A primer set, kit and method for detecting ToBRFV

Abstract

The present invention belongs to the field of nucleic acid detection technology, and specifically relates to a primer set, a kit and a method for detecting ToBRFV. The primer set provided by the present invention can detect ToBRFV by loop-mediated isothermal amplification, and has the characteristics of rapidity, high specificity, high sensitivity, and easy operation. The amplified product can be efficiently and accurately displayed by nucleic acid gold-labeled test strips, fluorescent labels, enzymatic color change reactions, electrochemistry and other methods. At the same time, non-professionals can also operate and use the primer set for detection, which is conducive to promotion and application in actual quarantine.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a primer set, a kit and a method for detecting ToBRFV. Background Art

[0002] Tomato brown wrinkled fruit virus (ToBRFV) belongs to the Virgaviridae family and the Tobamovirus genus. The genome is a single-stranded positive-sense RNA consisting of about 6390 nucleotides. The virus particles are straight rod-shaped, about 18 nanometers wide, and vary in length, with the longer ones reaching 500 nanometers, the shorter ones being 250 nanometers, and most of them being about 300 nanometers in length. Tomato brown wrinkled fruit virus spreads quickly and has strong destructive power. It mainly infects Solanaceae plants such as tomatoes and peppers, causing mosaics on the leaves and brown wrinkled fruit spots on the fruits, causing the fruits to completely lose their commercial value, seriously threatening the production of Solanaceous vegetables such as tomatoes and peppers. Therefore, it is necessary to detect and monitor ToBRFV to prevent the spread of the virus.

[0003] Currently, ToBRFV detection methods include multiplex PCR and fluorescence quantification, which all require precision instruments or professional operators. The operations are complicated and the detection time is long, making it difficult to complete the detection quickly during on-site quarantine. Summary of the invention

[0004] The first object of the present invention is to provide a primer set for detecting ToBRFV, which comprises an outer primer and an inner primer, and the specific sequence is as follows:

[0005] Upstream outer primer F3:

[0006] 5'-CACGAAGGCCAGAAAGAC-3' (SEQ ID No. 1);

[0007] Downstream outer primer B3:

[0008] 5'-TCCGAATTCATCAGCAGG-3' (SEQ ID No. 2);

[0009] Upstream inner primer FIP:

[0010] 5'-CCTTTTGGAAATTTGGGACAACTTAGTATAGAATTATACCTTTCCAGGC-3' (SEQ ID No. 3);

[0011] Downstream inner primer BIP:

[0012] 5'-CAGACGAAGTTGTCTGTCACAGTAATGCAATAGCATACACCCTG-3' (SEQ ID No. 4).

[0013] The second object of the present invention is to provide a loop-mediated isothermal amplification reagent for detecting ToBRFV, which includes the primer set.

[0014] The third object of the present invention is to provide a kit for detecting ToBRFV, which comprises the primer set or the loop-mediated isothermal amplification reagent.

[0015] The fourth object of the present invention is to provide a method for detecting ToBRFV, which comprises: using the primer set, or the loop-mediated isothermal amplification reagent, or the kit to perform loop-mediated isothermal amplification on the sample to be tested.

[0016] The primer set provided by the present invention can detect ToBRFV by loop-mediated isothermal amplification, and has the characteristics of rapidity, high specificity, high sensitivity, and simple operation. The amplified product can efficiently and accurately display the results through nucleic acid gold-labeled test strips, fluorescent labeling, enzymatic color change reaction, electrochemistry, etc. At the same time, non-professionals can also operate and use the primer set for detection, which is conducive to promotion and application in actual quarantine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a nucleic acid result detection diagram in Example 2 of the present invention.

[0019] Figure 2 This is a detection diagram of the result of the sensitivity experiment in Example 3 of the present invention.

[0020] Figure 3 This is a result detection diagram of the sample experiment in Example 4 of the present invention; Figure 3 The samples corresponding to the results from left to right are: 1. Leaf 1, 2. Leaf 2, 3. Leaf 3, 4. Leaf 4, 5. Leaf 5, 6. Fruit 1, 7. Fruit 2, 8. Fruit 3, 9. Fruit 4, 10. Fruit 5, 11. Leaf 6, 12. Fruit 6.

[0021] Figure 4It is a result detection diagram of the sensitivity experiment in the comparative example of the present invention. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0023] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0024] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0025] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0026] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0027] The present invention relates to concentration values, and the meaning includes fluctuations within a certain range. For example, it can fluctuate within a corresponding precision range. For example, 2%, it is allowed to fluctuate within ±0.1%. For values ​​that are large or do not require too fine control, it is also allowed to have a greater fluctuation. For example, 100mM, it is allowed to fluctuate within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, it is allowed to have a fluctuation of ±10%.

[0028] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0029] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0030] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0031] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0032] In the present invention, the term "ToBRFV" refers to Tomato brown rugose fruit virus, which is a plant virus belonging to the genus Tobamovirus. ToBRFV mainly harms tomato (Solanum lycopersicum L.) and pepper (Capsicum) plants and can be transmitted through seeds, agricultural operations, insects and pollen.

[0033] In the present invention, the term "loop-mediated isothermal amplification (LAMP)" refers to a nucleic acid amplification technology performed under constant temperature conditions, which was proposed by Notomi et al. in 2000. LAMP technology uses Bst DNA polymerase with strand displacement activity and four specially designed primers to perform rapid amplification in the temperature range of 60-65°C, and can usually achieve up to 10^9 to 10^10 times of nucleic acid amplification within 15-60 minutes. This method does not require complex thermal cycle equipment, simplifies the operation process, and has high specificity and sensitivity, making LAMP widely used in the fields of pathogenic microorganism detection, infectious disease diagnosis, food safety detection, etc. The reaction process of LAMP technology includes three stages: replication initiation, cyclic amplification and extension. In the replication initiation stage, a LAMP primer binds to the complementary sequence of the double-stranded target gene, initiates DNA synthesis under the action of the strand displacement DNA polymerase, and forms a dumbbell-shaped complementary chain. In the cyclic amplification stage, a large amount of amplified DNA is produced by continuous strand displacement reactions. The final product of amplification is a mixture of DNA with different stem-loop structures and multi-ring cauliflower-like structures. These product DNAs are alternating inverted repeat sequences of the amplified target sequence.

[0034] The present invention relates to a primer set for detecting ToBRFV, comprising an outer primer and an inner primer, and the specific sequence is as follows:

[0035] Upstream outer primer F3:

[0036] 5'-CACGAAGGCCAGAAAGAC-3' (SEQ ID No. 1);

[0037] Downstream outer primer B3:

[0038] 5'-TCCGAATTCATCAGCAGG-3' (SEQ ID No. 2);

[0039] Upstream inner primer FIP:

[0040] 5'-CCTTTTGGAAATTTGGGACAACTTAGTATAGAATTATACCTTTCCAGGC-3' (SEQ ID No. 3);

[0041] Downstream inner primer BIP:

[0042] 5'-CAGACGAAGTTGTCTGTCACAGTAATGCAATAGCATACACCCTG-3' (SEQ ID No. 4).

[0043] The above-mentioned primer set can detect ToBRFV with high specificity and high sensitivity, and can be applied to LAMP detection to achieve efficient detection of ToBRFV.

[0044] In some embodiments, the primer set further includes a loop primer, the specific sequence of which is as follows:

[0045] Upstream loop primer LF:

[0046] 5'-TGTTGCCCCGCTCAA-3' (SEQ ID No. 5);

[0047] Downstream loop primer LB:

[0048] 5'-CTTCCAACTGTACG-3' (SEQ ID No. 6).

[0049] By adding the above-mentioned loop primer, the amplification process can be further accelerated.

[0050] In some embodiments, the 5' end of the upstream loop primer LF is modified with a fluorescent reporter group or a chemical marker, and the 5' end of the downstream loop primer LB is modified with a biotin affinity group.

[0051] In specific implementation, those skilled in the art can select specific fluorescent reporter groups in combination with common sense. In some specific embodiments, the fluorescent reporter groups include one or more of FAM (Fluorescein amidite), iHEX (Hexachloro-fluorescein), TET (Tetrachlorofluorescein), Cyanine dye, ROX (Carboxy-X-rhodamine), and Texas Red.

[0052] In the specific implementation, those skilled in the art can select specific chemical markers in combination with common sense. In some specific embodiments, the chemical marker is selected from one or more of the following: 1) enzyme markers: such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.; 2) chemiluminescent markers: such as fluorescein, rhodamine, digoxin, fluorescent markers (such as FITC), etc.; 3) radioactive markers: radioisotopes (such as ^125I), etc.; 4) quantum dots; 5) magnetic nanoparticles; 6) polymer markers. These chemical markers can be combined with antibodies, antigens or other biological molecules, so that the detection of target molecules is more sensitive and accurate. The selection of appropriate markers depends on the specific requirements of the experiment and the detection method.

[0053] In specific implementation, those skilled in the art can select a specific biotin affinity group in combination with common sense. In some specific embodiments, the biotin affinity group is selected from one or more of Biotin, Avidin, Streptavidin, and NeutrAvidin.

[0054] In some embodiments, the 5' end of the upstream loop primer LF is modified with a FAM group, and the 5' end of the downstream loop primer LB is modified with a Biotin group. This facilitates the detection of the amplification product of the primer set by a nucleic acid gold-labeled test strip, thereby further facilitating simple and accurate confirmation of the test results.

[0055] In the specific implementation, those skilled in the art may further modify the primers in the primer set in combination with the detection needs of fluorescent labeling, enzymatic color change reaction, electrochemical and other methods, which all fall within the protection scope of the present invention.

[0056] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain more embodiments of the primer set of the present invention.

[0057] The present invention also provides a loop-mediated isothermal amplification reagent for detecting ToBRFV, which includes the primer set.

[0058] In some specific embodiments, the loop-mediated isothermal amplification reagent further contains a suitable buffer to adjust the primer concentration and the final reaction conditions.

[0059] In some specific embodiments, the loop-mediated isothermal amplification reagent further contains one or more reagents in the following kit.

[0060] In some specific embodiments, the loop-mediated isothermal amplification reagent contains other components of the loop-mediated isothermal amplification reaction system described below except for the template.

[0061] The present invention also provides a kit for detecting ToBRFV, which comprises the primer set or the loop-mediated isothermal amplification reagent.

[0062] In some embodiments, the kit further comprises one or more of the following reagents: lysate, LAMP reaction buffer, Bst polymerase, Rt reverse transcriptase, MRI enzyme, dNTPs, Mg 2+ ,water.

[0063] In some embodiments, the kit further comprises a lysis solution, which comprises 100-300 nM Tris-HCl (pH 7-10), 0.1-3.0 Vol% Triton X-100, and water. The lysis solution of the present invention can quickly and effectively lyse common ToBRFV target plants such as tomatoes, peppers, and eggplants in a short time and release the nucleic acid to be detected, and is compatible with subsequent detection systems, thereby helping to further shorten the detection time and improve the detection efficiency.

[0064] In some embodiments, the kit further comprises a composite enzyme, wherein the composite enzyme comprises Bst polymerase, Rt reverse transcriptase and MRI enzyme in an enzyme activity unit ratio of 1-2:5-15:2-5.

[0065] In some embodiments, the kit further comprises: the lysate, LAMP reaction buffer, the complex enzyme, dNTPs, Mg 2+ and water.

[0066] Those skilled in the art can identify the specific components in the LAMP reaction buffer based on common sense.

[0067] In some specific embodiments, the kit further includes: the lysis solution, MgSO4, KCl, (NH4)2SO4, dNTPs, the complex enzyme, betaine and water.

[0068] In some specific embodiments, the water mentioned in the present invention is Nuclease-Free Water.

[0069] In some embodiments, the kit further comprises a nucleic acid gold-labeled test strip, wherein the nucleic acid gold-labeled test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water-absorbing pad, wherein the nitrocellulose membrane is coated with a detection line and a quality control line;

[0070] The detection line is coated with Biotin monoclonal antibody;

[0071] The quality control line is coated with sheep anti-chicken IgY antibody, and the gold label pad is coated with colloidal gold-labeled FAM monoclonal antibody and chicken IgY antibody.

[0072] In specific implementation, those skilled in the art can make corresponding replacements for the antibodies in the above-mentioned nucleic acid gold-labeled test strips in combination with the modified groups on the primers, and such a technical solution also falls within the protection scope of the present invention.

[0073] In some specific embodiments, the kit further comprises: a positive control and a negative control for verifying the accuracy and specificity of the reaction.

[0074] In some specific embodiments, the kit further comprises: a stabilizer and a preservative to ensure the stability of the kit during storage and use.

[0075] In some embodiments, the kit further comprises: extraction and dilution buffers. If the kit is designed to detect directly from complex samples, it may contain buffers for sample lysis and DNA release.

[0076] In some embodiments, the kit further comprises: instructions for providing detailed operating steps, reaction conditions, usage guidelines and precautions.

[0077] The other components in the kit mentioned in the present invention can be obtained through commercial channels, and can be provided in multiple or one reagent in the form of a single or combined form. For example, the Bst polymerase, Rt reverse transcriptase and MRI enzyme in the complex enzyme can be provided in one reagent in the form of a complex enzyme (i.e., an enzyme mixture), or can be provided separately in three reagents.

[0078] Those skilled in the art can combine the above-mentioned embodiments with common sense to obtain more embodiments of the kit of the present invention.

[0079] The present invention also provides a method for detecting ToBRFV, which comprises: using the primer set, or the loop-mediated isothermal amplification reagent, or the kit to perform loop-mediated isothermal amplification on the sample to be tested.

[0080] In some embodiments, the reaction system of the loop-mediated isothermal amplification is: 50~150nM MgSO4, 300~700mM KCl, 60~120mM (NH4)2SO4, 20~30μL template, 5~10nM upstream inner primer FIP, 5~10nM downstream inner primer BIP, 1~3nM upstream outer primer F3, 1~3nM downstream outer primer B3, 1~3nM upstream loop primer LF, 1~3nM downstream loop primer LB, 700~1000pM dNTPs, 10~20U Bst polymerase, 50~150U Rt reverse transcriptase, 20~50UMRI enzyme, 50~200pM betaine, and the rest is made up to 50μL with water.

[0081] In some embodiments, the reaction system of the loop-mediated isothermal amplification is: 100~150nM MgSO4, 500~700mM KCl, 60~80mM (NH4)2SO4, 20~30μL template, 6~10nM upstream inner primer FIP, 6~10nM downstream inner primer BIP, 1.5~2.5nM upstream outer primer F3, 1.5~2.5nM downstream outer primer B3, 1.5~2.5nM upstream loop primer LF, 1.5~2.5nM downstream loop primer LB, 700~900pM dNTPs, 15~20U Bst polymerase, 50~100U Rt reverse transcriptase, 20~40U MRI enzyme, 50~150pM betaine, and the rest is made up to 50μL with water.

[0082] In some embodiments, the method further comprises: using a lysis solution to lyse the sample to be tested to obtain a lysis product; using the lysis product as a template, configuring a loop-mediated isothermal amplification reaction system, and performing a loop-mediated isothermal amplification reaction.

[0083] In the specific implementation, an appropriate amount of leaves or fruits of the sample to be tested can be taken to grind out leaf pulp or fruit pulp for later use, 10-100 μL of the liquid to be tested is added to a tube containing 0.3-1 mL of lysis solution, lysed for 2-10 minutes and fully mixed for loop-mediated isothermal amplification reaction.

[0084] In some embodiments, the reaction temperature of the loop-mediated isothermal amplification is 60-65° C. As an example, the reaction temperature can be 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., or any value between 60 and 65° C.

[0085] In some specific embodiments, the reaction time of the loop-mediated isothermal amplification is 5 to 60 min, more preferably 20 to 40 min.

[0086] In some embodiments, the method further comprises: dropping the amplification product of the loop-mediated isothermal amplification reaction onto the sample pad of the nucleic acid gold-labeled test strip, and confirming the result by observing the color development of the nucleic acid gold-labeled test strip.

[0087] In some specific embodiments, the chromatography time (or reaction time or waiting time) of the nucleic acid gold-labeled test strip is 2 to 10 minutes.

[0088] In some specific embodiments, the method comprises: using a lysis solution to lyse the sample to be tested to obtain a lysis product;

[0089] Using the cleavage product as a template, configuring a loop-mediated isothermal amplification reaction system, and performing a constant temperature reaction at 60-65° C. for 5-60 minutes to obtain an amplification product;

[0090] The amplification product is added dropwise to the sample pad of the nucleic acid gold-labeled test strip, and the result is confirmed by observing the color development of the nucleic acid gold-labeled test strip.

[0091] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0092] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0093] Example 1

[0094] This example first provides a primer set for detecting ToBRFV, which includes an outer primer, an inner primer and a loop primer, and the specific sequences are as follows:

[0095] Upstream outer primer F3:

[0096] 5'-CACGAAGGCCAGAAAGAC-3' (SEQ ID No. 1);

[0097] Downstream outer primer B3:

[0098] 5'-TCCGAATTCATCAGCAGG-3' (SEQ ID No. 2);

[0099] Upstream inner primer FIP:

[0100] 5'-CCTTTTGGAAATTTGGGACAACTTAGTATAGAATTATACCTTTCCAGGC-3' (SEQ ID No. 3);

[0101] Downstream inner primer BIP:

[0102] 5'-CAGACGAAGTTGTCTGTCACAGTAATGCAATAGCATACACCCTG-3' (SEQ ID No. 4);

[0103] Upstream loop primer LF:

[0104] 5'-TGTTGCCCCGCTCAA-3' (SEQ ID No. 5);

[0105] Downstream loop primer LB:

[0106] 5'-CTTCCAACTGTACG-3' (SEQ ID No. 6);

[0107] The 5' end of the upstream loop primer LF is modified with a FAM group, and the 5' end of the downstream loop primer LB is modified with a Biotin group.

[0108] This embodiment further provides a kit for detecting ToBRFV, which includes the primer set, lysate, MgSO4, KCl, (NH4)2SO4, dNTPs, complex enzyme, betaine, enzyme-free water and nucleic acid gold-labeled test strips;

[0109] The lysis solution includes 140nM Tris-HCl (pH 7-10), 0.45 Vol% Triton X-100, and the remainder is enzyme-free water;

[0110] The complex enzyme is a mixture of Bst polymerase, Rt reverse transcriptase and MRI enzyme in an enzyme activity unit ratio of 2:5:2;

[0111] The nucleic acid gold-labeled test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water-absorbing pad; the nitrocellulose membrane is coated with a detection line and a quality control line; the detection line is coated with Biotin monoclonal antibody; the quality control line is coated with sheep anti-chicken IgY antibody; and the gold-labeled pad is coated with colloidal gold-labeled FAM monoclonal antibody and chicken IgY antibody.

[0112] Example 2

[0113] This embodiment provides a method for detecting ToBRFV, which uses the kit in Example 1, and the specific steps are as follows:

[0114] Step 1: Take an appropriate amount of leaves or fruits of the sample to be tested and grind them into leaf pulp or fruit pulp as the test liquid, mix 10 μL of the test liquid with 300 μL of lysis solution and shake well, let it stand at room temperature for 5 minutes, and obtain the lysis product;

[0115] Step 2: Using the cleavage product as a template, a loop-mediated isothermal amplification reaction system is prepared, wherein the reaction system of 50 μL contains: 120 nM MgSO4, 600 mM KCl, 70 mM (NH4)2SO4, 25 μL template, 8 nM upstream inner primer FIP, 8 nM downstream inner primer BIP, 2 nM upstream outer primer F3, 2 nM downstream outer primer B3, 2 nM upstream loop primer LF, 2 nM downstream loop primer LB, 700 pM dNTPs, 90 U complex enzyme (20 U Bst polymerase, 50 U Rt reverse transcriptase, 20 U MRI enzyme), 100 pM betaine, and the rest is supplemented to 50 μL with enzyme-free water; the reaction system is reacted at 62° C. in a PCR amplification instrument for 30 min to obtain an amplified product;

[0116] Step 3: Add the amplification product dropwise onto the sample pad of the nucleic acid gold-labeled test strip, let stand for 5 minutes, and confirm the result by observing the color development of the nucleic acid gold-labeled test strip.

[0117] from Figure 1 It can be seen that the TOBRFV gene band (the bottom band) has a strong color, the C line (the top band) has normal color, and the NTC is normal and uncontaminated.

[0118] Example 3

[0119] In this example, the sensitivity of the kit in Example 1 was tested by diluting the template in a 10-fold gradient from 10^5 to 10^1 copies / μL, and then adding the diluted template according to the methods of steps 1 to 3 of Example 2 to perform loop-mediated isothermal amplification and test the amplification product to verify the sensitivity of the present invention.

[0120] The results are as follows Figure 2 As shown, the results of the nucleic acid gold-labeled test strips showed that the templates of 10^5, 10^4, 10^3, and 10^2 copies were specifically amplified, and the strip colors were obvious. The experimental results showed that the detection sensitivity could reach 100 copies / test.

[0121] Example 4

[0122] This example provides a plant sample experiment of the kit in Example 1. Specifically, 10 tomato samples infected with ToBRFV (leaf 1, leaf 2, leaf 3, leaf 4, leaf 5, fruit 1, fruit 2, fruit 3, fruit 4, fruit 5) and 2 tomato samples not infected with ToBRFV (leaf 6, fruit 6) were tested according to the method of steps 1 to 3 of Example 2.

[0123] The results are as follows Figure 3As shown, the results of the nucleic acid gold-labeled test strips are from left to right: 1. Leaf 1, 2. Leaf 2, 3. Leaf 3, 4. Leaf 4, 5. Leaf 5, 6. Fruit 1, 7. Fruit 2, 8. Fruit 3, 9. Fruit 4, 10. Fruit 5, 11. Leaf 6, 12. Fruit 6. Among them, 1 to 10 are known positive samples, and the loop-mediated isothermal amplification method is also positive; 11 to 12 are known negative samples, and the loop-mediated isothermal amplification method is also negative. This result shows that this experiment was successful and verifies that the method of the present invention can detect ToBRFV virus samples.

[0124] Example 5

[0125] In this embodiment, the specificity of the kit in Example 1 is tested, and common viruses on tomatoes are tested. Tomato leaf samples carrying the following viruses are tested according to the method of steps 1 to 3 of Example 2. The tested viruses are tomato spotted wilt virus (TSWV), tomato mottle mosaic virus (ToMMV), tomato ringspot virus (ToRSV), tomato mosaic virus (ToMV), cucumber mosaic virus (CMV), pepper mild mottle virus (PMMoV), arabis mosaic virus (ArMV), tobacco mosaic virus (TMV), and tobacco ringspot virus (TRSV). The detection results of the loop-mediated isothermal amplification method are all negative. The results show that this experiment is successful, verifying that the method of the present invention does not cross-react with common viruses and has good specificity.

[0126] Comparative Example

[0127] This comparative example provides examples of primer sets screened and eliminated by the present invention and their effect data.

[0128] The primer set includes an outer primer, an inner primer and a loop primer, and the specific sequences are as follows:

[0129] Upstream outer primer F3:

[0130] 5'-CAGGGTGTATGCTATTGCA-3' (SEQ ID No. 7);

[0131] Downstream outer primer B3:

[0132] 5'-CAGCTTGTCTCCATCACG-3' (SEQ ID No. 8);

[0133] Upstream inner primer FIP:

[0134] 5'-CGTAACAAACATGGACATTTTTCCTCAGTATATACGATATACCTGCTGA-3' (SEQ ID No. 9);

[0135] Downstream inner primer BIP:

[0136] 5'-CCGCCTTCCACTTTTCCGAGAAAAACACGCGTTGATTTCG-3' (SEQ ID No. 10);

[0137] Upstream loop primer LF:

[0138] 5'-AAAGTGCCGCTCCGAATTCA-3' (SEQ ID No. 11);

[0139] Downstream loop primer LB:

[0140] 5'-CTCGAAGATTCACACGTCAACCTT-3' (SEQ ID No. 12);

[0141] The 5' end of the upstream loop primer LF is modified with a FAM group, and the 5' end of the downstream loop primer LB is modified with a Biotin group.

[0142] The sensitivity of the above primer set was tested by diluting the template 10-fold from 10^5 to 10^1 copies / μL, and then adding the diluted template to perform loop-mediated isothermal amplification and amplification product testing according to the method of steps 1 to 3 of Example 2 (except the primer set, other reagents and operations are the same as Example 2) to verify its sensitivity.

[0143] The results are as follows Figure 4 As shown, the results of the nucleic acid gold-labeled test strips showed that the templates of 10^5, 10^4, and 10^3 were specifically amplified, and the band colors were obvious. The experimental results showed that the detection sensitivity could reach 1000 copies / test, which was poorer than the sensitivity of the primer set of the present invention.

[0144] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A primer set for detecting ToBRFV, comprising an outer primer, an inner primer and a loop primer, the specific sequences of which are as follows: Upstream outer primer F3: 5′-CACGAAGGCCAGAAAGAC-3′; Downstream outer primer B3: 5′-TCCGAATTCATCAGCAGG-3′; Upstream inner primer FIP: 5'-CCTTTTGGAAATTTGGGACAACTTAGTATAGAATTATACCTTTCCAGGC-3'; Downstream inner primer BIP: 5'-CAGACGAAGTTGTCTGTCACAGTAATGCAATAGCATACACCCTG-3'; Upstream loop primer LF: 5′-TGTTGCCCCGCTCAA-3′; Downstream loop primer LB: 5′-CTTCCAAACGTGTACG-3′.

2. The primer set according to claim 1, wherein The 5' end of the upstream loop primer LF is modified with a FAM group, and the 5' end of the downstream loop primer LB is modified with a Biotin group.

3. A loop-mediated isothermal amplification reagent for detecting ToBRFV, comprising the primer set described in claim 1 or 2.

4. A kit for detecting ToBRFV, comprising the primer set of claim 1 or 2 or the loop-mediated isothermal amplification reagent of claim 3.

5. The kit according to claim 4, wherein The kit also includes one or more of the following reagents: lysate, LAMP reaction buffer, complex enzyme, dNTPs, Mg 2+ ,water; Wherein, the lysate comprises 100-300 nM Tris-HCl, 0.1-3.0 Vol% Triton X-100, and water; The complex enzyme comprises Bst polymerase, Rt reverse transcriptase and MRI enzyme in an enzyme activity unit ratio of 1-2:5-15:2-5.

6. The kit according to claim 4 or 5, wherein The kit also includes a nucleic acid gold-labeled test strip, which includes a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water-absorbing pad, wherein the nitrocellulose membrane is coated with a detection line and a quality control line; The detection line is coated with Biotin monoclonal antibody; The quality control line is coated with sheep anti-chicken IgY antibody, and the gold label pad is coated with colloidal gold-labeled FAM monoclonal antibody and chicken IgY antibody.

7. A method for detecting ToBRFV, comprising: The sample to be tested is subjected to loop-mediated isothermal amplification using the primer set according to claim 1 or 2, or the loop-mediated isothermal amplification reagent according to claim 3, or the kit according to any one of claims 4 to 6.

8. The method according to claim 7, wherein: The reaction system of the loop-mediated isothermal amplification is: 50-150nM MgSO4, 300-700mM KCl, 60-120mM (NH4)2SO4, 20-30μL template, 5-10nM upstream inner primer FIP, 5-10nM downstream inner primer BIP, 1-3nM upstream outer primer F3, 1-3nM downstream outer primer B3, 1-3nM upstream loop primer LF, 1-3nM downstream loop primer LB, 700-1000pM dNTPs, 10-20U Bst polymerase, 50-150U Rt reverse transcriptase, 20-50U MRI enzyme, 50-200pM betaine, and the rest is made up to 50μL with water.

9. The method according to claim 7 or 8, wherein: The method comprises: Using a lysis solution to lyse the sample to be tested to obtain a lysis product; Using the cleavage product as a template, configuring a loop-mediated isothermal amplification reaction system, and performing a constant temperature reaction at 60-65° C. for 5-60 minutes to obtain an amplification product; The amplification product is dripped onto the sample pad of the nucleic acid gold-labeled test strip, and the result is confirmed by observing the color development of the nucleic acid gold-labeled test strip.

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