KR102096630B1 - Specific primer set for detecting Banana streak virus, quarantine plant virus and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for specific detection of a quarantine plant virus, Banana streak virus , and its use, and the four primer sets of the present invention can specifically detect BSV, thus, BSV It is thought that it will be able to effectively prevent the introduction of the drug into Korea.

Description

Specific primer set for detecting Banana streak virus, quarantine plant virus and uses thereof}

The present invention relates to a primer set and its use for specifically detecting a banana striped virus, a quarantine plant virus.

The emergence and risk of outbreak viruses and new viral diseases are gradually increasing as ecosystem changes due to climate warming, the evolution and transformation of plants and viruses, and the expansion of agricultural products trade. In addition, as the average temperature continues to increase, insects mediated by viruses increase, and plant virus diseases are spreading. In particular, under the influence of free trade, various kinds of plants are imported into Korea, and thus, potential or unidentified pathogenic plant viruses are likely to be imported together through crops, feed, hardwood, seeds, vegetables, fruits, and herbal medicines. It is increasing. The designation of quarantine pathogens is a plant-based pathogen. Among pathogens that are not distributed in Korea, pathogens with high quarantine risks are assessed annually and subject to 'prohibited payment,' management level ', and' regulated quarantine ' It is divided into 100 categories (Animal, Plant and Fisheries Quarantine and Inspection Agency, 2013).

Accordingly, in order to prevent national disasters that may occur when new pests are introduced, to stabilize the production of agricultural and forestry industries, protect ecosystems, and make human life comfortable, governments around the world are gradually conducting plant quarantine policies at the government level. The trend is strengthening. In addition, the basic reason for plant quarantine is that the expense and inconvenience required to prevent the invasion of new pests are very economical compared to the cost or damages spent in controlling the infested pest.

Currently, in the detection of viruses, a PCR method having high detection sensitivity and convenience is most commonly used, and development of a species-specific primer is most important for PCR detection of pathogens. In addition, when detecting by the PCR test method, a test system is required to prepare for a case in which it is difficult to discriminate because the specific reaction intensity is low or to cause a non-specific reaction with another nucleic acid. Moreover, development of species-specific primers capable of detecting all isolates present in a virus species is required because detection may fail when using isolate-specific primers.

Banana streak virus is a pararetrovirus belonging to the Badnavirus family of the Caulimoviridae family, and it is composed of 30 x 150 nm average hepatic particles with an average size of 7.0 to 7.8 It has a genome consisting of kb circular double stranded DNA. As a disease caused by the banana streak virus, brightly colored streaks may form on the banana leaves, resulting in necrosis or cracking of the part of the banana (pseudostem). It is known to influence.

On the other hand, Chinese Patent Publication No. 104805221 discloses a 'RT-LAMP method for detecting banana virus', and European Patent No. 1056879 discloses a 'banana striped virus promoter and detection method', but the quarantine of the present invention A primer set for specifically detecting the plant virus banana striped virus and its use have not been described.

The present invention has been derived by the above-mentioned needs, the present inventors of the banana striped virus ( Banana streak virus ) polyprotein (polyprotein, PP) base sequence was produced using the base sequence, and among the prepared primer sets, it specifically binds to the genomic DNA of the banana-striped virus, and the related virus and host-like virus The present invention was completed by identifying a set of primers specific to the banana striped virus that does not bind and does not bind to the genomic DNA of the host plant.

In order to solve the above problems, the present invention is an oligonucleotide primer set of SEQ ID NO: 2 and 16; Oligonucleotide primer set of SEQ ID NOs: 3 and 15; Oligonucleotide primer set of SEQ ID NOs: 4 and 15; And at least one oligonucleotide primer set selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 5 and 15 to provide a primer set for specifically detecting Banana streak virus .

In addition, the present invention provides a kit for specifically detecting a banana striped virus containing the primer set.

In addition, the present invention provides a method for specifically detecting banana striped virus using the primer set.

Final selection, a total of four primer sets of the present invention can be detected in banana stripe virus (Banana streak virus) quarantine plant viruses specifically, since the detection of the banana stripe virus inspection target can be carried out easily and quickly, banana It is thought that the introduction of the striped virus into Korea can be effectively prevented.

Figure 1 shows the results of the primary selection of a primer set for the detection of banana streak virus ( Banana streak virus , BSV). M: DNA size marker, N: negative control.
Figure 2 shows the results of the secondary selection of the primer for BSV detection, the results for the virus in a flexible relationship and host-like relationship. CMV: Cucumber mosaic virus , DaMV: Dahlia mosaic virus , BBTV: Banana bunchy top virus , BBrMV: Banana bract mosaic virus .
3 shows the results of the third selection of the primers for BSV detection, and is a result of detecting the genomic DNA of host plants.
4 shows the end-point PCR results of the selected primers for BSV detection. 10 ^-1 : BSV genomic DNA stock solution 10 ^-1 dilution, 10 ^-2 : BSV genomic DNA stock solution 10 ^-2 dilution, 10 ^-3 : BSV genomic DNA stock solution 10 ^-3 dilution, 10 ^-4 : BSV genomic DNA Dilution of stock solution 10 ^-4 , 10 ^-5 : Dilution of stock solution of BSV genomic DNA 10 ^-5 , 10 ^-6 : Dilution of stock solution of BSV genomic DNA 10 ^-6 .

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 2 and 16; Oligonucleotide primer set of SEQ ID NOs: 3 and 15; Oligonucleotide primer set of SEQ ID NOs: 4 and 15; And at least one oligonucleotide primer set selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 5 and 15 to provide a primer set for specifically detecting Banana streak virus .

The primer set of the present invention preferably includes one or more, two or more, three or more primer sets selected from the group consisting of the four primer sets, preferably four primer sets, that is, SEQ ID NO: 2 And 16 oligonucleotide primer sets; Oligonucleotide primer set of SEQ ID NOs: 3 and 15; Oligonucleotide primer set of SEQ ID NOs: 4 and 15; And oligonucleotide primer sets of SEQ ID NOs: 5 and 15.

The primers are fragments of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequences of SEQ ID NOs: 2, 3, 4, 5, 15 and 16, depending on the sequence length of each primer. It may include an oligonucleotide consisting of. For example, the primer of SEQ ID NO: 2 (20 oligonucleotides) may include an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more consecutive nucleotides in the sequence of SEQ ID NO: 2. . In addition, the primer may also include addition, deletion or substituted sequences of the base sequences of SEQ ID NOs: 2, 3, 4, 5, 15 and 16. The oligonucleotides of SEQ ID NOs: 2, 3, 4 and 5 are forward primers, and the oligonucleotides of SEQ ID NOs: 15 and 16 are backward primers.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primers will depend on the desired DNA or RNA target complexity, as well as the conditions of primer use, such as temperature and ionic strength.

As used herein, oligonucleotides used as primers may also include nucleotide analogs, such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or It may include an intercalating agent.

In order to achieve another object of the present invention, the present invention provides an oligonucleotide primer set according to the present invention; And a reagent for performing an amplification reaction, to provide a kit for specifically detecting Banana streak virus .

In the kit of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, buffers, and the like. In addition, the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout that explains how to use the kit, for example, how to make PCR buffers, and the reaction conditions presented. The handbook includes a brochure in the form of a brochure or leaflet, a label attached to the kit and a description on the surface of the package containing the kit. In addition, the handbook includes information that is disclosed or provided through electrical media, such as the Internet.

In addition, the present invention

Isolating genomic DNA from plant samples;

Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer set; And

Provided is a method for specifically detecting a banana streak virus , comprising the step of detecting the amplification product.

The method of the present invention includes isolating genomic DNA from a plant sample. As a method for separating genomic DNA from the plant sample, a method known in the art may be used, for example, a CTAB method may be used, or a Wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, an amplification reaction may be performed using a primer set according to an embodiment of the present invention to amplify a target sequence.

In a method according to an embodiment of the present invention, the plant is not limited to bananas and plantains, and may be included within the scope of the present invention as long as it is a plant capable of being infected with a banana striped virus.

In one embodiment of the invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a material emitting fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling material is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer when amplifying the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. In addition, a label using a radioactive material, when a radioactive isotope, such as ³² P or ³⁵ S, is added to the PCR reaction solution during PCR, the amplification product is synthesized and radioactive is incorporated into the amplification product, so that the amplification product can be radioactively labeled. Combinations of one or more oligonucleotide primers used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for detecting the banana-striped virus comprises detecting the amplification product, and the detection of the amplification product is DNA chip, gel electrophoresis, capillary electrophoresis, radioactivity measurement, fluorescence It may be performed through measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, capillary electrophoresis can be performed. For capillary electrophoresis, for example, an ABi Sequencer can be used. In addition, gel electrophoresis can be performed, and the gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer, the target sequence is labeled with a detectable fluorescent labeling material, and the fluorescence thus labeled is measured using a fluorescence meter. can do. In addition, in the radioactive measurement method, when performing PCR, a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter or liquid flash. Radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example  1. BSV specific Primer  Design and combination

For the design of primers for the detection of Banana streak virus (BSV), polyprotein sequences of BSV registered in the National Center for Biotechnology Information (NCBI) (KP877622, KT895258, DQ859899, DQ451009, DQ092436, AY493509, AY750155, NC_007002, NC_007003, NC_003381, NC_008018) were downloaded. The downloaded sequences were multi-sorted using the Multalign program, and common sequences of BSV were searched. The primer design for BSV detection was designed using Primer 3 and NCBI primer-Blast program (Table 1).

Thereafter, 63 sets capable of PCR amplification were combined by combining these forward and reverse primers (Table 2).

Example 2. Selection of the optimal primer set for BSV detection

Polymerase chain reaction (PCR) was performed to select the optimal primer for BSV detection with a combination of 63 primer sets prepared in Example 1. PCR conditions are shown in Table 3 below.

PCR reaction conditions step Temperature (℃) time Reverse warrior 42 60 minutes Pre-denaturation 95 10 minutes denaturalization 95 45 seconds Combination 55 1 minute kidney 72 1 minute Return to the metamorphic stage and repeat 34 additional times Final height 72 5 minutes keep 4 ∞

First, primary selection using BSV gene, which is the target of primer development, was performed (FIG. 1), of which the PCR amplification products were predicted to be 778 bp, 769 bp, 701 bp, 652 bp, 609 bp, and 506 bp, respectively. Six primer sets of 18, 26, 35, 44, and 53 combinations were selected.

CMV (Cumcumber mosaic virus ), DaMV ( Dahlia mosaic virus ), BBTV ( Banaana bunchy top virus ), BBrMV ( Banaana bract mosaic virus ) in flexible and host-like relationship with BSV using six primer sets selected from the primary selection Secondary selection was conducted to confirm the presence or absence of cross-reactivity with (FIG. 2). In addition, PCR was performed to determine whether the six primer sets respond to the genomic DNA of the host plant (FIG. 3).

Through the above results, four primer sets (combinations 18, 26, 35 and 44) capable of effectively detecting BSV without reacting to the virus in the flexible relationship and host-like relationship, and to the genomic DNA of host plants. Was selected, and as a result of performing end-point PCR (dilution limit experiment) using the selected primer set, as shown in FIG. 4, the selected four primer sets detected up to 10 ^-4 against BSV's genomic DNA. By showing that it is possible, it was confirmed that the four primer sets of the present invention are primers with high sensitivity to the detection target virus, BSV.

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Claims (6)

Oligonucleotide primer set of SEQ ID NOs: 2 and 16; Oligonucleotide primer set of SEQ ID NOs: 3 and 15; Oligonucleotide primer set of SEQ ID NOs: 4 and 15; And Banana streak virus comprising the oligonucleotide primer set of SEQ ID NO: 5 and 15 primer set for specifically detecting. delete The oligonucleotide primer set according to claim 1; And a reagent for performing an amplification reaction, a kit for specifically detecting Banana streak virus . The kit of claim 3, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs and buffer. Isolating genomic DNA from plant samples;
Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer set of claim 1; And
A method for specifically detecting a banana streak virus , comprising detecting the amplification product. The method of claim 5, wherein the detection of the amplification product is performed by DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement or phosphorescence measurement.

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