KR20130049764A - Primer composition for loop-mediated isothermal amplification reaction for detecting curtovirus, and use thereof - Google Patents
Primer composition for loop-mediated isothermal amplification reaction for detecting curtovirus, and use thereof Download PDFInfo
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- KR20130049764A KR20130049764A KR1020120124503A KR20120124503A KR20130049764A KR 20130049764 A KR20130049764 A KR 20130049764A KR 1020120124503 A KR1020120124503 A KR 1020120124503A KR 20120124503 A KR20120124503 A KR 20120124503A KR 20130049764 A KR20130049764 A KR 20130049764A
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Abstract
Description
The present invention relates to a primer set for isothermal amplification reaction for detecting a cultovirus, a composition comprising the same, and a method for detecting a cultovirus using the composition, and more specifically four for isothermal amplification reaction for detecting a cultovirus. It relates to a primer set and a primer composition and detection method comprising the same.
Geminivirus is a representative plant virus having a single stranded DNA as a genome. The gemini virus is known to damage major economic crops domestically and globally, and is known to be mediated by insects such as white fly. In Korea, damage cases have been reported several times, especially in tomato farms in the southern region since the 2000s, causing damage to economic crops such as tomatoes, and the economic losses are increasing. Representative examples include the African cassava mosaic virus, which is known to cause great damage by infecting cassava used as a staple food in Africa, and the tomato yellow leaf curl virus reported in Korea. Etc. Geminiviruses are divided into four genus according to the structure of the genome, the type of mediator, and the host range (Begomovirus, Mastrevirus, Curtovirus, Topokucu) Topocuvirus)).
Among them, Curtovirus is known to be mediated by leaf hoppers and mainly has dicotyledonous plants such as beets, spinach, and the like. Cultoviruses also have a genome and express a total of seven viral proteins. The viruses belonging to the cultovirus were known as the various strains of the beet curly top virus (BCTV) up to 20 years ago, but since 20 years ago each of them is a biologically isolated species. Beet curly top virus (BCTV), beet severe curly top virus (BSCTV), beet mild curly top virus (BMCTV), etc. Separated by. Since then, new cultoviruses, such as spinach curly top virus, have been reported.
The cultovirus has not been reported in Korea, but a virus that caused great damage in spinach, sugar beet, etc. in the United States is a virus that can cause a big economic problem when the domestic influx. However, there is no system in Korea that can diagnose Cultovirus because it has not been reported in Korea. In this case, failure of early diagnosis can cause large-scale economic damage when Cultovirus is introduced into Korea. Therefore, if a diagnostic method is developed that can diagnose various viruses in the cultovirus at the same time, it will be able to block the domestic influx of cultovirus. Therefore, there is an urgent need to develop an efficient diagnostic method that can quickly and accurately diagnose various viruses in the genus Cultovirus that can be introduced into the country through import and export processes from the outside.
Conventional virus detection method is a series of amplification of the viral gene through a polymerase chain reaction (PCR), and then confirmed the amplified gene by electrophoresis, and finally a sequence of verifying the viral gene through sequencing Had to go through the process. Such conventional technical methods require specialized equipment such as polymerase chain reactors and skilled personnel who can operate the same. In addition, sequencing of amplification products for final identification is a process that requires high cost and high technology.
Such a virus detection method requires a complex and specialized process of amplifying a gene through a conventional polymerase chain reaction and confirming that it is a virus through sequencing of the amplified gene fragment. It takes a lot of time to perform such a series of processes, requires specialized equipment such as electrophoresis and sequencing, and it is not useful in the field that is not equipped with analytical equipment because it is not a visually identifiable detection method. As described above, in order to detect the cultovirus effectively in a short time, development of a method capable of real-time detection in the field without specialized equipment is required.
Loop-mediated isothermal amplification (LAMP) is similar to the conventional PCR method, but the conventional PCR method continuously amplifies genes by repeatedly performing three steps of denaturation, conjugation, and kidney. While it requires a change in temperature, isothermal amplification has the advantage of bonding and stretching at a fixed constant temperature. Instead of using Taq DNA polymerase, which is used in existing PCR methods, it uses Bst DNA polymerase, which has an exonuclease function, to induce degeneration of DNA double helix structure without depending on heat. Because it can. Therefore, isothermal amplification does not require a change in temperature during gene amplification, thus enabling gene amplification at a fixed temperature easily without specialized equipment.
The present invention has been made to solve the above problems in the prior art, an object of the present invention is to provide a primer composition for isothermal amplification reaction for detecting a cultovirus, and a method for detecting a cultovirus using the same.
However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
The present invention provides a primer set for isothermal amplification reaction for detecting Curtovirus consisting of SEQ ID NOs: 1 to 4.
In one embodiment of the present invention, the cultovirus is a beet curl top virus (Beet curly top virus; BCTV), beet severe curl top virus (BSCTV), and beet mild curl top virus (Beet mild) curly top virus (BMCTV).
In another aspect, the present invention provides a primer composition for isothermal amplification reaction for detecting a cultovirus (Curtovirus), including the primer set.
In one embodiment of the invention, the composition is characterized in that it further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and the reaction buffer.
The present invention also comprises the steps of extracting genomic DNA from plants; Amplifying a target sequence by isothermal amplification at 60 to 70 ° C. for 30 minutes to 2 hours using the composition according to claim 3 as a template of the genomic DNA; And it provides a cultovirus (Curtovirus) detection method comprising the step of detecting the amplification product.
In one embodiment of the present invention, the cultovirus is a beet curl top virus (Beet curly top virus; BCTV), beet severe curl top virus (BSCTV), and beet mild curl top virus (Beet mild) curly top virus (BMCTV).
In another embodiment, the step of detecting the amplification product is characterized by identifying the amplified DNA using electrophoresis or SYBR Green I.
In another embodiment of the present invention, a method for identifying DNA amplified using SYBR Green I is characterized by visual observation under natural light using SYBR Green I at a concentration of 1,000 to 10,000 times.
Due to the primer set for isothermal amplification reaction for detecting a cultovirus according to the present invention, the primer composition comprising the primer set, and a detection method using the same, it is possible to effectively detect a cultovirus from a plant sample without specialized equipment in a short time. Can be. In addition, high-density SYBR Green I can be used to quickly diagnose with the naked eye under natural light. Therefore, it is expected that the development of a diagnostic system to be introduced from overseas will enable early detection of viruses that can cause enormous damage.
1 is a diagram showing nucleotide sequence information of Cultovirus BCTV species (Species).
2 is a diagram showing nucleotide sequence information of Cultovirus BSCTV species (Species).
3 is a diagram showing nucleotide sequence information of Cultovirus BMCTV species (Species).
FIG. 4 is a diagram showing similar sequencing information of Cultovirus BCTV, BSCTV and BMCTV species and the location of universal primers for isothermal amplification.
Figure 5 shows the nucleotide sequence of the universal primer set for isothermal amplification.
6 is a view showing the results of electrophoresis of the gene amplified using each primer set.
7 is a diagram showing the results of confirming the gene amplified using each primer set using SYBR Green I.
The present inventors have completed the present invention as a result of studying a method capable of detecting in real time in the field without specialized equipment in order to effectively detect the cultovirus in a short time.
The present invention provides a primer set for isothermal amplification reaction for detecting Curtovirus consisting of SEQ ID NOs: 1 to 4.
The present inventors used loop-mediated isothermal amplification (LAMP) to detect cultovirus without specialized equipment in a short time. Unlike conventional PCR (polymerase chain reaction), isothermal amplification does not require temperature control to amplify genes, so it is possible to amplify genes without specialized equipment and to amplify high concentrations of genes in a short time. In order to utilize loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must function as one set. F3 and FIP , And B3 and BIP are primers that bind in the reverse direction in the 3 'direction. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).
The present inventors use PrimerExplorer V4 software to prepare primers that can be used to detect all Culttovirus BCTV species, BSCTV species and BMCTV species that have been reported in Korea using isothermal amplification. It was. The common parts between the three kinds of cultovirus gene sequences were searched, and primer sets were created based on the search sites (see FIG. 4). Therefore, the inventors recombined the primer sets expected to be applied to BSCTV species and BMCTV species based on the primer sets of BCTV species (see FIG. 5). Preferably, the primer set is composed of SEQ ID NOs: 1 to 4, but is not limited thereto as long as it is an isothermal amplification primer set capable of diagnosing a cultovirus.
The present invention also comprises the steps of extracting genomic DNA from plants; Using the above genomic DNA as a template, the isothermal amplification reaction was carried out at 60 to 70 ° C for 30 minutes to 2 hours using a composition containing a primer set, a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer to obtain a target sequence Amplifying; And it provides a cultovirus (Curtovirus) detection method comprising the step of detecting the amplification product. Although DNA can be extracted directly from a plant, it can also be applied to clinical specimens and cultured cells for detection.
In an embodiment of the present invention, it was confirmed that both BCTV species, BSCTV species and BMCTV species were detected by performing isothermal amplification using a universal primer set. When used at a concentration of 1,000 to 10,000 times, it was confirmed that the naked eye can be detected under natural light (see Example 7).
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[ Example ]
Example One. Cultovirus Gene collection
Since the virus belonging to the Cultovirus has not been reported in Korea, plant samples infected with Cultovirus can not be obtained in Korea. Therefore, the experiments were conducted using samples of Arabidopsis thaliana infected with bitcolitope virus, bitcivircolitope virus, and bitmildcolitope virus through the artificial infection system prepared by the inventors of the present invention. It is identical to the condition of infected plants that can be obtained in the field.
The isothermal amplification primers used in the present invention are three viruses (bits) in the cultovirus previously reported from Genbank, a biological nucleic acid information database provided by the National Center for Biotechnology Information (NCBI). Sequence information (Beebank curly top virus (BCTV), beet severe curly top virus (BSCTV), beet mild curly top virus (BMCTV) GenBank accession number: M24597.2, U02311.1, U56975.1) were analyzed to focus on the part containing similar base sequence.
Example 2. primer write
Primers were prepared using PrimerExplorer V4 to detect Curtovirus through loop-mediated isothermal amplification (LAMP). In order to use isothermal amplification, four primers (F3, B3, FIP, and BIP) must act as a set. In order to detect all known types of cultoviruses, a common part of three cultovirus gene sequences is used. A primer set prepared by searching and primed using PrimerExplorer V4 is shown in FIG. 5. Therefore, the inventors recombined the primer sets expected to be applied to BSCTV species and BMCTV species based on the primer sets of BCTV species (see FIG. 5).
As shown in FIG. 5, a specific primer sequence of the F3 and BIP primers of BCTV species was changed to prepare a primer set expected to be applied to all three types of viruses.
Example 3. Collection of plant specimens
Plant samples were collected to confirm that the primer set prepared in Example 2 can be used to detect cultovirus. In the present invention, gDNA was isolated from virus-infected plant samples using the Delaporta's method, which is used to quickly and easily separate genomic DNA (gDNA) from plants. An extraction buffer for gDNA separation was prepared in 50 mM Tris-HCl, pH8.0, 10 mM EDTA, pH8.0, 10 mM NaCl, 1% SDS. After blocking the plant tissue, put it in a bowl, put liquid nitrogen, and when nitrogen evaporates, the tissue is ground to a fine powder immediately and the tissue is extracted using a spatula, which is cooled in liquid nitrogen, in advance. Transferred to. After the powdered tissue was added, the tube cap was closed and shaken vigorously 7-8 times, followed by reaction at 65 ° C. for at least 10 minutes. After the reaction, 166ul of 5M potassium acetate solution was added, the cap was closed and vigorously mixed, followed by centrifugation at 4 ° C. and 8,000 × g for 10 minutes. The supernatant was collected using Pipette, filtered with miracloth, and placed in a tube containing the same amount of isopropanol as the supernatant. Close the lid and shake gently several times to precipitate the DNA, and centrifuged at 8,000 × g for 10 minutes to precipitate the DNA. The pellet was washed with 70% ethanol solution, the tube was left upside down on a paper towel to dry, and the DNA was dissolved in 50ul of sterile water or elution buffer (TE buffer).
Example 4. Isothermal amplification and validation
In order to confirm whether the primer set prepared in Example 2 can be used to detect a cultovirus, a primer composition for amplification reaction was prepared. 10uL (10X) Bst polymerase reaction buffer (20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 2mM MgSO 4 , 0.1% Triton X-100 ), 1.6ul of 10mM dNTPs (mixture of 10mM each of dATP, dTTP, dGTP, dCTP), 0.4ul of 10uM F3 and B3 primer, 1.6ul of 10uM FIP and BIP primer, 1ul of 20mM MgSO 4 , 1ul (8 Unit ) Bst polymerase, 1 ul of template genomic DNA, and 11.5 ul of distilled water were added to the reaction tube, followed by mixing. The prepared amplification reaction composition was subjected to isothermal amplification by reacting in a 65 ° C. reaction vessel for 1
In addition, the present inventors performed the above experiment with the reaction temperature of 62 ° C. and the same conditions.
As shown in FIG. 6, when the three universal primer sets were used, the cultovirus gene was not amplified in the first (lane 2 to lane 5) and the second primer set (lane 6 to lane 9), but in the third primer set. In the non-infected strain (lane 10), the gene was not amplified, whereas the BCTV species (lane 11), BMCTV species (lane 12) and BSCTV species (lane 13) were all used as a template was confirmed that the gene was amplified.
In addition, as shown in FIG. 7, even when stained with a high concentration of SYBR Green I and amplified gene (universal primer set sample), the gene is amplified in BCTV, BMCTV and BSCTV-infected strains to have a green color under natural light. I could confirm that.
Through the above results, it was confirmed that the primer sets of SEQ ID NOs: 1 to 4 can be used to detect all BCTV species, BMCTV species, and BSCTV species. Also, when SYBR GreenI was concentrated to 10,000 times, it was confirmed visually under natural light. Could confirm.
The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
<110> SUNGKYUNKWAN UNIVERSITY FOUNDATION FOR CORPORATE <120> Primer composition for loop-mediated isothermal amplification reaction for detecting Curtovirus, and use <130> PB12-11046 <150> KR 2011/0114821 <151> 2011-11-04 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Universal Curtovirus F3 primer <400> 1 aatcctcaaa gtgcktgg 18 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Universal Curtovirus B3 primer <400> 2 ccctggacat aattattcaa cat 23 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Universal Curtovirus FIP primer <400> 3 ttgtcacagg tctcctccat tccgaagaag aggaggacta 40 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Universal Curtovirus BIP primer <400> 4 aacaggactc ygaagttaaa gatgtaccat tattactaat ggtagatcct 50
Claims (8)
The cultovirus consists of beet curly top virus (BCTV), beet severe curly top virus (BSCTV), and beet mild curly top virus (BMCTV). A primer set, characterized in that selected from the group.
The composition is characterized in that it further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and the reaction buffer.
Amplifying a target sequence by isothermal amplification at 60 ° C. to 70 ° C. for 30 minutes to 2 hours using the composition according to claim 3 as a template of the gDNA; And
Curtovirus detection method comprising the step of detecting the amplified product.
The cultovirus consists of beet curly top virus (BCTV), beet severe curly top virus (BSCTV), and beet mild curly top virus (BMCTV). The detection method, characterized in that selected from the group.
The detecting of the amplified product is characterized in that for identifying the amplified DNA using electrophoresis or SYBR Green I.
The method for identifying the DNA amplified using the SYBR Green I, characterized in that the observation using the SYBR Green I in a concentration of 1,000 to 10,000 times with natural light under the naked eye.
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