KR20190050125A - 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 specifically detecting a quarantine-subjected plant virus, banana streak virus (BSV), and uses thereof. Since four primer sets of the present invention can specifically detect BSV, it is considered that the introduction of BSV into the country can be effectively prevented.

Description

[0001] The present invention relates to a primer set for specifically detecting a banana streak virus which is a quarantine plant virus, and a use thereof. Specific primer set for detecting Banana streak virus, quarantine plant virus and uses thereof,

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

The emergence and risk of emergent viruses and new viral diseases are increasing gradually due to ecosystem changes due to climate warming, evolution and variation of plants and viruses, and increased trade in agricultural products. In addition, as the average temperature continues to increase, virus-mediated insects are increasing and plant virus diseases are spreading. In particular, the effects of free trade have led to the importation of various types of plants into the country, and the potential for virulent or unidentified pathogenic plant viruses to be imported together through crops, feed, hardwood, seeds, vegetables, fruits and herbal medicines It is getting higher. The designation of the quarantine pathogen is a pathogen that takes the plant as a natural host, and the pathogen that has high quarantine risk among the non-dominant pathogens is evaluated every year through the risk assessment, (Animal, Plant and Fisheries Quarantine and Inspection Agency, 2013).

Therefore, in order to stabilize the production of agriculture and forestry industry, to protect the ecosystem, and to make human life comfortable by preventing the national disasters that may occur when new pest infestation is introduced, It is a trend to strengthen. In addition, the fundamental reason for plant quarantine is that the expense and inconvenience of preventing the entry of new pests is very economical compared to the costs and damages spent on controlling the infested pests.

At present, PCR method with high detection sensitivity and convenience is most commonly used for detecting viruses, and development of species-specific primers is most important for PCR detection of pathogens. In addition, in the case of detecting by PCR, there is a need for an inspection system in case of difficulty in discrimination due to low specific strength of the reaction or in case of causing nonspecific reaction with other nucleic acid. Furthermore, the detection of a isolate-specific primer can fail, and development of a species-specific primer capable of detecting all isolates present in the virus species is desired.

Banana stripe virus (Banana streak virus) is in the cowl remote bayireoseugwa (Caulimoviridae) balance particles between the Bard and viruses in (Badnavirus) para retro by viruses (pararetrovirus), on average, 30 x 150 nm size belonging to, and 7.0 ~ 7.8 It has a genome consisting of circular double stranded DNA. Symptoms caused by the banana stripes virus include bright streaks on the banana leaves that can necrose or break the pseudostem part of the banana, which inhibits the growth of the plant and results in a reduction in the production of the banana It is known to affect.

On the other hand, Chinese Patent Laid-Open No. 104805221 discloses 'RT-LAMP method for detecting banana virus' and EP-A-1056879 discloses 'banana stripe virus promoter and detection method' There is no description of a primer set and its use for specifically detecting a banana stripe virus which is a plant virus.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have found that Banana streak The primers were prepared using the polyprotein (PP) base sequence of the virus, and they were specifically bound to the genomic DNA of the banana stripe virus among the prepared primer sets. And confirming a primer set specific to a banana stripe virus that does not bind to genomic DNA of the host plant, thus completing the present invention.

In order to solve the above problems, the present invention provides oligonucleotide primer sets of SEQ ID NOS: 2 and 16; An oligonucleotide primer set of SEQ ID NOS: 3 and 15; An oligonucleotide primer set of SEQ ID NOS: 4 and 15; And a set of oligonucleotide primers of SEQ ID NOS: 5 and 15, and a set of oligonucleotide primers selected from the group consisting of oligonucleotide primers set forth in SEQ ID NOS: 5 and 15, as well as a primer set for specifically detecting Banana streak virus .

The present invention also provides a kit for specifically detecting a banana stripe virus comprising the primer set.

The present invention also provides a method for specifically detecting a banana stripe virus using the primer set.

Since the four primer sets finally selected in the present invention can specifically detect the banana streak virus , which is a quarantine plant virus, the detection of the banana streak virus as a quarantine target can be performed easily and quickly, It would be possible to effectively prevent the introduction of streak virus into Korea.

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

In order to accomplish the object of the present invention, the present invention provides oligonucleotide primer sets of SEQ ID NOS: 2 and 16; An oligonucleotide primer set of SEQ ID NOS: 3 and 15; An oligonucleotide primer set of SEQ ID NOS: 4 and 15; And a set of oligonucleotide primers of SEQ ID NOS: 5 and 15, and a set of oligonucleotide primers selected from the group consisting of oligonucleotide primers set forth in SEQ ID NOS: 5 and 15, as well as a primer set for specifically detecting Banana streak virus .

The primer set of the present invention preferably includes one or more, two or more, or three or more primer sets selected from the group consisting of the above-mentioned four primer sets, preferably four primer sets, that is, And 16 oligonucleotide primer sets; An oligonucleotide primer set of SEQ ID NOS: 3 and 15; An oligonucleotide primer set of SEQ ID NOS: 4 and 15; And oligonucleotide primer sets of SEQ ID NOS: 5 and 15, respectively.

Wherein the primer comprises at least 16, at least 17, at least 18, at least 19, at least 20 contiguous nucleotide fragments in the sequence of SEQ ID NOS: 2, 3, 4, 5, 15 and 16 according to the sequence length of each primer Lt; / RTI > oligonucleotides. For example, a primer (20 oligonucleotides) of SEQ ID NO: 2 may comprise oligonucleotides 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 . The primers may also include additional, deleted or substituted sequences of the nucleotide 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 reverse primers.

In the present invention, a "primer" refers to a single strand 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 primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

According to another aspect of the present invention, there is provided an oligonucleotide primer set according to the present invention; And a reagent for carrying out an amplification reaction. The present invention also provides a kit for specifically detecting Banana streak virus .

In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In addition,

Isolating the genomic DNA from the plant sample;

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

And detecting the amplification product. The present invention also provides a method for specifically detecting Banana streak virus .

The method of the present invention comprises isolating genomic DNA from a plant sample. As a method for isolating genomic DNA from the plant sample, methods 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 separated 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 the target sequence.

In the method according to one embodiment of the present invention, the plant is not limited to banana, plantain, and may be included within the scope of the present invention if the plant is susceptible to the banana stripe virus.

In one embodiment of the invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. One or more oligonucleotide primer combinations used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for detecting the banana stripe virus comprises the step of detecting the amplification product, wherein the detection of the amplification product is performed using a DNA chip, gel electrophoresis, capillary electrophoresis, Measurement, or phosphorescence measurement. As one method of detecting the amplification product, capillary electrophoresis can be performed. Capillary electrophoresis, for example, can use the ABi Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and 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), multibody nucleotide sequences of BSV (KP877622, KT895258, DQ859899, registered at the National Center for Biotechnology Information (NCBI) DQ451009, DQ092436, AY493509, AY750155, NC_007002, NC_007003, NC_003381, NC_008018). Downloaded nucleotide sequences were multiplexed using the Multalign program and the common sequences of BSVs were searched. The design of the BSV detection primer was designed using primer 3 and NCBI primer-blast program (Table 1).

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

Example 2. Selection of optimal primer set for BSV detection

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

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

First, the first selection using the BSV gene as a primer development target (FIG. 1) was performed. The PCR amplification product sizes were estimated to be 778 bp, 769 bp, 701 bp, 652 bp, 609 bp, and 506 bp, respectively , 18, 26, 35, 44, and 53 combinations were selected.

( Cucumber mosaic virus ), DaMV ( Dahlia mosaic virus ), Banana bunchy top virus (BBTV), Banana bract mosaic virus (BBrMV), and the like, which are in a flexible and host relationship with BSV using six primer sets selected from the first selection, (Fig. 2). The results of the second selection are shown in Fig. PCR was performed to determine whether the six primer sets reacted with the genomic DNA of the host plant (Fig. 3).

The results show that there are four primer sets (combinations 18, 26, 35 and 44) that are able to effectively detect BSV without reacting to viruses in parental and host-like relationships, and genomic DNA of host plants, 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 were detected up to 10 ^-4 for genomic DNA of BSV It was confirmed that the four primer sets of the present invention were primers having high sensitivity to the BSV to be detected.

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Claims (6)

An oligonucleotide primer set of SEQ ID NOS: 2 and 16; An oligonucleotide primer set of SEQ ID NOS: 3 and 15; An oligonucleotide primer set of SEQ ID NOS: 4 and 15; And a set of oligonucleotide primers of SEQ ID NOs: 5 and 15, wherein the set of oligonucleotide primers is selected from the group consisting of oligonucleotide primers set forth in SEQ ID NOs: 5 and 15, and a primer set for specifically detecting Banana streak virus . 2. The oligonucleotide primer set of claim 1, wherein the oligonucleotide primer set of SEQ ID NOs: 2 and 16; An oligonucleotide primer set of SEQ ID NOS: 3 and 15; An oligonucleotide primer set of SEQ ID NOS: 4 and 15; And a set of oligonucleotide primers of SEQ ID NOS: 5 and 15, respectively. An oligonucleotide primer set according to claims 1 or 2; And a reagent for carrying out an amplification reaction. &Lt; Desc / Clms Page number 19 &gt; 4. The kit according to claim 3, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer. Isolating the genomic DNA from the plant sample;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of claim 1 or 2; And
And detecting the amplification product. A method for detecting a Banana streak virus , comprising: 6. The method according to claim 5, wherein the detection of the amplification product is performed by DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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