KR20170001071A - PCR primer for multiplex diagnosing infectious 4 virus to Chinese cabbages and method for detecting virus using the same - Google Patents
PCR primer for multiplex diagnosing infectious 4 virus to Chinese cabbages and method for detecting virus using the same Download PDFInfo
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Abstract
Description
The present invention relates to a method of PCR diagnosis of viral diseases in Chinese cabbage. More specifically, the present invention relates to a method for diagnosing viral diseases caused by Chinese cabbage mosaic virus (TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CaMV), and a method for diagnosing cabbage virus disease using the primer.
The Chinese cabbage virus is one of the major diseases that cause damage to the cabbage cultivation in addition to the root nodule disease. The major virus causing virus in Chinese cabbage is turnip mosaic virus (TuMV), Ribgrass mosaic virus virus, RMV), Cucumber mosaic virus (CMV), and Caluliflower mosaic virus (CaMV). It is known that about 63% of cabbage virus diseases are caused by the combined infection of these viruses. The most effective way to reduce the damage caused by the cabbage virus disease is to develop varieties showing resistance to the virus. However, in order to develop resistant cultivars according to the characteristics of viruses, there must be a virus-resistant gene source, It is very difficult to cultivate varieties that are resistant to the consumer's preference. Therefore, researches on the prevention of damage of cabbage virus disease are actively carried out. In order to prevent the damage of the cabbage virus disease, it is checked whether or not the cabbage has been infected by the virus. Since virus infection of a plant can not be controlled by pesticides once it is infected, it is required to develop a method for early detection or efficient diagnosis of the viral disease, Accordingly, various technologies are being developed.
For example, Korean Patent Laid-Open Publication No. 2009-0055178 discloses the use of an oligonucleotide probe set hybridizing specifically to the genomic RNA of 30 plant viruses, including TuMV and RMV, or a cDNA thereof, to determine whether TuMV and RMV are infected Korean Patent No. 625019 discloses a PCR primer capable of diagnosing TuMV and RMV singly or simultaneously. However, when performing the PCR using the probe set, it can not be confirmed whether or not the nucleic acid extracted from the plant to be subjected to the PCR assay is well extracted enough to be suitable for the PCR assay. In other words, in the case of a plant in which a virus has not been infected, there is a possibility that the actual virus may not exist, but the reaction result may not be derived due to an error caused by the nucleic acid extraction. there was. Although the frequency of TuMV and RMV infection is high in Chinese cabbage virus disease, it can also be caused by other viruses, so that it can not be confirmed when the virus is infected with other viruses .
Under these circumstances, the present inventors have made extensive efforts to develop a method for diagnosing cabbage virus disease more accurately and simply. As a result, it has been found that four kinds of viruses such as turnip mosaic virus (TuMV), window plant mosaic virus (CMV) and CaMV virus (CaMV), it was confirmed that four viruses could be detected by one PCR reaction, In order to increase the reliability of the PCR assay, a primer capable of amplifying the reference gene, which is always expressed in all the cabbage plants, regardless of the virus infection, was prepared and then combined with the above four virus detection reactions. In case of infection, 5 kinds of primers including the reference gene worked well, The plants which are not infected with the virus also show the amplification of the reference gene, thereby improving the reliability of the diagnosis, thereby completing the present invention.
It is an object of the present invention to provide a composition for diagnosing cabbage virus disease.
Another object of the present invention is to provide a kit for the diagnosis of cabbage viral diseases comprising the above composition.
It is still another object of the present invention to provide a method for diagnosing cabbage virus disease using the above-described virus diagnostic composition or kit.
In order to develop a method for diagnosing cabbage virus disease more accurately and simply, the inventors of the present invention have conducted extensive studies to identify the causative agents of cabbage viruses such as Turnip mosaic virus (TuMV) and Ribgrass mosaic virus , RMV), cucumber mosaic virus (CMV), and cauliflower mosaic virus (CaMV) were also detected, and it was confirmed that most of the cabbage virus diseases can be detected. (TuMV, RMV and CMV) and one species of DNA virus (CaMV) that can be detected simultaneously by PCR without interfering with each other. PCR primers were developed. As a result of detecting the four kinds of viruses individually or in combination using the developed PCR primers, it was confirmed that four types of viruses could be diagnosed through one PCR.
Therefore, it was found that when the four kinds of primers provided in the present invention are used, it is possible to more accurately and simply diagnose viral infectious diseases caused by Chinese cabbage.
In order to accomplish the above object, the present invention provides, in one aspect, a composition for diagnosing cabbage virus disease comprising the primers of SEQ ID Nos: 1 to 8.
The term "primer " of the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a complementary template of a plant nucleic acid, Lt; RTI ID = 0.0 > nucleotide < / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.
In the present invention, the primers include the nucleotide sequences of SEQ ID NOS: 1 to 8, wherein the primers are selected from the group consisting of turnip mosaic virus (TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CMV) CaMV). ≪ / RTI > For example, a primer base pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2 may be used for detection of TuMV, a primer base pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 may be used for detection of RMV, 6 can be used for the detection of CMV, and the primer base pair consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8 can be used for detection of CaMV.
The term " Turnip mosaic virus (TuMV) "of the present invention means a single-stranded RNA virus, which is a type of potiovirus which is a cause of diseases caused by cruciferous plants. Usually, it has no epidermis and has an average length of 720 nm Of filamentous spiral capsids, with a single genome on a straight line and exhibiting a characteristic of being inactivated at 62 < 0 > C. The TuMV is infected through aphids, which cause symptoms such as necrosis, mosaicism, uneven pigmentation and wrinkles in infected plants, which can lead to Chinese cabbage mosaic disease.
In the present invention, the TuMV can be interpreted as a cause of cabbage virus disease and is amplified by a primer consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2 provided by the present invention to show an amplification product of 722 bp. ≪ / RTI >
The term " Ribgrass mosaic virus (RMV) "of the present invention is also referred to as " Holmes ribgrass virus (HRV) ", and is a kind of RNA virus having rod- This infection can lead to Chinese cabbage mosaic disease.
In the present invention, the RMV can be interpreted as a cause of cabbage virus disease and is amplified by a primer consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4 provided by the present invention to show an amplified product of 1,078 bp, Lt; / RTI >
The term " cucumber mosaic virus (CMV) "of the present invention refers to a kind of plant RNA virus having three kinds of positive sense RNA genomes (RNA1, 2 and 3) and one subgenomic (RNA4) . The CMV contains spherical particles having a diameter of 30 nm and is infected by aphids and the like. It is known that the host range can be used as a host for more than 190 plants including cucumbers, branches and plants. It can be onset.
In the present invention, the CMV can be interpreted as a cause of cabbage virus disease and is amplified by a primer consisting of the nucleotide sequences of SEQ ID NOS: 5 and 6 provided in the present invention to show an amplified product of 493 bp. ≪ / RTI >
The term " cauliflower mosaic virus (CaMV virus) "of the present invention is a kind of DNA virus belonging to Caulimovirus, which has a spherical shape with a diameter of 50 nm, Means a virus in which virus particles accumulate. The CaMV is known to be a causative agent of diseases that occur in cruciferous plants, and is known to be transmitted through juice and aphids. This infection can lead to Chinese cabbage mosaic disease.
In the present invention, the CaMV can be interpreted as a cause of cabbage virus disease and is amplified by a primer consisting of the nucleotide sequences of SEQ ID NOS: 7 and 8 provided in the present invention to show an amplification product of 1,467 bp, Lt; / RTI >
The term "Chinese cabbage mosaic disease" of the present invention refers to a disease that occurs in Chinese cabbage due to infection of a virus, and in general, the color of the leaf is dark and the color of light is speckled and mosaic symptoms are caused. The symptoms of the Chinese cabbage mosaic disease are known to vary slightly depending on the type of virus.
As another embodiment for achieving the above object, the present invention provides a kit for diagnosing cabbage virus disease comprising the composition.
The kit of the present invention can be used for detecting the four kinds of viruses from a Chinese cabbage tissue sample suspected of infecting the four viruses, but not limited to, the primers of SEQ ID Nos: 1 to 8, One or more other component compositions, solutions or devices suitable for use in the present invention may be included.
As a specific example, the kit for diagnosing cabbage virus disease of the present invention may be a kit containing essential elements necessary for performing PCR analysis or multiplex-PCR analysis. The kit may further comprise an enzyme such as a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varying), deoxynucleotides (dNTPs), Taq polymerase in addition to the respective primer pairs specific for each of the polynucleotides , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like.
In particular, a primer capable of amplifying the ACT1 gene, which is a gene expressed in Chinese cabbage, may be further included to confirm whether or not the gene has been normally extracted from each virus.
In another aspect of the present invention, the present invention provides a method for diagnosing cabbage virus disease using the composition or kit.
Specifically, the method for diagnosing the Chinese cabbage virus disease of the present invention comprises the steps of (a) detecting the viruses of (a) turnip mosaic virus (TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CMV), cauliflower mosaic virus Obtaining cDNA or gDNA from a tissue sample of a Chinese cabbage suspected of infecting a virus selected from the group consisting of: (b) obtaining a PCR amplification product from the cDNA or gDNA obtained using the composition or kit; And (c) analyzing the obtained amplification product to determine the infected virus. At this time, the cDNA can be obtained by reverse transcribing RNA from a tissue sample of the Chinese cabbage, and the gDNA can be obtained by directly extracting from a tissue sample of the Chinese cabbage. In addition, the composition and kit are the same as described above.
In the above method, when a 722 bp amplification product was obtained by the primers of SEQ ID NOS: 1 and 2, it was judged that the TuMV was infected with the Chinese cabbage, and an amplification product of 1,078 bp by the primers of SEQ ID NOS: 3 and 4 When obtained, it can be determined that the Chinese cabbage has been infected with RMV, and when a 493 bp amplification product by the primers of SEQ ID NOS: 5 and 6 is obtained, it can be determined that CMV has been infected with the Chinese cabbage, When an amplification product of 1,467 bp by the primer of SEQ ID NO: 8 is obtained, it can be judged that CaMV is infected to the Chinese cabbage.
By using the composition for diagnosing cabbage virus disease according to the present invention, it is possible to accurately and simply diagnose most viral infectious diseases caused by Chinese cabbage, as well as to identify causative microorganisms of cabbage virus disease. Therefore, Which can be widely used for effective control.
Fig. 1 is a diagram showing the results of a test for Chinese cabbage samples in which turnip mosaic virus (TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CMV) and cauliflower mosaic virus (CaMV) To 10 < / RTI > primers.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example
1: Diagnosis of Chinese cabbage virus
Primer
making
The present inventors have found that the mutation of Cucumber mosaic virus (CMV) and Cauliflower mosaic virus (CMV) together with Turnip mosaic virus (TuMV) and Ribgrass mosaic virus (RMV) In order to diagnose the major viral diseases caused by Chinese cabbage, the gene sequences of the four viruses were analyzed to detect mutual interference at the time of PCR, Each primer that can be minimized was prepared as follows.
TuMV-722F: 5'-GCAGGTGAAACGCTTGATGCAGGT-3 '(SEQ ID NO: 1)
TuMV-722R: 5'-ATCTGGATGTGTGCCTCTCTCGCA-3 '(SEQ ID NO: 2)
RMV-1078F: 5'-TGGCTAGTGCCGGATTTCGTTAAA-3 '(SEQ ID NO: 3)
RMV-1078R: 5'-TGCGTAATTTGGTTTACGCCACCC-3 '(SEQ ID NO: 4)
CMV-493F: 5'-CGGATGCTAACTTTAGAGTC-3 '(SEQ ID NO: 5)
CMV-493R: 5'-TCGTCTTTTGAATACACGA-3 '(SEQ ID NO: 6)
CaMV-1467F: 5'-ATGGCCGAATCAATTTTAGACAGA-3 '(SEQ ID NO: 7)
CaMV-1467R: 5'-TCAGTCTGAGTCTGAGTCTTCAGA-3 '(SEQ ID NO: 8)
In order to confirm whether gene extraction from each virus was normally performed, a primer capable of amplifying the ACT1 gene expressed in Chinese cabbage was prepared as follows.
ACT1-F: 5'-GGCTGATGACATTCAACCAATC-3 '(SEQ ID NO: 9)
ACT1-R: 5'-TTTCCCTAGTGTTGTTGGTAGG-3 '(SEQ ID NO: 10)
Example
2: Diagnosis of Chinese cabbage virus disease
(TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CMV), and cauliflower mosaic virus (CMV) were used to confirm whether or not the cabbage virus disease could be diagnosed using the primers prepared in Example 1, Mosaic virus (CaMV) extracted genomic RNA (gRNA) or genomic DNA (gDNA) from leaves of Chinese cabbage, individually or in combination.
A total of 5 μl of the mixture was prepared by mixing 100 ng of the extracted gRNA, 10 pM of the reverse primer and sterilized water, and the prepared mixture was reacted at 70 ° C for 5 minutes to obtain a reaction product. 2.5 mM MgCl 2, 2.5 mM dNTP, reverse transcriptase (ImProm II reverse transcriptase, Promega) and an RNase inhibitor were added to the cooled reaction product, and the mixture was incubated at 37 ° C for 1 For a period of time to obtain cDNA.
PCR was carried out using all of the primers of SEQ ID NOs: 1 to 10 using the synthesized cDNA or the extracted gDNA as a template to confirm whether or not each of the viruses was detected (FIG. 1). The PCR conditions were 94 ° C for 5 minutes, 40 cycles (94 ° C for 15 seconds, 58 ° C for 15 seconds and 72 ° C for 30 seconds), and 72 ° C for 5 minutes.
Fig. 1 is a diagram showing the results of a test for Chinese cabbage samples in which turnip mosaic virus (TuMV), window plant mosaic virus (RMV), cucumber mosaic virus (CMV) and cauliflower mosaic virus (CaMV) To 10 < / RTI > primers. As shown in Fig. 1, no viruses other than ACT1, which are always expressed regardless of the virus infection, were detected in the case of the virus (no control), but each virus was infected alone or in combination , It was found that each virus - specific amplification product could be detected.
Thus, it can be seen that the primers of SEQ ID NOS: 1 to 8 provided in the present invention can be used to diagnose cabbage virus disease.
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> PCR primer for multiplex diagnosing infectious 4 virus to Chinese cabbages and method for detecting the same virus <130> KPA150150-KR <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gcaggtgaaa cgcttgatgc aggt 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atctggatgt gtgcctctct cgca 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tggctagtgc cggatttcgt taaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgcgtaattt ggtttacgcc accc 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cggatgctaa ctttagagtc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tcgtcttttg aatacacga 19 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atggccgaat caattttaga caga 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tcagtctgag tctgagtctt caga 24 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggctgatgac attcaaccaa tc 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tttccctagt gttgttggta gg 22
Claims (7)
The primer includes a primer for detecting Turnip mosaic virus (TuMV) comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NOS: 1 and 2, a primer for detecting a turnip mosaic virus (TuMV), a pollen nucleotide comprising a nucleotide sequence of SEQ ID NOS: 3 and 4 A primer for detecting a virus (Ribgrass mosaic virus, RMV), a primer for detecting a cucumber mosaic virus (CMV) composed of a polynucleotide comprising the nucleotide sequences of SEQ ID NOS: 5 and 6, and a nucleotide sequence of SEQ ID NOs: 7 and 8 And a primer for detecting cauliflower mosaic virus (CaMV) comprising a polynucleotide comprising a polynucleotide.
Wherein the cabbage virus disease is cabbage mosaic disease.
(b) obtaining a PCR amplification product from the cDNA or gDNA obtained using the composition or kit; And
(c) analyzing the resulting amplification product to determine the infected virus.
Wherein said cDNA is obtained by reverse transcription of RNA extracted from a tissue sample of Chinese cabbage, said gDNA being obtained by extracting from a tissue sample of Chinese cabbage.
In the step (c), when a 722 bp amplification product is obtained, the Chinese cabbage is judged to be infected with TuMV, and when an amplification product of 1,078 bp is obtained, it is judged that the Chinese cabbage is infected with RMV and a 493 bp amplification product Wherein said cabbage is judged to be infected with CMV when it is obtained, and when an amplification product of 1,467 bp is obtained, it is judged that said cabbage is infected with CaMV.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109055620A (en) * | 2018-10-24 | 2018-12-21 | 四川农业大学 | Tri- kinds of CMV, SMV and BCMV viral primers and its detection method in soybean are detected simultaneously |
| KR20190053733A (en) | 2017-11-10 | 2019-05-20 | 대한민국(농촌진흥청장) | Primers for multiple detection of the virus in Rehmannia glutinosa and detection method by using the same |
| KR20200144909A (en) | 2019-06-19 | 2020-12-30 | 대한민국(농촌진흥청장) | Primer Set for Simultaneous detection of Three Kinds of Yam Viruses and Detecting Method Using The Same |
| CN117187439A (en) * | 2023-04-06 | 2023-12-08 | 三峡大学 | Detection primer, detection kit and detection method for paris polyphylla turnip mosaic virus |
| CN117187439B (en) * | 2023-04-06 | 2024-05-31 | 三峡大学 | Detection primer, detection kit and detection method for paris polyphylla turnip mosaic virus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100625019B1 (en) * | 2005-05-06 | 2006-09-20 | 대한민국 | DETECTING PRIMER SETS FOR TuMV RMV AND CMV INFECTING CRUCIFERAE |
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2015
- 2015-06-25 KR KR1020150090499A patent/KR101771964B1/en active IP Right Grant
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20190053733A (en) | 2017-11-10 | 2019-05-20 | 대한민국(농촌진흥청장) | Primers for multiple detection of the virus in Rehmannia glutinosa and detection method by using the same |
| CN109055620A (en) * | 2018-10-24 | 2018-12-21 | 四川农业大学 | Tri- kinds of CMV, SMV and BCMV viral primers and its detection method in soybean are detected simultaneously |
| CN109055620B (en) * | 2018-10-24 | 2021-11-16 | 四川农业大学 | Primer for simultaneously detecting CMV, SMV and BCMV in soybean and detection method thereof |
| KR20200144909A (en) | 2019-06-19 | 2020-12-30 | 대한민국(농촌진흥청장) | Primer Set for Simultaneous detection of Three Kinds of Yam Viruses and Detecting Method Using The Same |
| CN117187439A (en) * | 2023-04-06 | 2023-12-08 | 三峡大学 | Detection primer, detection kit and detection method for paris polyphylla turnip mosaic virus |
| CN117187439B (en) * | 2023-04-06 | 2024-05-31 | 三峡大学 | Detection primer, detection kit and detection method for paris polyphylla turnip mosaic virus |
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