PfAgo-based geminivirus dual detection test strip as well as preparation method and application thereof

Abstract

The invention discloses a geminivirus dual detection test strip based on PfAgo as well as a preparation method and application thereof, and belongs to the technical field of virus detection. On one hand, the invention provides a geminivirus dual detection test strip based on PfAgo and a construction method thereof, and on the other hand, the invention provides application of the test strip in TYLCCNV and PaLCuCNV virus detection. According to the invention, the ULPD anti-pollution detection system is innovatively developed by utilizing the capability of identifying and cutting uracil-containing DNA (Deoxyribose Nucleic Acid) of PfAgo enzyme. The system combines a dUTP-LAMP technology for target enrichment, and realizes rapid diagnosis through designed gDNA and an immune colloidal gold test strip.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a PfAgo-based twin virus dual detection test strip and a preparation method and 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) 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 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 the dependence on sgRNA and PAM (Protospacer adjacent motif) / PFS (Protospacer flanking site) sequences. 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 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 fluorescent equipment. In response to the above problems, this study developed a dual-target detection test strip that does not rely on a real-time fluorescence detector, 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 twin virus dual detection test strip and a preparation method and application technical solution thereof.

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

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

[0007] 1) Preparation of colloidal gold liquid

[0008] 2) Preparation of gold-labeled antibodies

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

[0012] b) Align and place the gold label pad on the NC membrane with a spacing of 2 mm, place the cut sample pad on top of the NC membrane, and place 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, and draw the lines at a speed of 5 μL / cm to ensure that the interval between the two lines is more than 5 mm;

[0014] d) After the streaking is completed, the NC membrane is placed in a 37°C environment to dry for 16 hours before use;

[0015] e) spraying the gold-labeled antibody evenly on the glass fiber membrane at an amount of 2 μL / cm to form a gold-labeled pad, and drying it at 37° C. for 30 min before use;

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

[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, which comprises the following steps:

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

[0022] S2 uses a UDG-LAMP amplification system to amplify the sample to be tested, wherein 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 to perform targeted cleavage, PfAgo protein, probe, MnCl2 and LAMP amplification product, and performs a reaction;

[0024] After the reaction in S4 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, 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 2.0 1 μL, UDG enzyme 0.5 μL and ultrapure water supplemented to 25 μL.

[0027] Further, the gDNA that guides the PfAgo protein for targeted cutting 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 PfAgo enzyme to recognize and cut uracil-containing DNA, and innovatively develops a PfAgo-based anti-pollution dual-channel detection technology-test strip method (Combining UDG-LAMP and PfAgoforNucleic Acid Detection Platform, ULPD) hereinafter referred to as ULPD-test strip method. The 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 detection speed and sensitivity (10 copies / μL), and can effectively avoid aerosol contamination problems during LAMP amplification. 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, which has significant advantages in resource-poor areas. However, this chapter only preliminarily establishes the working concentration of the colloidal gold test strip, and the optimal working concentration of the colloidal gold test strip 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the diagram for 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 a diagram of the optimization of conditions for the preparation of colloidal gold antibodies;

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

[0035] Figure 5 This is a schematic diagram of the specificity evaluation of ULPD test strip detection;

[0036] Figure 6 This is a schematic diagram of the 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 Chinese: A. Actual sample detection by ULPD test strip method (if C line, T1, and T2 are all colored, the sample is identified as TYLCCNV and PaLCuCNV infected; if the test strip shows that T2 line and C line are red and T1 line is not colored, the sample is identified as TYLCCNV infected; if the test strip shows that T1 line and C line are red and T2 line is not colored, the sample is identified as PaLCuCNV infected; if the test strip C line is colored and T1 and T2 are not colored, the sample is identified as not infected with PaLCuCNV and TYLCCNV); B. Actual sample detection by qPCR standard detection (CT value ≤38 is positive). DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the 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, protect from light, and store at 4°C.

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

[0047] 3) 0.2M K2CO3 (20 mL): Dissolve 0.552 g of K2CO3 in ultrapure water and make up to 20 mL. Filter with 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 with 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 storage solution, add 40 μL of 10% BSA, mix well, and use immediately.

[0051] 7) PB buffer (100 mL): 0.024 g of potassium dihydrogen phosphate, 0.358 g of disodium hydrogen phosphate dodecahydrate and ultrapure water were mixed to make up to 100 mL, the pH was adjusted to 7.4, and the solution was filtered through a 0.22 μm filter membrane and stored 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 and clean plant leaves and place them in a 1.5 mL centrifuge tube containing 4-6 steel balls. After quick freezing in liquid nitrogen, grind with a grinding rod for 5 minutes to ensure that the sample is fully broken.

[0066] 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 to mix well.

[0067] 3) Add 600 μL of chloroform to the centrifuge tube, shake and mix. Centrifuge at 12000 rpm for 10 min.

[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 min.

[0069] 5) Discard the supernatant, add 700 μL of 75% alcohol, mix by inversion, and centrifuge at 12,000 rpm for 2 minutes.

[0070] 6) Discard the supernatant and leave 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] 1.2.2UDG-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 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 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.

[0080] (3) UDG enzyme digestion

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

[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 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 supplemented 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.

[0085] 1.2.4 Preparation of colloidal gold liquid

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

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

[0088] 3) After boiling, add 1 mL of chloroauric acid solution. Increase the stirring speed, continue heating for 15 minutes, and then let the solution cool naturally at room temperature.

[0089] 4) Make the solution volume up to 100 mL and transfer to a conical flask. Store at 4°C away from light.

[0090] 1.2.5 Determination of optimal pH and antibody amount

[0091] In order to determine the optimal pH value of the prepared colloidal gold solution, 1 mL of colloidal gold solution was added to each of 6 1.5 mL centrifuge tubes. Then different volumes of 0.2M K2CO3 solution (0, 1, 2, 3, 4, 5, 6, 7 and 8 μL) were added to the centrifuge tubes in turn and shaken to mix. Add an excess of 20 μL rabbit anti-FAM / mouse anti-Dig protein to each centrifuge tube and mix slowly again. After mixing evenly, add 100 μL of 10% NaCl solution to each centrifuge tube. After adding, slowly mix the solution and let it stand for 2 hours. Observe the color of the solution and set the volume of K2CO3 solution that can keep the colloidal gold solution unchanged as the optimal pH.

[0092] Add 1 mL of colloidal gold solution to each of 6 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 value. 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 in sequence. After adding rabbit anti-FAM / mouse anti-Dig protein, mix slowly to ensure sufficient contact and reaction with colloidal gold. Place the centrifuge tube in a greenhouse for 20 minutes to observe color changes or precipitation. Add 100 μL of 10% NaCl solution to each centrifuge tube and mix slowly again. Return the centrifuge tube to room temperature and let it stand for 1 hour to further evaluate the binding of rabbit anti-FAM / mouse anti-Dig protein to colloidal gold. Select samples without color change and precipitation as the optimal labeling amount of rabbit anti-FAM / mouse anti-Dig protein antibody.

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

[0094] Take 1mL of colloidal gold solution in a 1.5mL centrifuge tube, add the optimal 0.2M K2CO3 adjustment solution and the optimized rabbit anti-FAM / mouse anti-Dig protein antibody labeling amount, and mix slowly to ensure sufficient binding. Add a total volume of 200μL of 10% BSA and mix slowly to block unbound sites. Subsequently, centrifuge in a refrigerated centrifuge at 4°C, 12000rpm, for 30min. After removing the supernatant, add 200μL of the pre-prepared working solution to the remaining precipitate to resuspend the colloidal gold particles. The prepared gold-labeled antibody is stored at 4°C for future use.

[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 to ensure the surface is clean and free of pollution.

[0097] 2) Align and place the gold pad on the NC membrane with a spacing of 2 mm. Place the cut sample pad on top of the NC membrane. Place absorbent paper on the other end 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 line (T1 and T2 lines), streptavidin (SA, 1 μg / μL) as the quality control line (C line), draw the lines at a speed of 5 μL / cm, and ensure that the interval between the two lines is more than 5 mm.

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

[0100] 5) The gold-labeled antibody was evenly sprayed on the glass fiber membrane at an amount of 2 μL / cm to form a gold-labeled pad, and then dried at 37°C for 30 min before use.

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

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

[0103] 1.2.8 Test strip probe quantity optimization

[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 vertically inserted 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 detection line was observed and recorded by naked eyes, and photos were taken to record the false positive situation of the detection line at each concentration.

[0105] 1.2.9 ULPD test strip sensitivity and specificity detection

[0106] To achieve test strip detection, 5'FAM-ssDNA-Biotin 3' labeled and 5'Dig-ssDNA-Biotin3' labeled reporter DNA probes were constructed. After LAMP pre-amplification, 4μL of product was added to the PfAgo system to a final volume of 30μL, which contained 2μL 10× reaction buffer, 2.5μM PfAgo, 2μM gDNA, test strip probe (≤20pmol) and 0.6mM MnCl2. After incubation at 95℃ for 30min, the reaction product was taken from the reaction tube and added to 75μL of Working Buffer. The lateral flow strip was placed in the reaction tube for 5min and the results were directly observed. The color intensity of the line was photographed and analyzed with 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 assays and PfAgo cleavage assays were digested by UDG enzyme to prevent aerosol contamination. For ULPD test strip assays, dual detection test strips were used for detection and 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 limitation of qPCR instrument, this chapter simultaneously designed a double detection test strip containing two target lines (T1 and T2 lines) and one control line (C line). Figure 1As shown in D, in this design, two gold-labeled antibodies, rabbit anti-FAM antibody-AuNPs and mouse anti-Dig antibody-AuNPs, are pre-embedded into the conjugate pad. At the same time, streptavidin, goat anti-rabbit antibody, and goat anti-mouse antibody are integrated into the C, T1, and T2 lines, respectively. When the PfAgo cleavage reaction product is dropped on the sample pad, the substrate binds to the antibody-AuNPs. When the PfAgo cleavage reaction product binds to the antibody-AuNPs and is fixed on the C line through the biotin-streptavidin interaction, the cleaved probe substrate with FAM / DIG label will stay on the T1 or T2 line due to the recognition of the goat anti-rabbit / goat anti-mouse antibody, showing red, thereby directly, quickly, and accurately displaying the detection results of TYLCCNV and PaLCuCNV. If the result is negative, only the C line 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 has no color, it indicates that the 5'-FAM-ssDNA-Biotin-3' reporter DNA is cut (TYLCCNV negative, PaLCuCNV positive). If T1 and 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 strip.

[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 changes 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 C and D, when the amount of 0.2M K2CO3 solution added was 5μL and the amount of mouse anti-Dig antibody was 5μg, the colloidal gold solution also showed a bright wine red color, which was the optimal reaction condition. Based on these observations, the synthesis conditions of different gold-labeled antibodies were determined. At the same time, the concentration of each substance in the designed test strip should follow the SA amount > probe amount > gold-labeled antibody. For this reason, this experiment preliminarily calculated the initial concentration of the test strip, and the SA concentration and the initial assembly concentration of the secondary antibody were 0.2μg / μL and 0.1μg / μL, respectively. The test strip was assembled, and the colloidal gold test strip was preliminarily established for the next experiment.

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

[0113] When using the test strips for testing, 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 strips, the concentration of the test strip probe was optimized. Figure 4 By observing the color depth of the T line on the test strip with the naked eye, it was found that when the probe amount of the test strip was 20pmol, the color of the T line was the lightest. The two probe amounts selected in subsequent experiments were both less than 20pmol, which improved 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 strip and PaLCuCNV-Probe test strip (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 B, the product amplified by dUTP-LAMP without PfAgo cleavage failed to activate the test strip signal, which further confirmed the key role of accurate PfAgo cleavage in detection. These results fully demonstrate the high specificity and accuracy of colloidal gold test strips in ULPD detection strategies.

[0116] 1.2.13 ULPD test strip detection sensitivity

[0117] In order to evaluate the detection limit of ULPD in the colloidal gold method, this experiment performed 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 4The color of the T1 and T2 lines on the test strip gradually deepens, while the color of the C line gradually fades. The limit of this detection method can reach 10 copies per reaction. The high sensitivity of ULPD is mainly due to the efficient amplification of dUTP-LAMP and the directional cutting ability of PfAgo.

[0118] 1.2.14 Actual sample testing of ULPD test strips

[0119] In order to compare the difference in detection effect between the ULPD test strip method and the existing RT-PCR detection method, this experiment conducted a new round of test strip tests on the above samples and compared the results with the new round of standard qPCR results. To ensure the reliability of the results, ULPD test strip analysis and standard qPCR analysis were performed on 10 double-identified tomato samples and 5 standard samples. The infection status of the samples was identified by observing the color changes on the test strips. Figure 7 A The experimental results showed that 2 samples were identified as carrying PaLCuCNV, 1 sample was infected with TYLCCNV, and 2 samples were infected with two 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 possible false positive problem in the detection system based on PfAgo, this embodiment verifies and utilizes the ability of PfAgo enzyme to recognize and cut uracil-containing DNA, and innovatively develops the ULPD anti-pollution detection system. The system combines dUTP-LAMP technology for target enrichment, and realizes 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 performs well in detection speed and sensitivity (as low as 10 copies / μL), and can effectively avoid the aerosol pollution problem in 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 fluorescence method on qPCR instrument, and has significant advantages in resource-poor areas. However, this embodiment only preliminarily establishes the working concentration of colloidal gold test strips, and the optimal working concentration of colloidal gold test strips has not yet been determined, and the concentration of SA amount, gold-labeled antibody amount and secondary antibody needs to be further optimized. In short, PfAgo has greater and more extensive 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 constructing a PfAgo-based twin virus dual detection test strip, characterized in that: The following steps are involved: 1) Preparation of colloidal gold liquid 2) Preparation of gold-labeled antibodies Take 1 mL of colloidal gold solution, add K2CO3 adjustment solution, rabbit anti-FAM / mouse anti-Dig protein antibody according to the addition amount of 0.2M K2CO3 solution 5μL, rabbit anti-FAM antibody 4μg, 0.2M K2CO3 solution 5μL, mouse anti-Dig antibody 5μg, and mix slowly to ensure sufficient binding; add 10% BSA in a total volume of 200μL, mix slowly to block unbound sites; then, refrigerate centrifuge; After removing the supernatant, add 200 μL of working resuspending solution to the remaining precipitate to resuspend the colloidal gold particles. The prepared gold-labeled antibody is stored at 4°C for future 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 to ensure the surface is clean and free of pollution; b) Align and place the gold label pad on the NC membrane with a spacing of 2 mm, place the cut sample pad on top of the NC membrane, and place 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 the test lines, i.e., T1 and T2 lines, and 0.2 μg / μL streptavidin as the quality control line, i.e., C line, and draw the lines at a speed of 5 μL / cm to ensure that the interval between the two lines is more than 5 mm; d) After the streaking is completed, the NC membrane is placed in a 37°C environment to dry for 16 hours before use; e) spraying the gold-labeled antibody evenly on the glass fiber membrane at an amount of 2 μL / cm to form a gold-labeled pad, and drying it at 37° C. for 30 min before use; f) soaking the sample pad in the activation solution for 2 h, and then drying 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. A PfAgo-based Geminivirus dual detection test strip prepared by the method of claim 1.

3. Application of the test strip as claimed in claim 2 in TYLCCNV and PaLCuCNV virus detection.

4. A method for detecting TYLCCNV and PaLCuCNV viruses using the test strip according to claim 1, characterized in that: The following steps are involved: S1 extracts DNA from the sample to be tested; S2 uses a UDG-LAMP amplification system to amplify the sample to be tested, wherein 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; S3 establishes a PfAgo system including gDNA that guides PfAgo protein to perform targeted cleavage, PfAgo protein, probe, MnCl2 and LAMP amplification product, and performs a reaction; After the reaction in S4 is completed, the above detection system is tested using a test strip.

5. The method according to claim 4, characterized in that 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 IDNO.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.

6. The method according to claim 5, characterized in that The UDG-LAMP amplification system in step S2 includes: 2.5 μL of 10×IsothermalAmplification 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 DNATemplate, 2.01 μL of 8000 U / mL Bst t, 0.5 μL of UDG enzyme, and ultrapure water supplemented to 25 μL.

7. The method according to claim 4, characterized in that The gDNA that guides the PfAgo protein for targeted cutting in the 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.

8. The method according to claim 5, characterized in that 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.

9. The method according to claim 4 or 8, characterized in that The amount of probe in step S3 is less than 20 pmol.

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