Kit for visually detecting H5 subtype avian influenza virus based on RPA-CRISPR / Cas12b system and application

Abstract

The invention discloses a kit for visually detecting an H5 subtype avian influenza virus based on an RPA-CRISPR / Cas12b system and application of the kit, and belongs to the technical field of molecular detection. The kit comprises an RT-RPA (Reverse Transcription-Recombinase Polymerase Amplification) primer pair and sgRNA1 The RT-RPA primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2; and the nucleotide sequence of the sgRNA1 is as shown in SEQ ID NO. 7. According to the kit, nucleic acid amplification and detection are synchronously completed through a tubular reaction system, the detection sensitivity reaches 1 copy / mu L, the coincidence rate with fluorescent RT-PCR is 100%, and no cross reaction exists on H7, H9, NDV and IBV. The kit does not need a complex instrument, is suitable for rapid screening on a basic level site, and provides an efficient solution for prevention and control of avian influenza.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and in particular to a kit and application for visually detecting H5 subtype avian influenza virus based on an RPA-CRISPR / Cas12b system. Background Art

[0002] Avian influenza viruses belong to the influenza A virus genus. They are classified into different subtypes based on the antigenic differences in the surface proteins hemagglutinin (HA) and neuraminidase (NA). To date, 18 HA subtypes and 11 NA subtypes have been identified, theoretically resulting in a total of 198 subtype combinations. However, subtypes H17 and H18 have only been isolated from bats. AIVs are classified according to their pathogenicity: highly pathogenic, low pathogenic, and non-pathogenic. Highly pathogenic avian influenza is caused by some strains of the H5 and H7 subtypes.

[0003] In addition to culling infected and infected livestock at epidemic foci, measures to control highly pathogenic avian influenza (H5) include the efficient and convenient sampling and screening of poultry breeding bases and wild bird habitats, which can effectively prevent the spread of the virus. Conventional H5 diagnostic techniques include: virus isolation and identification, which require a biosafety level 3 laboratory and biosafety-trained personnel, and are time-consuming; hemagglutination and hemagglutination inhibition tests, which are highly subjective; RT-PCR amplification times; and real-time fluorescence RT-PCR testing, which is expensive and requires skilled operators. Therefore, providing a convenient, rapid, and easy-to-use CRISPR / Cas12b-H5 subtype influenza virus detection kit that does not require specialized equipment is of great significance for detecting H5 subtype influenza viruses at ports and at the grassroots level. Summary of the Invention

[0004] The present invention aims to provide a kit and application for visually detecting the H5 subtype of avian influenza virus based on the RPA-CRISPR / Cas12b system, addressing the aforementioned problems in the prior art. The kit provided by the present invention has a sensitivity of 1 copy / μL, a 100% consistency with fluorescent RT-PCR, and no cross-reactivity with H7, H9, NDV, or IBV.

[0005] In order to solve the above problems, the present invention provides the following solutions:

[0006] The present invention provides a kit for detecting H5 subtype avian influenza virus, comprising an RT-RPA primer pair and sgRNA1; the RT-RPA primer pair comprises a forward primer whose nucleotide sequence is shown in SEQ ID NO.1 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.7.

[0007] Furthermore, the kit also includes AaCas12b protein and a fluorescently labeled ssDNA reporter probe.

[0008] Furthermore, the working concentration of the AaCas12b protein is 50 nM.

[0009] Furthermore, the sequence of the ssDNA reporter probe is FAM-TTATT-BHQ1.

[0010] Furthermore, the kit is a one-tube reaction system.

[0011] Furthermore, the one-tube reaction system includes: an RPA amplification layer and a CRISPR detection layer; the RPA amplification layer contains a primer pair, template DNA, RPA buffer and magnesium acetate; the CRISPR detection layer contains AaCas12b, sgRNA1, ssDNA probe and buffer, which are pre-placed in the reaction tube cap.

[0012] The present invention also provides a method for rapid detection of H5 subtype avian influenza virus for non-diagnostic purposes, comprising the following steps:

[0013] The H5 HA gene in the sample to be tested was amplified using the RT-RPA primer pair; the amplified product was mixed with the sgRNA1, the AaCas12b protein and the ssDNA reporter probe, and incubated at 42° C. for 30 minutes; the presence of green fluorescence was determined to be the presence of H5 subtype avian influenza virus.

[0014] Furthermore, the RT-RPA primer pair amplification was performed at 42° C. for 15 minutes.

[0015] Furthermore, the excitation wavelength of the fluorescent signal is 485 nm, and the emission wavelength is 520 nm.

[0016] The present invention also provides an application of the kit in preparing a product for on-site screening of H5 subtype avian influenza virus.

[0017] The present invention discloses the following technical effects:

[0018] The detection method of the present invention is designed by optimizing H5 HA gene-specific primers (RPA-F1 / RPA-R1) and sgRNA1, combining RT-RPA isothermal amplification with the trans-cleavage activity of Cas12b, and significantly improving the detection sensitivity (10 times higher than traditional RT-PCR) and specificity (no cross-reaction). The one-tube design avoids the risk of contamination by opening the lid, and the detection time is shortened to 45 minutes to complete the visual interpretation; after the sample is incubated in the portable device for 2 minutes, the positive and negative properties of the sample can be judged by the display screen, and the detection time is shortened to 17 minutes. In the clinical simulation test, the test results of 50 samples were completely consistent with those of fluorescent RT-PCR, confirming its reliability and practicality. The technology of the present invention is particularly suitable for grassroots units and port sites with limited resources, and provides efficient and low-cost technical support for the early detection and prevention and control of H5 subtype avian influenza epidemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Screening test results for RPA and sgRNA;

[0021] Figure 2 These are the detection results after AaCas12b optimization; 1-5: AaCas12b added at concentrations of 50nM, 75nM, 25nM, 125nM, and 100nM; 6: negative control;

[0022] Figure 3 These are the test results after sgRNA optimization; 1-5: sgRNA added at concentrations of 3 ng / μL, 2 ng / μL, 1 ng / μL, 5 ng / μL, and 4 ng / μL; 6: negative control;

[0023] Figure 4 Visualization of sensitivity test results for RPA-CRISPR / Cas12b-H5; 1-7: 3.12×10 1 ng / μL-3.12×10 -5 ng / μL; 8: negative control;

[0024] Figure 5 is the result of RPA-CRISPR / Cas12b-H5 fluorescence detection; 1-8: 1.0×10 6 ~1.0×10 -1 copies / μL; 9: negative control;

[0025] Figure 6 is the result of RT-PCR H5 fluorescence detection; among them, 1-7: 1.0×10 6 ~1.0×10 0 copies / μL; 8: negative control;

[0026] Figure 7 is the fluorescence intensity detection result of RPA-CRISPR / Cas12b-H5; 1-8: 1.0×10 6 ~1.0×10 - 1 copies / μL; Negative: negative control;

[0027] Figure 8 This is the visualization of RPA-CRISPR / Cas12b-H5 specificity detection results. The unmarked tube is a blank tube (nothing added);

[0028] Figure 9 The results of RPA-CRISPR / Cas12b-H5 fluorescence detection; 1: H5 positive control; 2-5: H7, H9, NDV, IBV; 6: negative control;

[0029] Figure 10 This is the specific detection result of RPA-CRISPR / Cas12b-H5 fluorescence intensity;

[0030] Figure 11 This is the reproducibility test result of the RPA-CRISPR / Cas12b H5 subtype influenza molecular detection kit;

[0031] Figure 12 Visualization of clinical test results for RPA-CRISPR / Cas12b-H5; N and P represent negative and positive, respectively, and the unmarked tubes are blank tubes (nothing added);

[0032] Figure 13 This is the result of RPA-CRISPR / Cas12b-H5 fluorescence detection;

[0033] Figure 14 The clinical test results of RPA-CRISPR / Cas12b-H5 fluorescence intensity; N and P represent negative and positive, respectively. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] 1. Materials and Methods

[0041] 1.1 Recombinant plasmid and sample

[0042] The conserved region of the H5 HA gene (1533–1658 bp) was selected and synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized gene was cloned into the pUC57 vector, resulting in pUC57-H5 HA. The culture was incubated overnight for 16 hours, and pUC57-H5 HA was extracted using a plasmid extraction kit to obtain the recombinant plasmid standard, pUC57-H5 HA. The full gene sequence of H7 HA was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the pUC57 vector, resulting in pUC57-H7HA. The culture was incubated overnight for 16 hours, and pUC57-H7 HA was extracted using a plasmid extraction kit. The H9 vaccine strain and the NDV LaSota vaccine strain were purchased from Harbin Pharmaceutical Group Bio-Vaccine Co., Ltd. IBV RNA was stored in the laboratory of the Animal Inspection Institute of the Chinese Academy of Quality Inspection and Testing Sciences. Sterile water containing no nucleic acids was used as a negative control. With the exception of the recombinant plasmid standard, pUC57-H5 HA, all other products were specific quality control products.

[0043] 1.2 Main Reagents

[0044] 10×AaCas12b Buffer, AaCas12b, ssDNA and RT-RPA amplification kit (basic type) were purchased from Beijing Xunshi Biotechnology Co., Ltd.

[0045] 1.3 Design, screening and synthesis of RT-RPA primers and sgRNA

[0046] Three sgRNAs were designed using NCBI Blast to analyze conserved regions of H5 HA and principles of sgRNA design. Corresponding RPA primer pairs were designed based on the regions where the sgRNAs were located. The designed primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The sgRNA-specific sequences were synthesized by Beijing Xunshi Biotechnology (see Table 1). The sgRNA consists of two regions: the target sequence and the scaffold sequence. The optimal length of the sgRNA is approximately 100 bases. The scaffold sequence, which binds to the Cas12b protein, is approximately 80 bases long, while the target sequence is 20 bases long.

[0047] Table 1 RPA primer pairs and sgRNA sequences

[0048]

[0049]

[0050] 1.4RPA and sgRNA Optimization

[0051] In order to avoid aerosol contamination of nucleic acid products after RPA amplification, which may cause false positives, the present invention has developed a one-tube RPA-CRISPR / Cas12b-H5 detection kit to avoid the problem of aerosol contamination when the lid is opened after amplification. RPA reaction system: add 25μL amplification buffer A to each dry powder tube; take 12.5μL of liquid after dissolving the dry powder and place it at the bottom of an eight-tube strip, add 2μL of upstream and downstream primers respectively, add 2.0μL of template, and finally add 1.0μL of magnesium acetate, add water to make up to 25μL, cover the eight-tube strip cap and centrifuge to mix. CRISPR / Cas12b reaction system: 1.2μL (100nM) AaCas12b, 0.6μL (2ng / μL) sgRNA, 3μL (10×AaCas12b Buffer), 0.3μL ssDNA probe, and add water to 10μL. Add the prepared CRISPR / Cas12b system to the cap of the eight-tube strip of RPA reaction system to prevent the CRISPR system from falling into the tube. Do not centrifuge. Then immediately place the reaction tube in a handheld thermostat at 42°C and incubate for 15 minutes. After the RPA reaction, briefly centrifuge to mix the RPA amplified product and CRISPR system. Vortex for 10 seconds to thoroughly mix. Place the reaction tube in a portable fluorescence reader and incubate at 42°C for 30 minutes, collecting the fluorescence signal every minute. Select the RPA primers and sgRNA with the highest sensitivity.

[0052] 1.5AaCas12b and sgRNA optimization

[0053] The amount of AaCas12b and sgRNA reaction system was optimized using the optimized RPA primers and sgRNA. The added amount and the corresponding final concentration are shown in Table 2.

[0054] Table 2 Optimization of AaCas12b and sgRNA system

[0055]

[0056] 1.6 Sensitivity test

[0057] The optimized RPA primer pair, sgRNA, and AaCas12b and sgRNA reaction system were selected to establish the RPA-CRISPR / Cas12b H5 subtype influenza molecular detection method. The positive standard of recombinant plasmid pUC57-H5HA (1.0×10 6 ~1.0×10 -1 copies / μL) to evaluate the sensitivity of the detection method.

[0058] 1.7 Specificity test

[0059] The established RPA-CRISPR / Cas12b H5 subtype avian influenza molecular detection method was used to detect nucleic acids of specific quality control products H7, H9, NDV and IBV to evaluate the specificity of the detection method.

[0060] 1.8 Clinical simulation trial

[0061] Fifty chicken livers (100 g each) were collected to simulate clinical samples, and different concentrations of a positive standard were added and mixed thoroughly. The simulated samples were tested using standard real-time fluorescence RT-PCR. Thirty of the simulated samples tested positive, while 20 tested negative. The established RPA-CRISPR / Cas12b H5 subtype avian influenza virus kit was used to test the 50 simulated clinical samples to evaluate the clinical applicability of the assay and its compatibility with real-time fluorescence RT-PCR.

[0062] 1.9 Repeatability test

[0063] The recombinant plasmid positive standard pUC57-H5 HA was diluted 10-fold (1.0×10 6 ~1.0×10 3 The established RPA-CRISPR / Cas12b H5 subtype avian influenza molecular detection method was used to perform three replicate tests on these four concentration gradients to evaluate the specificity of the detection method.

[0064] 1.10 Result Evaluation

[0065] One method uses a gel imaging device or a flashlight with a 485nm excitation wavelength to illuminate the results of the sensitivity test, specificity test, clinical simulation test, and repeatability test. The presence of green fluorescence is considered positive, while the absence of green fluorescence is considered negative. Another method uses a portable fluorescence PCR instrument to detect the virus. A fluorescence value greater than 0 is considered positive, while a value less than or equal to 0 is considered negative.

[0066] 2. Results

[0067] 2.1 Screening and Optimization of RPA Primer Pairs and sgRNA

[0068] The RPA-CRISPR / Cas12b-H5 kit was used to detect H5 positive standards, and a pair of RPA primers RPA-F1 / RPA-R1 and sgRNA1 ( Figure 1). RPA-F1 / RPA-R1 and sgRNA1 were used to optimize the dosage of AaCas12b and sgRNA in the CRISPR / cas12b reaction system. When the dosage was 50nM and 3ng / μL, respectively, the fluorescence signal of the standard positive sample was strong ( Figure 2 and Figure 3 ).

[0069] 2.2 Sensitivity test

[0070] The recombinant plasmid standard pUC57-H5 HA was diluted 10-fold to a concentration of 1.0×10 6 ~1.0×10 -1 The optimized RPA1 primers and sgRNA1 were used as templates for detection. The results showed that fluorescence signals could be detected at all gradient concentrations, indicating that the lower limit of detection of H5 subtype influenza molecules can reach 1.0 copies / μL ( Figure 4-Figure 7 The recombinant plasmid standard was tested using a standard real-time fluorescence RT-PCR method, and the results showed that the lower limit of detection of H5 subtype influenza molecules could reach 10 copies / μL, indicating that the kit developed by the present invention has good detection sensitivity.

[0071] 2.2 Specificity test

[0072] The optimized RPA-CRISPR / Cas12b H5 subtype influenza molecular detection kit was used to detect H7, H9, NDV, and IBV nucleic acids. The results showed that this method can specifically amplify the H5 subtype influenza virus recombinant plasmid standard, and there is no amplification curve or fluorescence signal for H7, H9, NDV, and IBV nucleic acids ( Figures 8-10 ). This shows that the detection method has good specificity.

[0073] 2.3 Repeatability test

[0074] 1.0×10 6 ~1.0×10 3 The four concentration gradients of 100 copies / μL were used as templates and the detection was repeated three times. The results showed that all four concentration gradients could be detected and the amplification curve had a high degree of fit, indicating that this method has good repeatability ( Figure 11 ).

[0075] 2.4 Clinical simulation trial

[0076] The RPA-CRISPR / Cas12b H5 subtype influenza molecular detection kit was used to test 50 simulated samples. The results showed that 30 simulated samples were positive and 20 simulated samples were negative, which was 100% consistent with the standard real-time fluorescence RT-PCR test. Figure 12-14 ). This shows that the kit has good applicability.

[0077] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A kit for detecting H5 subtype avian influenza virus, characterized in that: It includes an RT-RPA primer pair and sgRNA1; the RT-RPA primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.

7.

2. The kit according to claim 1, wherein The kit also includes AaCas12b protein and a fluorescently labeled ssDNA reporter probe.

3. The kit according to claim 2, wherein The working concentration of the AaCas12b protein is 50 nM.

4. The kit according to claim 2, wherein The sequence of the ssDNA reporter probe is FAM-TTATT-BHQ1.

5. The kit according to claim 1, wherein The kit is a one-tube reaction system.

6. The kit according to claim 5, characterized in that The one-tube reaction system includes: an RPA amplification layer and a CRISPR detection layer; the RPA amplification layer contains a primer pair, template DNA, RPA buffer and magnesium acetate; the CRISPR detection layer contains AaCas12b, sgRNA1, ssDNA probe and buffer, which are pre-placed in the reaction tube cap.

7. A method for rapid detection of H5 subtype avian influenza virus for non-diagnostic purposes, characterized in that: The following steps are involved: Use the RT-RPA primer pair described in claim 1 to amplify the H5 HA gene in the sample to be tested; mix the amplified product with the sgRNA1 described in claim 1, the AaCas12b protein described in claim 2, and the ssDNA reporter probe, and incubate at 42° C. for 30 minutes; the presence of green fluorescence determines the presence of H5 subtype avian influenza virus.

8. The detection method according to claim 7, characterized in that The RT-RPA primer pair amplification was performed at 42°C for 15 minutes.

9. The detection method according to claim 7, characterized in that The excitation wavelength of the fluorescent signal is 485 nm, and the emission wavelength is 520 nm.

10. Use of the kit according to any one of claims 1 to 6 in preparing a product for on-site screening of H5 subtype avian influenza virus.

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