Novel one-pot RPA-CRISPR detection method for monkey pox virus based on sucrose-assisted multiphase solution system

Abstract

The invention discloses a novel one-pot RPA-CRISPR method for detecting monkey pox virus based on a sucrose-assisted multiphase solution system, sucrose is added in RPA reaction to increase the density of the RPA reaction, the RPA reaction is placed at the bottom of a tube, and the CRISPR reaction with lower density is kept in a water phase and located at the top of the RPA reaction; in a one-tube reaction process, newly accumulated RPA amplicons trigger non-specific cutting of Cas12a for ssDNA-FQ, so that a fluorophore is released, a real-time fluorescence signal is detected by using a fluorescence detector, and end point fluorescence can be visualized under a blue light source; compared with the prior art, the one-pot RPA-CRISPR method of the multiphase solution system is constructed by using the sucrose to increase the RPA reaction density, rapid, convenient and reliable detection of the monkey pox virus is realized, prevention and control of the monkey pox epidemic situation are facilitated, and a good example is provided for actual field detection application of the sucrose-assisted multiphase solution system.

Description

Technical Field

[0001] The present invention relates to the technical field of viral genotyping detection, namely, a real-time detection technology for the monkeypox F3L gene, and specifically to a novel one-pot RPA-CRISPR method for detecting monkeypox virus based on a sucrose-assisted multiphase solution system. Background Art

[0002] Monkeypox virus (MPV) is the causative agent of monkeypox, a zoonotic disease with symptoms similar to smallpox, including rash, fever, headache, and muscle aches. Given the risk of viral evolution and mutation, prompt identification of MPV infection and isolation of patients are crucial to prevent outbreaks, protect vulnerable populations, and limit the emergence of dangerous MPV variants. Early diagnostic methods are crucial for preventing the spread of monkeypox and safeguarding public safety. Nucleic acid testing, due to its rapid response and diagnostic certainty, has become an essential tool for early diagnosis of monkeypox. However, current methods for detecting MPV nucleic acid are less than satisfactory. PCR-based assays are generally the gold standard for pathogen nucleic acid detection, but they require precise thermal cyclers and rely on laboratory conditions. Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are suitable for on-site testing but are prone to nonspecific amplification and may suffer from instability, making them unreliable for point-of-care (POCT) testing. CRISPR / Cas12a-based methods have been widely used for early diagnosis of pathogen infections. The method relies on the recognition of pathogen-specific sequences by the Cas12a-crRNA complex, which then activates Cas12a to cut the signal-generating probe. The method is highly specific because the activation of Cas12a depends on a good match between the rationally designed crRNA and the target sequence of the pathogen. A highly sensitive monkeypox detection method combining RPA with CRISPR-Cas12a has been reported, with sensitivity reaching the single-copy level. However, this method uses a two-step procedure, which is not only complicated to operate but also poses the risk of aerosol contamination. Due to the incompatibility between amplification and Cas cleavage, the further developed RPA-CRISPR one-pot method sacrificed sensitivity, which has reduced the sensitivity to a dozen copies. In this study, a new one-pot RPA-CRISPR method based on a sucrose-assisted multiphase solution system was established for the detection of monkeypox virus. Summary of the Invention

[0003] This paper establishes a novel one-pot RPA-CRISPR method for detecting monkeypox virus (MPV) based on a sucrose-assisted multiphase solution system. By exploiting the density difference between sucrose and water, the RPA and CRISPR reactions can be spatially separated within the same tube, achieving simultaneous reactions. Specifically, sucrose is added to the RPA reaction to increase its density, placing it at the bottom of the tube; the less dense CRISPR reaction remains in the aqueous phase and is located at the top of the RPA reaction. During the one-tube reaction, slow and dynamic diffusion occurs between the two phases. The newly accumulated RPA amplicons trigger nonspecific cleavage of ssDNA-FQ by Cas12a, leading to the release of the fluorophore. The real-time fluorescence signal is detected using a fluorescence detector, and the end-point fluorescence can be visualized under a blue light source. This method boasts ultra-high sensitivity down to a single copy: existing RPA-CRISPR one-pot methods have a sensitivity of more than ten copies per reaction, while the sensitivity of this method is a single copy per reaction, making it 10 times more sensitive than existing RPA-CRISPR one-pot methods. The entire reaction is completed within 60 minutes at 37°C. This detection method demonstrated good specificity and effectively distinguished monkeypox from other orthopoxviruses. The test results were visible to the naked eye under ultraviolet or blue light, making it well-suited for use at home or in limited healthcare settings. This method addresses the incompatibility between the Cas12a cleavage reaction and the RPA reaction, demonstrating good specificity, accuracy, and the rapidity and convenience crucial for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox and establishes a paradigm for the development of a one-pot RPA-CRISPR assay using a multiphase solution system. The specific technical solution is as follows: A novel one-pot RPA-CRISPR method for detecting monkeypox virus based on a sucrose-assisted multiphase solution system includes adding sucrose to the RPA reaction to increase its density and placing it at the bottom of the tube, while the less dense CRISPR reaction remains in the aqueous phase and is located on the top of the RPA reaction. During the one-tube reaction, the newly accumulated RPA amplicons trigger the nonspecific cleavage of ssDNA-FQ by Cas12a, resulting in the release of fluorescent groups. The real-time fluorescent signal is detected using a fluorescence detector, and the end-point fluorescence can be visualized under a blue light source. Furthermore, the detection steps are as follows: (1) Preparation of viral genomic DNA; (2) Expression and purification of Cas12a; (3) crRNA preparation; (4) Sucrose multiphase-assisted RPA-CRISPR one-pot reaction system; (5) Monitor the fluorescence signal and plot the data.

[0004] Furthermore, the specific steps of the sucrose multiphase-assisted RPA-CRISPR one-pot reaction system are as follows: a. Prepare RPA premix and CRISPR premix separately; b. Gently mixing the two premixed solutions to form a sucrose-assisted multiphase reaction system; c. Incubate the mixture in step b at 37°C for 60 min; d. Monitor the FAM fluorescence signal, read and record it every 1 min.

[0005] Furthermore, the RPA premix in step a was prepared by adding 2 μL each of 10 μM forward and reverse primers, 18 μL of buffer A, and 3.25 μL of sucrose (50%, v / v) to a tube of freeze-dried enzyme powder to obtain an RPA premix. The CRISPR premix in step a was prepared by adding a final concentration of 100 nM Cas12a, 1 μM ssDNA-FQ, and 100 nM crRNA to a total volume of 40 μL CRISPR premix, and adding 1× NEB buffer 2.1. Finally, the volume was filled to 40 μL with deionized water to obtain a CRISPR premix.

[0006] Furthermore, the preparation method of the sucrose-assisted multiphase reaction system in step b is as follows: 18µL RPA premix, 1µL monkeypox virus F3L standard plasmid and 1µL B buffer are added to the bottom of the reaction tube and gently mixed; then 20µL CRISPR premix is slowly added to the top of the above mixture to form a sucrose-assisted multiphase reaction system.

[0007] Furthermore, the forward primer and reverse primer, namely RPA-F and RPA-R, have gene sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the crRNA gene sequence is shown in SEQ ID NO.3, and the ssDNA-FQ gene sequence is shown in SEQ ID NO.4.

[0008] Furthermore, the method for expression and purification of the Cas12a includes pET-28b-Cas12a plasmid construction, protein expression, and protein purification.

[0009] The above technical solution can achieve the following beneficial effects: Compared to existing technologies, the one-pot RPA-CRISPR method for detecting monkeypox virus using a multiphase solution system demonstrates sensitivity of single copy per reaction, an order of magnitude higher than existing one-pot RPA-CRISPR methods. This detection method exhibits good specificity and effectively distinguishes monkeypox from other orthopoxviruses. The application of this one-pot RPA-CRISPR method has three advantages: (i) High sensitivity reduces the risk of false negative results and facilitates early diagnosis of infection; (ii) The amplification-cleavage strategy further reduces potential false positives and provides better diagnostic accuracy; (iii) Single-tube reaction avoids exposure of amplification materials to the environment, reducing the risk of cross-contamination; From a point-of-care (POCT) perspective, this method completes the entire reaction at 37°C within 60 minutes, and the test results can be observed directly with the naked eye under ultraviolet or blue light. The method requires only a set of pipettes, a microcentrifuge, a low-power heating block, and a blue light source or portable fluorescence reader. These materials are easy to assemble, making the method well-suited for home or limited healthcare settings.

[0010] In summary, this study utilized sucrose to increase the RPA reaction density, establishing a one-pot RPA-CRISPR method for a heterogeneous solution system, enabling rapid, convenient, and reliable detection of monkeypox virus. This approach contributes to the prevention and control of monkeypox outbreaks and provides a valuable example for the practical application of sucrose-assisted heterogeneous solution systems for field detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the sucrose-assisted multiphase RPA-CRISPR one-pot reaction method.

[0012] Figure 2 are sensitivity and specificity.

[0013] (A) Sensitivity test. Fluorescence curves and endpoint fluorescence signals for reactions using different standard plasmid template amounts are shown. (B) Bar graph of endpoint fluorescence intensity for sensitivity test. Error bars represent the standard error of two replicate experiments. P < 0.001, **P < 0.01, and n.s.P = 0.2. (C) Specificity test. Fluorescence curves and endpoint fluorescence signals for reactions using different templates are shown. (D) Bar graph of endpoint fluorescence intensity for specificity test. Error bars represent the standard error of two replicate experiments.

[0014] Figure 3 The verification is performed using simulated samples.

[0015] Real-time fluorescence curves and endpoint fluorescence signal images of reactions using (A) a simulated skin swab sample containing a standard plasmid, (B) a simulated throat swab sample containing a standard plasmid, (C) a simulated blood sample containing a standard plasmid, and (D) a simulated skin swab sample containing a pseudovirus. The amount of positive target added to the reaction is indicated. NTC stands for no template control. DETAILED DESCRIPTION

[0016] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative work are within the scope of protection of the present invention.

[0017] 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 application. As used herein, unless the context clearly indicates otherwise, the singular form is also 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.

[0018] The following is combined with Figure 1-3 The present invention is described in detail to facilitate those skilled in the art to understand the present invention.

[0019] A novel one-pot RPA-CRISPR method for detecting monkeypox virus based on a sucrose-assisted multiphase solution system. Sucrose is added to the RPA reaction to increase its density and placed at the bottom of the tube, while the less dense CRISPR reaction remains in the aqueous phase and is located on the top of the RPA reaction. During the one-tube reaction, the newly accumulated RPA amplicons trigger the nonspecific cleavage of ssDNA-FQ by Cas12a, resulting in the release of the fluorescent group. The real-time fluorescent signal is detected using a fluorescence detector, and the endpoint fluorescence can be visualized under a blue light source.

[0020] As a preferred solution, the detection steps are as follows: (1) Preparation of viral genomic DNA; (2) Expression and purification of Cas12a; (3) crRNA preparation; (4) Sucrose multiphase-assisted RPA-CRISPR one-pot reaction system; (5) Monitor the fluorescence signal and plot the data.

[0021] As a preferred embodiment, the specific steps of the sucrose multiphase assisted RPA-CRISPR one-pot reaction system are as follows: a. Prepare RPA premix and CRISPR premix separately; b. Gently mixing the two premixed solutions to form a sucrose-assisted multiphase reaction system; c. The mixture in step b was heated at 37°C for 60 min; d. Monitor the FAM fluorescence signal, read and record it every 1 minute.

[0022] As a preferred embodiment, in step a, an RPA premix is prepared by adding 2 μL of a 10 μM forward primer, 2 μL of a 10 μM reverse primer, 18 μL of buffer A, and 3.25 μL of sucrose (50%, v / v) to a tube of lyophilized enzyme powder to prepare an RPA premix; Prepare CRISPR master mix: Add 100 nM Cas12a, 1 µM ssDNA-FQ, 100 nM crRNA to a total volume of 40 µL CRISPR master mix, add 1× NEB buffer 2.1, and finally add deionized water to 40 µL.

[0023] As a preferred embodiment, in step b, a sucrose-assisted multiphase reaction system is prepared: 18 μL of RPA premix, 1 μL of monkeypox virus F3L standard plasmid, and 1 μL of B buffer are added to the bottom of the reaction tube and gently mixed; then 20 μL of CRISPR premix is slowly added to the top of the above mixture to form a sucrose-assisted multiphase reaction system.

[0024] As a preferred embodiment, the forward primer and reverse primer gene sequences are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the crRNA gene sequence is the sequence shown in SEQ ID NO.3, and the ssDNA-FQ gene sequence is the sequence shown in SEQ ID NO.4, and the sequences are shown in Table 1 below:

[0025] Table 1 Primers and crRNA sequences As a preferred embodiment, the method for expression and purification of Cas12a includes pET-28b-Cas12a plasmid construction, protein expression, and protein purification. Example

[0026] 1. Reagents and Instruments TIANamp Virus RNA Kit and DNA extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; T7 High Efficiency Transcription Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; RPA Basic Nucleic Acid Amplification Kit was purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; Universal SYBR qPCR Master Mix, HiScript 1st Strand cDNA Synthesis Kit, and FastPure® Gel DNA Extraction Mini Kit were purchased from Nanjing Novogene Biotechnology Co., Ltd.; Qubit 4 Fluorometer was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Roche LightCycler 480 II qPCR Instrument was purchased from Switzerland; Nanodrop Lite Spectrophotometer was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Blue Light Transilluminator was purchased from Tianjin Novogene Bioinformatics Technology Co., Ltd.; Monkeypox Virus Nucleic Acid Detection Kit was purchased from Beinachuanglian Biotechnology Research Institute (Zhengzhou, China); 0.1M TCEP solution: weigh 2.51 g of tris(2-carboxyethyl)phosphine into a beaker, add water and stir until dissolved, finally adjust the volume to 100 mL, filter, and store at room temperature; The SnakeSkin 10K MWCO dialysis tubing was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Cas12a Lysis buffer: add 25 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM TCEP to a beaker, mix well, and place in a 4 ° C refrigerator for use; Cas12a Elute buffer: add 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM TCEP, 500 mM imidazole to a beaker, mix well, and place in a 4 ° C refrigerator for use; Cas12a Storage buffer: add 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM TCEP, 5% glycerol to a beaker, mix well, and place in a 4 ° C refrigerator for use; 1 mL Ni affinity prepacked columns were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0027] 2. Preparation of Viral Genomic DNA A standard plasmid containing the monkeypox virus F3L gene fragment (GenBank No. ON568298 / ON563414, 46168-46630 nt) was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Pseudovirus samples representing monkeypox virus and other human-associated orthopoxviruses (including cowpox virus, smallpox virus, and buffalopox virus), as well as DNA standards representing the N and ORF genes of SARS-CoV-2, were purchased from Fubaiao Biotechnology Co., Ltd. (Shanghai, China). Genomic DNA of Pseudomonas aeruginosa (ATCC 9027) was purchased from the American Type Culture Collection (ATCC). Skin swab samples were collected from consenting healthy individuals, immersed in 2 mL of virus stock solution (BeyondSky Biotechnology, Shanghai, China), and aliquoted into 200 µL aliquots. Mock samples were prepared by mixing the desired amount of monkeypox pseudovirus with the aliquots. The number of monkeypox pseudovirus copies in each mock sample was quantified by qPCR. Briefly, the purchased monkeypox pseudovirus (10 8 particles / mL) to determine the correlation between copy number and Ct value. Mock samples were subsequently tested using the same qPCR kit to confirm the copy number of each sample. DNA was extracted from the samples using a DNA extraction kit. qPCR experiments were performed on a Roche LightCycler 480 II qPCR instrument.

[0028] 3. Expression and purification of Cas12a (1) Construction of pET-28b-Cas12a plasmid The Cas12a gene sequence was derived from a standard plasmid (Addgene plasmid no. 90096) and synthesized by General Biotech (Anhui, China). The Cas12a fragment was amplified from the plasmid 6×His-MBP-TEV-Cas12a, and the pET28b vector was linearized with restriction endonucleases. The Cas12a gene fragment was constructed in the pET28b vector, and the His tag was constructed at the N-terminus.

[0029] (2) Protein expression Transformation: The extracted pET28b-6×His-Cas12a plasmid was transformed into BL21 (DE3) pLysS expression competent cells, incubated at 37°C and 200 rpm for 1 h, and then plated onto LB solid medium containing Kana (50 μg / mL) and Chl (100 μg / mL); Activation: Pick 10 single clones and culture them in LB liquid medium containing 50µg / mL kanamycin and 35µg / mL chloramphenicol. Incubate at 37°C, 200rpm for 4h. Expansion: Take 10 mL of activated bacterial solution and add it to 500 mL of LB medium containing the corresponding resistance, and culture it at 37°C until the OD 600 0.6-0.8; Induction: Add IPTG to a final concentration of 0.5 mM and place at 23 °C, 220 rpm shaker for 8 h to induce Cas12a protein expression; Harvest the bacteria: centrifuge the cells at 7500 x g for 5 min at 4°C, collect the precipitate and store at -80°C for later purification.

[0030] (3) Protein purification The stored cells were resuspended in Buffer A (25mM Tris-HCl (pH 7.5), 500mM NaCl, 1mM TCEP), PI (1000X), and AEBSF (4000X). The cells were disrupted using a low-temperature high-pressure homogenizer at 1000-1500 MPa until the cells were no longer viscous. The suspension was transferred to a 50mL centrifuge tube and centrifuged at 15,000g for 45 minutes at 4°C in a high-speed centrifuge to separate the supernatant and precipitate. The supernatant was collected and passed through a 0.22µM aqueous filter. Affinity purification was performed on an AKTA Prime-Plus instrument using a 1mL Ni-affinity prepacked column. Sample loading: The supernatant was passed through the Ni-affinity prepacked column at a flow rate of 1mL / min. Wash: The column was adjusted to 95% lysis buffer + 5% elution buffer using the instrument, and 10 column volumes were washed until UV absorption reached equilibrium. Elute: Adjust the elution buffer concentration from 5% to 100% using the instrument, and perform a linear gradient elution over 10 column volumes. Collect the solution based on the UV absorbance peak. Identify the collected protein solution by SDS-PAGE (6%). Identify the protein solution by SDS-PAGE, transfer it to The SnakeSkin 10K MWCO dialysis tubing, and dialyze it overnight in Cas12a Storage buffer. Purified Cas12a is stored in 20mM Tris-HCl (pH 8.0), 200mM NaCl, 1mM TCEP, and 50% glycerol at -20°C.

[0031] Example 1: crRNA preparation crRNA was designed based on the available PAM motif (TTTN) within the RPA amplicon sequence of the monkeypox virus F3L gene. For crRNA production, two reverse-complementary ssDNA fragments were synthesized, which form a dsDNA fragment containing the T7 promoter, the crRNA backbone sequence, and 21 bp downstream of the corresponding PAM motif upon annealing. The annealed dsDNA served as a template for in vitro transcription using the T7 High Yield RNA Transcription Kit. The transcribed crRNA was purified by phenol-chloroform extraction, dissolved in DEPC water, and quantified using a Qubit 4 fluorometer. The crRNA gene sequence is shown in SEQ ID NO. 3.

[0032] Example 2: Sucrose-assisted multiphase RPA-CRISPR one-pot reaction system The RPA master mix and CRISPR master mix are prepared separately, where: 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 18 μL of buffer A, and 3.25 μL of sucrose (50%, v / v) were added to a tube of lyophilized enzyme powder (Hangzhou Zhongce Biotechnology Co., Ltd.) to obtain an RPA premix. The gene sequences of the forward primer and reverse primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. To a total volume of 40 μL CRISPR premix, a final concentration of 100 nM Cas12a, 1 μM ssDNA-FQ, and 100 nM crRNA were added, and 1× NEB buffer 2.1 was added. Finally, the volume was supplemented with deionized water to 40 μL to obtain a CRISPR premix. The ssDNA-FQ gene sequence is as shown in SEQ ID NO. 4; The sucrose-assisted heterogeneous RPA-CRISPR one-pot reaction system was prepared as follows: 18 μL of RPA premix, 1 μL of monkeypox virus F3L standard plasmid, and 1 μL of buffer B were added to the bottom of the reaction tube and gently mixed; then, 20 μL of CRISPR premix was slowly added to the top of the above mixture to form a sucrose-assisted heterogeneous reaction system; the reaction was carried out at 37°C for 60 min on a Roche LightCycler 480 II qPCR machine (Basel, Switzerland), and the FAM signal was read every 1 min. Figure 1 As shown, the endpoint signal is visualized under a blue light source and the image is captured by a smartphone camera.

[0033] Example 3: Sensitivity and specificity of the sucrose-assisted one-pot RPA-CRISPR method To evaluate the sensitivity of the sucrose-assisted one-pot RPA-CRISPR assay, different concentrations of monkeypox virus standard plasmid were used for testing. The fluorescence curve and endpoint fluorescence results showed that the detection limit of this method reached 100 copies / reaction. Figure 2 As shown in A and B, further significance analysis showed that the sensitivity of the detection method was reliable.

[0034] The specificity of the sucrose-assisted one-pot RPA-CRISPR method was confirmed by detecting a series of common orthopoxviruses and coronaviruses, including cowpox virus, buffalopox virus, vaccinia virus, smallpox virus, SARS-CoV-2 N gene, SARS-CoV-2 ORF gene, and Pseudomonas aeruginosa. The results of fluorescence curves and end-point fluorescence showed that the method has good specificity for monkeypox virus. Figure 2 C and D.

[0035] Example 4: Validation of the Sucrose-Assisted One-Pot RPA-CRISPR Method Using Simulated Samples Since monkeypox virus is transmitted through close contact with body fluids, body fluids from blood samples, skin swabs, and throat swabs were mixed with monkeypox standard plasmids to prepare three mock samples. In addition, a mock sample was prepared by mixing skin swabs with monkeypox pseudovirus. These samples were tested to evaluate the reliability of the sucrose-assisted one-pot RPA-CRISPR method for clinical field detection. The fluorescence curves and endpoint fluorescence results showed that all four types of mock samples could detect single copy sensitivity, demonstrating the potential of the one-pot method in the clinical diagnosis of monkeypox virus, see Figure 3 .

[0036] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention are within the scope of protection of the claims attached to this application.

Claims

1. A novel one-pot RPA-CRISPR method for detecting monkeypox virus based on a sucrose-assisted multiphase solution system, characterized by: Sucrose is added to the RPA reaction to increase its density and placed at the bottom of the tube. The CRISPR reaction with lower density remains in the aqueous phase and is located on the top of the RPA reaction. During the reaction in one tube, the newly accumulated RPA amplicons trigger the non-specific cleavage of ssDNA-FQ by Cas12a, resulting in the release of the fluorescent group. The real-time fluorescent signal is detected using a fluorescence detector, and the endpoint fluorescence can be visualized under a blue light source.

2. The method of claim 1, wherein the method comprises: The detection steps are as follows: (1) Preparation of viral genomic DNA; (2) Expression and purification of Cas12a; (3) crRNA preparation; (4) Sucrose multiphase-assisted RPA-CRISPR one-pot reaction system; (5) Monitor the fluorescence signal and plot the data.

3. The method of claim 2, wherein the method comprises: The specific steps of the sucrose multiphase-assisted RPA-CRISPR one-pot reaction system are as follows: a. Prepare RPA premix and CRISPR premix separately; b. Gently mixing the two premixed solutions to form a sucrose-assisted multiphase reaction system; c. The mixture in step b was heated at 37°C for 60 min; d. Monitor the FAM fluorescence signal, read and record it every 1 minute.

4. The method of claim 3 for detecting monkeypox virus using a novel one-pot RPA-CRISPR method based on a sucrose-assisted multiphase solution system, characterized in that: Preparation of the RPA premix in step a: Add 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 18 μL of A buffer and 3.25 μL of sucrose (50%, v / v) to a tube of freeze-dried enzyme powder to obtain an RPA premix; Preparation of the CRISPR premix in step a: Add a final concentration of 100 nM Cas12a, 1 μM ssDNA-FQ, 100 nM crRNA to a total volume of 40 μL CRISPR premix and add 1× NEB buffer 2.1, and finally use deionized water to make up the volume to 40 μL to obtain a CRISPR premix.

5. The method of claim 3 for detecting monkeypox virus using a novel one-pot RPA-CRISPR method based on a sucrose-assisted multiphase solution system, characterized in that: In step b, a sucrose-assisted multiphase reaction system was prepared by adding 18 μL of RPA premix, 1 μL of monkeypox virus F3L standard plasmid, and 1 μL of B buffer to the bottom of the reaction tube and gently mixing; then, 20 μL of CRISPR premix was slowly added to the top of the above mixture to form a sucrose-assisted multiphase reaction system.

6. The method of claim 4 for detecting monkeypox virus using a novel one-pot RPA-CRISPR method based on a sucrose-assisted multiphase solution system, characterized in that: The gene sequences of the forward primer and reverse primer are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the crRNA gene sequence is the sequence shown in SEQ ID NO.3, and the ssDNA-FQ gene sequence is the sequence shown in SEQ ID NO.

4.

7. The method of claim 2, wherein the method comprises: The method for expression and purification of the Cas12a includes pET-28b-Cas12a plasmid construction, protein expression, and protein purification.

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