KR20140086234A - Primer composition for loop-mediated isothermal amplification reaction for detecting Cucumber Mosaic Virus, and use thereof - Google Patents

Abstract

The present invention relates to a set consisting of four primers for isothermal amplification reaction for detecting cucumber mosaic virus, a composition comprising the same, and a method for detecting cucumber mosaic virus using the composition. By using the primer set of the present invention, it is possible to effectively detect all five mutant strains of the cucumber mosaic virus in a short time without professional equipments by using the isothermal amplification method. In addition, SYBR Green I can be diagnosed quickly under natural light under high light intensity. Therefore, it is expected that viruses that cause enormous damage to cultivators of crops such as cucumber, watermelon, and red pepper can be detected early, and a more rapid and efficient cucumber mosaic virus diagnosis system can be constructed.

Description

A primer composition for use in isothermal amplification reaction for detecting cucumber mosaic virus, and a primer composition for use in the cucumber mosaic virus,

The present invention relates to a set consisting of four primers for isothermal amplification reaction for detecting cucumber mosaic virus, a composition comprising the same, and a method for detecting cucumber mosaic virus using the composition.

Cucumber mosaic virus is a virus that infects lily, asteraceae, bacillus, and the like, and is a virus that contains foreign RNA in spherical particles having a diameter of 28 nm.

Cucumber mosaic viruses are very broad, with 39 or 117 species of hosts known to date, and weed or crops are widely distributed, so there are infectious sources everywhere. It is also transmitted non-exclusively by more than 80 species of aphids. It is very susceptible to infection by contact during the work because of the easy transmission of juice. There is no soil infection or seed transmission, but it is possible to spread the graft. Aphids are widely distributed throughout Korea, and in particular, they are susceptible to rapid spread by causing more than 80% of infections caused by peach aphids and cotton aphids. In Korea, silver or vinyl tape is installed in houses or open fields to prevent aphid deposits. However, since there is no treatment after the onset of the disease, there is no way to treat only the infected objects. The main host plants are the major crops such as tomatoes, eggplant, pepper, cucumber, melon, melon and lettuce, so the damage is great.

On the other hand, an electron microscope or a serological method was mainly used as a conventional virus diagnosis method. Electron microscopy can detect the presence of virus but it is almost impossible to diagnose the species as a morphological feature. Among the serological methods, ELISA (Enzyme-Linked Immunosorbent Assay) is about 1,000 times lower than the most commonly used diagnostic methods or Polymerase Chain Reaction (PCR) It is often the case that accurate diagnoses fail due to unexpected nonspecific reactions. In recent years, PCR methods with high detection sensitivity and convenience have been widely used for the diagnosis of viruses. However, it is very important to develop specific primers for the diagnostic method using PCR. It must be confirmed by electrophoresis and finally subjected to a sequence of DNA sequencing. In addition, this method requires specialized equipment such as a thermocycler and a professional manpower to operate it, and sequencing of the amplification product for final confirmation is a process requiring high cost and high technology. In addition, this process is time-consuming to perform and is not visually detectable. Therefore, the ability to use analytical equipment in the field is significantly reduced. In order to effectively detect the virus in such a short time, development of a method capable of real-time detection on the spot without professional equipments is required.

The isothermal amplification method (LAMP) is similar to the conventional PCR method, but the conventional PCR method repeatedly performs three steps of denaturation, splicing, and elongation to amplify the gene, The isothermal amplification method has the advantage of being able to bond and stretch at a fixed temperature. This is because the Taq DNA polymerase ( Taq DNA It is because the polymerase) may result in place of the nucleic acid ends hydrolysis (denaturation of the double helix of DNA without depending exonuclease) by using the Bst DNA polymerase (Bst DNA polymerase) that has a function that uses the heat. Therefore, isothermal amplification does not require a temperature change during amplification of the gene, which makes gene amplification possible at fixed temperature with no special equipment.

The inventors of the present invention have developed a primer set for isothermal amplification reaction that can detect various species of cucumber mosaic virus without any specialized equipment in the field as a result of studying for easy detection of cucumber mosaic virus which is a big problem in a farmhouse Thereby completing the present invention.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object thereof is to provide a primer composition for isothermal amplification reaction for detecting cucumber mosaic virus and a detection method using the same.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can 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 Cucumber mosaic virus (CMV) comprising SEQ ID NOS: 1 to 4.

In one embodiment of the present invention, the cucumber mosaic virus is selected from the group consisting of GenBank accession numbers AB079891.1, AB369269.1, JQ612526.1, JN054635.1, and AF103992.1 strain . Since the primer of the present invention is designed for a region having a very small variation, it is theoretically detectable in almost all variants.

The present invention provides a primer composition for isothermal amplification reaction for detecting Cucumber mosaic virus (CMV) comprising the above primer set.

In one embodiment of the present invention, the composition further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer. However, it may further include a configuration necessary for using the primer of the present invention for detection.

The present invention

Extracting total RNA from the plant;

Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA);

Amplifying the target sequence by performing an isothermal amplification reaction at 60 ° C to 65 ° C for 30 minutes to 2 hours using the cDNA as a template and a composition comprising the primer; And

And detecting the amplified product. ≪ RTI ID = 0.0 > Cucumber < / RTI & mosaic virus , CMV) detection method.

In one embodiment of the present invention, the cucumber mosaic virus is selected from the group consisting of GenBank accession numbers AB079891.1, AB369269.1, JQ612526.1, JN054635.1, and AF103992.1 strain. .

The temperature for carrying out the isothermal amplification reaction of the present invention is most preferably 62 ° C.

Examples of plants that can be infected with the cucumber mosaic virus include, but are not limited to, pepper, tomato, spinach, and the like.

In another embodiment of the present invention, the step of detecting the amplification product is characterized by confirming 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.

Using the primer set for isothermal amplification reaction for detecting cucumber mosaic virus according to the present invention, the composition including the primer set, and the detection method using the primer set, the cucumber mosaic virus can be effectively detected from the plant sample in a short time without professional equipments have. In addition, SYBR Green I can be diagnosed quickly under natural light under high light intensity. Therefore, it is possible to detect a virus that can cause enormous damage to crop farmers such as watermelon, cucumber, and red pepper in an early stage, so that a quick and efficient cucumber mosaic virus diagnosis system can be constructed and economic loss It is anticipated that it will be possible to prevent it.

Figure 1 shows the sequence of the coat protein of CMV used in the preparation of the primer of the present invention.
FIG. 2 is a diagram showing a primer set prepared by using PrimerExplorer V4 based on a search region and a common portion between five types of CMV gene sequences.
3 is a diagram showing the nucleotide sequence of the primer set of the present invention.
FIG. 4 is a graph showing the result of electrophoresis of the amplified gene using the primer set of the present invention. FIG.
FIG. 5 is a diagram showing the result of checking the gene amplified using the primer set of the present invention under a natural light source using SYBR Green I. FIG.
FIG. 6 is a graph showing the result of checking the amplification product in FIG. 4 under a UV light source.

The present inventors have completed the present invention by studying a method for detecting cucumber mosaic virus in real time in a field without professional equipment in order to effectively detect cucumber mosaic virus in a short time.

The present invention provides a primer set for isothermal amplification reaction for detecting cucumber mosaic virus consisting of SEQ ID NOS: 1 to 4. The primer set of the present invention was prepared using a sequence of a coat protein (virion protein) as a template rather than a whole viral sequence. Coat protein is a protein that is essential for the virus to be made into an individual. It is known that some viruses are involved in the movement of viruses within the plant. That is, coat protein is used as a template because it is an unconditionally produced protein if the virus is infected. The genbank number of variants detectable by the present invention represents the full sequence of the virus variant. Sequences of the coat protein coding region of each mutant actually used in the preparation of the primer of the present invention are attached to the sequence listing.

The present inventors used loop-mediated isothermal amplification (LAMP) to detect cucumber mosaic virus in a short time without professional equipments. Unlike conventional polymerase chain reaction (PCR), the isothermal amplification method does not require temperature control to amplify the gene, so it can amplify the gene without specialized equipments and it is possible to amplify the gene at a high concentration in a short time. In order to use loop-mediated isothermal amplification (LAMP), four primers (F3, B3, FIP, and BIP) must act as one set, and F3 and FIP bind in five directions Primer, and B3 and BIP are primers that bind in the reverse direction in three directions. In addition, FIP and BIP are primers that contain the nucleotide sequence of F2 (or B2) and F1c (or B1c).

The present invention also relates to a method for producing a plant, comprising extracting total RNA (total RNA) from a plant; Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA); Isothermal amplification reaction was carried out at 60 ° C to 65 ° C for 30 minutes to 2 hours using the above cDNA as a template using a primer set of the present invention, a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer Amplifying the target sequence; And detecting the amplified product. The present invention also provides a method for detecting cucumber mosaic virus. DNA can be extracted directly from red pepper suspected of being infected, but it can also be applied to specimens and cultured cells that have been artificially infected for research purposes.

In one embodiment of the present invention, it has been confirmed that cucumber mosaic virus can be detected by performing isothermal amplification using the universal primer set of the present invention. When SYBR Green I is used at a concentration of 1,000 to 10,000 times, electrophoresis (See Example 4), under natural light, without going through the steps of FIG.

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  1. Cucumber Mosaic Virus Gene Collection

From the Rural Development Administration, cucumber mosaic virus ( Cucumber mosaic virus , CMV) naturally infected red pepper ( Capsicum I have an annuum . angulosum) was taken and the experiment was carried out.

The primers for isothermal amplification used in the present invention were prepared from 5 strains of CMV reported previously from Genbank, a biological nucleic acid information database provided by the National Center for Biotechnology Information (NCBI) And the nucleotide sequence information (GenBank accession number: AB079891.1, AB369269.1, JQ612526.1, JN054635.1, AF103992.1). Respectively.

Example  2. primer  write

To detect CMV by loop-mediated isothermal amplification (LAMP), a primer was prepared using PrimerExplorer V4. In order to use the isothermal amplification method, four primers (F3, B3, FIP, and BIP) should act as one set. In order to detect all kinds of known CMVs, a common part between five kinds of CMV gene sequences is searched Primer was prepared using PrimerExplorer V4 mainly at the site of detection (Fig. 2). As shown in Fig. 3, a universal primer set which is expected to be applied to all viruses of CMV virus was prepared.

Example  3. Collection of plant specimens

Plant samples were collected to confirm that the primer set produced in Example 2 could be used to detect CMV. In the present invention, total RNA was isolated from virus-infected plant samples and then total cDNA was synthesized by reverse transcription.

The extraction buffer used for total RNA isolation was TRI REAGENT (catalog number: TR 118) from MRC. The plant tissue is placed in a mortar and the liquid nitrogen is added, and when the nitrogen is evaporated, the tissue is grinded to a fine powder and the tissue is extracted with 1 ml of extraction buffer using a spatula preliminarily cooled in liquid nitrogen Transferred to a tube. After putting the powdered tissue into the tube, the tube lid was closed and reacted at 4 ° C for 5 minutes. Then, 200 μl of chloroform was added thereto, followed by vigorous shaking for 15 seconds, followed by reaction at room temperature for 10 minutes or more. After reacting, centrifuge at 8,000 × g at 4 ° C for 10 minutes. Collect 500 μl of the supernatant using a pipette, carefully transfer it to a new tube, and add the same amount of isopropanol to the supernatant. The reaction was allowed to proceed at room temperature for 10 minutes. The reaction tubes were centrifuged at 4 ° C and 8,000 × g for 10 minutes to precipitate RNA. The pellet was washed with a mixture of ethanol and DEPC treated water (0.1% diethyl pyrocarbonate aqueous solution) in a ratio of 7.5: 2.5, and centrifuged at 4 ° C and 8,000 × g for 10 minutes. After removing the tube, the tube was placed upside down on the test tube holder, and the RNA pellet was dissolved in 30 μl of DEPC treated water. The concentration of RNA extracted through a spectrophotometer was measured.

Reverse transcription was performed to synthesize cDNA based on total RNA obtained from the above experiment. Reverse transcription was performed using M-MLV Reverse Transcriptase (BIONEER) and random primers according to the manufacturer's instructions. RT-PCR was performed by a 3-step method. The tubes containing total RNA and random primer were reacted at 70 ° C for 10 minutes, and then the temperature was lowered to 4 ° C. After adding dNTP and buffer, After 10 minutes of reaction, the temperature was lowered to 4 ° C, and the reaction was allowed to proceed at 37 ° C for 1 hour, followed by 10 minutes of enzyme inhibition at 70 ° C.

Example  4. Isothermal amplification and validation

To confirm that the primer set prepared in Example 2 was usable for detecting CMV, a primer composition for amplification reaction was prepared.

For the preparation of the primer composition, 2 uL of 10X (10X) Bst 10 mM dNTPs (dATP, 10 mM Tris-HCl, 10 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X- dTTP, dGTP, dCTP mixture was mixed by 10 mM _{each), 20 mM MgSO 4, 1} ul of 0.4 ul of 10 uM F3 and B3 primer, 1.6 ul of 10 uM FIP and BIP primer, 1 ul (8 Unit) Bst polymerase , 1 μl of 1/10 diluted template cDNA, and 11.5 μl of distilled water were added to the reaction tube and mixed. The prepared amplification reaction composition was reacted at 40 ° C. for 30 seconds and reacted in a reaction vessel at 62 ° C. for 1 hour and 30 minutes to perform isothermal amplification. After the reaction was completed, enzyme activity was inhibited at 80 DEG C for 5 minutes. 5 μl of the total 20 μl reaction was electrophoresed to confirm that the gene was amplified. The results are shown in Fig. As shown in Figure 4, the universal primer set 3 as the template of plants with CMV virus different in the case of using (turnip mosaic virus sulfide (Turnip yellow mosaic virus ; TYMV), fake virus 2 ( Broad bean wilt virus 2; BBWV2), sugar beet sulfide virus ( Beet western yellows virus ; BWYV) was performed by isothermal amplification method to detect only CMV. In the absence of viral gene (lane 5), the gene was not amplified. When CMV (lane 4) was used as a template, the CMV gene was amplified, but the gene was not amplified in other types of viruses (lane 1, 2, 3).

Then, SYBR Green I concentrated to 1,000 times with the same reagent was added at a rate of 1 μl per 20 μl of the reaction, and the color development was confirmed by natural light. The results are shown in FIG. 5, and the result of color development reaction in ultraviolet light is shown in FIG. 6 . The significance was verified by comparing the results of the electrophoresis of SYBR Green I with the results of amplification of the viral gene. As shown in FIG. 5, even when the SYBR Green I was concentrated at a high concentration and the amplified gene (universal primer set sample) was stained, it was confirmed that the gene was amplified in the CMV infected strain and greens green under natural light. Samples infected with other viruses were found not to be green. As shown in Fig. 6, the result of coloring could be confirmed by green fluorescence on ultraviolet light.

From the above results, it was confirmed that the primer set used in this experiment can be used to specifically detect only CMV. In addition, when SYBR Green I is concentrated at 10,000 times, it is confirmed that the primer set can be visually observed under natural light I could.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the embodiments described above are in all respects illustrative and not restrictive.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Primer composition for loop-mediated isothermal amplification          reaction for detecting Cucumber Mosaic Virus, and use thereof <130> PB12-11093 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 657 <212> RNA <213> coat protein coding region of CMV AB079891.1 strain <400> 1 atggacaaat ctgaatcaac cagtgctggt cgtagccgtc gacgtcgtcc gcgtcgtggt 60 tcccgctccg cttcctcctc cgcggatgct aactttagag tcttgtcgca gcagctttcg 120 cgacttaata agacgttagc agctggtcgt ccaactatta accacccaac ctttgtaggg 180 agtgagcgtt gtaaacctgg atacacgttc acatctatta ccctaaagcc accaaaaata 240 gaccgcgggt cttattatgg taaaaggttg ttattacctg attcagtcac ggaatatgat 300 aagaagcttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cagtccgtaa agttcctgcc tcctcggact tatccgttgc cgctatctca 420 gctatgtttg cggacggagc ctcaccggta ctggtttatc agtatgctgc atctggagtc 480 caagctaaca acaaattgtt gtatgatctt tcggcgatgc gcgctgatat aggcgacatg 540 agaaagtacg ccgtcctcgt gtactctaaa gacgatgcac tcgagatgga cgagctagta 600 cttcatgttg acgtcgagca ccaacgcatt cccacgtctg gggtgctccc agtctga 657 <210> 2 <211> 657 <212> RNA <213> coat protein coding region of CMV AB369269.1 strain <400> 2 atggacaaat ctgaatcaac cagtgctggt cgtaaccgtc gacgtcgtcc gcgtcgtggt 60 tcccgctccg ccccctcctc ctcggatgct aactttagag tcttgtcgca gcagctttcg 120 cgacttaaca agacgttagc agctggtcgt ccaactatta accacccaac ctttgtaggg 180 agtgagcgct gtaaacctgg atacacgttc acatctatta ccctaaagcc accaaaaata 240 gaccgcgggt cttattatgg taaaaggttg ttattacctg attcagtcac ggaatatgat 300 aagaaacttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cagtccgtaa agttcctgcc tcctcggact tatccgttgc cgccatctct 420 gctatgtttg cggacggagc ctcaccggta ctggtttatc agtatgctgc atctggagtc 480 caagctaaca acaaattgtt gtatgatctt tcggcgatgc gcgctgatat aggcgacatg 540 agaaagtacg ccgtcctcgt gtattcaaaa gacgatgcgc tcgagacgga cgagttggta 600 cttcatgttg acgtcgagca ccaacgcatt cccacgtctg gggtgctccc agtctga 657 <210> 3 <211> 657 <212> RNA <213> coat protein coding region of CMV JQ612526.1 strain <400> 3 atggacaaat ctgaatcaac cagtgctggt cgtaaccgtc gacgtcgtcc gcgtcgtggt 60 tcccgctccg cttcctcctc cgcggatgcc aactttagag tcctgtcgca gcaactttcg 120 cgacttaata agacgttggc agctggtcgt cctaccctta accacccaac ctttgtgggg 180 agtgaacgtt gtaaacctgg gtacacgttc tcatctatta ccctgaagcc accgaaaata 240 gaccgcgggt cttattatgg taaaaggttg ttactacctg attcagtcac ggaattcgat 300 aagaagcttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cggtccgtaa agttcctgcc tcctcggacc tgtccgtcgc cgccatctct 420 gctgtgtttg cggacggagc ctcaccggta ctggtttatc agtatgctgc atccggagtc 480 caagccaaca ataaattgtt gtatgatctt tcggcgatgc gcgctgatat tggcgacatg 540 cgaaagtacg ccgttctcgt gtattcaaaa gacgatgctc tcgagacgga tgagttagta 600 cttcatgttg acatcgagca ccaacgcatt cccacatctg gggtgctacc agtctga 657 <210> 4 <211> 657 <212> RNA <213> coat protein coding region of CMV JN054635.1 strain <400> 4 atggacaaat ctgaatcaac cagtgccggt cgtaatcgtc gacgtcgtcc gcgtcgcggt 60 tcccgctccg cttcctcctc cgcggatgct acatttagag tcctgtcgca acagctttcg 120 cgacttaata agacgttagc agctggtcgt cctactatta accacccaac ctttgtgggt 180 agtgatcgtt gtaaacctgg gtacacgttc acctcgatta ccctgaagcc accgaaaata 240 gacaaaggtt cttattatgg caaaaggttg ttacttcctg attcagtcac tgagttcgat 300 aagaagcttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cggtccgtaa agttcctgcc tcctcggacc tgtccgtttc cgccatctct 420 gctatgttcg cggacggagc ctcaccggta ctggtttatc agtacgctgc atctggcgtt 480 caagccaaca acaaattgtt gtatgatctt tcagtggtgc gcgctgatat tggtgacatg 540 agaaagtacg ccgtgctcgt gtattcaaaa gacgatgcgc tcgagacgga tgagctagta 600 cttcatgttg acatcgagca ccaacgcatt cccacgtctg gggtgctccc agtttga 657 <210> 5 <211> 657 <212> RNA <213> coat protein coding region of CMV AF103992.1 strain <400> 5 atggacaaat ctgaatcaac cagtgctggt cgtaaccgtc gacgtcgtcc gcgtcgtggt 60 tcccgctccg ccccctcctc cgcggatgcc aactttagag tcttgtcgca gcaactttcg 120 cgactcaata agacgttgtc agctggtcgt ccaactatta accacccaac ctttgtaggg 180 agtgaacgct gtaaacctgg atacacgttc acatctatta ccctaaagcc accaaaaata 240 gaccgcgggt cttattatgg taaaaggttg ttattacctg attcagtcac agaatatgat 300 aagaaacttg tttcgcgcat tcaaattcga gttaatcctt tgccgaaatt tgattctacc 360 gtgtgggtga cagtccgtaa agttcctgcc tcctcggact tatccgttgc cgccatctct 420 gctatgtttg cggacggagc ctcaccggta ctggtttatc agtatgctgc atctggagtc 480 caagctaaca acaaattgtt gtatgatctt tcggcgatgc gcgctgatat aggcgacatg 540 agaaagtacg ccgtcctcgt gtattcaaaa gacgatgcgc tcgagacgga cgagctggta 600 cttcatgttg acgtcgagca ccaacgcatt cccacgtctg gggtgctccc agtctga 657

Claims (8)

Cucumber mosaic virus ( Cucumber) , consisting of SEQ ID NOS: 1 to 4 A primer set for isothermal amplification reaction for detecting mosaic virus (CMV).
The method according to claim 1,
The cucumber mosaic virus is selected from the group consisting of GenBank accession numbers AB079891.1, AB369269.1, JQ612526.1, JN054635.1, and AF103992.1 strain.
A cucumber mosaic virus ( Cucumber) , comprising the primer set of claim 1 mosaic virus , CMV).
The method of claim 3,
Characterized in that the composition further comprises a DNA polymerase for isothermal amplification reaction, dNTPs, and a reaction buffer.
Extracting total RNA from the plant;
Performing a reverse transcription reaction using the whole RNA as a template to synthesize a total cDNA (total cDNA);
Amplifying the target sequence by performing the isothermal amplification reaction at 60 ° C to 65 ° C for 30 minutes to 2 hours using the cDNA according to the third aspect of the present invention as a template; And
And detecting the amplified product. &Lt; RTI ID = 0.0 &gt; Cucumber &lt; / RTI & mosaic virus , CMV) detection method.
6. The method of claim 5,
Wherein the cucumber mosaic virus is selected from the group consisting of GenBank accession numbers AB079891.1, AB369269.1, JQ612526.1, JN054635.1, and AF103992.1 strain.
6. The method of claim 5,
Wherein the step of detecting the amplification product comprises the step of detecting amplified DNA using electrophoresis or SYBR Green I.
8. The method of claim 7,
The method for identifying the amplified DNA 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.

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