TYLCCNV and PaLCuCNV virus detection kit based on PfAgo and its application

Abstract

The TYLCCNV and PaLCuCNV virus detection kits based on PfAgo and their applications belong to the technical field of virus detection. The present invention provides a TYLCCNV and PaLCuCNV virus detection kit based on PfAgo on the one hand, and provides an application of the TYLCCNV and PaLCuCNV virus detection kits in anti-pollution dual fluorescence detection on the other hand. The present invention provides a new and reliable technical option for the field of nucleic acid detection and provides a new idea for the application of prokaryotic Ago protein in biotechnology.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a TYLCCNV and PaLCuCNV virus detection kit based on PfAgo and applications thereof. Background Art

[0002] Geminiviruses are a class of plant single-stranded DNA viruses transmitted by insect vectors, causing devastating damage to economic and food crops such as tomatoes worldwide. Tomato yellow leaf curl virus (TYLCCNV) and Chinese papaya leaf curl virus (PaLCuCNV) are both viruses belonging to the Begomovirus genus, which are mainly transmitted by whiteflies and can infect a variety of crops with important economic value. TYLCCNV infection can cause severe leaf curling, yellowing and growth retardation in crops, seriously affecting a large reduction in crop yield. The disease characteristics of PaLCuCNV-infected plants are wrinkled and curled leaves, rolling downward or inward, and forming an inverted cup shape. The leaves become leathery, hard, reduced in size, with thickened veins and serrated petioles. In addition, infected plants will experience leaf drop, fail to bloom or fruit, and have limited growth in the late stages of infection.

[0003] Nucleic acid detection technology based on the CRISPR / Cas (Clustered regularly interspaced short palindromicrepeats / CRISPR-associated proteins system) system has made significant progress in rapid diagnosis. This method has the advantages of high sensitivity and high specificity, but is limited by the sequence dependence of sgRNA and PAM (Protospaceradjacent motif) / PFS (Protospacer flanking site). In contrast, Argonaute protein, a nuclease similar to Cas protein, shows potential in nucleic acid detection. Among them, the PfAgo protein derived from Pyrococcus furiosus can bind to 5'phosphorylated single-stranded DNA (guide DNA, gDNA) at high temperature to form a PfAgo / gDNA complex, and cleave the complementary target DNA in a directed manner. The nucleic acid detection technology developed based on PfAgo has gotten rid of the limitation of PAM / PFS sequence and has great advantages in the field of multi-target in vitro nucleic acid detection. However, the application of this technology in the rapid detection of plant viruses is still in the early stages of research. Although plant virus detection technology based on RT-RPA and PfAgo has been developed, this technology still has the risk of aerosol contamination during isothermal amplification and is limited by its dependence on fluorescent equipment.

[0004] In response to the above problems, the present invention further explores the recognition mechanism of PfAgo for uracil-containing nucleic acids, clarifies the compatibility of PfAgo with double-stranded DNA containing uracil and guide DNA (gDNA), and develops a new method for nucleic acid anti-contamination detection based on PfAgo by combining it with the UDG-LAMP nucleic acid amplification strategy. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to design and provide a TYLCCNV and PaLCuCNV virus detection kit based on PfAgo and a technical solution for its application.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides a TYLCCNV and PaLCuCNV virus detection kit based on PfAgo, comprising:

[0008] A reagent for LAMP amplification, the reagent comprising a primer for TYLCCNV virus-specific amplification and a primer for PaLCuCNV virus-specific amplification;

[0009] A gDNA for guiding PfAgo protein to perform targeted cleavage, wherein the gDNA includes TYLCCNV-g1T, TYLCCNV-g2T, TYLCCNV-g3T, PaLCuCNV-g1T, PaLCuCNV-g2T, and PaLCuCNV-g3T, wherein the nucleotide sequence of TYLCCNV-g1T is shown in SEQ ID NO.11, the nucleotide sequence of TYLCCNV-g2T is shown in SEQ ID NO.12, the nucleotide sequence of TYLCCNV-g3T is shown in SEQ ID NO.13, the nucleotide sequence of PaLCuCNV-g1T is shown in SEQ ID NO.15, the nucleotide sequence of PaLCuCNV-g2T is shown in SEQ ID NO.16, and the nucleotide sequence of PaLCuCNV-g3T is shown in SEQ ID NO.17;

[0010] Fluorescent molecular beacons;

[0011] and PfAgo protein.

[0012] Further, the primers for TYLCCNV virus-specific amplification include TYLCCNV-F3, TYLCCNV-B3, TYLCCNV-FIP, TYLCCNV-BIP and TYLCCNV-LF, the nucleotide sequence of TYLCCNV-F3 is shown in SEQ ID NO.1, the nucleotide sequence of TYLCCNV-B3 is shown in SEQ ID NO.2, the nucleotide sequence of TYLCCNV-FIP is shown in SEQ ID NO.3, the nucleotide sequence of TYLCCNV-BIP is shown in SEQ ID NO.4 and the nucleotide sequence of TYLCCNV-LF is shown in SEQ ID NO.5; the primers for PaLCuCNV virus-specific amplification include PaLCuCNV-F3, PaLCuCNV-B3, PaLCuCNV-FIP, PaLCuCNV-BIP and PaLCuCNV-LB, the nucleotide sequence of PaLCuCNV-F3 is shown in SEQ ID NO.6, the nucleotide sequence of PaLCuCNV-B3 is shown in SEQ ID NO.7, the nucleotide sequence of PaLCuCNV-FIP is such as SEQ ID NO.8, the nucleotide sequence of PaLCuCNV-BIP is such as SEQ ID NO.9 and the nucleotide sequence of PaLCuCNV-LB is such as SEQ ID NO.10.

[0013] Furthermore, the fluorescent molecular beacon includes TYLCCNV-probe-fluorescence and PaLCuCNV-probe-fluorescence, the nucleotide sequence of the TYLCCNV-probe-fluorescence is shown in SEQ ID NO.14, and the nucleotide sequence of the PaLCuCNV-probe-fluorescence is shown in SEQ ID NO.18.

[0014] The second aspect of the present invention provides the use of any of the above-mentioned kits in the detection of TYLCCNV and PaLCuCNV viruses.

[0015] The third aspect of the present invention provides a PfAgo-based dual fluorescence detection method for Gemini virus anti-pollution, the method comprising the following steps:

[0016] S1 extracts DNA from the sample to be tested;

[0017] S2 uses UDG-LAMP amplification system to amplify the sample to be tested, and the UDG-LAMP amplification system includes 10×Isothermal Amplification Buffer, dNTPs, MgSO4, primers F3 / B3, primers FIP / BIP, primers LF / LB, DNA Template, Bst t 2.0, UDG enzyme and ultrapure water;

[0018] S3 establishes a PfAgo system including gDNA, PfAgo protein, fluorescent molecular beacon, MnCl2 and LAMP amplification product to carry out the reaction;

[0019] After the S4 reaction is completed, the above detection system is subjected to fluorescence detection to measure the fluorescence intensity.

[0020] Furthermore, the dNTPs in the UDG-LAMP amplification system of S2 include dTTP, dUTP, dATP, dGTP and dCTP, wherein the final concentration of dTTP and dUTP is 5:5.

[0021] Further, the Mg in the UDG-LAMP amplification system of S2 2+ The concentration is 8mM; the dNTPs concentration is 0.6 mM; the reaction temperature is 62-64 ℃; the reaction time is 40-50 min.

[0022] Furthermore, the Mn in the PfAgo system in S3 2+ The concentration is 0.6 mM; the gDNA concentration is 2 µM; and the PfAgo concentration is 2.5 µM.

[0023] The present invention explores the ability of PfAgo protein to recognize and cut uracil-containing DNA, and innovatively adopts the strategy of combining dUTP-LAMP amplification with PfAgo to establish a dual nucleic acid anti-contamination detection technology (Combining UDG-LAMP and PfAgoforNucleic Acid Detection Platform, ULPD). After verification, this method showed a detection sensitivity of up to 4.33-6.79 copies / μL. In practical applications, nucleic acid extracts of tomato leaves suspected of being infected with TYLCCNV and PaLCuCNV were tested, and the test results were consistent with the qPCR results. In addition, false positive contamination caused by aerosols was successfully prevented by the UDG digestion step, proving the effectiveness of the anti-contamination mechanism of the ULPD detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of ULPD detection principle.

[0025] Figure 2 Create a diagram for the UDG-LAMP system.

[0026] Figure 3 Figure 1 shows the optimization of LAMP conditions for PaLCuCNV. A. LAMP amplification of PaLCuCNV Mg 2+A. Optimization of concentration; B. Optimization of temperature for LAMP amplification of PaLCuCNV; C. Optimization of dNTP concentration for LAMP amplification of PaLCuCNV; D. Optimization of time for LAMP amplification of PaLCuCNV. NTC: negative control.

[0027] Figure 4 Figure 1 is the optimization diagram of LAMP conditions for TYLCCNV. Figure A. LAMP amplification of Mg by TYLCCNV 2+ A. A. Optimization of concentration; B. Optimization of temperature for LAMP amplification of TYLCCNV; C. Optimization of dNTP concentration for LAMP amplification of TYLCCNV; D. Optimization of time for LAMP amplification of TYLCCNV.

[0028] Figure 5 This is the key component optimization diagram of the PfAgo system, in which A. gDNA concentration optimization; B. Mn 2+ Concentration optimization; C. PfAgo concentration optimization.

[0029] Figure 6 This is a specificity evaluation diagram for ULPD multiplex detection, in which A. primer type is PaLCuCNV for dUTP-LAMP amplification of different types of templates; B. primer type is TYLCCNV for dUTP-LAMP amplification of different types of templates; C. primer type is PaLCuCNV+TYLCCNV for dUTP-LAMP amplification of different types of templates; D. ULPD fluorescence signal detection heat map of different primer types and template types.

[0030] Figure 7 This is the actual sample detection image of the ULPD fluorescence channel. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to the examples. Example

[0032] 1.1 Experimental Materials

[0033] 1.1.1 Experimental reagents

[0034] The reagents used in this example are shown in Table 1:

[0035] Table 1 Reagents used

[0036]

[0037] 1.1.2 Oligonucleotide sequences

[0038] The molecular beacons, gDNA and other ssDNA used in this application were purchased from Nanjing GenScript Biotechnology Co., Ltd. The 5' end of the molecular beacon was modified with different fluorescent groups (such as FAM, ROX fluorescent groups), and the 3' end was modified with corresponding quenching groups (such as BHQ1, BHQ2 quenching groups). The oligonucleotide sequences are shown in Tables 2 and 3:

[0039] Table 2 Oligonucleotide sequences used for LAMP amplification

[0040]

[0041] Table 3 Oligonucleotide sequences for PfAgo cleavage

[0042]

[0043] Note: P stands for phosphate group modification; FAM stands for biotin modification; ROX- stands for digoxigenin modification; BHQ1- stands for digoxigenin modification; BHQ2- stands for digoxigenin modification. 1 : Lowercase letters represent hairpin structures.

[0044] 1.2 Experimental methods

[0045] 1.2.1 Preparation of template DNA

[0046] 1) Collect fresh and clean plant leaves and place them in a 1.5 mL centrifuge tube containing 4-6 steel beads. After quick freezing in liquid nitrogen, grind with a grinding rod for 5 min to ensure that the sample is fully broken.

[0047] 2) Add 600 μL of CTAB to the centrifuge tube containing the sample, mix well, and incubate at 65°C for 20 min, inverting several times during the process.

[0048] 3) Add 600 μL of chloroform to the centrifuge tube and shake to mix. Centrifuge at 12,000 rpm for 10 min.

[0049] 4) Aspirate the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol (pre-cooled), mix well, and centrifuge at 12,000 rpm for 5 min.

[0050] 5) Discard the supernatant and add 700 μL of 75% alcohol. Mix by inverting and centrifuging at 12,000 rpm for 2 min.

[0051] 6) Discard the supernatant and leave the centrifuge tube at room temperature to allow the alcohol to evaporate completely.

[0052] 7) Add 50 μL of ultrapure water to the centrifuge tube and mix well to dissolve the DNA.

[0053] 1.2.2 UDG-LAMP amplification

[0054] (1) LAMP primer design

[0055] To design specific LAMP primers, we used NEB and input the target gene sequence. Primers for LAMP reaction were successfully designed.

[0056] (2) LAMP amplification

[0057] The LAMP amplification system is as follows:

[0058] Table 4 LAMP amplification system

[0059]

[0060] Incubate at 65°C for 40 min, and the amplified products are visualized by 3% TAE gel electrophoresis. For real-time LAMP fluorescence detection, add LAMP Fluorescent Dye to perform real-time fluorescence detection in a qPCR instrument.

[0061] (3) UDG enzyme digestion

[0062] 1) Add 0.5 μL of UDG enzyme to the next round of LAMP system.

[0063] 2) Set the reaction program as follows: 25°C, 15 min; 63°C, 40 min (to inactivate UDG enzyme and perform LAMP amplification).

[0064] 1.2.3 PfAgo cleavage experiment

[0065] The PfAgo reaction was set up in an 80 µL reaction system. First, 20 µL of LAMP reaction product, 4 µL of purified PfAgo, 1 µM probe, 0.6 mM MnCl2, and 2 µM 5' phosphorylated gDNA were combined. Subsequently, the reaction mixture was replenished by adding 8 µL of 10× reaction buffer. The reaction mixture was incubated at 95 °C for 30 min. The obtained products were analyzed by 3% TAE gel or fluorescent signal detection in CFX384 qPCR instrument.

[0066] 1.2.4PfAgo fluorescence detection analysis

[0067] The PfAgo reaction mixture consists of 2 µL 10× reaction buffer, 2 µM PfAgo, 2 µM gDNA, 1 µM fluorescent probe, and 0.6 mM MnCl2, and then the mixture is added to 4 µL LAMP product to prepare a total volume of 30 µL PfAgo reaction mixture. The reaction tube is placed in a qPCR instrument and incubated at 95°C, and the fluorescent signal is recorded or observed within 30 min.

[0068] 1.2.5 ULPD fluorescence detection sensitivity and specificity detection

[0069] Use 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 The sensitivity of the corresponding LAMP primers and the PfAgo cleavage sensitivity were compared with 0 copies of the target gene fragment template. For each LAMP primer, 100 copies of 5 common plant geminiviruses (extracted nucleic acids) were used for specificity determination. The amplification results of LAMP were displayed on TAE gel. For the ULPD assay, 4μL of LAMP amplicon was placed in the PfAgo cleavage reaction to verify the corresponding sensitivity of its detection. For each set of LAMP primers, 100 copies of 5 common plant geminiviruses (extracted nucleic acids) were amplified, and PfAgo fluorescence analysis experiments were performed for specificity determination. The test results were displayed on a qPCR instrument.

[0070] 1.2.6 Application of ULPD in actual sample testing

[0071] Ten tomato leaf samples were collected from various locations in Hangzhou. DNA was extracted using the CTAB method. All LAMP assays and PfAgo cleavage assays were digested by UDG enzyme to prevent aerosol contamination. For ULPD fluorescence assays, signals were collected at 95°C in the FAM and ROX channels of the qPCR instrument.

[0072] Experimental Results

[0073] 1.2.7 ULPD method design ideas

[0074] In order to solve the false positive problem, this example proposes a new nucleic acid detection system ULPD (Combining UDG-LAMP and PfAgo for Nucleic Acid Detection Platforms, ULPD), which introduces the UDG-LAMP system and combines PfAgo to achieve simultaneous detection of multiple targets and prevent contamination. For LAMP amplicons, in order to avoid interference caused by the stem-loop structure, the gDNA designed in the experiment is located in the non-stem-loop structure region.

[0075] ULPD detection mechanism such as Figure 1As shown. First, LAMP technology was used to enrich specific fragments of plant viruses. The specific sequences were the CP gene fragment of PaLCuCNV and the V1 gene fragment of TYLCCNV. Subsequently, under the guidance of three designed 5'-end phosphorylated gDNAs, the PfAgo protein was mediated to specifically recognize the non-stem-loop region of the amplified fragment. When the gDNA was successfully paired with one chain of the amplified fragment, PfAgo triggered the cleavage of the phosphodiester bond between the 10th and 11th bases of the target DNA from the 5' end to form a secondary gDNA, which continued to mediate PfAgo to cut the designed molecular beacon with fluorescent groups and quenching groups to release the fluorescent group, and release the fluorescent group for detection by the qPCR instrument ( Figure 1 Figure 2 (B). Different secondary gDNAs generated in this process are targeted to specific probes with different fluorescent labels, thus enabling the simultaneous detection of multiple viral targets. This dual recognition and cleavage process enhances the specificity and accuracy of the method. In addition, the use of UDG enzyme to process the sample before the next round of amplification further reduces the risk of false positives.

[0076] 1.2.8 Establishment of UDG-LAMP method

[0077] In order to prevent aerosol contamination caused by LAMP amplification in the ULPD system, the UDG-LAMP system was established in this example. Figure 2 A in the middle), specific primers were designed, and uracil nucleotides were integrated into the LAMP amplification product by adding dUTP to the raw material. Uracil-DNA glycosidase (UDG) can effectively remove DNA containing uracil to prevent aerosol contamination. However, the addition of too much dUTP will affect the LAMP amplification efficiency. For this reason, this example further explores the optimal mixing ratio of dUTP and other dNTPs to ensure maximum amplification efficiency. Different ratios of dTTP and dUTP were designed and tested for the specific fragments of the twin viruses TYLCCNV and PaLCuCNV. The experimental results show that ( Figure 2 In the figure (B), when the ratio of dTTP to dUTP is 5:5, this balance can effectively reduce contamination without significantly affecting the amplification efficiency. In addition, in order to evaluate the anti-contamination ability of UDG, the LAMP product with dTTP as the raw material (dTTP-LAMP) and the LAMP product with dUTP were treated with UDG and then subjected to LAMP amplification again. The experimental results showed that ( Figure 2 (C and D in the figure), dUTP-LAMP does not produce a positive signal after being digested with UDG enzyme, which verifies the effectiveness of UDG-LAMP in preventing contamination.

[0078] 1.2.9 Optimization of ULPD Method

[0079] To improve the performance of the ULPD reaction, this example systematically optimized the key components of the LAMP reaction and PfAgo system, including Mg 2+ concentration, dNTP concentration, reaction temperature, reaction time, and gDNA, Mn 2+ and PfAgo protein concentration. In the LAMP system, Mg 2+ The optimal concentration is 8mM (the test range includes 2, 4, 6, 8, and 10 mM); the optimal reaction temperature is 62-64 °C (48, 52, 54, 58, 62, 64, 66, 68, and 70 °C); the optimal dNTP concentration is 0.6 mM (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mM), and the optimal reaction time is 40-50 min (20, 30, 40, 50, 60, 70, and 80 min) (e.g. Figure 3 and Figure 4 In addition, the LAMP amplicon was directly added to the PfAgo system, and the key components of the PfAgo system were optimized. 2+ Mn at a concentration of 0.6 mM 2+ The optimal concentrations were (0, 0.2, 0.4, 0.6, 0.8, 1 mM); 2 µM gDNA concentration was optimal among all tested concentrations (0, 0.5, 1, 1.5, 2, 2.5, and 3 µM); a PfAgo concentration range of 0.5 µM to 3 µM was tested, and 2.5 µM PfAgo was determined to be the most effective concentration (e.g. Figure 5 as shown).

[0080] 1.2.10 Establishment of the fluorescence dual detection method and specific detection of ULPD

[0081] This example has successfully verified that the combination of dUTP-LAMP and PfAgo can achieve single target detection. In order to further verify whether dual detection can be achieved and the accuracy and specificity of the system, this example also designed multiple LAMP primers (Table 2), gDNA and molecular beacons (TYLCCNV-Probe-fluorescence and PaLCuCNV-Probe-fluorescence, Table 3), and formed them into a mixed system for detection. Figure 6 As shown in A, B, and C, by using different LAMP primer combinations for amplification, the specific detection of the two viruses was successfully achieved, and non-target viruses were excluded. Different dUTP-LAMP amplification products were added to the PfAgo cleavage system for specific cleavage. Figure 6As shown in D, the qPCR instrument analysis shows that the fluorescence signal can only be detected when the template and primer types are correct. It is worth noting that the dual fluorescence signal can be detected when the dual primers are present. This result confirms the specificity of the ULPD detection system and the feasibility of dual detection. In short, the detection system has good specificity and mixed sample detection capabilities, and has a wider application prospect in practical applications.

[0082] 1.2.11 Actual sample detection of ULPD fluorescence channel

[0083] After successfully verifying the performance of ULPD, this example further evaluated the ability of ULPD in detecting tomato samples suspected of being infected with the virus and analyzed the ULPD fluorescence detection method with the existing qPCR detection method. To ensure the reliability of the results, ULPD fluorescence channel analysis and standard qPCR analysis were performed on 10 double-identified samples and 5 standards. Figure 7 The test results at A in the middle show that 2 samples were identified as carrying PaLCuCNV, 1 sample was infected with TYLCCNV, and 2 samples were infected with both viruses. This result is consistent with the standard qPCR ( Figure 7 This method is consistent with the method in Figure 1, which proves that the detection method has extremely high sensitivity, specificity and accuracy.

[0084] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A TYLCCNV and PaLCuCNV virus detection kit based on PfAgo, characterized in that: include: A reagent for LAMP amplification, the reagent comprising a primer for TYLCCNV virus-specific amplification and a primer for PaLCuCNV virus-specific amplification; The primers for TYLCCNV virus-specific amplification include TYLCCNV-F3, TYLCCNV-B3, TYLCCNV-FIP, TYLCCNV-BIP and TYLCCNV-LF, the nucleotide sequence of TYLCCNV-F3 is shown in SEQ ID NO.1, the nucleotide sequence of TYLCCNV-B3 is shown in SEQ ID NO.2, the nucleotide sequence of TYLCCNV-FIP is shown in SEQ ID NO.3, the nucleotide sequence of TYLCCNV-BIP is shown in SEQ ID NO.4 and the nucleotide sequence of TYLCCNV-LF is shown in SEQ ID NO.5; the primers for PaLCuCNV virus-specific amplification include PaLCuCNV-F3, PaLCuCNV-B3, PaLCuCNV-FIP, PaLCuCNV-BIP and PaLCuCNV-LB, the nucleotide sequence of PaLCuCNV-F3 is shown in SEQ ID NO.6, the nucleotide sequence of PaLCuCNV-B3 is shown in SEQ ID NO.

7. NO.7, the nucleotide sequence of PaLCuCNV-FIP is SEQ ID NO.8, the nucleotide sequence of PaLCuCNV-BIP is SEQ ID NO.9, and the nucleotide sequence of PaLCuCNV-LB is SEQ ID NO.10; A gDNA for guiding PfAgo protein to perform targeted cleavage, wherein the gDNA includes TYLCCNV-g1T, TYLCCNV-g2T, TYLCCNV-g3T, PaLCuCNV-g1T, PaLCuCNV-g2T, and PaLCuCNV-g3T, wherein the nucleotide sequence of TYLCCNV-g1T is shown in SEQ ID NO.11, the nucleotide sequence of TYLCCNV-g2T is shown in SEQ ID NO.12, the nucleotide sequence of TYLCCNV-g3T is shown in SEQ ID NO.13, the nucleotide sequence of PaLCuCNV-g1T is shown in SEQ ID NO.15, the nucleotide sequence of PaLCuCNV-g2T is shown in SEQ ID NO.16, and the nucleotide sequence of PaLCuCNV-g3T is shown in SEQ ID NO.17; A fluorescent molecular beacon, wherein the fluorescent molecular beacon comprises TYLCCNV-probe-fluorescence and PaLCuCNV-probe-fluorescence, wherein the nucleotide sequence of the TYLCCNV-probe-fluorescence is shown in SEQ ID NO.14, and the nucleotide sequence of the PaLCuCNV-probe-fluorescence is shown in SEQ ID NO.18; and PfAgo protein.

2. Use of the kit as claimed in claim 1 in TYLCCNV and PaLCuCNV virus detection.

3. A PfAgo-based dual fluorescence detection method for Gemini virus anti-pollution, characterized in that: The following steps are involved: S1 extracts DNA from the sample to be tested; S2 uses UDG-LAMP amplification system to amplify the sample to be tested, wherein the UDG-LAMP amplification system includes 10×Isothermal Amplification Buffer, dNTPs, MgSO4, primers F3 / B3, primers FIP / BIP, primers LF / LB, DNA Template, Bst t 2.0, UDG enzyme and ultrapure water. 2+ The concentration is 8mM; the dNTPs concentration is 0.6 mM; the reaction temperature is 62-64 ℃; the reaction time is 40-50 min; S3 established a PfAgo system including gDNA, PfAgo protein, fluorescent molecular beacon, MnCl2 and LAMP amplification product. 2+ The reaction was carried out with a concentration of 0.6 mM, a gDNA concentration of 2 µM, and a PfAgo concentration of 2.5 µM. After the S4 reaction is completed, the above detection system is subjected to fluorescence detection to measure the fluorescence intensity.

4. A PfAgo-based dual fluorescence detection method for Gemini virus anti-pollution as claimed in claim 1, characterized in that: The dNTPs in the UDG-LAMP amplification system of S2 include dTTP, dUTP, dATP, dGTP and dCTP, wherein the final concentration of dTTP and dUTP is 5:5.

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