Dengue virus RPA-CRISPR-Cas12b detection system and detection method thereof

Abstract

The invention belongs to the technical field of dengue virus detection, and particularly discloses a dengue virus RPA-CRISPR-Cas12b detection system and a detection method of the dengue virus RPA-CRISPR-Cas12b detection system. The invention selects a conserved region of the dengue virus as an amplification and detection target, and provides a specific sgRNA (sequence as shown in SEQ ID NO.3) and an RPA primer for detecting the dengue virus. The RPA primer is used for carrying out RPA amplification on a to-be-detected sample, specific sgRNA is used for carrying out CRISPR reaction on an amplification product, and whether the to-be-detected sample contains the dengue virus or not can be judged according to the color change of a reaction system. The invention provides corresponding sgRNA and RPA primer sequences and application, and the provided RPA-CRISPR-Cas12b detection system and detection method have the technical advantages of simple operation, rapid and sensitive reaction, strong specificity and the like, and meet the application requirements of on-site instant visual detection of dengue viruses.

Description

Technical Field

[0001] This invention belongs to the technical field of dengue virus detection, specifically the dengue virus RPA-CRISPR-Cas12b detection system and its detection method. Background Technology

[0002] Dengue virus (DENV) infection has significant health impacts in tropical and subtropical countries worldwide. Increased infection rates greatly increase morbidity and mortality, most commonly due to dengue hemorrhagic fever and dengue shock syndrome. While developing an effective and durable vaccine has been a primary goal in controlling and preventing DENV infection, currently licensed vaccines have limitations. Therefore, early detection of DENV is crucial for implementing preventative measures against DENV infection.

[0003] Traditional methods for detecting DENV include reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and serological methods. However, these methods are highly dependent on specific DENV antibodies, require complex equipment and skilled operators, are costly, and are susceptible to cross-reactivity, thus limiting their application scenarios. Therefore, there is an urgent need to establish a detection system that achieves breakthroughs in sensitivity, specificity, and speed and portability, particularly suitable for on-site screening in the early stages of a viral outbreak, tiered medical services at the grassroots level, and travel health monitoring.

[0004] Isothermal nucleic acid amplification technology, due to its elimination of the need for repeated thermal denaturation and specialized instruments, offers faster reaction speeds and is suitable for rapid on-site detection, leading to its widespread application in life science research and many related fields. Currently, there are over ten isothermal nucleic acid amplification technologies, among which RPA (recombinase polymerase amplification) is an isothermal nucleic acid amplification technique that can rapidly amplify target nucleic acid sequences at 37-42℃ (10-20 minutes), showing broad application prospects in many fields such as disease diagnosis and pathogen identification. The CRISPR / Cas system, with its specific gene-targeting capabilities, has become an emerging diagnostic platform in recent years. Due to its high sensitivity and specificity, it is widely used for the detection of pathogens, cell-free tumor DNA, methylation, etc. Guided by sgRNA, the Cas12b protein activates its trans-cleavage activity upon recognizing a matching target sequence, thus indiscriminately cleaving single-stranded non-specific nucleic acid sequences. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a dengue virus RPA-CRISPR-Cas12b detection system and its detection method.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention selects a conserved region of dengue virus as the amplification and detection target, and uses DV dengue virus ID: MW512436.1 (10501-10723) as the target detection region. The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] In a first aspect, the present invention provides an sgRNA probe for detecting dengue virus, said sgRNA comprising a specific target sequence as shown in SEQ ID NO.2.

[0009] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.

[0010] Secondly, the present invention provides the application of the above-mentioned sgRNA probe in the preparation of a kit for detecting dengue virus.

[0011] Thirdly, the present invention provides a dengue virus RPA-CRISPR / Cas12b detection system, comprising an RPA amplification reaction system and a Cas12b detection reaction system; the Cas12b detection reaction system comprises sgRNA as shown in SEQ ID NO.3.

[0012] Furthermore, the RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO. 4-5.

[0013] Fourthly, the present invention provides a kit for visually detecting dengue virus, comprising sgRNA with nucleotide sequences as shown in SEQ ID NO.3 and RPA primer pairs as shown in SEQ ID NO.4-5.

[0014] Furthermore, the kit also includes AaCas12b enzyme protein, 10×AaCas12b Buffer, ssDNA reporter molecule, and RNase-free ddH2O (nuclease-free water).

[0015] Furthermore, the ssDNA reporter molecule is FAM-BQ1-labeled ssDNA: FAM-TTTTTTT-BQ1.

[0016] This invention also provides the application of the above-mentioned kit in the preparation of dengue virus detection reagents.

[0017] Fifthly, this invention provides a method for visual detection of dengue virus based on RPA-CRISPR / Cas12b, comprising the following steps: first, RPA amplification is performed on the sample to be tested, and the nucleotide sequences of the RPA primer pairs used are shown in SEQ ID NO. 4-5; then, CRISPR reaction is performed on the RPA amplification product using the Cas12b detection reaction system. After the CRISPR reaction is completed, the color change of the reaction system can be observed to determine whether the sample to be tested contains dengue virus; the Cas12b detection reaction system includes sgRNA with the sequence shown in SEQ ID NO. 3, AaCas12b enzyme protein, 10×AaCas12b Buffer, ssDNA reporter molecule, and nuclease-free water; the ssDNA reporter molecule is FAM-BQ1-labeled ssDNA: FAM-TTTTTTT-BQ1.

[0018] Furthermore, in the RPA amplification reaction system described in the step, the final concentrations of the forward and reverse primers are 640 nM to 1 μM, respectively; more preferably, they are 800 nM.

[0019] The amplification reaction conditions are as follows: temperature: 37℃~45℃, preferably 43℃; reaction time: 15min~60min.

[0020] Furthermore, in the Cas12b detection reaction system described in the steps, the final concentrations of sgRNA, AaCas12b enzyme protein, and ssDNA reporter molecule are 5 ng / μL, 0.06 μM, and 2 μM, respectively.

[0021] The CRISPR reaction conditions are as follows: temperature: 37℃~45℃, preferably 43℃; reaction time: 15min~60min.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention targets the dengue virus DV MW512436.1 (10501-10723) gene. The designed sgRNA can detect dengue virus without complex instruments, providing results within 30-40 minutes, and can detect as few as 1E0 copies / T per reaction. The RPA-CRISPR / Cas12b dual mechanism combines rapid amplification with high specificity, eliminating cross-reactivity and avoiding false positives. This invention offers multiple technical advantages, including ease of operation, rapid reaction, excellent sensitivity, and high specificity. It is particularly suitable for screening early-stage infection (1-2 days before symptom onset) or asymptomatic carriers, compensating for the lag of antibody-dependent colloidal gold test strips (which appear 4-5 days after infection), and meeting the application requirements for on-site, real-time visual detection. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the location of the DV target sgRNA designed in this invention.

[0025] Figure 2 The following are the fluorescence intensity measurements of the CRISPR reaction for the amplification products of each sgRNA and its corresponding template in this invention: A: sgRNA screening fluorescence change rate; B: sgRNA screening fluorescence curve; NTC is the negative control.

[0026] Figure 3 A schematic diagram of the DV target RPA primer positions designed for this invention.

[0027] Figure 4 The results show the screening results of RPA primers for the DV target; A: fluorescence change rate of RPA primer screening; B: fluorescence curves of different RPA primers at high concentrations; C: fluorescence curves of different RPA primers at low concentrations; NTC is the negative control.

[0028] Figure 5 The results are for DV sensitivity verification; A: Fluorescence growth rate per unit time for different template concentration groups; B: Fluorescence amplification curves for different template concentration groups; NTC is the negative control.

[0029] Figure 6 A: Fluorescence change rate during the DV target sensitivity verification phase; B, C: RPA-CRISPR fluorescence curves at high and low concentrations of the DV target; NTC is the negative control.

[0030] Figure 7 The results are as follows: A: Fluorescence change rate of real DV target samples; B: Fluorescence curve of real DV target samples.

[0031] Figure 8 This is the result of actual sample validation for dengue virus qPCR.

[0032] Figure 9 A: DV target specificity verification results; B: DV target specificity verification fluorescence change rate; PTC: nucleic acid of real sample No. 2 (type I dg2-157); NTC: negative control.

[0033] Figure 10 This is a diagram illustrating the positive / negative property judgment on the SynsorPocket-One platform.

[0034] Figure 11 These are preliminary test results for the SynsorPocket-One platform.

[0035] Figure 12 The results show the sensitivity verification of the SynsorPocket-One platform. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0038] Example 1: Experimental Materials and Methods

[0039] 1. Experimental Materials

[0040] 1.1 Experimental Apparatus

[0041] QPCR instrument - SLAN-96P (Shanghai Hongshi Medical Technology Co., Ltd.), PCR instrument (TC-S / 96 / G / H(b)BA) (Hangzhou Bori Technology Co., Ltd.), Qubit 3.0 fluorometer (Thermo Fisher Scientific), Nanodrop 2000 ultra-micro spectrophotometer (Thermo Fisher Scientific), metal bath (DH100-2) (Hangzhou Ruicheng Instrument Co., Ltd.).

[0042] 1.2 Experimental Reagents

[0043] SynSorAaCas 12b(C2c1)(XS-R-002)(Xunshi Biosciences), SynSor DNA / RNA Isothermal Rapid Amplification Reagent (XS-R-101)(Xunshi Biosciences), SynSor CRISPR ssDNAReporter (12b-FAM)(XS-R-201)(Xunshi Biosciences), SynSor sgRNA (XS-R-301)(Xunshi Biosciences), Gold MIX (Green)(TSE101)(Beijing Qingke Biotechnology Co., Ltd.), and the water used are all UltraPure™ Distilled Water, Dnase, Rnase Free (10977-015) (Invitrogen (Shanghai) Trading Co., Ltd.).

[0044] The SynSor CRISPR ssDNA Reporter is specifically: FAM-BQ1-labeled ssDNA: FAM-TTTTTTT-BQ1.

[0045] 2. Experimental methods

[0046] 2.1 Target selection and sgRNA design for dengue virus detection

[0047] Conserved regions of dengue virus were selected as amplification and detection targets. Dengue virus DV MW512436.1 (10501-10723) was chosen as the target detection region (SEQ ID NO.1). Combined with the host background genome to be avoided, this invention uses bioinformatics algorithms to score sgRNA design based on PAM position, GC% (gamma-to-mass ratio), internal dimer structure, fragment structural openness, base position preference, and specificity. The score can be understood as a success probability; generally, three candidate fragments with scores above 40 are retained. If the scores are generally below 40, the number of candidate fragments needs to be increased to improve the success probability. Sequences in the spacer that pose a risk of disrupting the backbone secondary structure are directly excluded from the candidate list to avoid risks to specificity and sensitivity.

[0048] Based on the above design principles, this invention designed sgRNAs for each selected region, followed by synthesis and validation. The spacer sequences of the designed sgRNAs were analyzed for coverage and specificity using NCBI primer-BLAST. The alignment results showed that the designed sgRNA spacer sequences achieved 100% coverage within the tested species, indicating good inclusiveness; the number of bases matching across species was <15 nt, demonstrating good specificity. To ensure specificity, this invention selected the sgRNAs listed in Table 1 for synthesis (sgRNA positions are shown in Table 1). Figure 1 ).

[0049] Table 1 Information on sgRNA used in this invention

[0050]

[0051]

[0052] *The underlined area at the bottom indicates the spacer region of the sgRNA.

[0053] 2.2 sgRNA Performance Verification

[0054] 2.2.1 PCR Primer Design

[0055] Based on the designed sgRNA location, PCR primers were designed within 100 bp upstream and downstream of the sgRNA (see Table 2). Primers were designed using commonly used PCR primer design software, with primer lengths controlled between 20-25 bp. If multiple sgRNA design sites exist within the same target fragment, the PCR amplification product (fragment length controlled within 500 bp) will contain all sgRNA binding sites, allowing all sgRNAs to be validated using the same template.

[0056] Table 2. Template amplification specific primers

[0057]

[0058] 2.2.3 Template amplification verification

[0059] PCR amplification was performed using template DNA and corresponding primers to obtain high-concentration PCR products. The specific amplification system is shown in Table 3, and the corresponding PCR reaction procedure is shown in Table 4.

[0060] Table 3 PCR System

[0061]

[0062] Table 4 PCR Procedure

[0063]

[0064] 2.2.4 sgRNA Validation

[0065] Prepare the CRISPR system according to the system in Table 5.

[0066] Table 5. CRISPR System Preparation

[0067]

[0068] Mix 1 μL of the PCR amplification product with the above CRISPR system, and incubate at 43℃ for 15 min in a qPCR instrument, collecting FAM fluorescence signals every minute. Based on the changes in the fluorescence signal curve, perform preliminary verification of sgRNA performance or specificity. The endpoint where the fluorescence value curves of the target amplification product and the negative control stop rising should be significantly different.

[0069] The formula for calculating the fluorescence intensity growth rate is as follows:

[0070]

[0071] In this invention, Fluorescence Slope represents the fluorescence growth rate, Rn represents the fluorescence signal at minute n (the fluorescence signal at minute 15 is used in this invention), and R0 represents the background fluorescence signal.

[0072] In this invention, the fluorescence intensity of the CRISPR reaction was measured for the amplification products of each sgRNA and its corresponding template. The fluorescence curves and fluorescence growth rate trends are shown below. Figure 2 The analysis results showed that the DV target DV-sgRNA-3 was effective and could be used for subsequent RPA primer screening.

[0073] 2.3 Primer Design and Validation

[0074] 2.3.1 RPA Primer Design

[0075] Within the target detection range, fragments of 30-35 bases in length were selected as candidate RPA primers. To ensure stability and specificity, the GC content of the RPA primers should be between 40% and 60%, and the RPA amplification fragment is typically 100-200 bp. Based on this primer design principle, three upstream and three downstream primers were designed for RPA primer validation. RPA primers as shown in Table 6 were designed for each template (primer positions are shown in Table 6). Figure 3 ), and then conduct subsequent synthesis and verification experiments.

[0076] Table 6 RPA Primer Table

[0077]

[0078] 2.3.2 RPA Primer Screening

[0079] RPA primers were screened using DV-sgRNA-3, which had shown good performance in previous validations. The specific screening steps are as follows:

[0080] 1) Thoroughly mix the isothermal amplification lyophilized bulbs with 48 μL of amplification buffer A;

[0081] 2) Take 12 μL of the liquid after dissolving the lyophilized bulbs and place it on the cap of the eight-tube strip, and add 1.6 μL of nucleic acid template;

[0082] 3) Take a new row of eight-tube strips and add 0.2 μL of upstream primer and 0.2 μL of downstream primer (primer concentration 100 μM) to each tube.

[0083] 4) Add 1.0 μL of magnesium acetate to the reaction tube. For multiple reactions, it is recommended to add magnesium acetate to the inside of the reaction tube in step 3 and slowly put on the eight-tube cap in step 2 to prevent the liquid inside the cap from falling out. Do not mix for now.

[0084] 5) Prepare a 10 μL CRISPR system according to the AaCas12b protein instructions, and add the prepared CRISPR system to the cap of the new eight-tube connector;

[0085] 6) Place the capped eight-tube bundle from step 4 onto a centrifuge and centrifuge briefly to throw all components to the bottom of the tube. Vortex for 10 seconds to mix thoroughly.

[0086] 7) Discard the cap of the eight-tube in step 6, and gently replace it with an eight-tube cap containing the CRISPR system to prevent the CRISPR system from falling into the tube. Do not centrifuge, and then immediately place the reaction tube in a constant temperature device and incubate at 43°C for 30 minutes.

[0087] 8) After the RPA process is completed, the RPA system is briefly centrifuged to mix with the CRISPR system, vortexed for 10 seconds to mix thoroughly, and then placed in an instrument capable of fluorescence reading and incubated at 43°C for 30 minutes, with fluorescence read every 1 minute.

[0088] The results of DV target primer screening are as follows: In this invention, upstream primers DV-RPA-F1, DV-RPA-F2, and DV-RPA-F3 and downstream primers DV-RPA-R1, DV-RPA-R2, and DV-RPA-R3 were synthesized for cross-primer performance verification (sequences are shown in Table 6). According to the primer verification results, the DV-RPA-F2 / R1 primer set has better detection performance at lower concentrations (see Table 6). Figure 4 Therefore, this invention subsequently used DV-RPA-F2 / R1 in combination with DV-sgRNA-3 to verify the performance of the DV target RPA-CRISPR detection system.

[0089] The formula for calculating the plasmid copy number in this invention is: N plasmid =N A ×C×10 -9 / (2710+N i )×660

[0090] In the above formula, N plasmid The calculated plasmid copy number concentration is expressed in copies / μL; N A is Avogadro's constant, 6.02E+23; C is the plasmid concentration after measurement, in ng / μL; N i 2710 represents the number of base insertions in the plasmid, 660 represents the fragment length of the puc57 vector, and 2710 represents the average molecular weight of the base pairs. This invention calculates the copy number concentration of the plasmid at a specified concentration using the above formula and then dilutes it to a specific concentration for different verifications.

[0091] 2.4 Validation of the testing system

[0092] 2.4.1 Validation of the sensitivity of the detection system

[0093] The plasmid template was serially diluted, with gradients including 1E3 copies / T, 1E2 copies / T, 1E1 copies / T, 1E0 copies / T, and 1E-1 copies / T. The primer and sgRNA combinations selected in section 2.3.2, along with the same CRISPR system (Table 5), were used to validate the sensitivity of the target. Each gradient was replicated three times.

[0094] Based on the sensitivity verification results ( Figure 5 Based on the analysis of significant differences, it was preliminarily determined that the DV target can detect a minimum of 1E0 copies / T of samples per reaction.

[0095] After completing the sensitivity verification for each target, this invention continued with 10 experimental replicates near the detection limit of each target to confirm the target sensitivity. Based on the previously verified sensitivity of each target, this invention selected the 1E1 and 5E0 copies / T concentration groups of the DV target for sensitivity confirmation. The amplification curves and fluorescence signal growth rates for the detection limit verification of each target are shown below. Figure 6 .

[0096] 2.4.2 Real Sample Validation

[0097] Based on the establishment and validation of the DV target detection system, this invention further validated the detection system using actual samples. This validation used 16 real nucleic acid samples provided to the client for verification, and simultaneously employed the detection method in SN / T 2301-2009 as a qPCR gold standard control (see...). Figure 8 Detailed sample information is shown in Table 7. The reaction system and primer / sgRNA combination used in the test are the same as in section 2.3.2. Detailed validation results are available in [link to validation results]. Figure 7 See Table 8. The results of real sample verification show that all samples tested positive, consistent with the qPCR results.

[0098] Table 7 Actual Sample Information

[0099] Sample ID Sample Name Sample concentration (ng / μL, NanoDrop method) 1 Type I YNH12 20.1 2 Type I dg2-157 60.6 3 Type II RL30 34.8 4 Type II RL27 9.4 5 Type II RL18 11.7 6 Type II RL16 4 7 Type II RL35 28.1 8 Type II RL25 24.7 9 Type III JH88 13.5 10 Type III JH669 3.7 11 Type III MN1308 38.1 12 Type III MN1303 42.5 13 Type III MN1302 37.66 14 Type IV WD17019 90.7 15 Type IV WD17041 986.7 16 Type IV WD17023 117.8 17 Chikungunya 2019.JK-11 6.5 18 Chikungunya JK-45 2.9 19 Novel filovirus WD133FV2 3.4 20 Novel filovirus WD133FV3 3.6

[0100] Table 8 Comparison of actual sample detection results and qPCR detection results

[0101]

[0102]

[0103] 2.4.4 Specificity Verification

[0104] This invention uses four cross-samples provided by the customer (2019.JK-11 (serum), JK-45, WD133F V2, WD133F V3, details see 7) for specificity testing. Sample 2 nucleic acid (type I dg2-157) is used as a positive control, the cross-species is used as the specificity verification test group, and water is used as the negative control template. The primer and sgRNA combinations selected in section 2.3.2, along with the same CRISPR system, are used to verify the specificity of the target. Each cross-reaction is validated twice.

[0105] In this invention, the results of the specificity verification of the DV target are shown in the figure below. Figure 9 The specificity verification results showed that the RPA-CRISPR detection method for the DV target developed in this invention could only obtain positive amplification in the target DV (nucleic acid of real sample No. 2 (type I dg2-157)), and the detection results in the other four cross-species were negative, indicating that the detection method for the DV target developed in this invention has good specificity.

[0106] Example 2: Development and Verification of the SynsorPocket-One Platform Detection System

[0107] 1. SynsorPocket-One platform chip fabrication process

[0108] Due to the special nature of the SynsorPocket-One platform, in-situ chip verification of the CRISPR system is required before verification can begin. The in-situ CRISPR system of this invention is shown in Table 9.

[0109] Table 9. CRISPR detection system configuration in the SynsorPocket-One process.

[0110]

[0111]

[0112] After preparing the CRISPR system, add 30 μL of the system to the detection well of the SynsorPocket-One platform and place the chip in a 50°C oven to dry for 1.5 hours. After drying, remove the chip, cover it with the protective film, and store it in a sealed aluminum foil bag under low humidity. Before use, unscrew the reagent protective cap and remove the chip protective cap above the sample dispensing port.

[0113] 2. SynsorPocket-One Platform Testing Process

[0114] In this part of the test, the RPA amplification system for the target was first prepared, with two RPA lyophilized beads added to the system. The amount of each component added and the final concentration in the RPA system in this procedure are shown in Table 10.

[0115] Table 10 RPA System Configuration for SynsorPocket-One Platform

[0116]

[0117] After thoroughly mixing all reaction components in the RPA, transfer the entire system to the SynsorPocket-One chip. Then, fully press down the raised portion of the test kit card, waiting for the sample liquid to completely fill the test wells. Following the directional indicators on the test kit's handle, insert the chip into the instrument's test card slot. Finally, press the instrument's start button. The Test indicator light should be constantly on. Once the indicator light illuminates (the maximum detection time for the Pocket platform system is 50 minutes), read the experimental results. Simultaneously, the platform can be connected to a computer for real-time fluorescence data acquisition and analysis.

[0118] 3. Fluorescence threshold verification on the SynsorPocket-One platform

[0119] To ensure the SynsorPocket-One platform can stably convert amplified fluorescence signals into positive and negative output signals, this invention first detected the platform's fluorescence threshold. This part of the study involved testing blank background samples under different instruments, operators, times, and environments to determine the LOB (Level of Birth) of the blank samples for this platform. Based on the statistical summary of LOB measurements at different time points, combined with the average LOB value and standard deviation of LOB values ​​at different experimental times, the threshold for different time points was determined. This threshold was subsequently integrated into the platform to determine the positive or negative results of the experiments.

[0120] 4. Results Analysis of SynsorPocket-One Platform

[0121] Based on the test results from the Pocket platform, the positive / negative judgment is as follows: Figure 10As shown: Positive (+): The SynsorPocket-One platform system will output a positive result in as little as 10 minutes according to the instrument's internal algorithm. At this time, the Positive red light on the instrument will be constantly lit, and the test result is positive; Negative (-): After the SynsorPocket-One platform completes all detection processes, if the fluorescence still does not exceed the threshold line, it will output a negative result. At this time, the Negative green light on the instrument will be constantly lit, and the test result is negative; Invalid: When an instrument or chip malfunction occurs, the Error red light on the instrument will be constantly lit, and the test result is invalid. The instrument and chip need to be replaced and the operation repeated.

[0122] 5. Feasibility Verification of the SynsorPocket-One Platform

[0123] Building upon the established RPA-CRISPR workflow, this invention first aims to verify the feasibility of the SynsorPocket-One platform. Preliminary verification will be conducted using 1E4 copy / T target samples and cross-samples, and the verification will be performed within the SynsorPocket-One platform's detection workflow. Test results are as follows... Figure 11 As shown.

[0124] 6. Sensitivity results of the SynsorPocket-One platform

[0125] Plasmid samples were serially diluted 10-fold, with the gradient set to 1E3 copy / T, 1E2 copy / T, 1E1 copy / T, and 1E0 copy / T. The target sensitivity was validated using the primer and sgRNA combinations selected in section 2.3 and the SynsorPocket-One platform detection workflow. The sensitivity validation results are shown below. Figure 12 Preliminary sensitivity test results show that the RPA-CRISPR SynsorPocket-One platform detection system in this invention can detect 1E2 copy / T samples.

[0126] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An sgRNA probe for detecting dengue virus, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

2. The use of the sgRNA probe according to claim 1 in the preparation of a kit for detecting dengue virus.

3. A dengue virus RPA-CRISPR / Cas12b detection system, characterized in that, It includes an RPA amplification reaction system and a Cas12b detection reaction system; the Cas12b detection reaction system includes the sgRNA as described in claim 1.

4. The dengue virus RPA-CRISPR / Cas12b detection system according to claim 3, characterized in that, The RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO.4-5.

5. A reagent kit for visually detecting dengue virus, characterized in that, It includes the sgRNA as described in claim 1 and the RPA primer pairs shown in SEQ ID NO. 4-5.

6. The reagent kit for visually detecting dengue virus according to claim 5, characterized in that, The kit also includes AaCas12b enzyme protein, 10×AaCas12b Buffer, ssDNA reporter molecule, and nuclease-free water; the ssDNA is FAM-TTTTTTT-BQ1.

7. The use of the kit according to claim 5 or 6 in the preparation of dengue virus detection reagents.

8. A real-time visual detection method for dengue virus based on RPA-CRISPR / Cas12b, characterized in that, Includes the following steps: First, the sample to be tested is amplified using RPA, and the nucleotide sequences of the RPA primer pairs used are shown in SEQ ID NO. 4-5. Then, the RPA amplification product is subjected to a CRISPR reaction using the Cas12b detection reaction system. After the CRISPR reaction is completed, the color change of the reaction system can be observed to determine whether the sample to be tested contains dengue virus. The Cas12b detection reaction system includes the sgRNA, AaCas12b enzyme protein, 10×AaCas12b Buffer, ssDNA reporter molecule, and nuclease-free water as described in claim 1. The ssDNA is FAM-TTTTTTT-BQ1.

9. The detection method according to claim 8, characterized in that, In the RPA amplification reaction system described in the steps, the final concentrations of the forward and reverse primers are 640 nM to 1 μM, respectively, and the amplification reaction conditions are: temperature 37℃ to 45℃, and reaction time 15 min to 60 min.

10. The detection method according to claim 8, characterized in that, In the Cas12b detection reaction system described in the steps, the final concentrations of sgRNA, AaCas12b enzyme protein, and ssDNA reporter molecule are 5 ng / μL, 0.06 μM, and 2 μM, respectively; the CRISPR reaction conditions are: temperature 37℃~45℃, reaction time 15 min~60 min.

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