Primer probe combination, kit and RPA-LFD method for detecting tomato brown fruit wrinkling disease

Abstract

The invention relates to the field of biological detection, and discloses a primer probe combination, a kit and an RPA-LFD method for detecting tomato brown fruit wrinkling disease. The primer probe combination provided by the invention has the characteristics of good specificity and high sensitivity, and can accurately identify the tomato brown fruit wrinkling disease in a sample suspected to be infected with the tomato brown fruit wrinkling disease. The RPA-LFD method for detecting the tomato brown fruit wrinkling disease is based on the primer probe combination, so that the detection result of the detection method is good in specificity and high in sensitivity, detection operation only needs several simple steps, and by combining visualization of a test paper card, the operation is simple, the consumed time is short, and the detection accuracy is high. The method can realize real-time and rapid detection of the tomato brown fruit wrinkling disease in the field or in the field.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a primer-probe combination, kit, and RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes. Background Technology

[0002] Tomato brown fruit wrinkling disease is caused by the tomato brown fruit wrinkling virus, which can be transmitted through seeds, aphids, etc., and is difficult to control once it spreads. Early symptoms are not obvious and can be easily confused with nutrient deficiencies and environmental stress. Later stages of the disease lead to deformed tomato fruits, reduced yield, and even total crop failure. Biodetection technologies (such as PCR and qPCR) can intervene early. However, PCR requires a complex reaction system, has a long detection time, and requires sophisticated and precise temperature-controlled equipment. Traditional nucleic acid extraction methods, such as column extraction or magnetic bead extraction, are cumbersome, complex, and time-consuming, requiring professional operators. qPCR detection, combined with traditional nucleic acid extraction to form PCR detection technology, is also cumbersome, time-consuming, requires sophisticated instruments and professional operators, and cannot achieve rapid, real-time detection, nor can it be performed in the field, thus limiting its use.

[0003] Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technology that has developed rapidly in recent years and is increasingly being used. It is a sensitive technique that enables rapid DNA amplification at a relatively constant temperature (40±2℃), shortening the isothermal amplification reaction time to 10–15 minutes. RPA-LFD technology, combining RPA with lateral flow dipsticks (LFD), enables rapid and simple visual detection of nucleic acids, showing promising application prospects in rapid on-site nucleic acid detection of pathogens.

[0004] It is evident that existing technologies still need improvement and enhancement. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a primer-probe combination, RPA-LFD kit, and RPA-LFD detection method for rapid detection of tomato brown wrinkled fruit disease in the planting area.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A primer-probe combination for detecting brown wrinkled fruit disease in tomatoes, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-AGTTGTCTGTCACAGTACCTTCCAAACGTG-3'; The nucleotide sequence of the R1 primer is as follows: 5'-Biotin-TCCGAATTCATCAGCAGGTATATCGTATG-3'; The nucleotide sequence of probe P1 is as follows: 5'-CCAGCAGGTGGAAAACACAGGCAGGGTGTATGCTATTGCATTGCACAGT-3'; The A base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

[0007] The primer-probe combination for detecting brown wrinkled fruit disease in tomatoes, wherein the fluorescent group is FAM.

[0008] An RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes, comprising the aforementioned primer-probe combination.

[0009] The RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes further includes lysis buffer, washing buffer, elution buffer, amplification lyophilized powder, activator, LFD test strip, and product dilution solution.

[0010] The RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes comprises the following: the lysis buffer includes guanidine thiocyanate at a concentration of 1–3 M, Tris-H at a concentration of 10–50 mM, and EDTA at a concentration of 10–30 mM; the pH of the lysis buffer is 5.0–8.0; the washing buffer is ethanol at a concentration of 60–75%; the eluent is Tris-H at a concentration of 5–20 mM and a pH of 7.0–8.0; and the product dilution buffer is Tris-H at a concentration of 20–50 mM and a pH of 7.0–8.0.

[0011] An RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes includes the following steps: S1. Sampling and processing of the samples to be tested; S2. Obtain the RNA from the sample to be tested; S3. Perform an RPA reaction on the RNA of the sample to be tested and the primer and probe combination to obtain the amplification product; S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test results.

[0012] The RPA-LFD method for detecting tomato brown fruit wrinkling virus, wherein step S1 specifically involves: taking tomato leaves suspected of being infected with tomato brown fruit wrinkling virus, with a sample size of 4-6 cm² surface area. 2 Or weigh 0.05–0.1g; then cut the tomato leaves into small pieces.

[0013] The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes, wherein step S2 specifically involves: placing the processed sample in a container, adding lysis buffer to the container, grinding the sample for 1-2 minutes until it becomes a paste, and then incubating the sample at 40-45°C for 5-10 minutes; centrifuging the sample and aspirating the supernatant, adding it to a container containing anhydrous ethanol, mixing thoroughly, transferring all the mixed liquid to a purification column fitted with a collection tube, centrifuging the purification column, adding washing buffer to the purification column, and centrifuging again; adding elution buffer to the purification column, and then allowing it to stand and centrifuging to obtain the RNA extract.

[0014] The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes, wherein step S3 specifically involves: mixing and diluting the primer-probe combination and activator to obtain a reaction agent; adding RNA extract to a reaction tube containing lyophilized amplification powder, then adding the reaction agent and mixing thoroughly; and incubating at 38–42°C for 12–15 min to obtain the amplification product.

[0015] The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes, wherein step S4 specifically involves: taking a sample of the amplification product, adding it to a container containing a product diluent, mixing it thoroughly to obtain the amplification product diluent; and adding the amplification product diluent to an LFD test strip to obtain the identification result.

[0016] Beneficial effects: This invention provides a primer-probe combination, kit, and RPA-LFD method for detecting tomato brown fruit wrinkle disease. The primer-probe combination, used in the RPA-LFD method for detecting tomato brown fruit wrinkle disease, offers advantages of high accuracy and high sensitivity. The RPA-LFD method for detecting tomato brown fruit wrinkle disease simplifies the column method, requiring only a few simple steps to extract RNA from the disease within 20-30 minutes. Furthermore, the RPA-LFD method is simple and convenient to operate, requiring no large-scale precision equipment, specialized facilities, or professional personnel. Overall, this invention creatively simplifies RNA extraction, and combined with RPA technology, it is simple and time-saving, enabling real-time and rapid detection of tomato brown fruit wrinkle disease in the field or outfield. Attached Figure Description

[0017] Figure 1This is a schematic diagram illustrating the specificity detection results of the primer-probe combination provided by the present invention.

[0018] Figure 2 This is a schematic diagram illustrating the sensitivity detection results of the RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes provided by this invention.

[0019] Figure 3 This is a schematic diagram showing the specificity detection results of the primer-probe combination in Example 1 and the primer-probe combination in the comparative example. Detailed Implementation

[0020] This invention provides a primer-probe combination, kit, and RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the scope of protection of the invention.

[0021] This invention provides a primer-probe combination for detecting brown wrinkled fruit disease in tomatoes, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-AGTTGTCTGTCACAGTACCTTCCAAACGTG-3', whose nucleotide sequence is shown in SEQ ID NO: 1. The F1 primer is a forward primer, 30 bp in length, which specifically targets the conserved region of tomato brown wrinkled fruit virus as the starting site for RPA amplification, ensuring that only the target sequence is specifically amplified.

[0022] The nucleotide sequence of the R1 primer is as follows: The nucleotide sequence of 5'-Biotin-TCCGAATTCATCAGCAGGTATATCGTATG-3' is shown in SEQ ID NO: 2. The R1 primer is a reverse primer, 29 bp in length, with biotin labeled at the 5' end. The core function of biotin modification is to facilitate subsequent detection of the amplified product, while the 29 bp length enhances the binding specificity to the template and reduces non-specific amplification.

[0023] The nucleotide sequence of probe P1 is as follows: The nucleotide sequence of the P1 probe is 5'-CCAGCAGGTGGAAAACACAGGCAGGGTGTATGCTATTGCATTGCACAGT-3', as shown in SEQ ID NO: 3. The A base of the P1 probe is replaced by a dSpacer at a position 30 bp from the 5' end. The P1 probe has a fluorescent group (FAM) attached to its 5' end and a dideoxycytidine (ddC) attached to its 3' end. The ddC blocks probe elongation, preventing it from participating in the RPA reaction as a primer, and ensures that the probe functions only by hybridizing with the amplification product. The dSpacer acts as a breakpoint in the detection system, facilitating subsequent signal release and improving detection sensitivity.

[0024] The present invention also provides an RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes, comprising the above-mentioned primer and probe combination.

[0025] Specifically, the RPA-LFD kit also includes lysis buffer, washing buffer, elution buffer, amplification lyophilized powder, activator, LFD test strip, and product dilution buffer. The lysis buffer is used to disrupt tomato sample cells, release viral nucleic acid, and inhibit nucleases, providing a high-quality detection template. The washing buffer is used to improve RNA purity. The elution buffer is mainly used to elute the RNA adsorbed on the purification column. The amplification lyophilized powder contains key RPA amplification substances (recombinase, polymerase, etc.), which, when mixed with the sample solution and activator, can rapidly amplify viral target nucleic acid under mild conditions by binding to specific primers and probes. The activator provides suitable reaction conditions, activates enzyme activity, initiates amplification, and is easy to operate and suitable for on-site detection. The LFD test strip visualizes the results; the T line is pre-coated with streptavidin, which binds to biotinylated amplification products for color development (indicating a positive result); the C line is pre-coated with an anti-fluorescent antibody to verify the validity of the test strip (no color development indicates an invalid result). The concentration of the amplified product was adjusted using the product dilution buffer to match the LFD test strip, while maintaining a stable pH in the system to ensure the smooth progress of the binding reaction and chromatography, and to ensure accurate results.

[0026] In this embodiment, the lysis buffer includes guanidine thiocyanate at a concentration of 1–3 M, Tris-H at a concentration of 10–50 mM, and EDTA at a concentration of 10–30 mM; the pH of the lysis buffer is 5.0–8.0; the washing buffer is ethanol at a concentration of 60–75%; the eluent is Tris-H at a concentration of 5–20 mM and a pH of 7.0–8.0 (prepared with DEPC-treated water); and the product diluent is Tris-H at a concentration of 20–50 mM and a pH of 7.0–8.0. In the lysis buffer, 1–3 M guanidine thiocyanate efficiently lyses sample cells and destroys viral RNA, while inhibiting RNase activity to protect RNA from degradation; 10–50 mM Tris-H maintains a stable pH range of 5.0–8.0, providing a suitable environment for RNA release; and 10–30 mM EDTA assists in inhibiting nucleases, synergistically ensuring the integrity of the RNA. The washing buffer is 60–75% ethanol, which removes residual proteins, salts, and other impurities from the purification column while preventing RNA loss and ensuring the purity of the eluted RNA. The elution buffer is 5–20 mM Tris-H at pH 7.0–8.0, which gently elutes the RNA adsorbed on the purification column, and its low-concentration buffer environment reduces interference with subsequent RPA amplification reactions.

[0027] This invention also provides an RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes, using the aforementioned RPA-LFD kit for identification, comprising the following steps: S1. Sampling and processing of the samples to be tested.

[0028] The specific procedure is as follows: Take tomato leaves suspected of being infected with Tomato Brown Wrinkled Fruit Virus, with a sample size of 4-6 cm². 2 Or weigh 0.05 to 0.1g; then cut it into pieces the size of 1 / 4 to 1 / 2 soybean with scissors.

[0029] S2. Obtain the RNA from the sample to be tested.

[0030] The specific operation of step S2 is as follows: Place the processed sample into a container with tweezers, add lysis buffer to the container, grind the sample for 1-2 minutes until it becomes a paste, and then incubate the sample at a temperature of 40-45℃ for 5-10 minutes; after centrifuging the sample, aspirate the supernatant and add it to a container containing anhydrous ethanol, mix well, transfer all the mixed liquid to a purification column with a collection tube attached, centrifuge the purification column, add washing buffer to the purification column, and centrifuge again; add elution buffer to the purification column, and then let it stand and centrifuge to obtain RNA extract.

[0031] S3. Perform an RPA reaction on the RNA of the sample to be tested and the primer-probe combination to obtain the amplification product. Specifically, mix and dilute the primer-probe combination and activator to obtain the reaction reagent; add the RNA extract to a reaction tube containing the lyophilized amplification powder, then add the reaction reagent and mix thoroughly; incubate at 38–42℃ for 12–15 min to obtain the amplification product. In outdoor locations such as fields, the reaction temperature can be simulated by holding the reaction tube in hand, further reducing the difficulty of detecting tomato brown wrinkled fruit disease.

[0032] S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test result. The specific operation is as follows: draw up the amplification product, add it to a container containing the product diluent, mix well to obtain the amplification product diluent; add the amplification product diluent dropwise to the LFD test strip to obtain the identification result.

[0033] To further illustrate the primer-probe combination, kit, and RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes provided by this invention, the following examples and comparative examples are provided.

[0034] Example 1 A primer-probe combination RPA-1 for detecting brown wrinkled fruit disease in tomatoes includes an F1 primer, an R1 primer, and a P1 probe. The nucleotide sequence of the F1 primer is as follows: 5'-AGTTGTCTGTCACAGTACCTTCCAAACGTG-3'; The nucleotide sequence of the R1 primer is as follows: 5'-Biotin-TCCGAATTCATCAGCAGGTATATCGTATG-3'; The nucleotide sequence of probe P1 is as follows: 5'-CCAGCAGGTGGAAAACACAGGCAGGGTGTATGCTATTGCATTGCACAGT-3'; The A base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

[0035] An RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes includes the following steps: (1) Sample collection and sample processing Tomato leaves suspected of being infected with Tomato Brown Fruit Curl Virus were sampled, with a surface area of ​​4–6 cm². 2 Or weigh 0.05 to 0.1g; then cut it into pieces the size of 1 / 4 to 1 / 2 soybean with scissors.

[0036] (2) RNA extraction Place the processed sample into a 1.5 mL centrifuge tube using tweezers. Use a pipette to add 400–500 μL of lysis buffer (which includes 1–3 M guanidine thiocyanate, 10–50 mM Tris-H, and 10–30 mM EDTA) into the centrifuge tube. Grind the sample with a pestle for 1–2 min until it becomes a paste. Then incubate the sample in a metal bath or constant temperature water bath at 40–45 °C for 5–10 min.

[0037] Centrifuge at 10,000 rpm for 60 seconds. Use a pipette to transfer 100–200 μL of the supernatant into a new centrifuge tube. Then add 50–100 μL of anhydrous ethanol to the centrifuge tube and mix well. Transfer all the liquid to a nucleic acid purification column (the purification column is fitted with a 2 mL collection tube, and the purification kit is a commercially available product). Centrifuge the purification column at 10,000 rpm for 30 seconds. Then add 100–150 μL of washing buffer (60–75% ethanol) to the purification column using a pipette and centrifuge at 10,000 rpm for 60–120 seconds.

[0038] Remove the purification column from the purification kit and place it in a new 2mL collection tube. Discard the original 2mL collection tube containing waste liquid. Use a pipette to add 100–200 μL of elution buffer (5–20 mM Tris-H at pH 7.0–8.0) to the purification column. Let the purification column stand for 1–3 minutes, and then centrifuge at 10,000 rpm for 30 seconds to obtain the RNA extract.

[0039] (2) RPA reaction Use commercially available RPA reagents as raw materials. The reagent kit includes lyophilized amplification powder and activator. Dissolve the pre-mixed reagents and activator from the reagent kit at room temperature 10 minutes in advance. After the reagents are dissolved, gently shake for 3-5 seconds to mix, and then centrifuge for 2-3 seconds.

[0040] Take out the reaction tube containing the amplification lyophilized powder, open the cap and add the reagents in order according to Table 1. For multiple samples, a premix solution other than the nucleic acid template can be prepared and then added to the amplification lyophilized powder.

[0041] Table 1 RPA Reaction Configuration System

[0042] After adding the above components to the lyophilized amplification powder, add 2 μL of activator to the cap of each reaction tube, cap the reaction tube, and then centrifuge at 1000–10000 r / min for 2–3 s to allow the activator to enter the reaction mixture. Immediately invert the reaction tube 8–10 times or shake to mix for 3–5 s, and then immediately centrifuge at 1000–10000 r / min for 2–3 s to allow all reagents to settle to the bottom of the tube.

[0043] Place the reaction tube containing the RPA reaction reagent in a metal bath at 38–42°C or other suitable instrument and keep it warm for 12–15 minutes to obtain the amplification product.

[0044] In the RPA reaction, the commercially available RPA reagent is specifically the chromatographic RPA (NFO probe) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd.

[0045] (3) LFD test strip detection Add 590 μL of product diluent to a new 1.5 mL centrifuge tube beforehand. After RPA incubation, carefully open the cap and pipette 10 μL of amplification product into a centrifuge tube containing 780 μL of product diluent. Tighten the cap and shake well by inverting the tube. Then, pipette 60–100 μL of the liquid and add it to the sample loading area (S) of the LFD test strip. Wait 1–3 minutes and observe the results. The results displayed within 1–5 minutes are considered accurate; results displayed after 5 minutes are not considered as a basis for determining positive or negative. Specifically, the product diluent is Tris-H with a concentration of 20–50 mM and a pH of 7.0–8.0.

[0046] Result interpretation: If there are lines at both C (control line) and T (test line) on the reagent card (even if the line is lighter in color, it is considered positive), the result is positive; if there is only a line at C, the result is negative; if there is no line at C (regardless of whether there is a line at T), the reagent card is invalid and needs to be replaced for testing.

[0047] Example 2 Specific detection Nine viruses with high genetic homology to tomato brown fruit wrinkling virus were selected, along with tomato brown fruit wrinkling virus, for a total of 10 pure viral liquid cultures for specificity testing. The 10 samples were, in order: tomato brown fruit wrinkling virus, tomato mottle mosaic virus, tomato mosaic virus, tomato yellow leaf curl virus, tomato chlorosis virus, tobacco mosaic virus, cucumber mosaic virus, cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, and zucchini green mottle mosaic virus. One negative control (nuclease-free water) was also included to avoid false positives. Information on the 11 samples is shown in Table 2.

[0048] Table 2 Sample Information Table

[0049] Viral RNA was extracted from the 10 viral liquid cultures mentioned above using the viral RNA extraction kit from Guangzhou Meiji Biotechnology Co., Ltd., and then mixed according to the formula in Table 3.

[0050] Table 3 RT-RPA Reaction Configuration System

[0051] After preparing the mix, add it to the lyophilized amplification powder, mix thoroughly by pipetting several times, add 2 μL of activator to the cap of the reaction tube, cap the reaction tube, centrifuge at low speed for 5-10 seconds, then invert the reaction tube to mix the liquid, centrifuge again at low speed for 5-10 seconds, place it in a metal bath at 40±2℃ for 12-15 minutes, after the incubation is complete, take out the amplification product and dilute it 60 times with the amplification product diluent, then take 80 μL of the diluted amplification product and add it to the LFD test strip for detection.

[0052] RPA Results and Analysis: Figure 1 The test samples are numbered 1-11 from left to right. Figure 1 As can be seen, among the 10 viruses tested, only the target sample, Tomato Brown Wrinkled Fruit Virus (the first LFD test strip), showed a positive result, while all other samples and negative controls showed negative results. This indicates that the primer-probe combination provided by this invention has good specificity and can accurately identify Tomato Brown Wrinkled Fruit Virus from other viruses with similar homology.

[0053] Example 3 Sensitivity test Tomato samples infected with Tomato Brown Curly Fruit Virus were selected. RNA extraction stock solution was obtained according to the RNA extraction method described in Example 1. The stock solution was diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times, resulting in 11 different template concentrations, along with one blank control. The samples were then tested using the RPA reaction and LFD test strip method described in Example 1. Sample information and results are shown in Table 4. Figure 2 As shown.

[0054] Table 4 Sample Information and Results

[0055] Analysis and Conclusion: From Table 4 and Figure 2It can be seen that even after the original template was diluted 64 times, a relatively clear band remained. The band weakened after a 128-fold dilution, and disappeared after a 256-fold dilution. This demonstrates that even trace amounts of virus infection can be detected. The extracted RNA from the original nucleic acid solution, measured using a micro-spectrophotometer, had a concentration of 10.56 nanograms per microliter. Based on the dilution factor, the detection sensitivity was calculated to be 0.83 × 10⁻⁶. -1 The nanograms per microliter indicates that the detection method of the present invention has extremely high sensitivity.

[0056] Comparative Example A primer-probe combination RPA-2 for detecting brown wrinkled fruit disease in tomatoes includes an F2 primer, an R2 primer, and a P2 probe. The nucleotide sequence of the F2 primer is: 5'-TATGCAGATGTGTCGTTGGTCAGATTGACT-3', whose nucleotide sequence is SEQ ID NO: 4.

[0057] The nucleotide sequence of the R2 primer is as follows: 5'-Biotin-ACTACGGTGTAATACTTCAATGTTTGGGTATG-3', whose nucleotide sequence is SEQ ID NO: 5.

[0058] The nucleotide sequence of the P2 probe is as follows: 5'-ATCATCGCAGGAGATAGTCCGCACGTTCTGGCTAGCTTTGTCAAGGCATA-3', whose nucleotide sequence is SEQ ID NO: 6; the C base of the P2 probe is replaced by dSpacer at a position 28 bp from the 5' end; the P2 probe is connected to a FAM fluorescent group at the 5' end and to ddC at the 3' end.

[0059] Positive samples of brown wrinkled tomato fruits and negative controls were selected and tested according to the method in Example 1. The primer and probe combinations provided in Example 1 and the comparative example were compared to detect the effectiveness of the target. Sample information is shown in Table 5. Test strip test results are also shown. Figure 3 As shown.

[0060] Table 5 Primer-probe combination sample information

[0061] Results analysis: From Figure 3It can be seen that samples 1-3 corresponding to primer probe RPA-1 all yielded positive results, while only one of samples 4-6 corresponding to primer probe RPA-2 was positive, and it was a weak positive. This indicates that when detecting positive samples of brown wrinkled tomato fruit, RPA-1 is significantly more effective than RPA-2, meaning that RPA-2 has a weaker detection effect.

[0062] It is understood that those skilled in the art can make equivalent substitutions or changes to the technical solution and inventive concept of the present invention, and all such changes or substitutions should fall within the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting brown wrinkled fruit disease in tomatoes, characterized in that, The primer-probe combination includes an F1 primer, an R1 primer, and a P1 probe; The nucleotide sequence of the F1 primer is as follows: 5'-AGTTGTCTGTCACAGTACCTTCCAAACGTG-3'; The nucleotide sequence of the R1 primer is as follows: 5'-Biotin-TCCGAATTCATCAGCAGGTATATCGTATG-3'; The nucleotide sequence of the P1 probe is as follows: 5'-CCAGCAGGTGGAAAACACAGGCAGGGTGTATGCTATTGCATTGCACAGT-3'; The A base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

2. The primer-probe combination for detecting tomato brown wrinkled fruit disease according to claim 1, characterized in that, The fluorescent group is FAM.

3. An RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes, characterized in that, The RPA-LFD kit includes the primer-probe combination as described in claim 1 or 2.

4. The RPA-LFD kit for detecting brown wrinkled fruit disease in tomato according to claim 3, characterized in that, The RPA-LFD kit also includes lysis buffer, washing buffer, elution buffer, amplification lyophilized powder, activator, LFD test strip, and product dilution solution.

5. The RPA-LFD kit for detecting brown wrinkled fruit disease in tomatoes according to claim 4, characterized in that, The lysis buffer comprises guanidine thiocyanate at a concentration of 1–3 M, Tris-H at a concentration of 10–50 mM, and EDTA at a concentration of 10–30 mM; the pH of the lysis buffer is 5.0–8.0; the washing buffer is ethanol at a concentration of 60–75%; the eluent is Tris-H at a concentration of 5–20 mM and a pH of 7.0–8.0; and the product diluent is Tris-H at a concentration of 20–50 mM and a pH of 7.0–8.

0.

6. An RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes, characterized in that, The detection using the RPA-LFD kit as described in claim 3 includes the following steps: S1. Sampling and processing of the samples to be tested; S2. Obtain the RNA from the sample to be tested; S3. Perform an RPA reaction on the RNA of the sample to be tested and the primer and probe combination to obtain the amplification product; S4. Dilute the amplification product and add it dropwise to the LFD test strip for detection to obtain the test results.

7. The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes according to claim 6, characterized in that, The specific operation of step S1 is as follows: Take tomato leaves suspected of being infected with Tomato Brown Wrinkled Fruit Virus, with a sample size of 4-6 cm² surface area. 2 Or weigh 0.05–0.1g; then cut the tomato leaves into small pieces.

8. The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes according to claim 7, characterized in that, The specific operation of step S2 is as follows: Place the processed sample into a container, add lysis buffer to the container, grind the sample for 1-2 minutes until it becomes a paste, and then incubate the sample at a temperature of 40-45℃ for 5-10 minutes; after centrifuging the sample, aspirate the supernatant and add it to a container containing anhydrous ethanol, mix well, transfer all the mixed liquid to a purification column with a collection tube attached, centrifuge the purification column, add washing buffer to the purification column, and centrifuge again; add elution buffer to the purification column, and then let it stand and centrifuge to obtain RNA extract.

9. The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes according to claim 8, characterized in that, The specific operation of step S3 is as follows: mix and dilute the primer probe combination and activator to obtain the reaction agent; add RNA extraction solution to the reaction tube containing amplification lyophilized powder, then add the reaction agent and mix well; incubate at 38-42℃ for 12-15 min to obtain the amplification product.

10. The RPA-LFD method for detecting brown wrinkled fruit disease in tomatoes according to claim 9, characterized in that, The specific operation of step S4 is as follows: aspirate the amplification product, add it to a container containing the product diluent, mix well to obtain the amplification product diluent; add the amplification product diluent to the LFD test strip to obtain the identification result.

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