KR101566480B1 - Method for detecting Tomato chlorosis virus using loop-mediated isothermal amplification assay - Google Patents

Abstract

The present invention relates to a primer set for isothermal amplification reaction, which comprises a primer set for detection of Tomato chlorosis virus , consisting of SEQ ID NOs: 1 to 4, 5 to 8, 9 to 12, 13 to 16, or 17 to 20 to provide. The primer set according to the present invention has an advantage of being able to diagnose the ToCV infection early, thereby preventing the economic loss of the farmhouse due to the infection, and promptly diagnosing the virus on the spot.

Description

[0001] The present invention relates to a method for detecting tomato cereus virus using isothermal amplification,

The present invention relates to a method for detecting ToC virus (ToCV), more specifically, a primer for isothermal amplification reaction for detecting ToC virus (ToCV) and a method for detecting Tomococcal virus (ToCV) .

ToCV is known to be carried by the usual bemi cyano other Bridge (Bemisia tabaci) as Crimean you virus (Crinivirus) in viruses belonging to the interrogating Claus virus (Closteroviridae).

Crops such as tomatoes and peppers infected with the virus are sulphated with leaves, small sized fruits are enlarged, and fruit yields are reduced.

In 2007, the National Plant Quarantine Service, under the Ministry of Agriculture and Forestry, designated the tomato pestilence virus as a management pest, and promptly diagnosed it as a diagnostic pest. The need for the development of

As a conventional virus diagnosis method, an electron microscope or a serological method was mainly used. Electron microscopy can detect the presence of virus but it is almost impossible to diagnose the species as a morphological feature. Among the serological methods, the enzyme-linked immunosorbent assay (ELISA) has a detection sensitivity about 1,000 times lower than that of the most commonly used diagnostic methods or the polymerase chain reaction (hereinafter referred to as PCR) And an unexpected nonspecific response of the test sample often leads to failure of accurate diagnosis. In recent years, PCR methods with high detection sensitivity and convenience have been widely used for the diagnosis of viruses. However, it is very important to develop specific primers for the diagnostic method using PCR. It must be confirmed by electrophoresis and finally subjected to a sequence of DNA sequencing. In addition, this method requires specialized equipment such as a thermocycler and a professional manpower to operate it, and sequencing of the amplification product for final confirmation is a process requiring high cost and high technology. In addition, this process is time-consuming to perform and is not visually detectable. Therefore, the ability to use analytical equipment in the field is significantly reduced.

In order to effectively detect the virus in such a short time, development of a method capable of real-time detection on the spot without professional equipments is required.

The iso-amplification method (LAMP) is similar to the conventional PCR method. However, since the conventional PCR method amplifies the gene while repeating the three steps of denaturation, splicing, and elongation, The isothermal amplification method has the advantage of being able to bond and stretch at a fixed temperature. This is because instead of using the DNA Taq polymerase used in the existing PCR method, the Bst DNA polymerase (Bst DNA polymerase) having the exonuclease function of the nucleic acid is used, Because it can cause the deformation of the spiral structure. Therefore, isothermal amplification does not require a temperature change during amplification of the gene, which makes gene amplification possible at fixed temperature with no special equipment.

Disclosure of Invention Technical Problem [8] In order to solve the above-described technical problem, the present invention provides a method for detecting tomato retrovirus.

The present invention provides a primer set for isothermal amplification for detecting tomato cerebro viruses.

In addition, the present invention provides a composition for isothermal amplification for the detection of tomato erosion virus.

In order to achieve the above object, the present invention provides a virus detection method capable of rapidly and rapidly detecting tomato pest virus.

In order to accomplish the above object, the present invention provides a primer set for isothermal amplification reaction for detecting Tomato chlorosis virus .

The inventors of the present invention have conducted research on a method capable of real-time detection in the field without professional equipments in order to easily and effectively detect the tomato cerebro virus in a short time, and as a result, the present invention has been completed.

The present inventors used loopmediated isothermal amplification (LAMP) to detect tomato retroviruses in a short time without professional equipments. Unlike conventional polymerase chain reaction (PCR), the isothermal amplification method does not require temperature control to amplify a gene. Therefore, it is possible to amplify a gene without professional equipments and it is possible to amplify a gene at a high concentration in a short time. In order to utilize loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must function as one set. Among them, F3 and FIP , And B3 and BIP are primers that bind in the reverse direction in the 3 'direction. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).

ToCV has been reported to cause a decrease in the yield of crops when it is infected with tomatoes and peppers, and because it has a strong infectious ability such as easy spreading by contact with powder and dilution about 10,000 times, Diagnosis is urgent.

However, up to now, temperature control for gene amplification has been necessary to diagnose ToCV. Therefore, there has been a problem that the results can be confirmed only in a laboratory equipped with specialized equipment. In order to solve the above problems, the inventors have developed a technology for diagnosing the tomato retrovirus using the isothermal amplification method which can be applied in the field.

It is preferable that the present invention is a primer set for isothermal amplification reaction for detecting tomato cerebro-virus consisting of SEQ ID NOS: 1 to 4, 5 to 8, 9 to 12, 13 to 16, or 17 to 20, Preferably, the primer set is composed of the primers set forth in SEQ ID NOS: 13 to 16, but is not limited thereto, as long as it is a primer set for isothermal amplification reaction capable of diagnosing tomato cerebro viruses.

The tomato retroviruses according to embodiments of the present invention may be selected from the group consisting of GenBank accession numbers KC311375.1, AB513443.1 and DQ234673.1.

According to still another embodiment of the present invention, the present invention provides a primer composition for isothermal amplification reaction comprising the primer set.

The primer composition is for detecting tomato retrovirus, and the composition may further comprise a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer.

In addition, the present invention relates to a method for producing a DNA fragment, which comprises the steps of extracting genomic DNA (gDNA) from tomato, firstly reacting the gDNA with the primer composition at 30 ° C to 50 ° C, followed by a second reaction at 70 ° C to 90 ° C Amplifying the target sequence by performing an isothermal amplification reaction method; And a step of detecting the amplified product.

The first reaction is performed for 20 to 40 seconds, and the second reaction can be performed for 30 to 90 minutes.

The step of detecting the amplified product according to the present invention is characterized by confirming amplified DNA using electrophoresis or SYBR Green I.

The primer set for isothermal amplification according to the present invention can diagnose the infection of ToCV early, thereby preventing the economic loss of the farm due to the infection.

The method of diagnosing virus according to the present invention does not require a step of changing the temperature for the amplification of the sequence, thereby enabling gene amplification at a fixed temperature easily without professional equipments

In addition, the specimen in which the virus is detected can be visually confirmed without special equipment, and can be detected with high specificity.

Through the virus diagnosis method according to the present invention, faster, simpler and more accurate diagnosis than conventional ToCV diagnosis method is possible.

Brief Description of the Drawings Fig. 1 is a diagram showing the nucleotide sequence of tomato retroviruses of GenBank accession number KC311375.1.
Fig. 2 is a diagram showing the nucleotide sequence of tomato retroviruses of GenBank accession number AB513443.1.
FIG. 3 is a diagram showing the nucleotide sequence of tomato retroviruses of GenBank accession number DQ234673.1. FIG.
FIG. 4 is a diagram showing gene base sequence similar regions of three strains of tomato retrovirus. FIG.
5 is a diagram showing the nucleotide sequence of a primer set for isothermal amplification of tomato retrovirus.
FIG. 6 is a diagram showing the nucleotide sequence of the most efficient primer set among the five primer sets.
FIG. 7 is a graph showing the result of electrophoresis of the amplified gene using the primer set of the present invention. FIG.
FIG. 8 is a diagram showing the result of checking the gene amplified using the primer set of the present invention under a UV light source using SYBR Green I. FIG.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One. Tomato retrovirus ( ToCV ) Gene collection

Tomato chlorotic virus (Tomato chlorosis In order to investigate the detection method of the virus, tomato clonorviruses were collected and cloned. Each virus is tomato chlorotic virus obtained from the outbreak site (Tomato chlorosis virus ; ToCV) Natural Infected Tomatoes ( Solanum lycopersicum ) as a sample. The isothermal amplification primers used in the present invention were prepared from three previously reported ToCV strains from Genbank, a biological nucleic acid information database provided by the National Center for Biotechnology Information (NCBI) (GenBank accession number: KC311375.1, AB513443.1, and DQ234673.1), respectively. The nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of SEQ ID NO:

Example 2 . primer  write

Primers were prepared using PrimerExplorer V4 to detect ToCV through loop-mediated isothermal amplification (LAMP). In order to use the isothermal amplification method, four primers (F3, B3, FIP, and BIP) should function as one set. In order to detect all kinds of ToCV known, a common portion between five ToCV gene sequences is searched A primer set prepared using PrimerExplorer V4 mainly at the search site is shown in FIG. As shown in Fig. 5, a universal primer set which was expected to be applied to all viruses of ToCV was prepared.

Example  3. Collection of plant specimens

Plant samples were collected to confirm that the primer set produced was usable to detect ToCV. In the present invention, total RNA was isolated from virus-infected plant samples and then total cDNA was synthesized by RT (reverse transcriptase) -PCR. The extraction buffer used for total RNA isolation was the TRI REAGENT (catalog No. TR 118) from MRC. After the plant tissue was placed in a mortar and liquid nitrogen was poured into the tube, the nitrogen was evaporated, and the tissue was immediately ground to a fine powder and the tissue was extracted with a 1 ml extraction buffer using a spatula preliminarily cooled in liquid nitrogen . After putting the powdered tissue, the tube lid was closed and reacted at 4 ° C for 5 minutes. 200ul of chloroform was added thereto, followed by vigorous shaking for 15 seconds, followed by reaction at room temperature for at least 10 minutes. After reacting, centrifuge for 10 minutes at 4 ℃ and 8,000 × g. Collect 500 μl of the supernatant using a pipette, carefully transfer it to a new tube, add the same amount of isopropanol as the supernatant, Lt; / RTI > The reaction tubes were centrifuged at 4 ° C and 8,000 × g for 10 minutes to precipitate RNA. The pellet was washed with a mixture of ethanol and DEPC treated water (0.1% diethyl pyrocarbonate aqueous solution) in a ratio of 7.5: 2.5, and centrifuged at 4 ° C and 8,000 × g for 10 minutes. , The tube was placed upside down on the test tube holder, and the RNA pellet was dissolved in 30 μl of DEPC treated water. The concentration of RNA extracted through a spectrophotometer was measured. RT-PCR was performed to synthesize cDNA based on the total RNA obtained from the above experiment. RT-PCR was performed using M-MLV Reverse Transcriptase (BIONEER) and random primers according to manufacturer's instructions. RT-PCR was performed by a 3-step method. The tubes containing total RNA and random primer were reacted at 70 ° C for 10 minutes, and then the temperature was lowered to 4 ° C. After adding dNTP and buffer, After 10 minutes of reaction, the temperature was lowered to 4 ° C, and the reaction was allowed to proceed at 37 ° C for 1 hour, followed by 10 minutes of enzyme inhibition at 70 ° C.

Example  4. Isothermal amplification and validation

A primer composition for amplification reaction was prepared to confirm that the prepared primer set can be used to detect ToCV. To prepare the primer composition, 2 μl of 10 × Bst DNA polymerase reaction buffer (20 mM Tris-HCl, 10 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X- 100), of 1.6ul 10mM dNTPs (dATP, dTTP, dGTP, dCTP mixture mixed by 10mM each), 10uM F3 and B3 primers 0.4ul, 10uM FIP and BIP primer of 1.6ul, 20mM MgSO of 1ul _4, 1ul (8 Unit) Bst DNA polymerase, 1 μl of 1/10 diluted template cDNA, and 9.4 μl of distilled water were added to the reaction tube and mixed. The prepared amplification reaction composition was allowed to react at 40 ° C for 30 seconds and then reacted in a reaction vessel at 62 ° C for 1 hour to perform isothermal amplification. After the reaction was completed, enzyme activity was inhibited at 80 DEG C for 5 minutes. 5ul of the total 20ul reaction was electrophoresed to confirm that the gene was amplified. The results are shown in Fig.

FIG. 7 also shows the results of assaying ToCV naturally infected tomatoes using the primer set of FIG. As a result of the test, it was confirmed that the primer set of FIG. 6 can commonly test the ToCV strain.

FIG. 8 shows SYBR Green I concentration of 1,000 times concentrated by the same reagent in the presence of 1ul per 20ul of the reaction mixture. The significance was verified by comparing the results of the electrophoresis of SYBR Green I with the results of amplification of the viral gene.

As a result of analyzing through the examples of the present invention, it was confirmed that all the five kinds of tomato retroviruses can be detected by performing the isothermal amplification method using the universal primer set of the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method for detecting Tomato chlorosis virus using loop-mediated          isothermal amplification assay <130> P13-145 <160> 23 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV1_F3 primer <400> 1 gttgggcttt gttgaacc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV1_B3 primer <400> 2 ggcctgacac atagacatg 19 <210> 3 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV1_FIP primer <400> 3 cctgagtcaa ggtgagagta tcactataac gagttaagga aattgaagg 49 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV1_BIP primer <400> 4 aagaggagtt cgagaagata ctcgccagcg aaaatcttat ccgt 44 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV2_F3 primer <400> 5 cccggatact attaattata acga 24 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV2_B3 primer <400> 6 tacaatggcc tgacacat 18 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV2_FIP primer <400> 7 ctcctcttcc tgagtcaagg tgtaaggaaa ttgaaggtac actc 44 <210> 8 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV2_BIP primer <400> 8 cgagaagata ctcgaatcct tttgcacatg tagaaaccag cgaa 44 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV3_F3 primer <400> 9 agatttaagt ggaaaactcc g 21 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV3_B3 primer <400> 10 caacgaacca tttatataat cgc 23 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV3_FIP primer <400> 11 ttcatgactt ctggcgtacc gggttagacc caaaatgtcc g 41 <210> 12 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV3_BIP primer <400> 12 ggactttatc aactccggtc ttattggtac caaacacatg gctt 44 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV4_F3 primer <400> 13 gacgaggtgc agaacttg 18 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV4_B3 primer <400> 14 attcgagtat cttctcgaac t 21 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV4_FIP primer <400> 15 aagcccaact ggaagatcga ggagggacga ttcgacat 38 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV4_BIP primer <400> 16 acgagttaag gaaattgaag gtacactctt cctgagtcaa ggtga 45 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV5_F3 primer <400> 17 ccactagggg tgatactctc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV5_B3 primer <400> 18 tgttattacc ggctgcttta 20 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV5_FIP primer <400> 19 ccgattattc gcctgcaaaa ggaccttgac tcaggaagag g 41 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Unuversal ToCV5_BIP primer <400> 20 tgagaccccg atgacggata ccttggttta caatggcc 38 <210> 21 <211> 774 <212> DNA <213> GenBank accession number KC311375.1 <220> <221> gene &Lt; 222 > (1) .. (774) <223> Tomato chlorosis virus isolate BJ coat protein gene <400> 21 atggagaaca gtgctgttgc aaacactggt gataacggtg gtgaccgcaa tcctctggtt 60 agaccgttag atgatggcgt agatgacgag gtgcagaact tgggcaggag ggacgattcg 120 acatctctca ttccggctaa tcctaatcga tcttccagtt gggctttgtt gaacccggat 180 actattaatt ataacgagtt aaggaaattg aaggtacact ccactagggg tgatactctc 240 accttgactc aggaagagga gttcgagaag atactcgaat ccttttgcag gcgaataatc 300 ggtgagaccc cgatgacgga taagattttc gctggtttct acatgtctat gtgtcaggcc 360 attgtaaacc aagggacctc agttaaagca gccggtaata acagtcttga aaactacttt 420 gaggtagatg gtgcgagatt taagtggaaa actccggatt tgataaatga ggttagaccc 480 aaaatgtccg atgttccaaa cgccatacgt cggtacgcca gaagtcatga aaagattatt 540 caggacttta tcaactccgg tcttattaag cctgattatc atttacaatt caaacatggc 600 gtattaccaa gccatgtgtt tggtaccggc gattatataa atggttcgtt gatgaatatc 660 tcagatgatc aacttatctc gaacctgctt atgaaaagaa acgctttgtg caagggtaac 720 gagggcaagg aactgtacaa cgttaaccaa cttgcatcga taactggttg ctaa 774 <210> 22 <211> 774 <212> DNA <213> GenBank accession number AB513443.1 <220> <221> gene &Lt; 222 > (1) .. (774) <223> Tomato chlorosis virus CP gene for coat protein <400> 22 atggagaaca gtgctgttgc aaacactggt gataacggtg gtggccgcaa tcctctggtt 60 agaccgttag atgatggcgt agatgacgag gtgcagaact tgggcaggag ggacgattcg 120 acatctctca ttccggctaa tcctaatcga tcttccagtt gggctttgtt gaacccggat 180 actattaatt ataacgagtt aaggaaattg aaggtacact ccactagggg tgatactctc 240 accttgactc aggaagagga gttcgagaag atactcgaat ccttttgcag gcgaataatc 300 ggtgagaccc cgatgacgga taagattttc gctggttttt acatgtctat gtgtcaggcc 360 attgtaaacc aagggacctc agttaaagca gccggtaata acagtcttga aaactacttt 420 gaggtagatg gtgcgagatt taagtggaaa actccggatt tgataaatga ggttagaccc 480 aaaatgtccg atgttccaaa cgctatacgt cggtacgcca gaagtcatga aaagattatt 540 caggacttta tcaactccgg tcttattaag cctgattatc atttacaatt caaacatggc 600 gtattaccaa gccatgtgtt tggtaccggc gattatataa atggttcgtt gatgaatatc 660 tcagatgatc aacttatctc gaacctgctt atgaaaagaa acgctttgtg caagggtaac 720 gagggcaagg aactgtacaa cgttaaccaa cttgcatcga taactggttg ctaa 774 <210> 23 <211> 774 <212> DNA <213> GenBank accession number DQ234673.1 <220> <221> gene &Lt; 222 > (1) .. (774) <223> Tomato chlorosis virus isolate IS coat protein gene <400> 23 atggagaaca gtgccgttgc aaacactggt gataacggtg gtggccgcaa tcctctggtt 60 agaccgttag atgatggcgt agatgacgag gtgcagaact taggcaggag ggacgatcca 120 acatctctca ttccggctaa tcctaatcga tcttccagtt gggctttgtt gaacccggat 180 actattaatt ataacgagtt aaggaaattg aaggtacact ccactagggg tgatactctt 240 accttgactc aggaagagga gttcgagaag atactcgaat ccttttgcag gcgaataatc 300 ggtgagaccc agatgacgga taagattttc gctggtttct acatgtccat gtgtcaggcc 360 attgtaaacc aagggacctc agttaaagca gccggtaata acagtcttga aaactacttt 420 gaggtagatg gtgcgagatt taagtgcaaa actccggatt tgataaatga ggttagaccc 480 aaaatgtccg atgttccaaa cgctatacgt cggtacgcca gaagtcatga aaagattatt 540 caggacttta tcaactccgg tcttataaag cctgattatc atttacaatt caaacatggc 600 gtattaccga gccatgtgtt tggtaccggc gattatataa atggttcgtt gatgaatatc 660 tcagatgatc aacttatctc gaacctgctt atgaaaagaa acgctttgtg caagggtaac 720 gagggcaagg aactgtacaa cgttaaccaa cttgcatcga taactggttg ctaa 774

Claims (10)

delete As a primer set for isothermal amplification reaction,
A primer set for detecting Tomato chlorosis virus , characterized in that the primer set consists of SEQ ID NOS: 13 to 16. 3. The primer set according to claim 2, wherein the tomato retrovirus is selected from the group consisting of GenBank accession numbers KC311375.1, AB513443.1 and DQ234673.1. A primer composition for isothermal amplification reaction comprising the primer set of claim 2 or 3. 5. The primer composition according to claim 4, wherein the primer composition is used for detecting tomato retrovirus. 5. The primer composition according to claim 4, wherein the composition further comprises a DNA polymerase for isothermal amplification, a plurality of dNTPs, and a reaction buffer. Extracting genomic DNA (gDNA) from the tomato;
A primer composition for detecting Tomato chlorosis virus consisting of SEQ ID NOS: 13 to 16 using the above gDNA as a template was subjected to a first reaction at 30 ° C to 50 ° C, followed by a second reaction at 70 ° C to 90 ° C Amplifying the target sequence by performing an isothermal amplification reaction method; And
And detecting the amplified product. [Claim 7] The method according to claim 7, wherein the tomato retrovirus is selected from the group consisting of GenBank accession numbers KC311375.1, AB513443.1 and DQ234673.1. 8. The process according to claim 7, wherein the first reaction is carried out for 20 to 40 seconds,
Wherein the second reaction is carried out for 30 minutes to 90 minutes. 8. The method according to claim 7, wherein the step of detecting the amplified product comprises detecting DNA amplified using electrophoresis or SYBR Green I.

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