KR102279791B1 - Specific primer set for detecting TEV and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for specifically detecting Tobacco etch virus (TEV) and uses thereof. Since the two primer sets of the present invention can specifically detect TEV, it is judged that the primer sets can be usefully utilized to reduce damage caused by TEV infection of major agricultural products.

Description

A primer set for specifically detecting TEV and uses thereof

The present invention relates to a primer set for specifically detecting TEV (Tobacco etch virus) and uses thereof.

Virus damage is spreading as the ecosystem changes due to climate change, the evolution and mutation of plants and viruses, and the number of virus vectors increase. In addition, the recent increase in international trade and imports and exports of various plants is increasing the possibility of introducing new plant viruses into the country.

Since 2000, several countries, including the United States, Australia, and China, have strengthened plant quarantine. This is because the cost to prevent the invasion of new pests is much more economical compared to the total cost of damage and control costs caused by the invasion of new pests.

Currently, in the detection of RNA viruses, the RT-PCR method with high detection sensitivity and convenience is most commonly used, and the development of species-specific primers is the most important for PCR detection of pathogens.

TEV (Tobacco etch virus) is a plant virus belonging to the genus Potyvirus of the Potyviridae, and mainly occurs widely in many species of the Solanaceae. When infected with TEV, specific necrotic plaques and spots caused by necrosis, collapse, or dehydration of the superficial tissue occur on the leaves of plants, appear grayish-white or grayish-brown, and may progress to general necrotic plaques in the late stages of infection. In addition, TEV, as a pathogen subject to quarantine, requires an effective diagnostic method capable of detecting it from imported plants.

Meanwhile, Korean Patent Application Laid-Open No. 2014-0076706 discloses 'a probe set for specifically diagnosing or detecting TEV and its use', and Korean Patent Registration No. 2096633 discloses 'a quarantine plant virus, banana bract mosaic virus. Although the 'a primer set for specifically detecting and its use' is disclosed, the primer set for specifically detecting TEV of the present invention and its use are not described. In addition, even if a PCR test method for the virus has been developed, since nucleotide sequence mutations occur continuously due to the characteristics of the virus, there is a need to upgrade to a PCR test method that can encompass such mutated sequences.

The present invention was derived from the above needs, and the present inventors designed a candidate primer set by collecting TEV nucleic acid information from a known database in order to develop a primer set capable of specifically detecting TEV, Nine candidate primer sets showing a specific reaction with the target virus gene were first selected, and 5 candidate primer sets showing no non-specific reaction to a related virus gene among the first selected primer sets were secondarily selected, 2 Two candidate primers were finally selected based on whether the primary selected primer sets had a non-specific response to the plant gene. In addition, as a result of analyzing the sensitivity to the finally selected primer set, the present invention was completed by confirming that ^{the primer sets according to the present invention can detect up to a viral RNA concentration of 10 −1 ng level.}

In order to solve the above problems, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 11; And it provides a primer set composition for specifically detecting Tobacco etch virus (TEV) comprising one or more oligonucleotide primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 2 and 14.

In addition, the present invention is the oligonucleotide primer set; And it provides a kit for specifically detecting TEV, comprising a reagent for performing an amplification reaction.

In addition, the present invention isolates total RNA from a plant sample; amplifying a target sequence by using the isolated total RNA as a template and performing RT-PCR using the oligonucleotide primer set according to the present invention; And it provides a method for specifically detecting TEV, comprising the step of detecting the amplification product.

The two primer sets capable of specifically detecting TEV (Tobacco etch virus) according to the present invention can detect TEV easily and quickly, so it is expected that it will be usefully utilized to reduce damage caused by TEV infection of major agricultural products. is judged

1 to 4 show the results of TEV sequence alignment and the positions of candidate primers for TEV detection.
5 and 6 are results of verification of candidate primer sets using TEV positive and negative samples.
7 is a verification result of a candidate primer set using four types of related viruses.
8 is a verification result of a candidate primer set using a TEV test target sample.
9 is a detection limit analysis result of a candidate primer set through an endpoint test.
10 shows the positions of the finally selected primer sets in the nucleotide sequence of the positive control clone prepared for the PCR test.
11 is a PCR reaction result of a clone containing a nucleotide sequence of a positive control prepared for PCR test.

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NOs: 1 and 11; And it provides a primer set composition for specifically detecting Tobacco etch virus (TEV) comprising one or more oligonucleotide primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 2 and 14.

The primer set composition of the present invention preferably includes one or more primer sets selected from the group consisting of the two primer sets, and preferably includes both primer sets.

The primer is an oligonucleotide consisting of a fragment of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NOs: 1, 2, 11 and 14, depending on the sequence length of each primer. may include. For example, the primer of SEQ ID NO: 1 (21 oligonucleotides) is an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 1 may include In addition, the primer may include a sequence of addition, deletion or substitution of the nucleotide sequences of SEQ ID NOs: 1, 2, 11 and 14.

The oligonucleotide primers of SEQ ID NOs: 1 and 2 of the present invention are forward primers, and the oligonucleotide primers of SEQ ID NOs: 11 and 14 are reverse primers.

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

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.

The present invention also provides the oligonucleotide primer set; And it provides a kit for specifically detecting TEV (Tobacco etch virus), including a reagent for performing an amplification reaction.

In one embodiment of the present invention, the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, and a buffer, and may further include a ribonuclease inhibitor, a thermostable polymerase, and the like. However, it is not limited thereto. The DNA polymerase may be a reverse transcriptase such as an RNA dependent DNA polymerase, a reverse transcriptase from a variety of sources, for example, avian myeloblastosis virus-derived reverse transcriptase. derived virus reverse transcriptase (AMV RTase), murine leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rous-associated virus 2 reverse transcriptase (RAV-2 RTase) ), but is not limited thereto. In addition, the buffer may include both a reverse transcription buffer and a PCR buffer, and the heat-resistant polymerase may be EF Taq polymerase.

In addition, the kit of the present invention may further include a positive control sequence consisting of the nucleotide sequence of SEQ ID NO: 20. The nucleotide sequence of SEQ ID NO: 20 is a primer so that the two primer sets (total of four oligonucleo primers) selected in the present invention can produce an amplification product having the same size as the expected amplification product size of each primer set without a non-specific reaction, respectively. As a nucleotide sequence artificially synthesized by adding a nucleotide sequence and an artificial nucleotide sequence (FIG. 10), it may be included in the kit in the form of a DNA fragment or a vector including the nucleotide sequence.

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

The present invention also comprises the steps of isolating total RNA from plant samples; amplifying a target sequence by using the isolated total RNA as a template and performing RT-PCR using the oligonucleotide primer set according to the present invention; And it provides a method for specifically detecting TEV (Tobacco etch virus), comprising the step of detecting the amplification product.

The method of the present invention comprises the step of isolating total RNA from a plant sample. A method for isolating total RNA from the sample may use any method known in the art, for example, a phenol extraction method may be used. Using the isolated total RNA as a template and using one or more oligonucleotide primer sets according to an embodiment of the present invention as primers, RT-PCR (reverse transcription polymerase chain reaction) may be performed to amplify the target sequence. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method according to an embodiment of the present invention, the plant may be, but is not limited to, plants of the family Solanaceae, golden berries, alfalfa, and the like, and any plant that can be infected with TEV may be included within the scope of the present invention.

In the method of the present 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 substance is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. ^{In addition, when a radioactive isotope such as 32} P or ³⁵ S is added to a PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The 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 TEV comprises detecting the amplification product, and the detection of the amplification product is performed by DNA chip, gel electrophoresis, capillary electrophoresis, radiometric measurement, fluorescence measurement, or It may be performed through phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorometer. can do. In addition, the radioactive measurement method is a ^{radioactive isotope such as 32} P or ³⁵ S added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter or liquid scintillation. The radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Primer set design for detecting TEV

Sequence alignment was performed using 149 TEV sequences available in NCBI GenBank (as of May 2020) ( FIGS. 1 to 4 ), and candidate primers for TEV-specific detection were designed (Table 1).

In order to verify the designed candidate primer set, it was necessary to test for specificity for the strain (target virus and related species) and to check whether non-specific products were generated for the test item. The closely related viruses used in the present invention are Alstroemeria mosaic virus (AlMV), Banana bract mosaic virus (BBrMV), Bean common mosaic virus (BCMV), and ChiVMV (Chilli veinal mottle virus). All of the target virus and related viruses were purchased from Agdia Inc. (USA) and used. As the plants to be tested, kaleidoscope, golden berry, alfalfa, red pepper, tobacco and tomato were used, and the plants were used in the experiment by purchasing seeds or seedlings judged to be virus-free.

Example 2. Validation of a primer set for detecting TEV

PCR was performed to verify and select the candidate primer set designed in Example 1. Total RNA samples were extracted using the RNeasy plant mini kit (QIAgen, Germany). PCR was performed using RT-PCR Premix (PLUTOS, Korea; Company P), AccuPower RT-PCR PreMix & Master Mix (Bioneer, Korea; Company B), SuPrimeScript RT-PCR Premix (GeNet Bio, Korea; Company G) and the composition and reaction conditions of the mixture are shown in the table below.

2-1. Confirmation of TEV species specificity

First, verification of candidate primers using TEV positive and negative samples was performed.

As a result of verifying 15 candidate primer sets using TEV-infected samples, primer set no. 9 primer sets (no. 1, 2, 4, 5, 6, 7, 11, 12, 14) except for 3, 8, 9, 10, 13, and 15 were first selected ( FIGS. 5 and 6 ) .

2-2. Validation of candidate primer sets using closely related viruses

As a result of checking whether a non-specific reaction was performed against four types of closely related viruses using the first 9 primer sets, the primer set no. In 2, 11, 12, and 14, a non-specific reaction was confirmed, and 5 (no. 1, 4, 5, 6, 7) primer sets excluding the primer sets were selected secondarily ( FIG. 7 ).

2-3. Validation of candidate primer sets using test target plants

As a result of confirming the non-specific reaction of the primer set to the 6 types of test target plants using the 5 primer sets selected secondarily, the primer set no. In 4, 5, and 6, a non-specific reaction was confirmed, and two (no. 1, 7) primer sets were finally selected except for this ( FIG. 8 ).

2-4. Detection limit analysis of candidate primer sets through endpoint testing

To confirm the detection limit of the two final selected primer sets, the sensitivity analysis was performed by varying the ^{viral total RNA template at concentrations of 100, 10, 1, 10 -1} , 10 ^-2 , 10 ^-3 , and 10 ^{-4 ng.} carried out. As a result, it was found that the final selected primer set showed some differences in sensitivity depending on the type of PCR premix, and could detect up to an RNA concentration of ^{10 −1 ng ( FIG. 9 ).} From the above results, it was confirmed that the primer set of the present invention was a marker with excellent sensitivity.

Example 3. Base sequence design and verification for producing positive control clones

The present inventors prepared a positive control clone that can be used as a positive control in the PCR test of the finally selected primer sets. The positive control clone is a primer so that the two primer sets (total of four oligonucleo primers) finally selected in the present invention can produce an amplification product having the same size as the expected amplification product size of each primer set without a non-specific reaction, respectively. It is a nucleotide sequence designed by adding a nucleotide sequence and an artificial nucleotide sequence (SEQ ID NO: 20, FIG. 10). The present inventors designed the base sequence of a positive control, and then prepared it in a cloned form into the pUCIDT-AMP vector (IDT, USA).

In order to confirm the efficiency of the prepared positive control clone, PCR was performed using ^{1 ng and 10 -1 ng of the positive control clone as a template.} The composition of the PCR mixture was the same as in Table 2 except for the N25 primer, and the reaction conditions were the same as in Table 3 except for the RT reaction. As a result, as shown in FIG. 11, it was found that the two primer sets finally selected in the present invention amplify the PCR product of the expected size, and the positive control clone prepared through this amplifies the PCR test of the present invention. It was found that it can effectively function as a positive control for the primer set.

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Claims (6)

oligonucleotide primer sets of SEQ ID NOs: 1 and 11; And a primer set composition for specifically detecting TEV (Tobacco etch virus) comprising the oligonucleotide primer sets of SEQ ID NOs: 2 and 14. The oligonucleotide primer set according to claim 1; and a reagent for performing an amplification reaction, a kit for specifically detecting Tobacco etch virus (TEV). The kit according to claim 2, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs and a buffer. The kit according to claim 2, wherein the kit further comprises a positive control sequence consisting of the nucleotide sequence of SEQ ID NO: 20. isolating total RNA from a plant sample;
using the isolated total RNA as a template and amplifying a target sequence by performing RT-PCR using the oligonucleotide primer set according to claim 1; and
A method for specifically detecting Tobacco etch virus (TEV), comprising detecting the amplification product. The method according to claim 5, wherein the detection of the amplification product is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

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