KR102213231B1 - Primer set for detecting of virus infected Pepper, and uses thereof - Google Patents

Abstract

The present invention relates to a primer set for detecting pepper mottle virus (PepMoV), pepper mild mottle virus (PMMoV), and cucumber mosaic virus (CMV), which are representative viruses of pepper, and to a detection method thereof. In case of using the primer set optimized for a recombinant-polymerase amplification method (RPA) of the present invention, it is possible to detect pepper mottle virus (PepMoV), pepper mild mottle virus (PMMoV) and cucumber mosaic virus (CMV) more rapidly and promptly than a conventional detection method using PCR, and it is very useful because pepper mottle virus (PepMoV), pepper mild mottle virus (PMMoV) and cucumber mosaic virus (CMV) can be rapidly and accurately diagnosed in one sample at once.

Description

Primer set for detecting of virus infected Pepper, and uses thereof

The present invention relates to a primer set for detecting pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), and cucumber mosaic virus (CMV), which are representative viruses of pepper, and a detection method thereof.

Plant viruses cause abnormal symptoms in crops, leading to a decrease in crop yield and quality, thereby lowering the economic value of crops. Unlike other pathogens that damage crops, plant viruses are known to be incapable of treatment and chemical control. Therefore, it is important to immediately remove and quarantine crops infected with plant viruses to prevent secondary damage caused by virus transmission, and rapid and accurate virus diagnosis is required to prevent damage and spread by plant viruses.

Pepper is one of the world's widely cultivated crops cultivated in the climate of a warm tropical region. It is mainly used as a spice in Korea, and is a major crop that is widely cultivated in small private gardens as well as large-scale farmers. There are 68 types of plant viruses that infect peppers worldwide, and 16 types of viruses have been reported in Korea. Plant viruses that infect peppers are a big problem because they cause great economic losses by inhibiting the growth of peppers and reducing their quality. In addition, plant viruses exist more in the form of complex infections in which several other types of viruses are infected than when one virus is infected alone. In general, peppers infected with virus complexes show more severe symptoms and decreased yields than peppers infected alone.

As a conventional virus diagnosis method, direct observation through an electron microscope, a serological method (Enzyme-Linked ImmunoSorbent Assay; ELISA), and a PCR polymerase chain reaction (PCR) technique were used. Direct observation through an electron microscope can confirm the presence of virus by the presence or absence of virus particles, but it is not possible to distinguish the end of the virus. ELISA is the most commonly used serological method, and its detection sensitivity is about 1000 times lower than that of PCR diagnostic method, and it is not suitable for accurate diagnosis because non-specific reactions are frequently found.

Therefore, in recent years, PCR diagnostic methods have been widely used to diagnose plant viruses. The PCR diagnostic method requires an average reaction time of 2 hours or more, including reverse transcription, in the case of RNA virus, and specialized equipment capable of temperature control is required for the action of the enzyme. In addition, it is very important to develop a specific primer suitable for the virus, and additional time and equipment are required when checking the amplified reaction product by electrophoresis. As such, a series of professional processes exist until the diagnosis is confirmed, and since it requires a professional manpower and time to execute it, its utility in the field is significantly reduced.

(001) Korean registered patent KR 10-0552633 (002) Korean registered patent KR 10-1354041

Accordingly, the present inventors have developed a primer set capable of simultaneously accurately detecting three types of viruses infecting peppers using Recombinase Polymerase Amplification (RPA), which complements the shortcomings of the existing PCR, and a detection method thereof. By establishing, the present invention was completed.

In order to solve the above problems, the present invention is a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And it provides a primer set for detecting a virus infected with pepper comprising at least one primer set selected from the group consisting of a primer set comprising the nucleotide sequence of SEQ ID NO: 5 and 6.

In addition, the present invention is a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And it provides a method for detecting a virus infected with pepper using one or more primer sets selected from the group consisting of a primer set comprising the nucleotide sequences of SEQ ID NOs: 5 and 6.

In addition, the present invention is a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And it provides a kit for detecting a virus infected with pepper comprising at least one primer set selected from the group consisting of a primer set comprising the nucleotide sequence of SEQ ID NO: 5 and 6.

If the primer set optimized for the recombinant-polymerase amplification method (RPA) of the present invention is used, pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), cucumber mosaic virus ( CMV) can be detected, and it is very useful because it quickly and accurately diagnoses pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV) and cucumber mosaic virus (CMV) in one sample at the same time.

Figure 1 is a schematic diagram showing the principle of the recombinant-polymerase amplification (Recombinase polymerase amplification, RPA).
Figure 2 is a picture showing the primer positions in the sequence of pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), cucumber mosaic virus (CMV): (A) PepMoV (B) PMMoV, (B) CMV.
3 is a photograph showing the results of electrophoresis of (A) Multiplex RT-PCR and (B) Multiplex RT-RPA amplification products using the prepared primers.
Figure 4 is a photograph showing the result of electrophoresis of (A) Multiplex RT-PCR and (B) Multiplex RT-RPA amplification products using the prepared primers.
Figure 5 shows the results of electrophoresis of the amplification product after performing Multiplex RT-RPA with RNA extracted from a plant inoculated with pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), and cucumber mosaic virus (CMV), respectively. This is the picture shown.
6 is a photograph showing the result of confirming the multiplex RT-RPA amplification product under natural light source and UV using SYBR Green I.

Hereinafter, the present invention will be described in detail.

The present invention uses a recombinant-polymerase amplification (RPA) method to compensate for the disadvantages of the conventional PCR (polymerase chain reaction). The RPA method is a technology that can confirm DNA and RNA amplification based on PCR technology. Unlike the existing PCR method, the RPA method can amplify the target sequence quickly and accurately using DNA binding protein and recombinase, and it uses isothermal mesophilic polymerase. Since amplification is possible even under isothermal conditions, viruses can be detected and diagnosed in a short time if there is no special equipment and only an isothermal device (see Fig. 1). Since nonspecific reactions often occur in this RPA method, it is important to prepare suitable primers to prevent this.

The preparation of these specific primers is more important in Multiplex RPA, which uses various primers in which non-specific reactions occur well. In addition, in RPA, which recommends that the length of the target amplicon is less than 500bp, it is possible to distinguish the length of three target amplicons, and it is very difficult to prepare a primer that does not cause a non-specific reaction.

In order to achieve the above object, the present invention is a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And a primer set (Table 1) for detecting a virus infected with peppers containing at least one primer set selected from the group consisting of a primer set including the nucleotide sequences of SEQ ID NOs: 5 and 6 was prepared, and the primer set was Multiplex No nonspecific reactions occur when performing RPA.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be copied, and can serve as an initiating point for the synthesis of a primer extension product. The length and sequence of the primers should allow the synthesis of the extension product to begin. The specific length and sequence of the primers 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.

In addition, the present invention comprises the steps of separating total RNA (Ribonucleic acid) from pepper samples; A primer set using the isolated RNA as a template and including the nucleotide sequences of SEQ ID NOs: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And amplifying a target sequence using at least one primer set selected from the group consisting of a primer set including the nucleotide sequences of SEQ ID NOs: 5 and 6; And it provides a method for detecting a virus infected with pepper comprising the step of detecting the amplification product.

In one embodiment of the present invention, the amplification reaction can be used in the recombinant-polymerase amplification method (RPA).

Viruses infected with the pepper are pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), or cucumber mosaic virus (CMV).

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling material. In one embodiment, the labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto.

The detection of the amplification product may be performed through a DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplified product, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. The detection of the amplification product can be easily confirmed with the naked eye under natural light source and UV using SYBR Green I (see FIG. 6).

In addition, the present invention is a primer set comprising the nucleotide sequence of SEQ ID NO: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And one or more primer sets selected from the group consisting of a primer set comprising the nucleotide sequences of SEQ ID NOs: 5 and 6; And it provides a kit for detecting a virus infected with pepper comprising a reagent for performing an amplification reaction.

In the kit of the present invention, the reagent for performing the amplification reaction may include a DNA binding protein, a recombinase, a mesophilic polymerase, and the like.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1> Pepper virus extraction

To secure pepper virus RNA, PepMoV-Vb1, PMMoV-Kr, and CMV-Rs1 were inoculated with a juice solution to tobacco ( Nicotiana benthamiana , N. tabacum cv. Samsun nn) and pepper ( Capsicum annuum ). RNA was extracted from leaves showing symptoms 21 days after inoculation and used in the experiment.

<Example 2> Preparation of primer set

In order to create a primer set for detecting a virus infected with pepper, the NCBI Basic Local Alignment Search Tool organized 13 isolates in the case of Pepper Mottle Virus (PepMoV) and 12 isolates in the case of Pepper weak Mottle Virus (PMMoV). I did. For this, a well-preserved region was determined through ClustalW multiple sequence alignment, and primers were prepared.

In addition, the size of the expected amplicon was prepared with a difference enough to distinguish viruses detected on an agarose gel for use in Multiplex RT-RPA that can detect three viruses in one reaction (Table 1). And Figure 2).

<Example 3> Primer set assay

In Example 1 above Using the primer set prepared in Example 2, RNA was extracted from cigarettes inoculated with Pepper Mottle Virus (PepMoV), Pepper Weak Mottle Virus (PMMoV), and Cucumber Mosaic Virus (CMV) alone. RT-PCR and RT-RPA reactions were carried out.

Specifically, RT-PCR performs reverse transcription at 42° C. for 1 hour and 92° C. for 5 minutes, PCR is performed at 95° C. for 3 minutes, 95° C. for 30 seconds, denaturation, and annealing at 65° C., Repeated elongation at 72°C for 35 cycles, and left at 72°C for 5 minutes. RPA was performed at 40° C. for 40 minutes. The amplification product was confirmed by loading on a 3% agarose gel for 50 minutes.

As a result, it was confirmed that all of the primer sets for detecting the virus infected with pepper prepared in Table 1 were normally reacted in PCR and RPA, and the target virus, Pepper Mottle virus (PepMoV), and Pepper weak Mottle virus ( PMMoV) and cucumber mosaic virus (CMV) were detected (Fig. 3).

In addition, it was confirmed whether a non-specific reaction occurred during Multiplex RT-RPA using a primer set mixed set for detecting a virus infected with pepper prepared in Table 1 above.

Specifically, in Example 1 500ng of RNA was extracted and mixed from tobacco inoculated with Pepper Mottle Virus (PepMoV), Pepper Weak Mottle Virus (PMMoV), and Cucumber Mosaic Virus (CMV) alone, followed by Multiplex RT-PCR and Multiplex RT-RPA. RT-PCR performed reverse transcription at 42°C for 1 hour and at 92°C for 5 minutes. PCR was performed at 95°C for 3 minutes, 95°C for 30 seconds, denaturation at 65°C, annealing at 65°C, and 72°C. The elongation was repeated 35 cycles and left at 72° C. for 5 minutes. RPA was carried out at 40°C for 40 minutes. Then, the amplification product was confirmed by loading on a 3% agarose gel for 50 minutes.

As a result, the primer mixture set for detecting a virus infected with pepper prepared in Table 1 was a target virus, pepper Mottle virus (PepMoV), and pepper weak mottle virus (PMMoV), without a non-specific reaction in multiplex RT-RPA. , Cucumber mosaic virus (CMV) was detected (Fig. 4).

In addition, 1,500 ng of RNA was extracted from cigarettes and peppers inoculated with pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), and cucumber mosaic virus (CMV), and multiplex RT-RPA was performed.

As a result, even in plants infected with the mixture, target viruses such as pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV), and cucumber mosaic virus (CMV) were detected without non-specific reaction in all reactions (Fig. 5).

In addition, observing the amplified product by electrophoresis requires several equipment and a lot of time. Therefore, the present inventor used SYBR Green I to visually check the RT-RPA diagnosis result more quickly and easily in order to reduce the time and resources consumed for electrophoresis.

In addition, the concentration of SYBR Green I optimized to observe this SYBR Green I reaction was analyzed.

Specifically, SYBR Green I 100X, 200X, and 400X were diluted in each of the RT-RPA amplified products under natural light and UV, and the reaction was confirmed under a natural light source and a UV light source.

As a result, all of the pepper mottle virus (PepMoV), the pepper weak mottle virus (PMMoV), and the cucumber mosaic virus (CMV) were reacted under both natural light and UV in SYBR Green I 100X (FIG. 6).

<110> KNU-Industry Cooperation Foundation <120> Primer set for detecting of virus infected Pepper, and uses thereof <130> PN1907-375 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA PepMoV F <400> 1 tacgtcaaca cgtgctcgcg aagcccatat 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA PepMoV R <400> 2 aggaacacaa ggagaaaaca cagagcgcca 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA PMMoV F <400> 3 gacggtggcc attagggcca gtataagtaa 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA PMMoV R <400> 4 ctcgtagtgt ttttccctcc acttaaatcg 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA CMV F <400> 5 ctgatatagg tgacatgaga aagtacgccg 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RPA CMV R <400> 6 ccatccagct tacggctaaa atggtcagtc 30

Claims (8)

A primer set including the nucleotide sequences of SEQ ID NOs: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And In a primer set for detecting a virus infected with pepper comprising at least one primer set selected from the group consisting of a primer set comprising the nucleotide sequence of SEQ ID NO: 5 and 6,
The virus infected with pepper is pepper mottle virus (PepMoV), pepper weak mottle virus (PMMoV) or cucumber mosaic virus (CMV). delete Separating total RNA (Ribonucleic acid) from the pepper sample;
A primer set using the isolated RNA as a template and including the nucleotide sequences of SEQ ID NOs: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And amplifying a target sequence using at least one primer set selected from the group consisting of a primer set comprising the nucleotide sequences of SEQ ID NOs: 5 and 6; And
In the method for detecting a virus infected with pepper comprising the step of detecting the amplification product,
The virus infected with the pepper is pepper Mottle virus (PepMoV), pepper weak Mottle virus (PMMoV), or cucumber mosaic virus (CMV). delete The method of claim 3, wherein the amplification is a recombinant-polymerase amplification method (Recombinase polymerase amplification, RPA). The method of claim 3, wherein the detection of the amplification product is performed through gel electrophoresis, fluorescence measurement, or phosphorescence measurement. A primer set including the nucleotide sequences of SEQ ID NOs: 1 and 2; A primer set comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; And one or more primer sets selected from the group consisting of a primer set comprising the nucleotide sequences of SEQ ID NOs: 5 and 6; And in a kit for detecting a virus infected with pepper comprising a reagent for performing an amplification reaction,
The chili pepper-infected virus is a chili pepper virus (PepMoV), a pepper weak mottle virus (PMMoV), or cucumber mosaic virus (CMV) a kit for detecting a virus infected with pepper.
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