Geminivirus double detection test strip based on pfago and preparation method and application thereof

Abstract

The application discloses a PfAgo-based geminivirus double detection test strip and a preparation method and application thereof, and belongs to the technical field of virus detection.The application provides, on one hand, a PfAgo-based geminivirus double detection test strip and a construction method thereof, and on the other hand, application of the test strip in TYLCCNV and PaLCuCNV virus detection.The application uses the ability of PfAgo enzyme to recognize and cut uracil-containing DNA to innovatively develop a ULPD anti-pollution detection system.The system combines dUTP-LAMP technology for target enrichment, and realizes rapid diagnosis through designed gDNA and immunocolloidal gold test strips.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a PfAgo-based Gemini virus dual detection test strip, a preparation method, and an application 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) both belong to the Begomovirus genus, are mainly transmitted by whiteflies, and can infect a variety of economically important crops. TYLCCNV infection can cause severe leaf curling, yellowing, and growth retardation in crops, seriously affecting crop yields. The disease characteristics of PaLCuCNV-infected plants are wrinkled and curled leaves that roll downward or inward, 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 bear fruit, and have restricted growth in the late stages of infection.

[0003] In recent years, nucleic acid detection technology based on the CRISPR / Cas (Clustered regularly interspaced shortpalindromic repeats / 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 its dependence on the sgRNA and PAM (Protospacer adjacent motif) / PFS (Protospacer flanking site) sequences. In contrast, Argonaute protein, a nuclease similar to Cas protein, has shown 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 temperatures to form a PfAgo / gDNA complex, which can direct the cleavage of the complementary target DNA. The nucleic acid detection technology developed based on PfAgo has broken away from the limitations of PAM / PFS sequences 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 its early stages of research. Although plant virus detection technologies based on RT-RPA and PfAgo have been developed, they still rely heavily on fluorescence equipment. To address this issue, this study developed a dual-target detection test strip that eliminates the need for real-time fluorescence detection, making on-site multi-target rapid detection of plant viruses possible. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a PfAgo-based Geminivirus dual detection test strip and a preparation method and application technical solution.

[0005] The present invention is specifically achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a method for constructing a PfAgo-based Geminivirus dual detection test strip, comprising the following steps:

[0007] 1) Preparation of colloidal gold liquid

[0008] 2) Preparation of gold-labeled antibodies

[0009] Take 1 mL of colloidal gold solution, add 5 μL of 0.2 M K2CO3 solution, 4 μg of rabbit anti-FAM antibody, 5 μL of 0.2 M K2CO3 solution, and 5 μg of mouse anti-Dig antibody, add K2CO3 adjustment solution and rabbit anti-FAM / mouse anti-Dig protein antibody, and mix slowly to ensure sufficient binding; add 10% BSA in a total volume of 200 μL and mix slowly to block unbound sites; then, refrigerate and centrifuge; after removing the supernatant, add 200 μL of working solution to the remaining precipitate to resuspend the colloidal gold particles. The prepared gold-labeled antibody is stored at 4°C until use;

[0010] 3) Assembly of colloidal gold test strips

[0011] a) Cut the NC film of appropriate size and stick it flat on the PVC base plate, making sure the surface is clean and free of contamination;

[0012] b) Align and place the gold label pads on the NC membrane with a spacing of 2 mm. Place the cut sample pad on top of the NC membrane and absorbent paper on the other end of the NC membrane to form a tight structure.

[0013] c) Use 0.1 μg / μL goat anti-mouse antibody and 0.1 μg / μL goat anti-rabbit antibody as the test lines (i.e., T1 and T2 lines), and 0.2 μg / μL streptavidin as the quality control line (i.e., C line). Mark the lines at a speed of 5 μL / cm, ensuring that the interval between the two lines is at least 5 mm;

[0014] d) After the streaking is completed, the NC membrane is dried at 37°C for 16 hours before use;

[0015] e) Spray the gold-labeled antibody evenly onto the glass fiber membrane at a volume of 2 μL / cm to form a gold-labeled pad, and dry it at 37°C for 30 minutes before use;

[0016] f) soaking the sample pad in the activation solution for 2 hours, and then drying it at 37°C for 2 hours;

[0017] g) Use a paper cutter to accurately cut the test strips into 4 mm width. After cutting, seal the test strips and place them in a dry environment.

[0018] The second aspect of the present invention provides a PfAgo-based Geminivirus dual detection test strip prepared by the above method.

[0019] The third aspect of the present invention provides the use of the above-mentioned test strip in the detection of TYLCCNV and PaLCuCNV viruses.

[0020] A fourth aspect of the present invention provides a method for detecting TYLCCNV and PaLCuCNV viruses using the above-mentioned test strips, comprising the following steps:

[0021] S1 extracts DNA from the sample to be tested;

[0022] S2 uses the UDG-LAMP amplification system to amplify the sample to be tested, and the UDG-LAMP amplification system includes 10×IsothermalAmplificationBuffer, dNTPs, MgSO4, primers for TYLCCNV virus-specific amplification, primers for PaLCuCNV virus-specific amplification, DNA Template, Bst t 2.0, UDG enzyme and ultrapure water;

[0023] S3 establishes a PfAgo system including gDNA that guides PfAgo protein for targeted cleavage, PfAgo protein, probe, MnCl2 and LAMP amplification product, and performs a reaction;

[0024] After the S4 reaction is completed, the above detection system is tested using a test strip.

[0025] Further, the primers for TYLCCNV virus-specific amplification in step S2 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 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.

[0026] Furthermore, the UDG-LAMP amplification system in step S2 includes: 10×Isothermal Amplification Buffer 2.5 μL, 10 mM dNTPs 3.5 μL, 100 mM MgSO4 1.6 μL, 10 μM primer F3 / B3 0.5 μL, 10 μM primer FIP / BIP 3.2 μL, 10 μM primer LF / LB 1 μL, DNA Template 1 μL, 8000 U / mL Bst t 2.01 μL, UDG enzyme 0.5 μL, and ultrapure water supplemented to 25 μL.

[0027] Further, the gDNA that guides the PfAgo protein for targeted cleavage in step S3 includes TYLCCNV-g1T, TYLCCNV-g2T, TYLCCNV-g3T, PaLCuCNV-g1T, PaLCuCNV-g2T, and PaLCuCNV-g3T, 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.

[0028] Furthermore, the probe in step S3 includes TYLCCNV-probe-test strip and PaLCuCNV-probe-test strip, the nucleotide sequence of the TYLCCNV-probe-test strip is shown in SEQ ID NO.14, and the nucleotide sequence of the PaLCuCNV-probe-test strip is shown in SEQ ID NO.18.

[0029] Furthermore, the amount of probe in step S3 is less than 20 pmol.

[0030] The present invention utilizes the ability of the PfAgo enzyme to recognize and cleave uracil-containing DNA to innovatively develop a PfAgo-based anti-pollution dual-channel detection technology - the test strip method (Combining UDG-LAMP and PfAgo for Nucleic Acid Detection Platform, ULPD), hereinafter referred to as the ULPD-test strip method. This system combines dUTP-LAMP technology for target enrichment and achieves rapid diagnosis through designed gDNA and colloidal gold test strips. In terms of system optimization, after adjusting the key reaction components, the ULPD test strip method performs well in terms of detection speed and sensitivity (10 copies / μL) and can effectively avoid the problem of aerosol contamination during the LAMP amplification process. At the same time, the application of colloidal gold test strips in this detection system further breaks away from the dependence of the fluorescence method on the qPCR instrument, which has significant advantages in resource-poor areas. However, this section only preliminarily established the working concentration of the colloidal gold test strip. The optimal working concentration of the colloidal gold test strip has not yet been determined, and its SA amount, gold-labeled antibody amount and secondary antibody concentration need to be further optimized. In short, PfAgo has greater and broader advantages based on CRISPR technology and has the opportunity to become the main force in the development of the next-generation nucleic acid detection platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram of the construction and system optimization of colloidal gold test strips;

[0032] Figure 2 is a graph of colloidal gold particles and their wavelength;

[0033] Figure 3 This is the condition optimization diagram for the preparation of colloidal gold antibody;

[0034] Figure 4 This is the optimization diagram of the test strip probe concentration in the colloidal gold test strip reaction;

[0035] Figure 5 Schematic diagram of specificity evaluation of ULPD test strips;

[0036] Figure 6 Schematic diagram of sensitivity evaluation of ULPD test strip detection;

[0037] Figure 7 This is a schematic diagram of actual sample testing using the ULPD test strip method. Figure 7 Middle: A. Actual sample testing using the ULPD test strip method (if the C line, T1, and T2 lines are all colored, the sample is identified as TYLCCNV and PaLCuCNV infected; if the test strip shows that the T2 line and C line are red and the T1 line is not colored, the sample is identified as TYLCCNV infected; if the test strip shows that the T1 line and C line are red and the T2 line is not colored, the sample is identified as PaLCuCNV infected; if the test strip C line is colored and both T1 and T2 are not colored, the sample is identified as uninfected with PaLCuCNV and TYLCCNV); B. Actual sample testing using the qPCR standard test (CT value ≤38 is positive). DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the following examples.

[0039] Example

[0040] 1.1 Experimental Materials

[0041] 1.1.1 Experimental Reagents

[0042] The reagents used in this example are as follows:

[0043] Table 1 Reagents used in this example

[0044]

[0045] 1) 1% chloroauric acid solution (200 mL): Dissolve 2 g of chloroauric acid in 200 mL of ultrapure water. Store at 4°C in the dark.

[0046] 2) 1% trisodium citrate solution (10 mL): Dissolve 0.1 g of trisodium citrate powder in ultrapure water to a volume of 10 mL. Filter through a 0.22 μm filter membrane and store at 4°C.

[0047] 3) 0.2 M K2CO3 (20 mL): Dissolve 0.552 g of K2CO3 in ultrapure water and dilute to 20 mL. Filter through a 0.22 μm filter and store at 4°C.

[0048] 4) 10% NaCl solution (20 mL): Dissolve 2 g of NaCl in 20 mL of ultrapure water, filter through a 0.22 μm filter, and store at 4°C.

[0049] 5) Storage reconstitution solution (14 mL): 10% BSA 1 mL, 50% trehalose 1 mL, 50% sucrose 2 mL, 0.2 M Tris-HCl 10 mL, mix well, adjust the pH to 8.2, and store at 4°C.

[0050] 6) Working Buffer (400 μL): Prepare 360 ​​μL of the storage solution, add 40 μL of 10% BSA, mix well, and use immediately.

[0051] 7) PB Buffer (100 mL): Mix 0.024 g of potassium dihydrogen phosphate and 0.358 g of disodium hydrogen phosphate dodecahydrate with ultrapure water to make up to 100 mL. Adjust the pH to 7.4, filter through a 0.22 μm filter, and store at 4°C.

[0052] 1.1.2 Experimental instruments

[0053] The instruments used in this example are shown in the following table:

[0054] Table 2 Instruments used in this example

[0055]

[0056] 1.1.3 Oligonucleotide sequences

[0057] The molecular beacons, gDNA, and other ssDNA used in this example were purchased from Nanjing GenScript Biotechnology Co., Ltd. The oligonucleotide sequences are as follows:

[0058] Table 3 Oligonucleotide sequences used for LAMP amplification

[0059]

[0060] Table 4 Oligonucleotide sequences for PfAgo cleavage

[0061]

[0062] Note: P represents phosphate group modification; Biotin represents biotin modification; Dig- represents digoxigenin modification.

[0063] 1.2 Experimental methods

[0064] 1.2.1 Preparation of template DNA

[0065] 1) Collect fresh, clean plant leaves and place them in a 1.5 mL centrifuge tube containing 4-6 steel balls. Quickly freeze in liquid nitrogen and grind with a grinding rod for 5 minutes to ensure the sample is fully broken down.

[0066] 2) Add 600 μL of CTAB to the centrifuge tube containing the sample, mix thoroughly, and incubate at 65°C for 20 min, inverting the tube several times to mix thoroughly.

[0067] 3) Add 600 μL of chloroform to the centrifuge tube and shake to mix. Centrifuge at 12,000 rpm for 10 minutes.

[0068] 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 minutes.

[0069] 5) Discard the supernatant and add 700 μL of 75% ethanol. Mix by inverting the tube and centrifuge at 12,000 rpm for 2 minutes.

[0070] 6) Discard the supernatant and place the centrifuge tube at room temperature to allow the alcohol to evaporate completely.

[0071] 7) Add 50 μL of ultrapure water to the centrifuge tube and mix well to dissolve the DNA.

[0072] 1.2.2 UDG-LAMP amplification

[0073] (1) LAMP primer design

[0074] To design specific LAMP primers, we used NEB (https: / / lamp.neb.com / #! / ) and input the target gene sequence. Primers for the LAMP reaction were successfully designed.

[0075] (2) LAMP amplification

[0076] The LAMP amplification system is as follows:

[0077] Table 5 LAMP amplification system

[0078]

[0079] Incubate at 65°C for 40 minutes, and visualize the amplified products on 3% TAE gel electrophoresis. For real-time LAMP fluorescence detection, add LAMP Fluorescent Dye to perform real-time fluorescence detection in a qPCR instrument.

[0080] (3) UDG enzyme digestion

[0081] 1) Add 0.5 μL of UDG enzyme to the LAMP system in the next round.

[0082] 2) The reaction program was set as follows: 25°C, 15 min; 63°C, 40 min (to inactivate UDG enzyme and perform LAMP amplification).

[0083] 1.2.3 PfAgo cleavage experiment

[0084] The PfAgo reaction was set up in an 80 μL reaction system. First, 20 μL of the 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 minutes. The resulting products were analyzed on a 3% TAE gel.

[0085] 1.2.4 Preparation of colloidal gold liquid

[0086] 1) Treat the relevant glassware, wrap it with tin foil and seal it for storage.

[0087] 2) Add 99 mL of ultrapure water to a flask equipped with a rotor and place it on a heated magnetic stirrer. Set the temperature and rotor speed to the desired setting. When the flask is about to boil, add 1 mL of trisodium citrate solution.

[0088] 3) After boiling, add 1 mL of chloroauric acid solution. Stir at an increased speed and continue heating for 15 minutes, then allow the solution to cool naturally at room temperature.

[0089] 4) Dilute the solution to 100 mL and transfer to a conical flask. Store at 4°C in the dark.

[0090] 1.2.5 Determination of optimal pH and antibody amount

[0091] To determine the optimal pH of the prepared colloidal gold solution, add 1 mL of colloidal gold solution to each of six 1.5 mL centrifuge tubes. Then, add different volumes of 0.2 M K2CO3 solution (0, 1, 2, 3, 4, 5, 6, 7, and 8 μL) to the centrifuge tubes in sequence and vortex to mix. Add an excess of 20 μL of rabbit anti-FAM / mouse anti-Dig protein to each centrifuge tube and slowly mix again. After mixing thoroughly, add 100 μL of 10% NaCl solution to each centrifuge tube. After addition, slowly mix the solution and let it stand for 2 hours. Observe the color of the solution and determine the volume of K2CO3 solution that can maintain the colloidal gold solution unchanged as the optimal pH.

[0092] Add 1 mL of colloidal gold solution to each of six 1.5 mL centrifuge tubes. Add a predetermined amount of K2CO3 to each centrifuge tube to adjust the colloidal gold solution to the optimal pH. Subsequently, add different amounts of rabbit anti-FAM antibody (0.6 mg / mL) / mouse anti-Dig antibody (0.5 mg / mL) (0, 1, 2, 3, 4, 5, and 6 μL) to each centrifuge tube. After adding the rabbit anti-FAM / mouse anti-Dig protein, mix slowly to ensure sufficient contact and reaction with the colloidal gold. Place the centrifuge tubes in a room temperature for 20 minutes and observe for color changes or precipitation. Then, add 100 μL of 10% NaCl solution to each centrifuge tube and mix slowly again. Return the centrifuge tubes to room temperature and let them stand for 1 hour to further evaluate the binding of the rabbit anti-FAM / mouse anti-Dig protein to the colloidal gold. The sample without color change and precipitation is selected as the optimal labeling amount of the rabbit anti-FAM / mouse anti-Dig protein antibody.

[0093] 1.2.6 Preparation and identification of gold-labeled antibodies

[0094] Place 1 mL of colloidal gold solution in a 1.5 mL centrifuge tube, add the optimal 0.2 M K₂CO₃ solution and the optimized amount of rabbit anti-FAM / mouse anti-Dig antibody labeling, and mix gently to ensure sufficient binding. Add 10% BSA to a total volume of 200 μL and mix gently to block unbound sites. Subsequently, centrifuge at 12,000 rpm in a refrigerated centrifuge at 4°C for 30 minutes. Remove the supernatant and add 200 μL of the pre-prepared working reconstitution solution to the remaining pellet to resuspend the colloidal gold particles. Store the prepared gold-labeled antibody at 4°C until needed.

[0095] 1.2.7 Assembly of colloidal gold test strips

[0096] 1) Cut the NC film of appropriate size and stick it flat on the PVC base plate, making sure the surface is clean and free of contamination.

[0097] 2) Align and place the gold label pads on the NC membrane, 2 mm apart. Place the cut sample pad on top of the NC membrane. Place absorbent paper on the other side of the NC membrane to form a tight structure.

[0098] 3) Use goat anti-mouse antibody (0.2 μg / μL) and goat anti-rabbit antibody (0.2 μg / μL) as the test lines (T1 and T2 lines), and streptavidin (SA, 1 μg / μL) as the quality control line (C line). Mark the lines at a speed of 5 μL / cm, ensuring that the interval between the two lines is greater than 5 mm.

[0099] 4) After the marking is completed, the NC membrane is placed in a 37°C environment to dry for 16 hours before use.

[0100] 5) Spray the gold-labeled antibody evenly on the glass fiber membrane at a volume of 2 μL / cm to form a gold-labeled pad, and dry it at 37°C for 30 min before use.

[0101] 6) Soak the sample pad in the activation solution for 2 hours, and then dry it at 37°C for 2 hours.

[0102] 7) Use a paper cutter to accurately cut the test strips into 4mm widths. After cutting, seal the test strips and store them in a dry environment.

[0103] 1.2.8 Optimization of test strip probe quantity

[0104] In order to minimize the false positive phenomenon in the test strip detection line, the test strip probe concentration was optimized. The specific experimental process is as follows: different concentrations of 5'-FAM-ssDNA-Biotin-3' probe (PaLCuCNV-probe-test strip) and 5'-Dig-ssDNA-Biotin-3' probe (TYLCCNV-probe-test strip) (concentrations are 80pmol, 60pmol, 40pmol, 20pmol, 0pmol) were added to 80μL of Working buffer, and the test strips were inserted vertically into the prepared reaction solutions and allowed to stand at room temperature for 2 minutes. Afterwards, the test strips were taken out, and the color development of the test line was observed and recorded with the naked eye, and photos were taken to record the false positives of the test line at each concentration.

[0105] 1.2.9 ULPD test strip sensitivity and specificity testing

[0106] To enable strip detection, reporter DNA probes labeled with 5'FAM-ssDNA-Biotin 3' and 5'Dig-ssDNA-Biotin 3' were constructed. After LAMP preamplification, 4 μL of the product was added to the PfAgo system to a final volume of 30 μL, containing 2 μL of 10× reaction buffer, 2.5 μM PfAgo, 2 μM gDNA, the strip probe (≤20 pmol), and 0.6 mM MnCl2. After incubation at 95°C for 30 minutes, the reaction product was transferred to 75 μL of working buffer. A lateral flow strip was placed in the reaction tube for 5 minutes, and the results were directly observed. The color intensity of the line was photographed and analyzed using ImageJ.

[0107] 1.2.10 Application of ULPD Test Strip Method in Actual Sample Testing

[0108] Ten tomato leaf samples were collected from various locations in Hangzhou. DNA was extracted using the CTAB method. All LAMP and PfAgo cleavage assays were digested with UDG enzyme to prevent aerosol contamination. For ULPD strip assays, dual-detection strips were used for analysis.

[0109] Results 1 Construction and system optimization of colloidal gold test strips

[0110] In order to match the PfAgo cleavage output signal and get rid of the limitations of the qPCR instrument, this chapter simultaneously designed a dual detection test strip containing two target lines (T1 and T2 lines) and one control line (C line). Figure 1As shown in Figure D, in this design, two gold-labeled antibodies, rabbit anti-FAM antibody-AuNPs and mouse anti-Dig antibody-AuNPs, are pre-embedded in the conjugate pad. Simultaneously, streptavidin, goat anti-rabbit antibody, and goat anti-mouse antibody are incorporated into lines C, T1, and T2, respectively. When the PfAgo cleavage reaction product is dropped onto the sample pad, the substrate binds to the antibody-AuNPs. When the PfAgo cleavage reaction product binds to the antibody-AuNPs and is immobilized on line C through a biotin-streptavidin interaction, the FAM / DIG-labeled cleaved probe substrate is recognized by the goat anti-rabbit / goat anti-mouse antibodies and retained on lines T1 or T2, displaying a red color. This directly, quickly, and accurately displays the TYLCCNV and PaLCuCNV detection results. If the result is negative, only line C is visible. When the 5'-Dig-ssDNA-Biotin-3' reporter is cleaved (TYLCCNV positive, PaLCuCNV negative), the C line and T2 line will appear red at the same time, and the T1 line will have no obvious color. If the C line and T1 line appear at the same time and the T2 line shows no color, it indicates that the 5'-FAM-ssDNA-Biotin-3' reporter DNA is cut (TYLCCNV negative, PaLCuCNV positive). If T1, T2 and C lines appear at the same time, and the C line becomes significantly lighter, it indicates that both TYLCCNV and PaLCuCNV are positive. The C line is not only an indicator of the quality of the test paper, but also used to verify the reliability of the test paper results. If the C line does not appear after incubation with complete DNA and RNA substrates, the result is invalid. Such a test strip design can directly, quickly and accurately evaluate the diagnostic results of TYLCCNV and PaLCuCNV from the test paper.

[0111] First, a colloidal gold solution was prepared. The prepared colloidal gold solution showed a bright wine red color, which was clear and transparent without any impurities ( Figure 2 ). In order to ensure the stability of the gold-labeled antibody in the test strip, the gold-labeled antibody was optimized in pH (0.2M K2CO3 addition) and antibody amount (rabbit anti-FAM antibody and mouse anti-Dig antibody addition) experiments. The optimal reaction pH and antibody addition amount were determined by observing the color change of the gold-labeled antibody. The experimental results showed that, as shown in 3A and B, when the addition amount of 0.2M K2CO3 solution was 5μL and the amount of rabbit anti-FAM antibody was 4μg, the colloidal gold solution showed a bright wine red color, which was the optimal reaction condition; Figure 3As shown in Figures C and D, when 5 μL of 0.2 M K₂CO₃ solution and 5 μg of mouse anti-Dig antibody were added, the colloidal gold solution also exhibited a bright wine-red color, indicating the optimal reaction conditions. Based on these observations, the synthesis conditions for different gold-labeled antibodies were determined. Furthermore, the concentrations of the various substances in the designed test strip should follow the rule of SA amount > probe amount > gold-labeled antibody. To this end, this experiment preliminarily calculated the initial concentrations of the test strip, with the SA concentration and the initial assembly concentration of the secondary antibody being 0.2 μg / μL and 0.1 μg / μL, respectively. The test strip was then assembled, initially establishing a colloidal gold test strip for subsequent experiments.

[0112] 1.2.11 Optimization of colloidal gold test strip probe concentration

[0113] When testing with this test strip, it was observed that different batches of test strips showed different degrees of false positives. In order to minimize the false positive performance of the T line (test line) on the test strip, the concentration of the test strip probe was optimized. Figure 4 By visually observing the color depth of the T line on the test strip, it was found that the T line was the lightest when the probe concentration on the test strip was 20 pmol. Subsequent experiments used two probe concentrations less than 20 pmol to improve the accuracy of the test strip detection.

[0114] 1.2.12 Specificity of ULPD Test Strips and Mixed Sample Testing

[0115] In order to verify the effectiveness of the test strip detection, the probes of the ULPD system were replaced in this example, namely TYLCCNV-Probe test strips and PaLCuCNV-Probe test strips (Table 4), and different types of cleavage products were added to verify it. Figure 5 As shown in A, under the synergistic effect of DNA template, probe and gDNA, the test strip signals for different targets were observed, proving the feasibility and accuracy of the test strip detection method. Figure 5 As shown in Figure B, products amplified only by dUTP-LAMP without PfAgo cleavage failed to activate the test strip signal, further confirming the critical role of accurate PfAgo cleavage in detection. These results fully demonstrate the high specificity and accuracy of the colloidal gold test strip in the ULPD detection strategy.

[0116] 1.2.13 Sensitivity of ULPD Test Strips

[0117] In order to evaluate the detection limit of ULPD by colloidal gold method, this experiment carried out ULPD detection analysis on TYLCCNV and PaLCuCNV virus samples with different concentrations. Figure 6 As the number of viral template copies increases from 0 to 10 4As the number of copies / μL increases, the T1 and T2 lines on the test strip gradually darken, while the C line gradually fades. The detection limit of this method can reach 10 copies per reaction. The high sensitivity of ULPD is primarily due to the efficient amplification of dUTP-LAMP and the targeted cleavage ability of PfAgo.

[0118] 1.2.14 Actual Sample Testing of ULPD Test Strips

[0119] To compare the ULPD test strip method with existing RT-PCR detection methods, a new round of test strip testing was conducted on the aforementioned samples and the results were compared with a new round of standard qPCR. To ensure the reliability of the results, ULPD test strip analysis and standard qPCR analysis were performed on 10 dual-identified tomato samples and 5 standard samples. Infection status of the samples was determined by observing color changes on the test strips. Figure 7 The results of A experiment showed that 2 samples were found to carry PaLCuCNV, 1 sample was infected with TYLCCNV, and 2 samples were infected with both viruses. This result was consistent with the standard qPCR (CT value, Figure 7 B) The results were consistent, proving the accuracy and effectiveness of the test strip detection method.

[0120] 1.3 Summary and Discussion

[0121] In order to solve the problem of false positives that may exist in the detection system based on PfAgo, this example verifies and utilizes the ability of the PfAgo enzyme to recognize and cut uracil-containing DNA, and innovatively develops a ULPD anti-pollution detection system. The system combines dUTP-LAMP technology for target enrichment and achieves rapid diagnosis through designed gDNA and immune colloidal gold test strips. In terms of system optimization, after adjustment of key reaction components, the ULPD test strip method has excellent performance in detection speed and sensitivity (as low as 10 copies / μL), and can effectively avoid the problem of aerosol contamination during the LAMP amplification process. At the same time, the application of colloidal gold test strips in this detection system further gets rid of the dependence of the fluorescence method on the qPCR instrument, and has significant advantages in resource-poor areas. However, this example only preliminarily established the working concentration of the colloidal gold test strips, and the optimal working concentration of the colloidal gold test strips has not yet been determined, and the SA amount, gold-labeled antibody amount and secondary antibody concentration need to be further optimized. In short, PfAgo has greater and broader advantages based on CRISPR technology and has the opportunity to become the main force in the development of a new generation of nucleic acid detection platforms.

Claims

1. A method for detecting TYLCCNV and PaLCuCNV viruses using a test strip, characterized in that: The following steps are involved: S1 extracts DNA from the sample to be tested; S2 amplifies the sample to be tested using a UDG-LAMP amplification system, wherein the UDG-LAMP amplification system includes 10×Isothermal Amplification Buffer, dNTPs, MgSO4, primers for TYLCCNV virus-specific amplification, primers for PaLCuCNV virus-specific amplification, DNA Template, Bst t 2.0, UDG enzyme, and ultrapure water; S3 establishes a PfAgo system including gDNA that guides PfAgo protein for targeted cleavage, PfAgo protein, probe, MnCl2 and LAMP amplification product, and performs a reaction; After the S4 reaction is completed, the PfAgo system is tested using a test strip; The test strip is constructed by the following steps: 1) Preparation of colloidal gold liquid 2) Preparation of gold-labeled antibodies Take 1 mL of colloidal gold solution, add K2CO3 solution and rabbit anti-FAM antibody at a rate of 5 µL of 0.2 M K2CO3 solution and 4 µg of rabbit anti-FAM antibody, and add K2CO3 solution and mouse anti-Dig antibody at a rate of 5 µL of 0.2 M K2CO3 solution and 5 µg of mouse anti-Dig antibody, and mix slowly to ensure sufficient binding; add 10% BSA to a total volume of 200 µL and mix slowly to block unbound sites; then, centrifuge at room temperature; After removing the supernatant, add 200 μL of working solution to the remaining precipitate to resuspend the colloidal gold particles. The prepared gold-labeled antibody is stored at 4°C until use. 3) Assembly of colloidal gold test strips a) Cut the NC film of appropriate size and stick it flat on the PVC base plate, making sure the surface is clean and free of contamination; b) Align and place the gold label pads on the NC membrane with a spacing of 2 mm. Place the cut sample pad on top of the NC membrane and absorbent paper on the other end of the NC membrane to form a tight structure. c) Use 0.1 μg / μL goat anti-mouse antibody and 0.1 μg / μL goat anti-rabbit antibody as test lines (T1 and T2), and 0.2 μg / μL streptavidin as control line (C). Mark the lines at a speed of 5 μL / cm, ensuring a spacing of at least 5 mm between the lines. d) After the streaking is completed, the NC membrane is dried at 37°C for 16 hours before use. e) Spray the gold-labeled antibody evenly onto the glass fiber membrane at a volume of 2 μL / cm to form a gold-labeled pad. Dry the pad at 37°C for 30 min before use. f) Soak the sample pad in the activation solution for 2 h, and then dry it at 37 °C for 2 h; g) Use a paper cutter to accurately cut the test strips into 4 mm width. After cutting, seal the test strips and place them in a dry environment.

2. The method according to claim 1, wherein The primers for TYLCCNV virus-specific amplification in step S2 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 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.

3. The method according to claim 1, wherein The UDG-LAMP amplification system in step S2 includes: 2.5 μL of 10×Isothermal Amplification Buffer, 3.5 μL of 10 mM dNTPs, 1.6 μL of 100 mM MgSO4, 0.5 μL of 10 μM primer F3 / B3, 3.2 μL of 10 μM primer FIP / BIP, 1 μL of 10 μM primer LF / LB, 1 μL of DNA Template, 1 μL of 8000 U / mL Bst t 2.0, 0.5 μL of UDG enzyme, and ultrapure water supplemented to 25 μL.

4. The method according to claim 1, wherein The gDNA that guides the PfAgo protein for targeted cleavage in step S3 includes TYLCCNV-g1T, TYLCCNV-g2T, TYLCCNV-g3T, PaLCuCNV-g1T, PaLCuCNV-g2T, and PaLCuCNV-g3T. 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.

5. The method according to claim 2, wherein The probes in step S3 include TYLCCNV-probe-test strip and PaLCuCNV-probe-test strip. The nucleotide sequence of the TYLCCNV-probe-test strip is shown in SEQ ID NO.14, and the nucleotide sequence of the PaLCuCNV-probe-test strip is shown in SEQ ID NO.

18.

6. The method according to claim 1 or 5, wherein: The amount of probe in step S3 is less than 20 pmol.

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