LAMP-CRISPR visual detection method for identifying monkey pox virus based on ATI gene as well as kit and application of LAMP-CRISPR visual detection method

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to an LAMP-CRISPR visual detection method for identifying a monkey pox virus based on an ATI gene and a kit and application of the LAMP-CRISPR visual detection method. Preparing an LAMP system and a CRISPR system, wherein the LAMP system and the CRISPR system are separated through mineral oil and air; carrying out LAMP amplification reaction to obtain an amplification product; adding an amplification product into a CRISPR (clustered regularly interspaced short palindromic repeats) system for enzyme digestion and visualization; wherein the reaction container comprises a PCR (Polymerase Chain Reaction) tube with the capacity of 200 mu L; the thickness of the mineral oil is 2.5-3mm; the air thickness ranges from 11 mm to 12 mm; the LAMP system is prepared at the bottom of the reaction container, the mineral oil is positioned on the upper surface of the LAMP system, and the CRISPR system is prepared in a circular groove of a tube cover of the PCR reaction tube. According to the detection method, a reaction system for visual detection is designed aiming at a monkey pox virus specific detection target, and an LAMP amplification system and a CRISPR detection system which are integrated into one tube are separated by utilizing mineral oil and liquid surface tension, so that not only is the detection convenience improved, but also the influence of high temperature during LAMP amplification on enzyme of the CRISPR detection system can be avoided.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid detection technology, and specifically to a LAMP-CRISPR visualization detection method for identifying monkeypox virus based on the ATI gene, a kit and an application thereof. Background Art

[0003] At present, the main methods for detecting monkeypox virus are PCR technology and loop-mediated isothermal amplification (LAMP) technology. Among them, LAMP is an in vitro isothermal amplification buffer nucleic acid fragment technology. Its basic principle is to design multiple primers for the 6 specific interval sequences of the target sequence, and use Bst DNA polymerase with displacement activity for constant temperature amplification at 65°C. The reaction can be completed within 60 minutes. LAMP technology has a series of advantages for viral nucleic acid detection. First, the LAMP primer set consists of 4 to 6 primers, which can specifically identify different regions in the DNA sequence; secondly, the DNA yield of LAMP (10μg / 25μL) is higher than that of traditional PCR (0.2μg / 25μL), and the nucleic acid signal can be amplified by 10 in a short time. 9 amplification effect; finally, compared with the traditional PCR technology that requires variable temperature operation, LAMP technology only requires a constant temperature to complete the detection of the target sequence, thereby reducing the requirements for experimental instruments.

[0004] At present, the PCR technology has a long detection time and requires special instruments and equipment, high-standard laboratory environment and professional technicians, which restricts its widespread use in underdeveloped or underdeveloped areas. The detection limit of LAMP technology is 20 copies / reaction. The commonly used detection method for LAMP reaction products cannot effectively distinguish between specific amplification and nonspecific amplification. At the same time, there is a risk of contaminating the laboratory environment. It is necessary to combine LAMP technology with other diagnostic technologies to improve sensitivity and detect both specific and nonspecific amplification.

[0005] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated protein (CRISPR-associated, Cas) system is an adaptive immune system of bacteria that can recognize exogenous substances and remove foreign substances through the endonuclease activity of Cas proteins. The CRISPR / Cas system has targeted recognition and trans-cleavage activity, which is conducive to the development of in vitro nucleic acid detection technology. Compared with the fluorescent probe-based LAMP detection, the combination of CRISPR and LAMP technology can effectively improve the detection sensitivity. At the same time, CRISPR RNA (crRNA) has a high single-base specific recognition ability and can distinguish between specific and nonspecific amplification. Combining the sensitivity, specificity and flexibility of CRISPR diagnostic technology with the ease of use, cost-effectiveness and speed of LAMP technology will provide point-of-care testing (POCT) in the future in the fields of pathogen detection, tumor detection, single nucleotide polymorphism (SNP) analysis, etc. At present, research has gradually developed to combine RPA technology or recombinase-mediated isothermal nucleic acid amplification technology (Recombinase Aided Amplification, RAA) with CRISPR technology for the diagnosis of monkeypox virus. However, there are no reports on the combined use of LAMP and CRISPR technology for the diagnosis of monkeypox virus. In addition, since the optimal temperature of the enzymes in LMAP technology and CRISPR technology is different, even if the two technologies are used in combination, step-by-step heating or a CRISPR enzyme with high heat resistance will be used. The step-by-step heating method requires opening the mouth of the reaction tube after the LAMP reaction, which is prone to contamination; choosing a heat-resistant CRISPR enzyme will limit the use of enzymes and easily increase production costs.

[0006] Therefore, a new detection method is needed to establish a LAMP-CRISPR nucleic acid detection system for monkeypox virus. Summary of the invention

[0007] The problem that the present application aims to solve is to provide a LAMP-CRISPR visual detection method for identifying monkeypox virus based on the ATI gene, a kit and an application thereof. The detection method targets monkeypox virus-specific detection targets, designs a reaction system for visual detection, and uses mineral oil and liquid surface tension to separate the LAMP amplification system and the CRISPR detection system integrated in one tube, which not only improves the convenience of detection, but also avoids the influence of high temperature during LAMP amplification on the enzyme of the CRISPR detection system.

[0008] In order to solve the above technical problems, this application adopts the following technical solutions:

[0009] On the one hand, the present application provides a LAMP-CRISPR visualization detection method for identifying monkeypox virus based on the ATI gene, comprising the following steps:

[0010] Extracting DNA from a sample to be tested; preparing a LAMP amplification system and a CRISPR detection system in a reaction container, wherein the LAMP amplification system and the CRISPR detection system are separated by mineral oil and air; using the DNA as a template, performing a LAMP amplification reaction in the LAMP amplification system to obtain an amplification product; and mixing the components in the reaction container, and performing an enzyme digestion reaction and visual detection;

[0011] Wherein, the reaction container comprises a PCR reaction tube with a capacity of 200 ml;

[0012] The thickness of the mineral oil is 2.5 to 3 mm;

[0013] The thickness of the air is 11 to 12 mm;

[0014] The LAMP amplification system is prepared at the bottom of the reaction container, the mineral oil is located on the upper surface of the LAMP amplification system, and the CRISPR detection system is prepared in the circular groove of the PCR reaction tube cover.

[0015] In some embodiments, the LAMP amplification system comprises the following components:

[0016]

[0017]

[0018] Wherein, in the primer set (10×), an outer primer pair F3 / B3 and an inner primer pair FIP / BIP are included, the concentration of the outer primer pair F3 / B3 is 2 μM, and the concentration of the inner primer pair FIP / BIP is 16 μM;

[0019] The nucleotide sequence of the forward outer primer F3 is shown in SEQ ID NO.1, the nucleotide sequence of the reverse outer primer B3 is shown in SEQ ID NO.2, the nucleotide sequence of the forward inner primer FIP is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse inner primer BIP is shown in SEQ ID NO.4.

[0020] In some embodiments, when performing the fluorescence detection, the CRISPR detection system includes NE Buffer (10×) 2 μL, LbCas12a protein (1 μM) 1 μL, crRNA (10 μM) 1.25 μL, single-stranded DNA probe (50 μM) with FAM / BHQ1 fluorescent labeling 1 μL, RNase inhibitor (40 U / μL) 0.25 μL and DEPC water 12.5 μL;

[0021] When performing the immunochromatographic test strip test, the CRISPR detection system includes NE Buffer (10×) 2 μL, LbCas12a protein (1 μM) 1 μL, crRNA (10 μM) 1.25 μL, single-stranded DNA probe (10 μM) with FAM / Biotin fluorescent labeling 1 μL, RNase inhibitor (40 U / μL) 0.25 μL and DEPC water 12.5 μL;

[0022] Wherein, the nucleotide sequence of the crRNA is shown in SEQ ID NO.5.

[0023] In some embodiments, the visual detection comprises one of fluorescence detection and immunochromatographic test strip detection.

[0024] In some embodiments, the method of mixing comprises flash centrifugation.

[0025] In some embodiments, the temperature of the LAMP amplification reaction is 60°C to 65°C, and the time of the LAMP reaction is 30 to 60 min; and / or, the temperature of the enzyme cleavage reaction is 37°C, and the time of the enzyme cleavage reaction is 5 to 25 min.

[0026] On the other hand, the present application provides a LAMP-CRISPR visualization detection kit for identifying monkeypox virus based on the ATI gene, characterized in that the kit comprises at least one of the LAMP amplification system in the LAMP-CRISPR visualization detection method of claim 2, the CRISPR detection system in the LAMP-CRISPR visualization detection method of claim 3, and mineral oil.

[0027] In some examples, the mineral oil includes ST275 Mineraloil produced by Bio-Technology Co., Ltd.

[0028] In some embodiments, the kit further comprises at least one of an immunochromatographic test strip and a portable small ultraviolet lamp.

[0029] On the other hand, the present application provides the use of the above-mentioned LAMP-CRISPR visualization detection method or the above-mentioned LAMP-CRISPR visualization detection kit in identifying the monkeypox virus ATI gene.

[0030] This application has the following beneficial effects:

[0031] (1) The LAMP-CRISPR visualization detection method provided in the present application comprises the following steps: after preparing the LAMP amplification system at the bottom of the reaction container, a layer of mineral oil is covered on the surface of the LAMP amplification system, and then the CRISPR detection system is prepared in the circular groove at the cover of the PCR reaction tube by utilizing the surface tension of the liquid. At the same time, the heat insulation effect of the mineral oil and air is utilized to prevent the system temperature from being heated to 65°C during the LAMP amplification reaction, thereby preventing the activity of enzymes related to the CRISPR detection system from being affected.

[0032] (2) Based on the monkeypox virus ATI gene, the present application combines the sensitivity, specificity and flexibility of LAMP technology with the ease of use, cost-effectiveness and speed of LAMP technology to achieve rapid and effective detection of monkeypox virus.

[0033] (3) The LAMP-CRISPR visualization detection kit provided in the present application integrates the LAMP amplification system and the CRISPR reaction system into one tube. After the LAMP reaction is completed, there is no need to open the lid to add the CRISPR detection system, thus avoiding the possibility of nucleic acid product and laboratory contamination.

[0034] (4) The LAMP-CRISPR visualization detection kit provided in this application can obtain the detection results without complex instruments, making the reading of the detection results simple and intuitive, and suitable for field sampling and rapid diagnosis.

[0035] (5) As monkeypox virus is an emerging pathogen, there has been no research on LAMP-CRISPR nucleic acid detection for monkeypox virus. This application combines LAMP amplification technology and CRISPR detection technology based on the ATI gene of monkeypox virus to provide a new detection method for nucleic acid detection of monkeypox virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the present disclosure, the drawings required for use in some embodiments of the present disclosure are briefly introduced below.

[0037] Figure 1 Agarose gel electrophoresis results after amplification of the five sets of primers designed in Example 1 (wherein, M: DL2000 DNA marker, 1, 2, 3, 4, 5 are five sets of LAMP primers respectively);

[0038] Figure 2The fluorescence signal curves and fluorescence imaging results after the five LAMP amplification systems and CRISPR detection systems reacted in Example 1 (where a: fluorescence signal curve, Fluoresecence is the fluorescence signal intensity, Time is the reaction time of the CRISPR detection system; b: fluorescence imaging of LAMP amplification systems of different volumes, LAMP volume is the volume of the LAMP amplification system);

[0039] Figure 3 The agarose gel electrophoresis results of the LAMP reaction products of Example 1 (wherein, M: DL2000 DNA marker, BC: blank control, ATI: experimental group containing monkeypox virus ATI gene);

[0040] Figure 4 The fluorescence signal curves and fluorescence imaging results after the reaction of the two CRISPR detection systems in Example 2 (a: fluorescence signal curve of CRISPR detection of ATI gene, b. UV fluorescence image of CRISPR detection of ATI gene);

[0041] Figure 5 This is a schematic diagram of the LAMP-CRISPR detection system of this application integrated into one tube (from bottom to top, LAMP amplification system - mineral oil - air - CRISPR detection system);

[0042] Figure 6 The fluorescence curve signal of CRISPR detection of ATI gene in Example 3 (N: blank control, P: experimental group containing monkeypox virus ATI gene);

[0043] Figure 7 Fluorescence imaging images of CRISPR detection at different times in Example 3;

[0044] Figure 8 The immunochromatographic test strip test results of the five LAMP amplification systems in Example 4 (C: reference line, T: test line);

[0045] Fig. 9 Fluorescence imaging of DNA templates of different concentrations after LAMP-CRISPR reaction. DETAILED DESCRIPTION

[0046] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0047] Example 1 Establishment of LAMP amplification system and primer selection

[0048] (1) Preparation of DNA samples

[0049] 4 μg of plasmid powder containing the monkeypox virus ATI gene fragment was added to 20 μL TE Buffer and diluted to a plasmid solution with a concentration of 100 ng / μL by adding DEPC water. After further confirmation of the plasmid concentration by Nanodrop analyzer, 10-fold gradient dilution was performed to prepare a series of DNA samples with a concentration gradient (8×10 0 ~8×10 8 ) copies / μL, and stored in a -20°C refrigerator, wherein the nucleotide sequence of the monkeypox virus ATI gene is shown in SEQ ID NO.6.

[0050] (2) Design and synthesis of primers for LAMP amplification system

[0051] For the ATI fragment of the monkeypox virus-specific gene, the LAMP primer design software Primer Explorer V5 (https: / / primerexplorer.jp / lampv5 / index.html) developed by Rongyan was used for primer design. The primer design software can generate 5 sets of LAMP primers. After the primer sequences are determined, they are sent to Bio-Industry for synthesis. The primer sequences are shown in Table 1. Agarose gel electrophoresis was used to screen each set of primers (such as Figure 1 As shown), it is also ensured that the gene fragment amplified by the screened primers contains the binding target of crRNA.

[0052] Table 1

[0053]

[0054]

[0055] The results showed that among the five primer sets, the fourth primer set had the brightest electrophoretic band and the LAMP reaction could achieve the best amplification efficiency. Therefore, the fourth primer set was selected for the establishment of the subsequent LAMP amplification system.

[0056] (3) Establishment and optimization of LAMP amplification system

[0057] The LAMP amplification system was prepared according to the formula in Table 2, wherein 10× Primers was the primer set 4, and the amplification reaction was carried out at 65°C for 30 min in a constant temperature water bath. After the amplification, 20 μL of CRISPR detection system was added, and the reaction was carried out at 37°C for 40 min in a fluorescence quantitative PCR instrument. The fluorescence signal curve was collected, and fluorescence imaging was performed on a Bio Rad company imager ( Figure 2 ).

[0058] Table 2

[0059]

[0060]

[0061] The LAMP amplification system was prepared according to the amplification system 2 in Table 2, wherein 10× Primers was the primer set 4, and the amplification reaction was performed at 55-70°C for 15-75 min in a constant temperature water bath. After the amplification, 20 μL CRISPR detection system was added, and fluorescence detection was performed at 37°C for 5 min. The detection results of different amplification times and temperatures were observed and recorded (as shown in Table 3)

[0062] Table 3

[0063]

[0064] According to the fluorescence intensity and detection, it can be seen that the optimal dosage of the LAMP amplification system is 12.5 μL, the temperature of the LAMP amplification reaction is 60-65°C, and the time of the amplification reaction is 30-60 min.

[0065] (4) Detection of LAMP amplification products by agarose gel electrophoresis

[0066] After amplification, weigh 1.5g of LAMP amplification product and add it to 100ml TBE (0.5×), mix well, and heat at high temperature in the microwave for 2min. After cooling, add 10μL of nucleic acid dye (10000×), shake well and pour into the mold, insert the comb, and wait for the gel to solidify naturally. After the gel solidifies, pull out the comb and place the gel in an electrophoresis tank containing TBE (0.5×). Take 10μL of the LAMP reaction product and add 2μL loading buffer (6×), load 10μL into each well, and add 2000bp DNAMaker as a band marker. Set the electrophoresis instrument to a constant voltage of 120V / 60min. After the electrophoresis is completed, take out the gel and place it in a gel imager to observe the bands (such as Figure 3 shown).

[0067] The results showed that the screened ATI primers could specifically complete LAMP amplification without any false positive reaction. This group of LAMP primers can be used to establish subsequent detection methods.

[0068] Example 2 Probe design and system establishment for CRISPR reaction

[0069] (1) crRNA sequence

[0070] The optimal sequence SEQ ID NO.5 provided by existing studies was used as the direct repeat sequence of the crRNA sequence. The crRNA spacer sequence SEQ ID NO.22 was designed based on the complementary pairing of the crRNA spacer sequence with approximately 21 bases downstream of the PAM sequence (TTTA\TTTT\TTTC\TTTG) on the target gene fragment. The designed crRNA sequence was sent to Bioengineering for synthesis. Since crRNA can also specifically recognize single-stranded DNA fragments, the designed crRNA sequence should not overlap with the LAMP primer.

[0071] (2) DNA probe design

[0072] The FAM / BHQ1 reporter groups are labeled at the 5' and 3' ends of the single-stranded DNA to design the fluorescent probe. Under normal circumstances, the fluorescent signal of FAM can be quenched by the BHQ1 quencher group. When crRNA recognizes the target nucleic acid fragment, the trans-cleavage activity of the Cas12a protein can be activated, thereby indiscriminately cleaving the single-stranded and double-stranded DNA fragments in the system. When the fluorescent probe is cut, the fluorescent group and the quencher group separate and emit a fluorescent signal.

[0073] (3) Establishment of CRISPR detection system

[0074] The CRISPR detection system was prepared according to the formula in Table 3, wherein the LAMP reaction product was prepared in Example 1. The components in the CRISP detection system were mixed and reacted at 37° C. for 40 min on a fluorescence quantitative PCR instrument. The fluorescence signal curve was collected and fluorescence imaging was performed on a Bio Rad company imager (eg Figure 4 shown).

[0075] Table 3

[0076]

[0077] The results showed that the designed crRNA and DNA probes can detect ATI gene fragments and emit visible fluorescence under ultraviolet light. Compared with the basic reaction system, CRISPR 2 has a higher fluorescence brightness, so CRISPR2 was selected as the CRISPR reaction system in the future.

[0078] Example 3 Establishment of a fluorescence-based LAMP-CRISPR one-tube detection method

[0079] Add 25 μL of LAMP amplification system containing primer set 4 to the bottom of the PCR reaction tube, then cover the prepared LAMP amplification system with 2.5 mm of mineral oil, and use the surface tension of the liquid to adsorb 20 μL of CRISPR 2 (such as Figure 5 The LAMP amplification system was reacted at 65°C for 30 min in a thermostat and then centrifuged. Enzyme digestion was performed at 37°C on a fluorescence quantitative PCR instrument and the fluorescence curve signal was collected (as shown in Figure 6 The fluorescence changes of the reaction system were recorded in real time every 5 min using a fluorescence imager (as shown in Figure 7 shown).

[0080] The results showed that after the amplification reaction was completed, the components in the PCR reaction tube were mixed and subjected to enzyme digestion reaction at 37°C for 5 to 25 minutes. The reaction results could be observed with the naked eye under ultraviolet light.

[0081] Example 4 Establishment of a LAMP-CRISPR One-Tube Detection Method for Immunochromatographic Test Strips

[0082] The single-stranded DNA probe labeled with FAM / BHQ1 fluorescence in CRISPR 2 was replaced with a single-stranded DNA probe labeled with FAM / Biotin fluorescence at an initial concentration of 10 μM to obtain CRISPR 3.

[0083] According to Table 2, add the LAMP amplification system containing primer set 4 to the bottom of 5 PCR reaction tubes, then cover the prepared LAMP amplification system with 2.7 mm mineral oil, and use the surface tension of the liquid to add 20 μL of CRISPR 3 into the circular groove on the cap of the PCR reaction tube 11.5 mm away from the mineral oil. After the LAMP amplification system is reacted at 65°C for 30 minutes in a constant temperature device, it is centrifuged instantaneously, and after a 5-minute enzyme digestion reaction at 37°C, 50 μL of the treated product is taken, and a test strip is inserted into it with the sample pad facing down. After being placed at room temperature for 5 to 10 minutes, the result is judged (such as Figure 8 The judgment standard for negative results is that the C line is colored and the T line is not colored, indicating that the nucleic acid probe is not cut by the Cas12a protein; the judgment standard for positive results is that the C line is colored and the T line is visible to the naked eye or the C line is not colored and the T line is visible to the naked eye, indicating that part of the nucleic acid probe is cut by the Cas12a protein or the nucleic acid probe is completely cut by the Cas12a protein; the judgment standard for invalid results is that both the C line and the T line are not colored.

[0084] The results showed that after the amplification reaction was completed, the components in the PCR reaction tube were mixed, and after the enzyme digestion reaction at 37°C for 5 to 25 minutes, the test strip was inserted and the result could be read in 5 to 10 minutes. When the LAMP amplification system was 6.25 μL, the positive result was most obvious.

[0085] Example 5 Sensitivity of the fluorescence-based LAMP-CRISPR one-tube detection method

[0086] 25 μL of LAMP amplification system containing primer set 4 was added to the bottom of each of the 9 PCR reaction tubes, where the concentration of the template DNA was 8×10 0 copies / μL, 8×10 1 copies / μL, 8×10 2 copies / μL, 8×10 3 copies / μL, 8×10 4 copies / μL, 8×10 5 copies / μL, 8×10 6 copies / μL, 8×10 7 copies / μL, 8×10 8 Then, cover the prepared LAMP amplification system with 3 mm mineral oil, and use the surface tension of the liquid to add 20 μL of CRISPR 2 into the circular groove on the cap of the PCR reaction tube 12 mm away from the mineral oil. After the LAMP amplification system is reacted at 65°C for 30 minutes in a constant temperature device, it is shaken and then subjected to enzyme digestion at 37°C for 5 minutes. Fluorescence imaging is performed using a portable small UV lamp (such as Fig. 9 shown).

[0087] The results showed that the minimum detection limit of the fluorescence-based LAMP-CRISPR one-tube detection method was 8×10 7 copies / μL.

[0088] In summary, the present application provides a new detection method for monkeypox virus, which is based on LAMP technology and CRISPR detection technology. It utilizes the heat insulation effect of mineral oil and air and the surface tension of liquid to integrate the LAMP amplification system and the CRISPR detection system in one tube. It can detect 8×10 7 The monkeypox virus ATI gene fragment with a concentration of more than 100 copies / μL can be detected without the use of large instruments and equipment. It can be judged by naked eyes only through portable ultraviolet lamps or immunochromatographic test strips. It is a detection method with great market value.

[0089] The above is only a preferred embodiment of the present disclosure. It should be understood that the present disclosure is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present disclosure, and should be within the scope of protection of the claims attached to the present disclosure.

Claims

1. A LAMP-CRISPR visualization detection method for identifying monkeypox virus based on the ATI gene, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested; A LAMP amplification system and a CRISPR detection system are prepared in a reaction container, wherein the LAMP amplification system and the CRISPR detection system are separated by mineral oil and air; Using the DNA as a template, performing a LAMP amplification reaction in the LAMP amplification system to obtain an amplification product; as well as Mixing the components in the reaction container, and performing enzyme cleavage reaction and visual detection; Wherein, the reaction container comprises a PCR reaction tube with a capacity of 200 μL; The thickness of the mineral oil is 2.5 to 3 mm; The thickness of the air is 11 to 12 mm; The LAMP amplification system is prepared at the bottom of the reaction container, the mineral oil is located on the upper surface of the LAMP amplification system, and the CRISPR detection system is prepared in the circular groove of the PCR reaction tube cover.

2. The LAMP-CRISPR visualization detection method according to claim 1, characterized in that: The LAMP amplification system includes the following components: Wherein, in the primer set (10×), an outer primer pair F3 / B3 and an inner primer pair FIP / BIP are included, the concentration of the outer primer pair F3 / B3 is 2 μM, and the concentration of the inner primer pair FIP / BIP is 16 μM; The nucleotide sequence of the forward outer primer F3 is shown in SEQ ID NO.1, the nucleotide sequence of the reverse outer primer B3 is shown in SEQ ID NO.2, the nucleotide sequence of the forward inner primer FIP is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse inner primer BIP is shown in SEQ ID NO.

4.

3. The LAMP-CRISPR visualization detection method according to claim 1, characterized in that: When performing the fluorescence detection, the CRISPR detection system includes NE Buffer (10×) 2 μL, LbCas12a protein (1 μM) 1 μL, crRNA (10 μM) 1.25 μL, single-stranded DNA probe (50 μM) 1 μL with FAM / BHQ1 fluorescent labeling, RNase inhibitor (40 U / μL) 0.25 μL and DEPC water 12.5 μL; When performing the immunochromatographic test strip test, the CRISPR detection system includes NE Buffer (10×) 2 μL, LbCas12a protein (1 μM) 1 μL, crRNA (10 μM) 1.25 μL, single-stranded DNA probe (10 μM) with FAM / Biotin fluorescent labeling 1 μL, RNase inhibitor (40 U / μL) 0.25 μL and DEPC water 12.5 μL; Wherein, the nucleotide sequence of the crRNA is shown in SEQ ID NO.

5.

4. The LAMP-CRISPR visualization detection method according to claim 1, characterized in that The visual detection includes one of fluorescence detection and immunochromatographic test paper detection.

5. The LAMP-CRISPR visualization detection method according to claim 1, characterized in that: The mixing method includes flash centrifugation.

6. The LAMP-CRISPR visualization detection method according to claim 1, characterized in that: The temperature of the LAMP amplification reaction is 60° C. to 65° C., and the time of the LAMP reaction is 30 to 60 min; and / or, the temperature of the enzyme cleavage reaction is 37° C., and the time of the enzyme cleavage reaction is 5 to 25 min.

7. A LAMP-CRISPR visualization detection kit for identifying monkeypox virus based on the ATI gene, characterized in that: The kit comprises the LAMP amplification system in the LAMP-CRISPR visualization detection method of claim 2, one of the CRISPR detection systems in the LAMP-CRISPR visualization detection method of claim 3, and mineral oil.

8. The LAMP-CRISPR visualization detection kit according to claim 7, characterized in that The kit also includes at least one of a portable small-sized ultraviolet lamp and an immunochromatographic test strip.

9. Use of the LAMP-CRISPR visualization detection method according to any one of claims 1 to 6 or the LAMP-CRISPR visualization detection kit according to any one of claims 7 to 8 in identifying the ATI gene of monkeypox virus.

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