are the results of no target, MP DNA alone, H1N1 RNA alone, and a mixture of MP DNA and H1N1 RNA, respectively.

[0109] From the experimental results in this example, it can be seen that the lateral flow analysis (LFA) detection platform designed in this example, which integrates a three-stage amplification system of recombinase polymerase amplification (RPA), CRISPR / Cas12a, and catalytic hairpin assembly (CHA) cycles, can be used to detect Mycoplasma pneumoniae DNA (MP DNA).

[0110] Example 3

[0111] Lateral flow test strips detect targets at different concentrations:

[0112] Targets of varying concentrations were prepared and amplified by RPA. For H1N1 RNA detection, the H1N1 RNA RPA product (2 μL) was mixed with the corresponding CRISPR / Cas12a-1 system (2 μL) and BS-1 (1 μM, 1 μL) and incubated at 37°C for 30 minutes. The corresponding initiator strand (IS-1) was then added to the above solution and reacted for 10 minutes. Subsequently, hairpin DNA capable of undergoing CHA cycling (FAM-H1 (2 μL, 2 μM) and Biotin-H2 (2 μL, 2 μM) were added and reacted for 5 minutes. The MP DNA nucleic acid detection process was consistent with the above. After the reaction, the solutions were mixed and 1× PBS buffer (60 μL) was added. A test strip was inserted into the final solution, and the test results were visually read after 5 minutes.

[0113] The results are as follows Figure 6 As shown, the results showed that the color intensity of TL1 and TL2 gradually increased with the increase of H1N1 RNA and MP DNA concentrations, indicating that this strategy can sense the changes in target concentration and independently detect different types of targets.

[0114] Example 4

[0115] The T1 and T2 bands of the lateral flow test paper were quantified, and the results were as follows: Figure 7 As shown in the figure, it can be seen that in the range of 0.1 to 1000 fM, the target concentration is linearly correlated with the TL signal. According to the linear relationship, the detection limits of this detection method for H1N1 RNA and MP DNA are 10 aM and 25 aM, respectively.

[0116] Example 5

[0117] In order to verify the anti-interference ability of the CRISPR / Cas-based isothermal rapid visualization method for detecting H1N1 influenza virus and Mycoplasma pneumoniae, the target nucleic acid doped with saliva was detected. Under the optimal reaction conditions (same as the feasibility of the lateral flow test paper in Examples 1 and 2 (4) to detect the target nucleic acid through the detection system), the visualization detection results are as follows: Figure 8As shown, saliva does not affect the detection performance of the test strip, and the red bands of T1 and T2 gradually increase with the increase of the corresponding target concentration, which is consistent with the experimental results in PBS buffer, indicating that the strategy performs well under changes in target concentration.

[0118] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A CRISPR / Cas-based isothermal rapid visual detection composition for H1N1 influenza virus and Mycoplasma pneumoniae, characterized in that include: Compositions for visually detecting H1N1 influenza virus and compositions for visually detecting Mycoplasma pneumoniae; Among them, the composition for visual detection of H1N1 influenza virus includes: The first RPA front primer, the sequence of which is shown in SEQ ID NO.1; The first post-RPA primer, the sequence of which is shown in SEQ ID NO. 2; The first CRISPR RNA, the sequence of which is shown in SEQ ID NO. 3; The first blocking strand for blocking hairpin assembly, the sequence of which is shown in SEQ ID NO.4; The first initiator strand that catalyzes hairpin assembly; the sequence is shown in SEQ ID NO.5; The first probe of the hairpin assembly had the sequence FAM-AAACTCAGCTTGTCAAATATGGTGTGATTTGACAAGCT; The second probe was assembled by hairpin assembly, and the sequence was Biotin-ACTTGTCAAATCACACCATATTTGACAAGCATGGTGTGATTT; The composition for visually detecting Mycoplasma pneumoniae comprises: The second RPA front primer, the sequence of which is shown in SEQ ID NO.6; The second post-RPA primer, the sequence of which is shown in SEQ ID NO.7; A second CRISPR RNA, the sequence of which is shown in SEQ ID NO. 8; A second blocking strand for blocking hairpin assembly, the sequence of which is shown in SEQ ID NO.9; The second initiator strand catalyzes hairpin assembly; the sequence is shown in SEQ ID NO.10; The third probe for hairpin assembly had the sequence TAMRA-AAAAAAATACAATAGCAAACACCATAGGTGTGAGTGGTGTTTGC; The fourth probe was assembled by hairpin assembly, and the sequence was Dig-AAAAAAACACCACTCACACCTATGGTGTTTGGGTGTGAGTG.

2. Use of the composition according to claim 1 in any of the following: A1) Detection of H1N1 influenza virus and Mycoplasma pneumoniae; A2) Preparation of products for detecting H1N1 influenza virus and Mycoplasma pneumoniae; Preferably, the product comprises a kit.

3. A kit for visually detecting H1N1 influenza virus and Mycoplasma pneumoniae, characterized in that: The kit comprises the composition of claim 1.

4. The kit according to claim 3, wherein The kit also includes a lateral flow test paper, which is sequentially provided with a conjugation pad, a T1 line, a T2 line, a quality control C line and an absorption pad along the transmission direction, wherein the conjugation pad includes a first specific antibody and a second specific antibody, the first specific antibody is colloidal gold-labeled streptavidin; the second specific antibody is a colloidal gold-labeled anti-TAMRA antibody; the T1 line is coated with an anti-FAM antibody; and the T2 line is coated with an anti-Dig antibody.

5. The kit according to claim 3, wherein The kit also includes reagents for recombinase polymerase amplification and Cas12a nuclease.

6. The kit according to claim 5, wherein The reagents for recombinase polymerase amplification include reaction buffer, magnesium acetate, water and lyophilized powder.

7. A method for visually detecting H1N1 influenza virus and Mycoplasma pneumoniae, characterized in that: Including visual detection of H1N1 influenza virus and visual detection of Mycoplasma pneumoniae; Among them, the method of visually detecting H1N1 influenza virus includes: Amplifying the target RNA using a first RPA front primer and a first RPA rear primer to obtain a first amplification product; The first amplified product is sequentially incubated with the first CRISPR RNA and the first blocking strand for blocking hairpin assembly for a first time, incubated with the first priming strand for catalyzing hairpin assembly for a second time, and incubated with the first hairpin assembly probe and the second hairpin assembly probe for a third time to obtain an incubated product; Immerse the conjugate pad of the lateral flow test paper in the incubated product and read the result after 4 to 6 minutes; If the T1 line is red and the quality control C line is blue, it is a positive result; if only the quality control C line is blue, it is a negative result; if the quality control C line does not show color, it indicates that the experiment has failed; Visual methods for detecting Mycoplasma pneumoniae include: Amplifying the target DNA using a second RPA front primer and a second RPA rear primer to obtain a second amplification product; The second amplified product is sequentially incubated with the second CRISPR RNA and the second blocking strand for blocking hairpin assembly for a first time, incubated with the second priming strand for catalyzing hairpin assembly for a second time, and incubated with the third hairpin assembly probe and the fourth hairpin assembly probe for a third time to obtain an incubated product; Immerse the conjugate pad of the lateral flow test paper in the incubated product and read the result after 4 to 6 minutes; If the T2 line is red and the quality control C line is blue, it is a positive result; if only the quality control C line is blue, it is a negative result; if the quality control C line does not show color, it indicates that the experiment has failed.

8. The method according to claim 7, wherein In the method of visual detection of H1N1 influenza virus, The first incubation condition is 36-40°C for 28-40 min, preferably 37°C for 30 min; Alternatively, the second incubation is performed at 36-40° C. for 8-12 min, preferably at 37° C. for 10 min.

9. The method according to claim 7, wherein: In the method for visually detecting H1N1 influenza virus, the third incubation is performed at 36-40° C. for 4-6 minutes, preferably at 37° C. for 5 minutes.

10. The method according to claim 7, wherein: In the method of visual detection of Mycoplasma pneumoniae, The first incubation condition is 36-40°C for 28-40 min, preferably 37°C for 30 min; Alternatively, the second incubation is performed at 36-40°C for 8-12 min, preferably at 37°C for 10 min; Alternatively, the third incubation is performed at 36-40° C. for 4-6 min, preferably at 37° C. for 5 min.

Images