KR101062726B1 - RT-PCR method using N-primer and RT-PCR kit including N-primer - Google Patents

Abstract

The present invention relates to a RT-PCR method using an N-primer and a kit for RT-PCR including an N-primer, and more particularly, a primer optionally including some bases having a sequence having a high degree of reaction association with a virus. Viruses can be detected quickly and accurately only by the presence of a small number of viral RNAs in plants infected with viruses, and infected as a result of reverse transcriptase polymerase chain reaction, which has a detection limit of 1,000 times higher than that of antisera. Diagnosis can be made.

N-Primer, Plant, Virus Diagnostic, RT-PCR, One-Step RT-PCR

Description

RT-PCR method using N-primer and RT-PCR kit including N-primer {RT-PCR method using N-primer and kit for RT-PCR comprising N-primer}

The present invention is an RT-PCR method using an N-primer, comprising RNA as a template, and annealing the N-primer to the template to synthesize a first strand cDNA, and one-step using N-primer ( one-step) RT-PCR method and kit for RT-PCR comprising N-primer and reagents required for RT-PCR.

Viruses are very small (about 20 nm), and they vary in shape and do not have the full cellular structure of any other organism. Viruses also paralyze other organisms because they lack metabolism. This is why most viruses are pathogens. By viral parasitic host, plant viruses (TMV, potato atrophy virus, tomato virus, etc.) and animal viruses (influenza virus, AIDS virus, measles virus, rabies (rabies) virus) and bacterial viruses (bacteria bacteriophage; T1) -T7 phage, T2 and T4 are commonly used in genetic studies). In addition, it is divided into RNA virus (TMV, influenza virus, etc.) and DNA virus (bacteriophage) according to the type of nucleic acid.

Viruses are the smallest and simplest living things, but also the most threatening to humans. The scary thing about viruses is that they constantly change their genes, making them unidentified. Even if a vaccine is developed to increase immunity to the virus, the virus changes its genetic information and emerges in a new shape. This mutation rate is a million times faster than other microorganisms. Therefore, it is very difficult to predict when a new virus will appear new and to respond.

Viruses that cause various animal and plant diseases have been recognized as pathogens closely related to humans. They have the characteristics of eukaryotes and prokaryotes. The situation is going deep. Therefore, experimental limits due to the lack of genetic information of the virus can be seen everywhere. This requires methodological detection to obtain and experiment virus quantitatively and qualitatively.

ELISA method (enzyme-binding antibody method) has been widely used as a method for diagnosing viruses, and recently, virus assay technology using RT-PCR (reverse transcription-polymerase chain reaction) has been put to practical use. RT-PCR method has been confirmed to be 100 to 1,000 times higher sensitivity than the ELISA method.

In the conventional RT-PCR technique, the experiment was performed in two stages by performing a PCR reaction after a reverse transcription (RT) reaction. However, the recently developed one-step RT-PCR method has a reverse transcription reaction and a PCR reaction. The addition of the primer and the enzyme is carried out in one tube (tube), there is an advantage that can reduce the experimental step and the risk of contamination.

However, the existing RT-PCR technique uses specific forward and reverse primers to diagnose the virus. However, this method is not effective for the virus diagnosis of a plant virus having one genomic RNA or a sample of low plant virus density. It was.

The present invention has been made in accordance with the above requirements, and in the present invention, despite the inter-virus variation, the RT-PCR is more efficiently performed using an N-primer having a broad detection spectrum across all viral species. It has been found that plant viruses can be diagnosed and the present invention has been completed.

In order to solve the above problems, the present invention provides a RT-PCR method using an N-primer comprising a step of synthesizing a first strand cDNA by using RNA as a template and annealing the N-primer to the template. to provide.

The present invention also provides a one-step RT-PCR method using the N-primer.

The present invention also provides a kit for RT-PCR comprising N-primer and reagents required for RT-PCR.

The specific N-primer for diagnosing the virus according to the present invention has the advantage of planning an experiment regardless of the virus genotype for all species of viruses known so far, and thus can be widely applied to various experiments using plants and all viruses. Providing a range can lead to breakthrough method transformations and applications in dealing with viruses and eukaryotes in medicine and industry.

In particular, in the case of pathogens diagnosed by one-step RT-PCR, N-primers are added instead of the reverse primers used for cDNA synthesis. There is an advantage to this. N-primers can be converted to cDNA by the N-primer for replication of plant viruses in the cells of the diseased plant, and because of the high synthesis rate of full-length cDNA, the limit of detection can be raised and primers can be used. The width of the combination can also be widened.

In addition, the synthesis of cDNA by specific primers and the diagnosis by PCR cannot be made when unidentified or complex infected samples are handled by several different pathogens. However, since all RNAs are converted to cDNA by N-primer, various types of pathogens can be diagnosed from complex infected samples, and synthesized cDNAs can be stored semi-permanently, so that newly identified pathogens can be diagnosed later. Do.

In order to achieve the object of the present invention, the present invention comprises the steps of synthesizing a first strand cDNA by using RNA as a template and annealing the N-primer to the template; And

It provides a RT-PCR method using an N-primer comprising the step of performing PCR by adding a forward primer and a reverse primer to the synthesized first strand cDNA.

In order to design common primers for detecting viruses, total genome genetic information of all known viruses was analyzed. The analysis was performed on the entire genetic region of the virus, including mRNA replication by virus group, presence or absence of poly A tail, and the role and function of proteins that are central to viral composition. The production of sub-genome RNA during mRNA replication could influence the quantitative detection of the virus, and the capsid or coat protein is mainly a part of the nucleic acid fragment that specifically reacts to the virus species as a part that satisfies specific primer conditions. Could be designed to match. However, if these two regions were the 3 'end portion or the same portion of the genome, there was no problem in the design of the ideal primer of a virus, but it was partially different from virus to virus, and the related nucleic acid sequences also varied. By providing such various infrastructures, N-primers were finally selected by repeating N sequences to match all viruses, considering random sequence of sequences and focusing on breaking existing infrastructure.

Viruses, eukaryotes, and prokaryotes provided in the present invention were compared and tested with representative samples. The primer design determined the repetition of the N base sequence to 25mers (25mers), and all comparative experiments were performed with the clarity of the target bands by the RT-PCR reaction of the existing primers and the N-primers by RT-PCR. This was validated and described in the following examples in connection with their usefulness in virus detection.

N-primers are primers in which all G, A, T, and C can be located at each nucleotide position, for example, the number of combinations of primers that can be expected with an N-primer of 25 nucleotides is 4 ^25. . The length of the N-primer is preferably 5 to 50 nucleotides, more preferably 20 to 30 nucleotides, and most preferably 25 nucleotides. This is because the RT-PCR efficiency may be lowered if the length of the N-primer is outside the above range.

The RT-PCR method using the N-primer of the present invention can use the reagents and methods used in the general RT-PCR method except using a specific N-primer.

The method for synthesizing the first strand cDNA may use a method commonly used in the art, and for example, the first strand cDNA may be synthesized using a reverse transcriptase, an RNase block ribonuclease inhibitor, or the like. Examples of reverse transcriptases include reverse transcriptases from various sources, such as avian myeloblastosis virus-derived virus reverse transcriptase (AMV RTase), mouse leukemia virus-derived reverse transcriptase ( murine leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rous-associated virus 2 reverse transcriptase (RAV-2 RTase).

RNA, which is a nucleic acid used as a template in the method of the present invention, may be isolated or prepared from any sample that may contain nucleic acid. Examples of samples that may include nucleic acids include, but are not limited to, whole blood, serum, buffy coat, urine, feces, cerebrospinal fluid, semen, saliva, tissues (such as tumor tissue or lymph nodes), and cell culture fluids. Microorganisms such as samples from organisms, such as mammalian cell cultures or bacterial cell cultures, samples containing nucleic acids such as viroids, viruses, bacteria, fungi, yeast, plants and animals, microorganisms such as viruses or bacteria Samples that are expected to be contaminated or infected with (eg, food or biological agents), and samples that may include organisms such as soil and wastewater. Nucleic acids can be prepared from the above-mentioned materials using, for example, cell lysis using a detergent, sonication, glass beads or shaking / stirring using a French press. The RNA is preferably of virus origin, more preferably of plant virus, most preferably of monopartite genomic plant virus with one genomic RNA.

RT-PCR method using the N-primer of the present invention is used for virus diagnosis, preferably plant virus diagnosis, more preferably monopartite genomic plant virus having one genomic RNA or plant virus diagnosis of a sample having low plant virus density. Can be.

The invention also comprises N-primer, forward primer and reverse primer, reverse transcriptase, heat resistant polymerase and reaction buffer, comprising RNA as a template and performing RT-PCR, in which N- Provided is a one-step RT-PCR method using primers.

One-step RT-PCR method of the present invention comprises the first step of synthesizing the first strand cDNA first; And RT-PCR is a method of performing RT-PCR through a two-step process of performing PCR by adding a forward primer and a reverse primer to the synthesized first strand cDNA. Therefore, one-step RT-PCR is to perform RT-PCR by adding N-primers, forward primers and reverse primers, reverse transcriptases, heat resistant polymerases and reaction buffers necessary for reverse transcription and polymerase chain reaction to one tube. Forward primers and reverse primers are primer combinations designed to produce the desired PCR product. Thus, depending on the target product, the sequence will be different. The reaction buffer includes both reverse transcription buffer and PCR buffer, and the heat resistant polymerase may use EF Taq polymerase, and the like, and the reverse transcriptase may use MuLV reverse transcriptase as described above.

The length of the N-primer used in the one-step RT-PCR method is preferably 5 to 50 nucleotides, more preferably 20 to 30 nucleotides, and most preferably 25 nucleotides. This is because the one-step RT-PCR efficiency may be lowered if the length of the N-primer is outside the above range.

The RNA used in the one-step RT-PCR method is as described above, and the RNA is preferably from virus, more preferably from plant virus, most preferably from a monopartite genomic plant virus with one genomic RNA. .

The one-step RT-PCR method using the N-primer of the present invention is used for virus diagnosis, preferably plant virus diagnosis, more preferably monopartite genomic plant virus having one genomic RNA or plant virus diagnosis of a sample having low plant virus density. Can be used.

In order to achieve another object of the present invention, the present invention provides a kit for RT-PCR comprising a reagent necessary for N-primer and RT-PCR. The length of the N-primer is preferably 5 to 50 nucleotides, more preferably 20 to 30 nucleotides, and most preferably 25 nucleotides. This is because the RT-PCR efficiency may be lowered if the length of the N-primer is outside the above range.

Reagents required for RT-PCR may be, for example, but not limited to, forward primers and reverse primers, reverse transcriptases, ribonuclease inhibitors, heat resistant polymerases, reaction buffers, and the like, and the reaction buffers include both reverse transcription buffer and PCR buffers. It includes all, the heat-resistant polymerase can be used such as EF Taq polymerase, reverse transcriptase can be used as described above MuLV reverse transcriptase.

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The kit for RT-PCR can be used for virus diagnosis, preferably plant virus diagnosis, more preferably monopartite genomic plant virus having one genomic RNA or plant virus diagnosis of a sample of low plant virus density.

The kit for RT-PCR can also be used for one-step RT-PCR.

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

<Example 1: RT with with the existing specific nucleotide sequence primer and loop primer of the present invention - PCR reaction>

Comparative RT-PCR photographs of specific primers and N-primers in soybean virus are shown in FIG. 1. The soybean mosaic virus used for the present invention used a virus disease plant sample selected by re-inoculation after virus isolation from the Academy of Agricultural Science and Technology. A primer having a nucleic acid sequence specific to the virus is produced in a coat protein of interest, and is a region that plays a role in forming a viral coat, and is generally used as a nucleic acid fragment used in a virus detection spectrum. . The designed N-primer is a nucleic acid fragment synthesized oligo (GCAT mixed bases) oligo from bioneer.

Total RNA isolation method used for the present invention is as follows.

Total RNA was isolated using the Trizol solution (Tri-reagent; TR118, MRC co.) Extraction method. 100 mg of the infected plant was put in a mortar and pulverized with liquid nitrogen, and then pulverized. The crushed plant tissue was placed in a container at which the temperature was dropped, and 1 ml of trizol solution was added and mixed by vortexing. 200 μl chloroform was added thereto, mixed, left to stand at room temperature for 15 minutes, centrifuged at 13,000 rpm for 5 minutes at 4 ° C., an aqueous layer containing RNA was collected, and 200 μl chloroform was added thereto. After adding and mixing, the mixture was allowed to stand at room temperature for 15 minutes, centrifuged at 13,000 rpm for 5 minutes at 4 ° C, and the aqueous layer was collected. After mixing the isopropanol of about 80% of the volume of the obtained aqueous layer, the mixture was allowed to stand at room temperature for 20 minutes, centrifuged at 13,000 rpm for 20 minutes, dried the isopropanol solution of precipitate, and 1 ml of 70% ethanol was added to shake the precipitate. do. The precipitate suspended in 70% ethanol was centrifuged at 13000 rpm for 15 minutes at 4 ° C to discard the supernatant, and the 70% ethanol solution of the precipitate was dried and dissolved in 100 µl of distilled water for freezing.

Reverse transcription-polymerase chain reaction (RT-PCR) method using the extracted total RNA is as follows.

Into 30 μg total RNA, add 1/10 μg of the existing specific reverse primer (SEQ ID NO: 1), and compare the N-primer to 1/10 μg in 30 μg total RNA, and add distilled water. The reaction solution was made into 12 [mu] l, respectively, and reacted at 70 [deg.] C. for 10 minutes, and then stood on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. MuLV reverse transcriptase 2 units were added, reverse transcription was carried out at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting half of each cDNA synthesized by reverse transcription and using 1 μl of the template as a template to conduct a polymerase chain reaction to derive a target detection spectrum band. 2 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole forward primer (SEQ ID NO: 2 or SEQ ID NO: 3), 5 pmole reverse primer (SEQ ID NO: 1) 5 pmole, 10 mM dNTPs 0.4 μl, 1 unit of EF Taq polymerase was added, and distilled water was added to the final 20 μl reaction solution. PCR conditions were denatured for 1 minute at 94 ° C., followed by 30 seconds at 94 ° C., 30 ° at 58 ° C., annealing. Second, the extension was performed 35 times 30 seconds at 72 ℃, PCR products were analyzed on 1% agarose gel (agarose gel).

As a result, as can be seen in Figure 1, the PCR product is more specific in the case of RT-PCT using the N-primer than in the case of RT-PCT using the reverse transcription-specific primer, almost no non-specific band, It can be seen that a large amount of specific bands are detected.

< Example  2: Tobacco green mammoth virus Soybean virus  Genomic 3 ′ Terminal Specific Nucleic Acid Fragments Or target ( target 3 'terminal specific nucleic acid fragment of the reverse  primer) and N- With primer  Of the target area RT - PCR  Reaction>

Tobacco green mammoth mosaic virus and soybean mosaic virus used for the present invention used a virus disease plant sample selected by re-inoculation after virus isolation from the Agricultural Science and Technology Institute. Primers having a nucleic acid sequence specific for the virus are selected from the entire genome 3 'end portion of the virus, and the target region is a portion of the entire genome of the virus or a portion of the nucleotide sequence that will be host-specific in the virus. Regions containing infectious protein (P1 Protein, HC-Pro Protein) and envelope proteins were prepared. N-primer was identified in the same manner as previously prepared.

(1) RT-PCR for detecting a portion of the total nucleic acid of Tobacco malignant mosaic virus (FIG. 2A)

Total RNA extraction method of the present experiment is the same as above, reverse transcription-polymerase chain reaction (RT-PCR) method and conditions are as follows.

The existing specific 3 ′ terminal nucleic acid fragment (SEQ ID NO: 4) was added to 30 μg total RNA at 1/10 μg of the total RNA amount, and the N-primer to be compared was also placed at 30 μg total RNA at 1/10 μg, and distilled water The reaction solution was added to 12 µl each, and reacted at 70 ° C for 10 minutes, and then allowed to stand on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. Two units of MuLV reverse transcriptase were added, reverse transcription was performed at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, followed by polymerase chain reaction to derive a target detection spectrum band using 1 μl as a template. 2 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole of forward primer (SEQ ID NO: 5 or SEQ ID NO: 6) in the target region, reverse primer (SEQ ID NO: 4 or sequence) No. 7) 5 pmole, 0.4 μl of 10 mM dNTPs, 1 unit of EF Taq polymerase were added, and distilled water was added to the final 20 μl reaction solution. PCR conditions were denatured at 94 ° C for 1 minute, denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension at 72 ° C for 30 seconds at 30 times. The product was analyzed on 1% agarose gel.

As a result, as shown in FIG. 2A, the PCR product was more specific in the case of RT-PCT using N-primer than in case of RT-PCT using reverse transcription-specific primer. It can be seen that the nonspecific bands are almost eliminated, and a large amount of specific bands are detected.

(2) RT-PCR for detecting soybean mosaic virus infection mediating protein (P1 Protein, HC-Pro Protein) and envelope protein (FIG. 2B)

Total RNA extraction method of the present experiment is the same as above, reverse transcription-polymerase chain reaction (RT-PCR) method and conditions are as follows. The existing specific 3 ′ terminal nucleic acid fragment (SEQ ID NO: 1) was added to 30 μg total RNA at 1/10 μg of the total RNA amount, and the N-primer to be compared was also added at 30 μg total RNA at 1/10 μg, and distilled water The reaction solution was added to 12 µl each, and reacted at 70 ° C for 10 minutes, and then allowed to stand on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. Two units of MuLV reverse transcriptase were added, reverse transcription reaction was performed at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, followed by polymerase chain reaction to derive a target detection spectrum band using 1 μl as a template. 2 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), forward primer of target region (SEQ ID NO: 8-SEQ ID NO: 10 or SEQ ID NO: 13, 14, 16, 17) 5 pmole, reverse primer (SEQ ID NO: 11 or SEQ ID NO: 12, 15, 18, 19) 5 pmole, 0.4 μl of 10 mM dNTPs, 1 unit of EF Taq polymerase was added to the distilled water and the final 20 μl reaction solution was carried out. PCR conditions were denatured at 94 ° C for 1 minute, denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension at 72 ° C for 30 seconds at 30 times. The product was analyzed on 1% agarose gel.

As a result, as shown in FIG. 2B, the PCR product was more specific in the case of soybean virus RT-PCT in the case of RT-PCT using N-primer than in case of RT-PCT using reverse transcription-specific primer. It can be seen that the nonspecific bands are almost eliminated, and that a large amount of specific bands are detected.

< Example  3: Soybean virus  Genomic 3 'terminal specific nucleic acid fragment or 3' terminal specific nucleic acid fragment of a target region ( reverse primer ) And N- With primer  Of the target area One step RT - PCR  Reaction>

Total RNA extraction method according to the present experiment is the same, reverse transcription-polymerase chain reaction (RT-PCR) method and conditions are as follows.

1 μg total RNA, 10 pmole of the forward primer (SEQ ID NO: 5 or SEQ ID NO: 6) of the target region, 10 pmole of the reverse primer (SEQ ID NO: 4 or SEQ ID NO: 7), 1 μl of RNA degrading enzyme inhibitor (RNase inhibitor), MuLV reverse transcriptase (reverse) transcriptase) 200 units, 5-fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 1.4 μl, 10 mM dNTP 0.5 μl, EF Taq polymerase 1 unit , 1.5 fold of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3) and 0.8 μl of DMSO were added to distilled water to make 20 μl of reaction solution. 20 pmole was added to 1 μg of total RNA, and the reaction solution and distilled water were added to the final 20 μl reaction solution.

RT-PCR conditions were synthesized cDNA for 1 hour at 42 ℃, denatured for 10 minutes at 94 ℃, 30 seconds at denature 94 ℃, 30 seconds at 58 ℃ annealing (Extension) Was performed 35 times for 1 minute at 72 ° C., and PCR products were analyzed on 1% agarose gel (FIG. 3).

As a result, as shown in FIG. 3, the PCR product was almost similar to the one-step RT-PCT using the reverse transcription-specific primer and the one-step RT-PCT using the N-primer in the case of soybean virus RT-PCT. It can be seen that it is detected.

< Example  4: Eukaryotic  Conventional nucleic acid fragments and N- commonly used for experiments of organisms and prokaryotes Of primer RT - PCR  Reaction>

(1) Comparative experiment using prokaryotic bacteria

Bacterial Burkholderia glumae is a bacterium that affects yield by causing bacterial rice blight on growing rice grains. This sample, which is being tested in Seoul National University and Korea Institute of Biotechnology Plant Genetic Laboratories, was separated into single colonies and used for subculture.

Random octamers or hexamers oligos and specific reverse primers of target regions have been commonly used for the general prokaryotic reverse transcription reaction. When the experiment using the oligonucleotide (oligo) with a reverse primer and N nucleotide sequence of interest of which obtained by performing the following purposes bacteria Burkholderia Part 11 ORF (open reading frame) of the entire genome of the glumae was subjected to a polymerase chain reaction to see the result (Fig. 4A).

The bacterial RNA isolation method used for the present invention is as follows.

Single colonies are incubated for 16 hours in liquid antibiotic medium (Rifampicin 50 μg / ml). Take 600 µl of the above culture and incubate in 60 ml of liquid medium. When the OD (Optimal Density) value reaches 0.6 at 600 nm, the culture is stopped. 7.5 ml ethanol / phenol spot solution (5% acid phenol in ethanol) is added. The supernatant is removed by centrifugation and 3 ml of acetate / SDS buffer is added to the precipitate. Mix evenly to release the precipitate, transfer 10ml to a 15ml tube, and add 3ml of phenol heated to 65 ℃. Mix evenly and transfer 1 ml each to an eppendorf tube (e-tube) and perform centrifugation (13,000 rpm) for 10 minutes. Take only the clear part of the upper layer, add the same amount of phenol and mix, and perform centrifugation (13,000 rpm) for 10 minutes. Take only the clear part of the upper layer, add 1/5 chloroform, mix, and perform centrifugation (13,000 rpm) for 10 minutes. Add 100% ethanol in an amount corresponding to 3 volumes of the supernatant and hold for 30 minutes at -70 ° C. Centrifuge (13,000 rpm) for 10 minutes and add 70% ethanol to the resulting precipitate and wash. The obtained precipitate was once again dissolved in acetate / SDS buffer, subjected to ethanol precipitation, and then dissolved in 100 μl of DEPC water. Treat DNase and react at 37 ° C for 20 minutes. 6 μl of 3M Na-acetate (pH5.2) is added to extract with phenol. Take the supernatant and extract with phenol: chloroform: isoamyl alcohol (25: 24: 1). Take the supernatant and extract with chloroform. Ethanol is run and the precipitate is dried and dissolved in DEPC water.

Reverse transcriptase-polymerase chain reaction (RT-PCR) using the extracted RNA RNA is as follows.

Into the 30 μg total RNA, add 1/10 μg of the existing specific reverse primer (SEQ ID NO: 21) to the total RNA amount, and the N-primer for comparison is also added in 30 μg of the total RNA at 1/10 μg, and distilled water is added. Each reaction solution was prepared into 12 µl and reacted at 70 ° C for 10 minutes, and then allowed to stand for 30 minutes on ice. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( Add 2µl of RNase inhibitor) and add distilled water to make 20µl of reaction solution. Add 2 units of MuLV reverse transcriptase, perform reverse transcription reaction at 42 ° C for 1 hour, treat at 70 ° C for 10 minutes, and cool with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, and then using 1 μl as a template to conduct a polymerase chain reaction to derive the desired detection spectrum band. 2 μl 10 fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole forward primer (SEQ ID NO: 20), 5 pmole reverse primer (SEQ ID NO: 21) 5 pmole, 0.4 μL 10 mM dNTPs, EF 1 unit of Taq polymerase was added thereto, added with distilled water, and the reaction solution was performed with a final 20 μl reaction solution. PCR conditions were denatured at 95 ° C for 1 minute, then denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension for 72 seconds at 30 ° C for 30 seconds and 72 ° C. The reaction was stopped after holding for 5 minutes at, and PCR products were analyzed on 1% agarose gel.

(2) Comparative experiment using liver tissue of experimental rat which is a eukaryotes

Liver tissues of healthy rats were isolated from the Korea Institute of Bioscience and Biotechnology. The target region for the polymerase chain reaction is the actin moiety, and oligo dT nucleic acid fragments suitable for the poly A tail of eukaryotes have been commonly used in experiments. Comparison of the detection of the actin moiety by reverse transcription reaction with the designed N-primer showed the possibility of using N-primer in eukaryotes. The result is shown in FIG. 4B.

Rat liver RNA separation method used for the present invention is as follows.

Liver RNA was isolated using the Trizol solution (Tri-reagent; TR118, MRC co.) Extraction method. 0.5 g of rat liver was removed, placed in an e-tube, and 300 µl of tri-reagent was added and crushed finely by an electric grinder. When the ground rat liver tissue was released into the solution and there was no lump, 200 µl of tri-reagent was added. Mix by vortexing and leave at room temperature. 100 μl chloroform was added to the mixture, followed by standing at room temperature for 15 minutes, followed by centrifugation at 4 ° C. at 13,000 rpm for 5 minutes. The aqueous layer containing RNA was collected, mixed with 100 µl chloroform, mixed for 15 minutes at room temperature, and then centrifuged at 13,000 rpm for 5 minutes at 4 ° C to obtain a clear aqueous layer. About 80% of the volume of the obtained aqueous layer wasopropanol was mixed, allowed to stand at room temperature for 20 minutes, and then centrifuged at 13,000 rpm for 20 minutes at 4 ° C. The isopropanol solution of the precipitate was dried and 1 ml of 70% ethanol was added to shake the precipitate. The precipitate suspended in 70% ethanol was centrifuged at 13000 rpm for 15 minutes at 4 ° C. The 70% ethanol solution of the precipitate was dried, dissolved in 100 µl of distilled water and stored frozen.

Reverse transcription-polymerase chain reaction (RT-PCR) method using the extracted total RNA is as follows.

1/10 μg of the total RNA amount is added to the existing specific reverse primer (SEQ ID NO: 23) or the oligo dT ₁₈ nucleic acid fragment in 30 μg total RNA, and the N-primer to compare is 1/10 μg in the 30 μg total RNA. Distilled water was added to make 12 µl of the reaction solution, and the mixture was allowed to react at 70 ° C for 10 minutes, and then allowed to stand on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. Two units of MuLV reverse transcriptase were added, reverse transcription was performed at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, followed by polymerase chain reaction to derive a target detection spectrum band using 1 μl as a template. 2 μl 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole forward primer (SEQ ID NO: 22), 5 pmole reverse primer (SEQ ID NO: 23) 5 pmole, 0.4 μl 10 mM dNTPs, EF 1 unit of Taq polymerase was added thereto, added with distilled water, and the reaction solution was performed with a final 20 μl reaction solution. PCR conditions were denatured at 95 ° C for 1 minute, denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension for 72 seconds at 72 ° C for 25 seconds, followed by 72 ° C. The reaction was stopped after holding for 5 minutes at. PCR products were analyzed on 1% agarose gel.

As a result, as can be seen in Figure 4, the proteomic and eukaryotic RT-PCT in the case of RT-PCT using reverse transcription-specific primers and the RT-PCT in the case of N-primer RT-PCT almost similar It can be seen that it is detected.

< Example  5: N- in adverse conditions in viruses and plants primer  Active gradient>

(1) Changes in N-primer activity under adverse conditions in pepper plants (FIG. 5A)

Bujiang pepper used for the present invention was grown in a greenhouse was used to select a healthy tissue. The polymerase chain reaction of the actin was observed by reverse transcription with oligo dT (oligo dT) primer commonly used for plant reverse transcription and N-primer. The usefulness of the N-primer nucleic acid fragments was demonstrated by the change of RNA from healthy tissue to the condition of poor sample.

Total RNA isolation method used for the present invention is as follows.

Total RNA was isolated using the Trizol solution (Tri-reagent; TR118, MRC co.) Extraction method. 100 mg of the healthy plant was put in a mortar into a liquid nitrogen and finely crushed using a pestle. The ground plant tissue was placed in a container at which the temperature was dropped, and 1 ml of trizol solution was added and mixed evenly by vortexing. 200 μl chloroform was added and mixed, and then allowed to stand at room temperature for 15 minutes, followed by centrifugation for 5 minutes at 13,000 rpm at 4 ° C. The aqueous layer containing RNA was collected, mixed with 200 µl chloroform, mixed for 15 minutes at room temperature, and then centrifuged at 13,000 rpm for 5 minutes at 4 ° C to obtain a clear aqueous layer. About 80% of the volume of the obtained aqueous layer wasopropanol was mixed, allowed to stand at room temperature for 20 minutes, and then centrifuged at 13,000 rpm for 20 minutes at 4 ° C. The isopropanol solution of the precipitate was dried and 1 ml of 70% ethanol was added to shake the precipitate. The precipitate suspended in 70% ethanol was centrifuged at 13000 rpm for 15 minutes at 4 ° C. 70% ethanol solution of the precipitate was dried and dissolved in 100ul distilled water and stored frozen.

The total RNA extracted is frozen at -70 ° C for 30 minutes after 1 hour room temperature (stress 1), frozen at -70 ° C for 30 minutes after 1 hour at room temperature (stress 2), and -70 ° C for 30 minutes after 1 hour at room temperature. RNA samples up to stress 5 were isolated by the method of freezing (stress 3) and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as follows.

For 5 μl of each RNA sample, 1 μg of oligo dT primer was added or 1 μg of N-primer was added to identify the primer amount. Distilled water was added to make 12 µl of the reaction solution, and the mixture was allowed to react at 70 ° C for 10 minutes, and then allowed to stand on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. Two units of MuLV reverse transcriptase were added, reverse transcription was performed at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, followed by polymerase chain reaction to derive a target detection spectrum band using 1 μl as a template. 2 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole of forward primer (SEQ ID NO: 24), 5 pmole of reverse primer (SEQ ID NO: 25) 5 pmole, 0.4 μl of 10 mM dNTPs, EF 1 unit of Taq polymerase was added thereto, added with distilled water, and the reaction solution was performed with a final 20 μl reaction solution. PCR conditions were denatured at 94 ° C for 1 minute, denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension at 72 ° C for 30 seconds at 30 times. The product was analyzed on 1% agarose gel.

(2) N-primer activity gradient under adverse conditions in virus (FIG. 5B)

For the present invention, a virus disease plant sample selected by re-inoculation after separating pepper mild mottle virus from the Academy of Agricultural Science and Technology was used. The virus was diagnosed by reverse transcription with a specific reverse primer commonly used for virus reverse transcription and the designed N-primer, but the reaction of N-primer nucleic acid fragments under adverse conditions of RNA samples was observed by reverse transcription polymerase chain reaction. Sensitive reactivity of N-primer nucleic acid fragments to changes from healthy tissue RNA samples to poor samples has proved their usefulness as nucleic acid fragments used in virus detection spectra.

Total RNA was isolated using the Trizol solution (Tri-reagent; TR118, MRC co.) Extraction method. 100 mg of the diseased plant was put into a mortar and pulverized finely by adding liquid nitrogen, and the ground plant tissues were placed in a container at which the temperature was dropped, and 1 ml of trizol solution was added and mixed by vortexing. 200 μl chloroform was added and mixed, and then allowed to stand at room temperature for 15 minutes, followed by centrifugation for 5 minutes at 13,000 rpm at 4 ° C. The aqueous layer containing RNA was collected and mixed by adding 200 µl chloroform, and then allowed to stand at room temperature for 15 minutes, followed by centrifugation at 13,000 rpm for 5 minutes at 4 ° C to obtain a clear aqueous layer. About 80% of the volume of the obtained aqueous layer wasopropanol was mixed, allowed to stand at room temperature for 20 minutes, and then centrifuged at 13,000 rpm for 20 minutes at 4 ° C. The isopropanol solution of the precipitate was dried and 1 ml of 70% ethanol was added to shake the precipitate. The precipitate suspended in 70% ethanol was centrifuged at 13000 rpm for 15 minutes at 4 ° C. The 70% ethanol solution of the precipitate was dried, dissolved in 100 µl of distilled water and stored frozen.

The extracted total RNA was frozen at -20 ° C for 15 hours (stress 1) after 9 hours at room temperature (stress 1), frozen at 15 hours -20 ° C after 9 hours at room temperature (stress 2), and 15 hours -20 ° C after 9 hours at room temperature. RNA samples were isolated from each other (stress 3) and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as follows.

For 5 μl of each RNA sample, 1 μg of reverse specific primer (SEQ ID NO: 27) was added or 1 μg of N-primer was added to compare the amount of primers.

Distilled water was added to make 12 µl of the reaction solution, and the mixture was allowed to react at 70 ° C for 10 minutes, and then allowed to stand on ice for 30 minutes. Reverse transcription was performed in the gastric reaction solution with 5 fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 4 μl, 10 mM dNTP 2 μl, RNA degrading enzyme inhibitor ( 2 μl of RNase inhibitor) was added and distilled water was added to make 20 μl of the reaction solution. Two units of MuLV reverse transcriptase were added, reverse transcription was performed at 42 ° C. for 1 hour, treated at 70 ° C. for 10 minutes, and cooled with ice. PCR was performed by diluting 1/2 of each cDNA synthesized by reverse transcription, followed by polymerase chain reaction to derive a target detection spectrum band using 1 μl as a template. 2 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3), 5 pmole forward primer (SEQ ID NO: 26) 5 pmole, reverse primer (SEQ ID NO: 27) 5 pmole, 0.4 μl 10 mM dNTPs, EF 1 unit of Taq polymerase was added thereto, added with distilled water, and the reaction solution was performed with a final 20 μl reaction solution. PCR conditions were denatured at 94 ° C for 1 minute, denatured at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and extension for 72 seconds at 72 ° C. PCR products were analyzed on 1% agarose gel.

As a result, as can be seen in Figure 5, RT-PCT using reverse transcription-specific primer or oligo dT primer in the case of RT-PCT under adverse conditions in viruses and plants and RT-PCT using N-primer It can be seen that the PCR product in is detected almost similarly.

< Example  6: Tobacco Ringspot Virus  ( TRSV From soybeans infected TRSV For the diagnosis of One step RT - PCR  Reaction>

Total RNA extraction method according to the present experiment is the same, reverse transcription-polymerase chain reaction (RT-PCR) method and conditions are as follows.

10 pmole of reverse primer (SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34), 10 pmole, RNA degrading enzyme inhibitor (RNase inhibitor) 1 μl, MuLV reverse transcriptase 200 units, 5-fold reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl ₂ , 150 mM KCl, 5 mM dithiothreitol, pH 8.5) 1.4 μl, 0.5 μl of 10 mM dNTP, 1 unit of EF Taq polymerase, 1.5 μl of 10-fold PCR buffer (100 mM Tris-HCl, 15 mM MgCl ₂ , 500 mM KCl, pH 8.3) and 0.8 μl of DMSO were added to the reaction solution. 20 μl of the prepared N-primer was added to 1 μg of total RNA, and the reaction solution and distilled water were added to the final 20 μl reaction solution.

RT-PCR conditions were synthesized cDNA for 1 hour at 42 ℃, denatured for 10 minutes at 94 ℃, 30 seconds at denature 94 ℃, 30 seconds at 55 ℃ bonding, (Extension) Was performed 30 times for 2 minutes at 72 ° C., and PCR products were analyzed on a 1% agarose gel (FIG. 6).

In FIG. 6, panel A is the result when the reverse transcriptase is included in the one step RT-PCR reaction solution and panel B is not included. The reason for making panel B is to check whether or not there is contamination because the virus of the present inventors handles a lot of the virus.

In panel B, the reactions tested without the addition of reverse transcriptase or N-primers (lanes 5, 6, 7, and 8) showed that there was already a large amount of soybean-derived RNA in the total RNA sample, but cDNA synthesized by reverse transcriptase Since it could not be generated, it can be seen that the DNAs shown in the RT-PCR reaction were derived from genomic DNA carried in small amounts as a total RNA sample. However, if there is no reverse transcriptase but N-primer is included in the reaction solution (lanes 1, 2, 3, 4), no amplified DNA can be seen at all. The reason is that even in RT-PCR reaction, N-primer seems to increase specificity by action we do not know. In many cases this can be observed. Reactions using specific primers generally appear clean, but N-primers seem to play a role in correcting a large number of non-specific reactions.

As can be seen in panel A, it was found that PCR products were detected almost similarly for one-step RT-PCT with reverse transcription-specific primers for TRSV RT-PCT and one-step RT-PCT with N-primer. Can be.

1 is a photograph of the N-primer RT-PCR reaction in soybean virus.

Lane 1: polymerase chain reaction with coat protein detection primer (SEQ ID NO: 2 and SEQ ID NO: 1) after reverse transcription-specific primer reaction, lane 2: polymerization with coat protein detection primer (SEQ ID NO: 3 and SEQ ID NO: 1) after reverse transcription-specific primer reaction Enzyme chain reaction, lane 3: polymerase chain reaction with the envelope protein detection primer (SEQ ID NO: 2 and SEQ ID NO: 1) after reverse transcription N-primer reaction, lane 4: envelope protein detection primer (SEQ ID NO: 3 and after reverse transcription N-primer reaction) Polymerase chain reaction with SEQ ID NO: 1).

2 is an N-primer RT-PCR reaction picture for detecting various regions of virus.

A: Tobacco Locosa Mosaic Virus RT-PCR

Left lanes 1 to 4 were polymerase chain reactions with the envelope protein detection primer after reverse transcription-specific primer reaction, and right lanes 1 to 4 were polymerase chain reactions with the envelope protein detection primer after the reverse transcription N-primer reaction. Primer combinations used in the lanes are as follows:

Lane 1; SEQ ID NO: 5 and SEQ ID NO: 4, Lane 2: SEQ ID NO: 5 and SEQ ID NO: 7,

Lane 3: SEQ ID NO: 6 and SEQ ID NO: 4, lane 4: SEQ ID NO: 6 and SEQ ID NO: 7.

B: soybean virus RT-PCR

Left lanes 1 to 6 were polymerase chain reactions with HC-Pro protein detection primer after reverse transcription-specific primer reaction, and right lanes 1 to 6 were polymerase chain reactions with HC-Pro protein detection primer after reverse transcription N-primer reaction. Left lanes 7 to 8 are polymerase chain reactions with CP protein detection primers after reverse transcription-specific primer reactions, and lanes 7 to 8 on the right lanes are polymerase chain reactions with CP protein detection primers after reverse transcription N-primer reactions. Left lanes 9-11 are polymerase chain reactions with P1 protein detection primers after reverse transcription-specific primer reactions, and right lanes 9-11 are polymerase chain reactions with CP protein detection primers after reverse-transcription N-primer reactions. The primer combinations used in each lane are as follows:

Lane 1: SEQ ID NO: 10 and SEQ ID NO: 12, lane 2: SEQ ID NO: 8 and SEQ ID NO: 12, lane 3: SEQ ID NO: 8 and SEQ ID NO: 11, lane 4: SEQ ID NO: 9 and SEQ ID NO: 12, lane 5: SEQ ID NO: 10 And SEQ ID NO: 11, lane 6: SEQ ID NO: 9 and SEQ ID NO: 11, lane 7: SEQ ID NO: 13 and SEQ ID NO: 15, lane 8: SEQ ID NO: 14 and SEQ ID NO: 15, lane 9: SEQ ID NO: 16, and SEQ ID NO: 18, lane 10: SEQ ID NO: 16 and SEQ ID NO: 19, lane 11: SEQ ID NO: 17 and SEQ ID NO: 19.

3 is an N-primer one-step RT-PCR reaction picture for detecting various regions of soybean virus.

Left lanes 1 to 6 were polymerase chain reactions with HC-Pro protein detection primers after reverse transcription-specific primer reaction, and right lanes 1 to 6 were polymerase chain reactions with HC-Pro protein detection primers after reverse transcription N-primer reaction. Left lanes 7-9 were polymerase chain reactions with P1 protein detection primers after reverse transcription-specific primer reactions, and left lanes 7-9 were polymerase chain reactions with P1 protein detection primers after reverse-transcription N-primer reactions. The primer combinations used in each lane are as follows:

Lane 1: SEQ ID NO: 10 and SEQ ID NO: 12, lane 2: SEQ ID NO: 8 and SEQ ID NO: 12, lane 3: SEQ ID NO: 8 and SEQ ID NO: 11, lane 4: SEQ ID NO: 9 and SEQ ID NO: 12, lane 5: SEQ ID NO: 10 And SEQ ID NO: 11, Lane 6: SEQ ID NO: 9 and SEQ ID NO: 11, Lane 7: SEQ ID NO: 16 and SEQ ID NO: 18, Lane 8: SEQ ID NO: 16 and SEQ ID NO: 19, Lane 9: SEQ ID NO: 17, and SEQ ID NO: 19.

4 is an N-primer RT-PCR photograph of eukaryotes and prokaryotes.

A: Prokaryotes (Bacteria) Burkholderia glumae , B: eukaryotes (rat) liver tissue

5 is a diagram showing the change in N-primer activity under adverse conditions in viruses and plants (A: pepper RT-PCR, B: PMMV RT-PCR).

6 is an N-primer one-step RT-PCR reaction photograph for detection of TRSV.

Lanes 1 to 4 performed RT-PCR using N-primers and specific primers, and lanes 5 to 8 performed RT-PCR using specific primers without N-primers. The combination is as follows:

Lane 1: TRSV CP-U (SEQ ID NO: 28) + TRSV CP-L (SEQ ID NO: 29) + N25 (500 bp)

Lane 2: YTRSV2-1U (SEQ ID NO: 30) + YTRSV2-2L (SEQ ID NO: 31) + N25 (684 bp)

Lane 3: YTRSV2-2U (SEQ ID NO: 32) + YTRSV2-2L (SEQ ID NO: 31) + N25 (1,080 bp)

Lane 4: YTRSV2-3U (SEQ ID NO: 33) + YTRSV2-3L (SEQ ID NO: 34) + N25 (1,542 bp)

Lane 5: TRSV CP-U (SEQ ID NO: 28) + TRSV CP-L (SEQ ID NO: 29) (500 bp)

Lane 6: YTRSV2-1U (SEQ ID NO: 30) + YTRSV2-2L (SEQ ID NO: 31) (684 bp)

Lane 7: YTRSV2-2U (SEQ ID NO: 32) + YTRSV2-2L (SEQ ID NO: 31) (1,080 bp)

Lane 8: YTRSV2-3U (SEQ ID NO: 33) + YTRSV2-3L (SEQ ID NO: 34) (1,542 bp).

<110> Korea Research Institute of Bioscience and Biotechnology <120> RT-PCR method using N-primer and kit for RT-PCR comprising          N-primer <130> PN08178 <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> specific reverse primer for reverse transcription from coat          protein 9191bp <400> 1 taactcccga gagagctgca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer from coat protein 8922 bp <400> 2 tgggtgatga tggatggaga 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from coat protein 8773 bp <400> 3 ctcgagcaac aagaacacag t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> specific reverse primer for reverse transcription from coat          protein 6192bp <400> 4 tttggtatca gccaccctga a 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer from coat protein 5669 bp <400> 5 ccttatacaa tcaactctcc ga 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer from coat protein 5847 bp <400> 6 tgagatttcc tgcatcggat tt 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from coat protein 6073 bp <400> 7 tgcttgattg aacatgccag tt 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer from HC-Pro protein 1082 bp <400> 8 ccgtggttgg aaaaaggtgt t 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer from HC-Pro protein 1124 bp <400> 9 ggagaatcat gaatgcacca tt 22 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer from HC-Pro protein 1202bp <400> 10 tccagttaag aaactatcat gtaa 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from HC-Pro protein 1534 bp <400> 11 tcttccacca ctgtgtcatt g 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from HC-Pro protein 1681 bp <400> 12 gaatgcctgc cacgctcttc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer from coat protein 8773 bp <400> 13 ctcgagcaac aagaacacag t 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer from coat protein 8922 bp <400> 14 tgggtgatga tggatggaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from coat protein 9191 bp <400> 15 taactcccga gagagctgca 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer from P1 protein 144 bp <400> 16 atgattggaa gcatggcgat tt 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer from P1 protein 601 bp <400> 17 accaactcca aagaagacta aa 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from P1 protein 603 bp <400> 18 tttagtcttc tttggagttg gt 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from P1 protein 893 bp <400> 19 yccataagtt atatcatccg ca 22 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer from ORF11 region in Burkholderia glumae <400> 20 cgaggttctg gccgctcag 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from ORF11 region in Burkholderia glumae <400> 21 ccgtcttcag cgtgtcctcg 20 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer from actin of mouse <400> 22 tgacggggtc acccacactg tgcccatcta 30 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from action of mouse <400> 23 ctagaagcat tgcggtggac gatggaggg 29 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer from actin of hot pepper <400> 24 ttggactctg gtgatggtgt g 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from actin of hot pepper <400> 25 aacatggttg agccaccact g 21 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer from PMMV diagnosis primer <400> 26 attgggcaga actcggagtc atcgg 25 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer from PMMV diagnosis primer <400> 27 gctccgagaa gtgccgaca 19 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TRSV CP-U primer <400> 28 gctgtgacgg ttgttcccg 19 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TRSV CP-L primer <400> 29 cgaaagtgtc acccaattga at 22 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YTRSV 2-1U primer <400> 30 atggagcccc tcttatggca 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> YTRSV 2-2L primer <400> 31 acacatcaaa tgagcagtgc tg 22 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> YTRSV 2-2U primer <400> 32 gcttctaccg gggaagtgc 19 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> YTRSV 2-3U primer <400> 33 gctgtgacgg ttgttcccg 19 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> YTRSV 2-3L primer <400> 34 ttaaaacaaa gtggcggagc g 21  

Claims (11)

Synthesizing a first strand cDNA by using RNA from a plant virus having one genomic RNA as a template, and annealing an N-primer having a length of 25 to 30 nucleotides to the template; And Reverse Transcription (PCR) -PCR, which is used for diagnosing plant virus of a low plant virus density sample using N-primer, comprising performing PCR by adding a forward primer and a reverse primer to the synthesized first strand cDNA. Way. As a template, RNA from a plant virus having one genomic RNA was used as a template, and RT-N was added by adding 25 to 30 nucleotides of N-primer, forward primer and reverse primer, reverse transcriptase, heat resistant polymerase and reaction buffer in one tube. (Reverse Transcription)-One-step Reverse Transcription (PCR) -PCR method used for diagnosing plant virus of a low plant virus density sample using N-primer, comprising performing PCR. delete delete delete delete Reverse (RT) is used to diagnose plant viruses in plant viruses or low-density plant viruses with one genomic RNA, including reagents required for N-primers of 25 to 30 nucleotides in length and reverse transcription (PCR). Transcription) -kit for PCR. delete delete delete 8. The kit of claim 7, wherein the kit is used for one-step RT-PCR.

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