KR101457274B1 - Primer composition for loop-mediated isothermal amplification reaction for detecting Broad Bean Wilt Virus 2, and use thereof - Google Patents

Abstract

The present invention relates to a set consisting of four primers for isothermal amplification reaction, a composition comprising the same, and a method for detecting fowlpox virus 2 using the composition. The detection method according to the present invention can detect all five mutant strains of the fake virus 2 effectively in a short time by using the isothermal amplification method without professional equipments. In addition, SYBR Green I can be diagnosed quickly under natural light under high light intensity. Therefore, it is anticipated that it will be possible to detect viruses that are causing enormous damage to cultivators of crops such as red pepper in the early stage, and to construct a more rapid and efficient virus detection system.

Description

[0002] Primer compositions for isothermal amplification reactions for detecting bean curd fowl virus 2, and uses thereof [0002]

The present invention relates to a set consisting of four primers for isothermal amplification reaction, a composition comprising the primer, and a method for detecting falciparum fowl virus 2 using the composition.

Forged virus 2 ( Broad Bean Wilt Virus 2 ) belongs to the Fabavirus , and its particle shape is spherical and is a small virus with a diameter of 25 nm. It is reported that viruses infect most branches and crops except tomatoes and cause crops to be harvested when infected with plants such as pepper. In early infected crops, weak mosaics appear as veins of clear veins, and when developed, the leaves are shrunken. Gradually, the entire crop turns yellow (yellowing), development is sluggish, and the tip of the fruit is pointed and browned.

Dwarf virus 2 has a wide host range and weeds and crops are widely distributed. It is also transmitted non-exclusively by aphids, which is very susceptible to infection by contact during the operation because of easy juice penetration. It was originally known as a virus that caused great damage in the US, Europe, Australia, and Japan. However, it is a major problem in pepper and spinach in Korea. Rural Development Administration has confirmed that weeds such as Gomari, Sorghum, Oxaloptera, and Gagatcheon can be an infectious source of fowlpox virus 2 and control such weeds, but it is not easy to control because of the common weeds. In addition, in the early symptom of the infection with Vigorous fowl virus 2, the newly emerged leaf is similar to the case when Mg and Mn are temporarily deficient and it is more difficult to judge the infection. There is no way to treat infected individuals only by picking them up and burning them. There is a possibility that the aphid, which is a kind of aphids, is spreading all over Korea and spread rapidly.

On the other hand, an electron microscope or a serological method was mainly used as a conventional virus diagnosis method. 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, ELISA (Enzyme-Linked Immunosorbent Assay) is about 1,000 times lower than the most commonly used diagnostic methods or Polymerase Chain Reaction (PCR) It is often the case that accurate diagnoses fail due to unexpected nonspecific reactions. 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 isothermal amplification method (LAMP) is similar to the conventional PCR method, but the conventional PCR method repeatedly performs three steps of denaturation, splicing, and elongation to amplify the gene, The isothermal amplification method has the advantage of being able to bond and stretch at a fixed temperature. This is because DNA Taq polymerase ( Taq DNA It is because the polymerase) may result in place of the nucleic acid ends hydrolysis (denaturation of the double helix of DNA without depending exonuclease) by using the Bst DNA polymerase (Bst DNA polymerase) that has a function that uses the heat. Therefore, isothermal amplification does not require a temperature change during amplification of the gene, which makes it possible to amplify the gene at fixed temperature with no special equipment.

The inventors of the present invention have conducted studies to easily detect the fungal virus 2, which is a serious problem in a farm, and as a result, it has found that a primer set for isothermal amplification reaction which can detect all kinds of fungal virus 2 Thereby completing the present invention.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a primer composition for isothermal amplification reaction, do.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention relates to a vaccine composition comprising a vaccine composition comprising a vaccine composition according to Broad Bean Wilt Virus 2 ) is provided.

In one embodiment of the present invention, the fungus virus 2 is selected from the group consisting of GenBank accession numbers JQ855708.1, JF704084.1, HQ283389.1, AF228423.1, and AF225954.1 strain. do. Since the primer of the present invention is designed for a region having a very small variation, it is theoretically detectable in almost all variants.

The present invention provides a primer composition for isothermal amplification reaction for detecting broad bean virus 2 (Broad Bean Wilt Virus 2) comprising the above primer set.

In one embodiment of the present invention, the composition further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer. However, it may further include a configuration necessary for using the primer of the present invention for detection.

The present invention

Extracting total RNA from the plant;

Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA);

Amplifying the target sequence by performing an isothermal amplification reaction at 60 ° C to 65 ° C for 30 minutes to 2 hours using the cDNA as a template and a composition comprising the primer; And

Broad bean forged virus comprising the step of detecting the amplified products 2 (Broad Bean Wilt Virus 2) detection method.

The temperature for carrying out the isothermal amplification reaction of the present invention is most preferably 62 ° C.

Examples of plants that can be infected with Fusarium spp. 2 include, but are not limited to, fenugreek, spinach, parsley, lycopene, petunia, and window plantlets.

In one embodiment of the present invention, the fungus virus 2 is selected from the group consisting of GenBank accession numbers JQ855708.1, JF704084.1, HQ283389.1, AF228423.1, and AF225954.1 strain. do.

In another embodiment of the present invention, the step of detecting the amplification product is characterized by confirming amplified DNA using electrophoresis or SYBR Green I.

In another embodiment of the present invention, a method for identifying DNA amplified using SYBR Green I is characterized by visual observation under natural light using SYBR Green I at a concentration of 1,000 to 10,000 times.

By using the primer set for isothermal amplification reaction according to the present invention, the primer composition including the primer set, and the detection method using the primer set, it is possible to effectively detect the fake virus 2 from the plant sample in a short time without professional equipments. In addition, SYBR Green I can be diagnosed quickly under natural light under high light intensity. Therefore, it is possible to detect viruses that can cause enormous damage to crop farmers such as pepper in the early stage, thereby enabling to construct a more rapid and efficient fake virus 2 diagnosis system and to prevent economic loss due to virus infection It is expected to be possible.

Figure 1 shows the sequence of polyprotein of BBWV2 used in the preparation of the primer of the present invention.
Fig. 2 is a diagram showing a primer set prepared by using PrimerExplorer V4 based on a search region and a common portion between five types of BBWV2 gene sequences.
3 is a diagram showing the nucleotide sequence of the primer set of the present invention.
FIG. 4 is a graph showing the result of electrophoresis of the amplified gene using the primer set of the present invention. FIG.
FIG. 5 is a diagram showing the result of checking the gene amplified using the primer set of the present invention under a natural light source using SYBR Green I. FIG.
FIG. 6 is a graph showing the result of checking the amplification product in FIG. 4 under a UV light source.

The present inventors have completed the present invention by studying a method capable of detecting in real time on the spot without professional equipments in order to effectively detect the bean curd virus 2 in a short time.

The present invention provides a set of primers for isothermal amplification reaction for detecting Drosophila virus 2 consisting of SEQ ID NOS: 1 to 4. The primer set of the present invention was prepared by using the sequence of polyprotein as a template rather than the entire viral sequence. BBWV2 produces a protein called giant polyprotein that is nearly identical to the entire genome length, resulting in the production of a variety of proteins that play a role as they are cut into pieces. That is, polyprotein is used as a template because it is an unconditionally produced protein if the virus is infected. The genbank number of variants detectable by the present invention represents the full sequence of the virus variant. The sequence of the polyprotein coding region of each mutant actually used in the preparation of the primer of the present invention is attached to the sequence listing.

The present inventors used loop-mediated isothermal amplification (LAMP) in order to detect fake virus 2 in a short time without professional equipments. Unlike conventional polymerase chain reaction (PCR), the isothermal amplification method does not require temperature control to amplify the gene, so it can amplify the gene without specialized equipments and it is possible to amplify the gene at a high concentration in a short time. In order to use loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must act as one set, and F3 and FIP bind in five directions Primer, and B3 and BIP are primers that bind in the reverse direction in three directions. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).

The present invention also relates to a method for producing a plant, comprising extracting total RNA (total RNA) from a plant;

Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA); Isothermal amplification reaction was carried out at 60 ° C to 65 ° C for 30 minutes to 2 hours using the above cDNA as a template using a primer set of the present invention, a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer Amplifying the target sequence; And a step of detecting the amplified product. DNA can be extracted directly from red pepper suspected of being infected, but it can also be applied to specimens and cultured cells that have been artificially infected for research purposes. Since the fake virus 2 is an RNA virus, it synthesizes cDNA by reverse transcription after extracting total RNA from the plant, and amplifies it by using a primer.

In one embodiment of the present invention, it was confirmed that it is possible to detect the fowlpox virus 2 by performing the isothermal amplification method using the universal primer set of the present invention. Also, when using SYBR Green I at a concentration of 1,000 to 10,000 times, (See Example 4) under natural light without going through a process such as the above.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  One. Forgery virus  2 gene collection

From the RDA, pepper naturally infected with broad bean wilt virus (BBWV2) ( Capsicum annuum L.) was taken and the experiment was carried out.

The primers for isothermal amplification used in the present invention were prepared from five strains of BBWV2 previously reported from Genbank, a biological nucleic acid information database provided by the National Center for Biotechnology Information (NCBI) And the nucleotide sequence information (GenBank accession number: JQ855708.1, JF704084.1, HQ283389.1, AF228423.1, AF225954.1). Respectively.

Example  2. primer  write

To detect BBWV2 through loop-mediated isothermal amplification (LAMP), a primer was prepared using PrimerExplorer V4. In order to use the isothermal amplification method, four primers (F3, B3, FIP, and BIP) should act as one set. In order to detect all kinds of known BBWV2, a common part between five kinds of BBWV2 gene sequences is searched Primer was prepared using PrimerExplorer V4 mainly at the site of detection (Fig. 2). As shown in Fig. 3, a universal primer set which is expected to be applied to all viruses of the BBWV2 virus was constructed.

Example  3. Collection of plant specimens

Plant samples were collected to confirm that the primer set produced in Example 2 could be used to detect BBWV2. In the present invention, total RNA was isolated from virus-infected plant samples and then total cDNA was synthesized by reverse transcription.

The extraction buffer used for total RNA isolation was TRI REAGENT (catalog number: TR 118) from MRC. The plant tissue is placed in a mortar and the liquid nitrogen is added, and when the nitrogen is evaporated, the tissue is grinded to a fine powder and the tissue is extracted with 1 ml of extraction buffer using a spatula preliminarily cooled in liquid nitrogen Transferred to a tube. After putting the powdered tissue into the tube, the tube lid was closed and reacted at 4 ° C for 5 minutes. Then, 200 μl of chloroform was added thereto, followed by vigorous shaking for 15 seconds, followed by reaction at room temperature for 10 minutes or more. After reacting, centrifuge at 8,000 × g at 4 ° C for 10 minutes. Collect 500 μl of the supernatant using a pipette, carefully transfer it to a new tube, and add the same amount of isopropanol to the supernatant. The reaction was allowed to proceed at room temperature for 10 minutes. 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. After removing the tube, 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.

Reverse transcription was performed to synthesize cDNA based on total RNA obtained from the above experiment. Reverse transcription was performed using M-MLV Reverse Transcriptase (BIONEER) and random primers according to the 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

To confirm that the primer set prepared in Example 2 was usable for detecting BBWV2, a primer composition for amplification reaction was prepared.

10 mM 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), 1.6 ul of 10 mM dNTPs (mixture of 10 mM each of dATP, dTTP, dGTP and dCTP), 0.4 ul of 10 uM F3 and B3 primer, 1.6 ul of 10 uM FIP and BIP primer, 1 ul of 20 _{mM MgSO 4, 1 ul (8} Unit) Bst polymerase, 1 μl of 1/10 diluted template cDNA, and 11.5 μl of distilled water were added to the reaction tube and mixed. The prepared amplification reaction composition was reacted at 40 ° C. for 30 seconds and reacted in a reaction vessel at 62 ° C. for 1 hour and 30 minutes to perform isothermal amplification. After the reaction was completed, enzyme activity was inhibited at 80 DEG C for 5 minutes. 5 μl of the total 20 μl reaction was electrophoresed to confirm that the gene was amplified. The results are shown in Fig. As shown in Figure 4, the universal primer set the mold in three different plant viruses when used with BBWV2 (turnip mosaic virus sulfide (Turnip yellow mosaic virus ; TYMV), sugar beet sulfide virus ( Beet western yellows virus ; BWYV), cucumber mosaic virus (Cucumber mosaic virus; underwent isothermal amplification method with CMV) were tested naeneunji detect only specific BBWV2. In the absence of viral gene (lane 5), the gene was not amplified. When BBWV2 (lane 2) was used as a template, the BBWV2 gene was amplified but the gene was not amplified in other types of viruses (lane 1, 3, 4).

Then, SYBR Green I concentrated to 1,000 times with the same reagent was added at a rate of 1 μl per 20 μl of the reaction, and the color development was confirmed by natural light. The results are shown in FIG. 5, and the result of color development reaction in ultraviolet light is shown in FIG. 6 . 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 shown in FIG. 5, even when the SYBR Green I was concentrated at a high concentration and the amplified gene (universal primer set sample) was stained, it was confirmed that the gene was amplified in the infected strain infected with BBWV2 and greens green under natural light. Samples infected with other viruses were found not to be green. As shown in Fig. 6, the result of coloring could be confirmed by green fluorescence on ultraviolet light.

From the above results, it was confirmed that the primer set used in this experiment can be used to specifically detect only BBWV2, and when SYBR GreenI is concentrated at 10,000 times, it can be visually confirmed under natural light there was.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the embodiments described above are in all respects illustrative and not restrictive.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Primer composition for loop-mediated isothermal amplification          reaction for detecting Broad Bean Wilt Virus 2, and use thereof <130> PB12-11091 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 3195 <212> RNA <213> polyprotein coding region of BBWV2 JQ855708.1 strain <400> 1 atgaatcctg agttagtggc cgtgttggat aggtatctat ctgagatcgc aagcagttta 60 tttttaggtt ggattataaa ttttttctta gtttcctttt gttctgctaa gagttgtttc 120 ttattgtggg ccgcatttct ttacatcaat tattacatat tgagattcga atttgcatat 180 atcgttgtgc ccttccttaa aacgatatat tcaaatagct ctcagtacca cactgttgat 240 tgggaaaacg cttacacggc acttcccaaa aatttgtggg agcaaataac tgattataat 300 tactgtttca atttcccaaa gcccattgta gagggttttg tgtcagattt ctcacctcgt 360 ttcacatttg aagagcttgt cgctatgaat gaggcaaata ttactccagt tcacacaata 420 ccaagagaga ccttgctcaa aagggcaagt gattataaat tggctgtgga gagtaaaaag 480 tccatattgc ccaaagttca agatttgtat gagatggaca aatggcatgc cttaaagagc 540 agattgaaca agaatgcgcc tagttatgtt gtaacttcag agattgcagt tggagccatg 600 tcaggtgcag ggaatgtgaa attggcgctg cctgtagtgg aaaaatacac tgaggaagta 660 gcagatgata gattgcctga caaagttcgc gccaaagccg atcaaataat ggttgcggcc 720 attgaattgg tggcagatgg cttcgcctcc gttaattctg atgttactat ggcaggtgcg 780 ctctatgata agcgccacaa gacgattgct agctctttca aaggagcttt tgcatctaga 840 gcgagtggag tcccttctca tgtcatttat tatccaatgc ataggattcc ttcaaatgat 900 gatcctaata caaccttgga actctccatg gttagtcgtg attctgattt tgatgagggt 960 tttacgttgg ctaatatctc agcacgtact ctttatgttc gtgcaaaagg acctgaaaag 1020 gtgactgaaa caaggcatct cttgaaggcc aagactgaag atgtggtgaa ggcacaacaa 1080 tttgcaagtg aagctcaagt tgtgtttgcc actcctaggc tctttcctga agtcaatttg 1140 gacaactaca atttgcctgg acctagcaat gtgcagcaaa cagaggccat aacaactgat 1200 agaggaatat tatttccgaa gccaaaattc aaaggaaatg aagtggtgct taactacaca 1260 ggaccagcga agattaatac tcatggctca cagagatttg gaaagaaaga ttcttctagt 1320 gatcaattcg tcaggagtgt tgaagatcta ggatgcttgt ctgacgaaga tggcaaggat 1380 tatagatatg gtcaaggctt gatggaggag gatgttctga atgtgcaaac gaacactttt 1440 gctatagagt cagccacgga gaccatgcgt ttactgttta gtggatatgc gagcattcct 1500 ttaaatgtag tacctgggac taaagttact gtggcttatc tgaatgagtt gtccaaacac 1560 agtgccgttc atactggttt gttgaacatg ttgagtaaaa tcccaggttc tttgaaggtc 1620 aaaatcaatt gccaggtggc tcctacgtgt ggtatagggt tggcagttag ctacgttgag 1680 ggcaatgaga gcgcaaatct gggctcaagt ttgggacggt tgttaggcat tcaacactat 1740 aagtggaacc cggccataga accgttcgtg gaatttgttt tcaaaccttt ttcctgtgcg 1800 gattggtgga acatgcacta cctgggatct ctaaaatatg ctcctgtggt ggtcattcag 1860 acactttcta agtggctgaa tgctccaaag gttgatgctc gaattagttt tgcaatctat 1920 tatgaacctt ccattgtgtt gcccaaacag atagcaactt tggagcatgc cccagcgttt 1980 atgtttcgta aggaactggg aactctggct ttcaagcaag gggagcgtgt ggcatattct 2040 ttcgaaatca attttggcaa accacaaact gacgggaaag aggtaacttc aacttttgcc 2100 tcttcttatt gtggtctcag tcaatatatg caatcagatg tcattttgga tttcactctt 2160 atgagtagcc ctatgattgg aggaactttc tcgattgcat atgttgctgg tgcctacatt 2220 gaaaagattg gaaatatgca agtccttgat tcattgcccc acattgattt cacgttctcg 2280 gcaggatcta agagcacccg ttccgtgcgc tttcctaaag aggtttttgg ggtgtatcag 2340 gcacttgata gatgggattt agactcagca agaggagatg atgtttcagg caattttgtg 2400 ctctaccaga gagatacagt gtccagtgct ttggaaggtg aacttacatt caggatagct 2460 gcccgtttat ccggagatat taatttcatt ggagtcagtg cgggttatcc aacgacgata 2520 acgcgcatag ggaaaggcaa gactgtagga aggtcacttg atcctgagat caggaaacct 2580 ctgagataca tgcttggaca agcccatgca acgcccaaag attttagctt agtgcgtttt 2640 gtgatgggcc gttggaagta caaggctggt ttgtatcctg gaagtaagtc ggatgaggac 2700 atccatccat tctccctgaa gatgcgcctt gatggatcta aaagcagtga gaattttgag 2760 attatacatt ccccttttgt acgcctgttg cagaactgtg catggatgag aggcactctg 2820 aaattttata ttgttgctcg tgccagttct gattacatga gttacagaag aacgtctcag 2880 ttgacggttt cagcccatga gaatagtctc agttcaaatc aattctacag tggagttttg 2940 acgagtccaa gtggtgagtt aagcttttct agagaagtgg ttggccctgt tgatggtttt 3000 gcttccatgg gttggaatgt tcgtgggagc aagaagtttt acaaaattca tgtggagatg 3060 ggaaatgttc acgagtatga tactgtggtg ttgtacggac aatttggctc tgatgtcgaa 3120 tttgctggcc aacagaaagg aggacattac ttactggaga aggaaactcc cattttcaaa 3180 accattaaat attaa 3195 <210> 2 <211> 3195 <212> RNA <213> polyprotein coding region of BBWV2 JF704084.1 strain <400> 2 atgcgtcccg aacttgttgc agcgctagat agatatttct cagaaatcat aagttgcttc 60 tttctgggct ggttactaaa ctttctgctt gtttggcttt gttccacaaa aagcactttt 120 cttttatggt cagtattttt atacatttgt tactacatat tgaggattga atttgcatat 180 atcgttgcac ccttcttaaa aacgatatac acaaatagtt ctcagtatca tactgtagac 240 tgggcaaacg cttatacggc attgcctaaa gggttgtggg agcaaatcac cgattacaat 300 tactgttaca atttccctcc tccttgcgtc gaaggttttg tgtctgattt ttcaccgaga 360 ttcacactta aagaacttga aatcatgaat gaggcaaata tcactccagt gcatactatt 420 ccaaaagaca cattgtttaa ttgcgggggt ggttatgggg tcgctgtgga gagcaagaaa 480 tccatcttac caagagtgca agatttgtat gaaatggata attggcacaa cttgagaagc 540 aaattgagca aaaatgttcc tagttacgtc acaacgtcag agattgctgt tggtgcaatg 600 tcaggtgcag gaaacaccaa attggcgata ccagtcgtgg agaagtacac tgaggaagtg 660 gctgatgaca ggttaccaga caaggttcgt gccaaagctg atcagataat ggtcgcagct 720 attgagttgg tggcggatgg ttttgcttca gtcaattcag acgttaccat ggctggtgca 780 ctctatgaca agcgccacaa gaccattgca agttctttca agggagcttt tgcatctcgg 840 gcaagtggag tcccttcaca tgtaatttat tttccaatgc atagagtccc agcatgtgat 900 gatccgaaca ctaccttgga actttcaatg gtaagtcgag attcagattt tgatgagggt 960 tacacactgg ccaacatatc agctcgcacc ctttacgttc gagcaaaagg acctgaaaag 1020 gtgactgaaa caaggcatct tttgaaagcc aaaactgagg atgtggttaa agcgcgccag 1080 tttgctagcg aggcacaagt tgttttcgct acgccacgat tatttcctga agtaaatctg 1140 gataattaca atttacctgg gcctagtaat gcgcaacata cagaggctat caccactgat 1200 agaggaatat tgtttccaaa gccaaaattc aaaggtaatg aagtggtact caattacaca 1260 ggttcaggga aaatcaggaa cgttggctct cagagatttg agaagaaaac cgccactggt 1320 gagcaatttg tcagaagtgt tgatgatctt ggatgcttgt ctgatgagga tggcaaagat 1380 tatagatatg gtcaaggtct gatggaggaa gatgttttga atgtgcaaac aaataatttt 1440 gccatagagt ccgcgacaga gaccatgcgc ttgctgttca gtgggtatgc aagcattcca 1500 ctgaacgtga tacctggaac aaaaattact gtggcttact taaatgaact gtctaaacat 1560 agcgctgtgc atactggttt gctgaacatg ttgagtaaga tccctggttc tttaaaagtc 1620 aaaattaatt gccaggtggc tccgacatgc ggcattggac tggcagtcag ctatgttgaa 1680 ggaaatgaaa gtgcaaactt gggttctagt ctgggacgct tgttgggcat tcagcactac 1740 aagtggaatc cagccataga gccatatgtg gagtttgttt tcaaaccttt ttcttgtgca 1800 gattggtgga atatgcacta cctgggatct ttcaagtttg cccctgtggt ggtaatacaa 1860 acgctttcca agtggttgaa tgctccgaag gtagatgcca gaataagttt tgcaatctac 1920 tatgaacccc ccgtcatgct gcctaagcaa atagcaactc ttgaacatgc cccagcgttc 1980 atgtttcgca aagagttggg gacactggct ttcaaacaag gggagcgtgt ggcttattct 2040 tttgaagtca attttggcaa accgcagaca gatgggaagg aagtgacttc aacttttgcc 2100 tcttcatatt gtggtcttag tcagtatatg caatcggatg ttattttaga ttttactctt 2160 atgagtagtc ccatgattgg aggaactttc tcaattgcat atgttgctgg agcctacatt 2220 gaaaaggttg ggaacatgca gattcttgat tcactgcccc atatcgattt caccttttct 2280 tctggatcta aaagcacgcg ttctgtgcgc tttcctaaag aagtctttgg agtgtaccaa 2340 gcgcttgata gatgggattt ggactcggca agaggggatg atgtctcagg aaacttcgta 2400 ctttatcaaa gggacgcggt gtcgagcgct ttggagggag agcttacatt cagaatagca 2460 gctcgtttat ctggagacat cagttttaca ggtgtgagtg caggataccc aacaacgatc 2520 acacgcatag ggaagggcaa gactataggg agatcacttg atcctgaaat cagaaagcct 2580 ttgaggtata tgcttgggca agcgcatgcg acgcccaaag actttagttc agtgcgtttt 2640 gtgatgggcc attggaaata taaggcaggc ttgtatcctg ggagcaaatc agatgaagac 2700 attcatcctt tctctttgaa aatgcgccta gatggatcta agagcagcga aaactttgaa 2760 attatacact ctccttttgt gcgtttactg caaaattgtg catggatgag aggaactttg 2820 aagttttacg tggtggctcg tgccagctct gattatatga gttaccgcag aacttcccaa 2880 ttaacagttt cagctcatga aaatagcctt agttccaacc aattctatag tggagttttg 2940 acgagtccaa gtggtgagtt gaatttctcc agagaggtgg ttggtcccgt ggatggtttt 3000 gcttctatgg gctggaatgt tcgtgggagc aagaagttct ataagataca cgtggagatg 3060 gggaatgttc atgagtatga tacagtagtg ttgtatggac aatttagctc tgacgtcgaa 3120 tttgctggcc aacagaaagg tgggcattac ttgctggaga aggaaactcc catgtttaag 3180 acaattaagt attaa 3195 <210> 3 <211> 3195 <212> RNA <213> polyprotein coding region of BBWV2 HQ283389.1 strain <400> 3 atgcgtcctg agcttgttgc agttttagac agatactttt cagagatcat aagttgtttt 60 ttcctgggtt ggttaataaa ttttctactt gtttggtttt gttccacaaa aagtgctttc 120 ttgttgtggg cagctttctt gtacgtttct tattacatat tgaggtttga atttgcgtat 180 atcgttgcgc cctttcttaa aacgatatac acaaatagct ctcaatatca cgttgttgat 240 tgggaaaacg cttacacggc acttcccaaa aatttgtggg agcaaataac tgattataat 300 tactgtttca atttcccaaa gcccattgta gagggttttg tgtcagattt ctcacctcgt 360 ttcacatttg aagagcttgt cgctatgaat gaggcaaata ttactccagt tcacacaata 420 ccaagagaga ccttgctcaa aagggcaagt gattataaat tggctgtgga gagtaaaaag 480 tccatattgc ccaaagttca agatttgtat gagatggaca aatggcatgc cttaaagagc 540 agattgaaca agaatgcgcc tagttatgtt gtaacttcag agattgcagt tggagccatg 600 tcaggtgcag ggaatgtgaa attggcgctg cctgtagtgg aaaaatacac tgaggaagta 660 gcagatgata gattgcctga caaagttcgc gccaaagccg atcaaataat ggttgcggcc 720 attgaattgg tggcagatgg cttcgcctcc gttaattctg atgttactat ggcaggtgcg 780 ctctatgata agcgccacaa gacgattgct agctctttca aaggagcttt tgcatctaga 840 gcgagtggag tcccttctca tgtcatttat tatccaatgc ataggattcc ttcaaatgat 900 gatcctaata caaccttgga actctccatg gttagtcgtg attctgattt tgatgagggt 960 tttacgttgg ctaatatctc agcacgtact ctttatgttc gtgcaaaagg acctgaaaag 1020 gtgactgaaa caaggcatct cttgaaggcc aagactgaag atgtggtgaa ggcacaacaa 1080 tttgcaagtg aagctcaagt tgtgtttgcc actcctaggc tctttcctga agtcaatttg 1140 gacaactaca atttgcctgg acctagcaat gtgcagcaaa cagaggccat aacaactgat 1200 agaggaatat tatttccgaa gccaaaattc aaaggaaatg aagtggtgct taactacaca 1260 ggaccagcga agattaatac tcatggctca cagagatttg gaaagaaaga ttcttctagt 1320 gatcaattcg tcaggagtgt tgaagatcta ggatgcttgt ctgacgaaga tggcaaggat 1380 tatagatatg gtcaaggctt gatggaggag gatgttctga atgtgcaaac gaacactttt 1440 gctatagagt cagccacgga gaccatgcgt ttactgttta gtggatatgc gagcattcct 1500 ttaaatgtag tacctgggac taaagttact gtggcttatc tgaatgagtt gtccaaacac 1560 agtgccgttc atactggttt gttgaacatg ttgagtaaaa tcccaggttc tttgaaggtc 1620 aaaatcaatt gccaggtggc tcctacgtgt ggtatagggt tggcagttag ctacgttgag 1680 ggcaatgaga gcgcaaatct gggctcaagt ttgggacggt tgttaggcat tcaacactat 1740 aagtggaacc cggccataga accgttcgtg gaatttgttt tcaaaccttt ttcctgtgcg 1800 gattggtgga acatgcacta cctgggatct ctaaaatatg ctcctgtggt ggtcattcag 1860 acactttcta agtggctgaa tgctccaaag gttgatgctc gaattagttt tgcaatctat 1920 tatgaacctt ccattgtgtt gcccaaacag atagcaactt tggagcatgc cccagcgttt 1980 atgtttcgta aggaactggg aactctggct ttcaagcaag gggagcgtgt ggcatattct 2040 ttcgaaatca attttggcaa accacaaact gacgggaaag aggtaacttc aacttttgcc 2100 tcttcttatt gtggtctcag tcaatatatg caatcagatg tcattttgga tttcactctt 2160 atgagtagcc ctatgattgg aggaactttc tcgattgcat atgttgctgg tgcctacatt 2220 gaaaagattg gaaatatgca agtccttgat tcattgcccc acattgattt cacgttctcg 2280 gcaggatcta agagcacccg ttccgtgcgc tttcctaaag aggtttttgg ggtgtatcag 2340 gcacttgata gatgggattt agactcagca agaggagatg atgtttcagg caattttgtg 2400 ctctaccaga gagatacagt gtccagtgct ttggaaggtg aacttacatt caggatagct 2460 gcccgtttat ccggagatat taatttcatt ggagtcagtg cgggttatcc aacgacgata 2520 acgcgcatag ggaaaggcaa gactgtagga aggtcacttg atcctgagat caggaaacct 2580 ctgagataca tgcttggaca agcccatgca acgcccaaag attttagctt agtgcgtttt 2640 gtgatgggcc gttggaagta caaggctggt ttgtatcctg gaagtaagtc ggatgaggac 2700 atccatccat tctccctgaa gatgcgcctt gatggatcta aaagcagtga gaattttgag 2760 attatacatt ccccttttgt acgcctgttg cagaactgtg catggatgag aggcactctg 2820 aaattttata ttgttgctcg tgccagttct gattacatga gttacagaag aacgtctcag 2880 ttgacggttt cagcccatga gaatagtctc agttcaaatc aattctacag tggagttttg 2940 acgagtccaa gtggtgagtt aagcttttct agagaagtgg ttggccctgt tgatggtttt 3000 gcttccatgg gttggaatgt tcgtgggagc aagaagtttt acaaaattca tgtggagatg 3060 ggaaatgttc acgagtatga tactgtggtg ttgtacggac aatttggctc tgatgtcgaa 3120 tttgctggcc aacagaaagg aggacattac ttactggaga aggaaactcc cattttcaaa 3180 accattaaat attaa 3195 <210> 4 <211> 3195 <212> RNA <213> polyprotein coding region of BBWV2 AF228423.1 strain <400> 4 atgcgtcctg aacttgttgc agcgttagat agatacttct cagaaatcat aagttgcttc 60 ttcttgggtt ggttattaaa ttttctgctt gtttgtttct gttccacaaa aagcactttt 120 cttttgtggt cagtattttt gtacatttgt tactacatat tgaggattga atttgcatat 180 atcgttgcac ccttctttaa aacgatatac gcaaatagtt ctcagtatca tactgtagac 240 tgggcaaacg catatacggc attgcctaaa ggattgtggg agcaaatcac tgattacgat 300 tactggtaca atttccctcc tcctcgcgtt gaaggttttg tgtctgattt ttcaccgaga 360 ttcacactta aagaacttga aatcatgaat gaggcaaata ttactccagt gcatactatt 420 ccaaaagaca cattgcttaa aagagcgagt gattacaagt tggctgtgga gagcaagaaa 480 tccatcttac caagagtgca agatttgtac gaaatggaca agtggcacaa cttgagaagt 540 aaattgagca aaaatgctcc tagctacgtc acgacgtcag agattgccgt tggtgcgatg 600 tcaggtgcag gaaacattaa attggcgata ccagttgtgg agaagtacac tgaggaagtg 660 gcagatgaca ggttgccaga caaggttcgc gctaaagctg atcagataat ggtcgcagct 720 attgagttgg tggcggatgg tttcgcttca gtcaattcag acgtcaccat ggctggtgca 780 ctctacgaca agcgccacaa gaccattgca agttctttta aaggagcttt tgcatctcgg 840 tgcatgtgat gatccaaata caactttgga gctctcaatg gtaagtcgag attcagattt tgatgagggc 960 tacacattgg ctaacatatc agctcgcact ctgtacgtgc gtgcaaaagg acctgaaaag 1020 gtgactgaaa caagacatct tttgaaagcc aaaactgagg atgtggtcaa agcgcgccaa 1080 tttgctagcg aggcacaggt tgtttttgct acgcctcgac tgttccctga agtaaatttg 1140 gacgattaca atttacctgg gcctagcaat gcgcaacata cagaggctat taccactgac 1200 agaggaatat tgtttccaaa gccaaaattc aaaggtaatg aagtggtact caactataca 1260 ggttcaggga aaatcaggaa cattggctct cagagatttg agaaagagac caccaatggt 1320 gagcagtttg tcagaagtgt cgatgatctt ggatgcttgt ctgatgaaga tggcaaagac 1380 tatagatatg gtcaaggtct gatggaggaa gatgttttga atgtgcaaac aaataacttt 1440 gccatagaat ctgctacaga gaccatgcgc ttactgttca gtggatatgc aagtattcca 1500 ctaaacgtaa tacctggaac aaaaattact gtggcttact taaatgaact ttccaaacat 1560 agtgctgtgc atactggttt gctaaacatg ttgagtaaga tccctggctc tttaaaagtc 1620 aaaattaatt gccaggtggc tccgacatgc ggtattgggc tggcagttag ctatgttgag 1680 ggaaatgaaa gcgcgaactt aggttctagt ctgggacgtt tgttaggcat tcagcactac 1740 aagtggaatc cggccataga gccatatgtg gagtttgttt ttaaaccatt ttcttgtgca 1800 gattggtgga atatgcatta tctgggatct tttaagtctg cccccgtggt ggtaatacaa 1860 acgctttcca aatggttgaa tgctccgaag gtagatgcca gaataagttt tgcaatctac 1920 tatgaaccca ccgtcatgct gcccaagcaa atagcaactc ttgaacatgc accagcattc 1980 atgtttcgca aagagttggg gacattagct ttcaaacaag gggagcgtgt ggcttattcc 2040 tttgaagtta attttggcaa accgcagaca gatgggaagg aagtgacttc aacttttgcc 2100 tcttcatatt gtggcctcag ccaatatatg caatcggatg taattctaga ttttactctt 2160 atgagtagcc ctatgattgg agggactttc tcaattgcat atgttgctgg agcctacatt 2220 gaaaaggttg ggaacatgca aattcttgac tcgctgcccc acatcgactt tactttctcc 2280 tctggctcta aaagcacacg ttctgtgcgc tttcctaaag aagtctttgg agtataccaa 2340 gcgcttgata gatgggattt ggactcggca agaggggatg atgtctcagg taactttgta 2400 ctttaccaaa gggatgcggt ttcgagcgct ttggaaggag agcttacatt cagaatagca 2460 gctcgtttgt ctggagatat cagtttcaca ggtgtgagtg cgggttaccc aacgacgatc 2520 acacgcatag ggaagggcaa aactatagga agatcacttg atcctgaaat cagaaagccc 2580 ttgaggtata tgctcgggca agcgcatgtg acacccgaag attttagttc agtgcgcttc 2640 gtgatgggcc attggaagta taaggcaggc ttgtatcctg ggagcaaatc agacgaagac 2700 attcatccct tttccttgaa aatgcgccta gatggatcta agagcagcga gaactttgaa 2760 attatacact ccccttttgt gcgtctgttg cagaattgtg catggatgag aggaactttg 2820 aagttttatg tggtggcccg tgccagttct gattatatga gttaccgcag aacttcccaa 2880 ttaactgtct cagctcatga aaatagtctt agctccaatc agttttatag tggagtgtcg 2940 acgagtccaa gtggtgagtt gaatttctcc agagaagtgg ttggtcccgt ggatggtttt 3000 gcttctatgg gctggaatgt tcgcgggagc aagaagtttt ataagataca cgtggagatg 3060 gggaatgttc atgagtatga tacagtagtg ttgtacggac aatttggctc taatgtcgaa 3120 tttgctggcc aacagaaagg tgggcattac ctgctggaga aggaaactcc catatttaag 3180 actattaagt attaa 3195 <210> 5 <211> 3195 <212> RNA <213> polyprotein coding region of BBWV2 AF225954.1 strain <400> 5 atgaatcctg agttggttgc cgcattagat aggtatctat ctgaaattgc aagcagtcta 60 tttttgggtt ggattataaa tcttttctta gttttctttt actcttctaa aagttgtttt 120 ttattgtggg ccgcatttct ttacgtcaat tattacatat tgagattcga atttgcatat 180 atcgttgtgc ccttccttga aacgatatac tcaaatagtt ctcaatacca cactgttgat 240 tgggagaacg cttacacggc gcgttccaaa agcttgtggg aacaaataac tgattacaac 300 tactgtttta atttcccaaa acccgttgtg gaaggctttg tgtcagattt ttcgcctcgt 360 ttcgcacttg aagagcttat tgctatgaac gaggcaaata ttactccagt ccacacaatc 420 ccgagagaga tcttgctcaa aaaagcaagt gactataaat tggctgtgga gagtaaaaag 480 tccatactgc caaaagttca agacttatat gagatggaca aatggcatgc cttgagaagt 540 aggttgagca agaatgcgcc caattacgtt gtaccctcag agattgcagt tggggccatg 600 tcaggtgctg gaaacgtgaa attggcgctg cccgtggtgg aaaaatacac tgaagaagtg 660 gcagatgata gattgcctga caaagttcgc tccaaagccg atcaaataat ggttgcggcc 720 attgaattgg tggcagatgg ctttgcctca gtcaattctg atgttactat ggcaggtgcg 780 ctttatgata agcgccacaa aacaattgct agttctttca aaggtgcttt cgcatccagg 840 gcaagtggag tcccatctca tgttatttat tatccaatgc atagggttcc ttcgaatgat 900 gatcctaaca caaccttaga gctttcaatg gttagtcgtg actctgattt tgatgagggt 960 ttcacgttgg ctaatatctc agtacgtact ttgtatgttc gtgcaaaagg acctgaaaag 1020 gtgacggaaa caaggcatct cttgaaggct aagaccgaag atgtggtgaa agcacaacaa 1080 tttgcgagtg aggcacaagt tgtatttgcc acccctcggc tatttcctga agtcaatctg 1140 aacaattaca atttacctgg acctagcaac gtgcagcaaa cagaggcaat caccaccaat 1200 ggaggaattc ttttcccgaa gccaaaattt aaagggaatg aagtggtgct caactacaca 1260 gggccaacga gagttggaaa tgttagtgcg cagaggcctg agaagcgaga gttcagcagc 1320 aaatcacatg tgggaagcac tgatgatctt ggatgtctgt cagatgagga tggcagagat 1380 tatagacatg gtcagggttt gatggaggag gatgttttgg acgttcagac taacaatttt 1440 gccattgagt ctgccacaga gactatgcgc ttgttgttta gtggttgcgc aagtattcct 1500 ctgaacgtta tacctgggac gaagcttaca gtggcctatt taaatgaact atccaagcat 1560 aatgctgtgc acactggatt gttgaatatg cttagcaaag ttccaggttc tttgaaggtc 1620 aagataaatt gccaggttgc tcccacttgc gggattggat tggcagttag ctacgtcgaa 1680 ggcaatgaaa gcgtgaactt gggatctagc ctggggcgct tgttgggtat tcagcattac 1740 aagtggaatc cggctataga gccttatgtg gaatttgttt tcaagccctt ctcctgcgca 1800 gattggtgga atatgcatta tttggggtca tgtaagtatg cacccgtgat ggtcgttcaa 1860 acattgtcca aatggttgaa tgctccaaaa gtggatgcca agatgagctt cgccatttat 1920 tatgaaccca atgtgatttt gcccaagcaa atagcgacct tggaccacgc cccagcgttc 1980 atgttccgca aggaactggg gacgctagct tttaagcaag gggagcgggc agcatattct 2040 tttgaagtta attttggcaa acctcagaca gatggaaagg aagtgacttc tacttttgcc 2100 tcatcttatt gtggtttgag tcagtatatg caatctgatg tgattttaga ttttactctc 2160 atgagcagtc ctatgattgg aggcactttt tcagttgcat atgttgcagg ggcatatatt 2220 gagaaagttg gtgacatgca aattcttgat tcattggccc catgtgattt cacattttct 2280 tcaggtacca agagcacgcg ttccgtgcga tttccgaaag agatttttgg ggtgcttcag 2340 gcgttggata ggtgggattt aaactcagca aggggagatg atgtttcagg caattttgtg 2400 atttaccaaa gagatgcagt ctcaagtgct cttgaagggg aattaacgtt ccgaattgct 2460 gctcgtttgt ctggggacat cgattttgtt ggtgttagtg caggctatcc aacaacaatt 2520 acacggattg gcaaaggcaa gacgcaagga agatcgcttg gtcccgaaat cagaaagcct 2580 ttaaggtaca tgcttggcca gtcccattca acaccattgg attttagttc agtgcgcttt 2640 gtaatgggtc actggaagta caaagctggt ttgtatccag ggagtaaatc ggatgaagac 2700 attcacccat attccctcaa aatgcgcctt gatggctcaa agagcagcga gaattttgaa 2760 attatccatt ccccttttgt tcgattattg caaaattgtg catggatgaa aggaactctg 2820 aacttttatg ttgtggcacg agcaagctct gattacatga gttacagaag gacctctcag 2880 ttgacagtct cagctcatga gaatagtctt agctccaacc aattctacag tggagtcttg 2940 acaagcccta gtggtgagtt agggttctcc agagagattg taggtccagt tgatggtttt 3000 gcatccatgg gctggaatgt gcgagggagc aaaaagtttt ataaaattca tgtagaaatg 3060 gggaatgttc atgagtacga aactgtgacg ctatatgggc gatttggtcc taatgtggag 3120 tttgctggcc agcagaaagg tggtcactat atgcttgaga aggaagctcc gacatttaag 3180 gcactcaaat attga 3195

Claims (8)

1. A primer set for isothermal amplification reaction for detecting Broad Bean Wilt Virus 2 (BBWV2) comprising SEQ ID NOS: 1 to 4, wherein said bacteriophage 2 comprises GenBank accession numbers JF704084.1, HQ283389.1 , AF228423.1, and AF225954.1 strain. &Lt; Desc / Clms Page number 13 &gt;
delete Comprising the primer set of claim 1, broad bean forged virus 2 (Broad Bean Wilt Virus 2, BBWV2).
The method of claim 3,
Characterized in that the composition further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer.
Extracting total RNA from the plant;
Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA);
Amplifying the target sequence by performing the isothermal amplification reaction at 60 ° C to 65 ° C for 30 minutes to 2 hours using the cDNA according to the third aspect of the present invention as a template; And
Broad bean forged virus comprising the step of detecting the amplified products 2 (Broad Bean Wilt Virus 2, BBWV2) detection method.
delete 6. The method of claim 5,
Wherein the step of detecting the amplification product comprises the step of detecting amplified DNA using electrophoresis or SYBR Green I.
delete

Images