KR102057077B1 - Primer composition for detecting living modified fluorescent aquarium fish and method of detecting living modified fluorescent aquarium fish by using the same - Google Patents

Abstract

The present invention, as a composition for detecting fluorescently labeled genes of genetically modified fluorescent ornamental fish, provides a primer composition including primer sets each corresponding to a plurality of fluorescently labeled genes expressing different fluorescent colors, and being capable of determining genetically modified fluorescent ornamental fish by multiplex PCR. The primer composition can determine various genetically modified fluorescent ornamental fish rapidly with high sensitivity, and maximize the convenience of use as a dried premix type composition.

Description

Primer COMPOSITION FOR DETECTING LIVING MODIFIED FLUORESCENT AQUARIUM FISH AND METHOD OF DETECTING LIVING MODIFIED FLUORESCENT AQUARIUM FISH BY USING THE SAME}

The present technology relates to a technology for monitoring genetically modified organisms, and specifically, a primer composition for effectively discriminating genetically modified (LM) fluorescent ornamental fishes expressing various fluorescent colors by a single polymerase chain reaction (PCR) process. And a method for identifying a genetically modified fluorescent ornamental fish using the same.

Recently, research on monitoring genetically modified organisms in the marine and fisheries sector including land and land has been actively conducted, and fish and microalgae transgenic organisms have been approved by FDA or patented and can be detected as the possibility of inflow is very high. The need for developing kits is increasing.

Development of genetically modified fish began in the mid-1980s, and in the 2000s, LM ornamental fishes with fluorescent red, green and orange colors were developed and sold in Taiwan and the United States. Recently, the types of purple and pink are increasing, so it is urgent to develop technology to strengthen border inspection. Further, the conventional method for detecting gene polymerase reaction can detect only one gene at a time and has limitations in terms of sensitivity, speed, and convenience.

An object of the present invention is to provide a primer composition for genetically modified fluorescent ornamental fish which can efficiently and quickly determine living modified fluorescent ornamental fish expressing various colors by multiplex PCR with only a kind of detection kit. .

It is another object of the present invention to provide a method for rapidly determining genetically modified fluorescent ornamental fish by performing multiplex PCR using the above primer composition.

The primer composition for genetically engineered fluorescent ornamental fish in one embodiment of the present invention is a composition for detecting the fluorescently labeled gene of the genetically modified fluorescent ornamental fish, each primer set corresponding to a plurality of fluorescently labeled genes expressing different fluorescent colors It includes, can be determined by multiplexing PCR whether or not genetically modified fluorescent ornamental fish.

The primer composition may be a dry type composition comprising a primer in the form of a dry matter.

The primer set may include a first primer set for detecting a fluorescent label gene expressing an orange-based fluorescent color; A second primer set for detecting a fluorescently labeled gene expressing green fluorescent color; A third primer set for detecting a fluorescently labeled gene expressing a purple or pink series of fluorescent colors; A fourth primer set for detecting a fluorescently labeled gene expressing a blue fluorescent color; A fifth primer set for detecting a fluorescently labeled gene expressing red fluorescent color; And a sixth primer set (IPC) for detecting internal positive control (IPC).

Meanwhile, each of the primer sets includes forward and reverse primers, and the first primer set includes primers of SEQ ID NO: 1 and primers of SEQ ID NO: 2, and the second primer set includes primers of SEQ ID NO: 3 and SEQ ID NO: A primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 6, wherein the third primer set comprises a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8; The fifth primer set comprises primers of SEQ ID NO: 9 and primers of SEQ ID NO: 10, and the sixth primer set comprises primers of SEQ ID NO: 11 and primers of SEQ ID NO: 12.

Method for determining a genetically modified fluorescent ornamental fish according to an embodiment of the present invention comprises the steps of collecting a DNA sample from the fluorescent ornamental fish; Mixing the DNA sample and the primer composition for discriminating the genetically modified fluorescent ornamental fish; And performing and analyzing the multiplexing PCR.

On the other hand, the annealing temperature of the PCR is preferably 61 to 63 ℃.

The primer composition of the present invention can easily determine whether or not genetic modification by a single multiplexing PCR detection for a variety of fluorescent ornamental fish.

In addition, the primer composition consists of a dry type premix (premix) form can serve as a detection kit for maximum ease of use, not only each fluorescent gene but also a positive control group (internal positive control (IPC) at a time) Can be detected.

Furthermore, the primer composition of the present invention is capable of precise gene detection capable of identifying fish genes and distinguishing fluorescent genes at the same time as at least 10 ng of samples.

1 is a diagram showing the results of electrophoretic analysis and primers selected according to the primary set for each primer set candidate group for fluorescence gene detection.
Figure 2 shows the results of further electrophoresis analysis and the optimum annealing temperature for the primer set candidate group for primary fluorescent fluorescent detection.
3 is a view showing the electrophoresis results of the IPC primer candidate set and the final selected IPC primer set through it.
4 is a view showing the results of electrophoresis according to the primer mixing concentration.
5 is a view showing the results of electrophoresis according to the annealing time at 62 ℃ temperature.
6 is a view showing the results of electrophoresis according to extension time under a temperature of 72 ℃.
FIG. 7 is a diagram showing electrophoresis results of four test group mixtures having different concentration ratios of a target primer and an IPC primer.
8 is an electrophoresis result showing the performance change before and after drying for the premix prototype.
9 is a diagram showing the results of electrophoresis according to three batches for the prototype.
10 shows electrophoresis results for PC products and positive markers.
FIG. 11 shows electrophoresis results for positive markers in three different configurations.
12 is a diagram showing electrophoresis results for validating positive markers.

Hereinafter, the primer composition for genetically modified fluorescent ornamental fish discrimination of the present invention and the genetically modified fluorescent ornamental fish discrimination method using the same will be described in detail with reference to the accompanying drawings and various experiments. However, the following descriptions are intended to explain the technical idea of the present invention, and the technical idea of the present invention is not limited by the following description or experiment. The spirit of the present invention can be interpreted or limited only by the claims that follow.

Fluorescent gene information

In order to design a composition for determining a genetically modified fluorescent ornamental fish according to an embodiment of the present invention, each of the fluorescent gene information should be confirmed first. Table 1 below is a list of standard materials of identified fluorescent genes. As shown in Table 1, 17 kinds of genomic DNAs were identified from the sequencing results of the applicant's marine biological resource center, and five kinds of plasmid DNA information were purchased from Takara.

Primer Design and Primary Screening for Fluorescent Gene Detection

The nucleotide sequence was analyzed for the obtained nucleotide sequence, and a primer candidate group as shown in Table 2 was derived.

As a result of pink fluorescence gene analysis, the pink fluorescence gene was found to have 90% homology with the purple fluorescence gene (TurboFP635) of Table 1.

In addition, as a result of various experiments, it is judged that it is difficult to design a primer capable of specifically detecting pink color because it is distinguished from purple color. As described above, pink fluorescent gene and purple fluorescent gene have 90% homology, so pink Fluorescent ornamental fish was designed under the premise that it could be detected simultaneously with a purple fluorescent gene detection primer.

That is, the primer design for the pink color was excluded and the primer was designed to simultaneously detect pink and purple colors with the purple fluorescent gene detection primer.

As an IPC primer, a candidate group was designed by analyzing the nucleotide sequence of the fluorescent coronary S7 ribosomal protein gene. However, the design and selection of the primer set for IPC will be described later in detail with respect to the primer set for fluorescent gene detection.

For the candidate primers above, PCR was performed using 1 ng of plasmids and 10 ng of genomic DNA corresponding to the primers, followed by electrophoresis, and a set of primary primers for fluorescent gene detection was selected. Table 3 below shows the above PCR conditions.

1 is a diagram showing the results of electrophoresis analysis for each fluorescent gene candidate primer set candidate group and accordingly the primary selected primer set.

1, primer sets 7 and 18 for the red fluorescent gene, primer set 4 for the purple fluorescent gene, primer set 2 for the green fluorescent gene, primer set 1 for the orange fluorescent gene, As for the blue fluorescent gene, primer sets 3, 11 and 12 were selected first.

Final Screening of Primer Set for Fluorescent Gene Detection

In order to select the optimum annealing temperature for the primary selected primer set, electrophoresis results were confirmed after PCR reaction of each primer on each fluorescent gene vector and genomic DNA.

Figure 2 shows the results of further electrophoresis analysis and the optimum annealing temperature for the primer set candidate group for primary fluorescent fluorescent detection.

Referring to FIG. 2, primer sets 7 for red fluorescent genes and 11 primer sets for blue fluorescent genes were selected. The primer set for the remaining colors was the same as the primary screening result because a single primer set candidate was selected as the primary screening result. In addition, the optimum annealing temperature was found to be approximately 61 to 63 ℃. In the final selection process, it was confirmed that each target primer specifically reacted with each fluorescent coronary gDNA but no specific binding.

Table 4 shows the selected primer sequences, each of the final designs and selected sequences were assigned a sequence listing number as follows.

IPC primer set candidate design and screening

Table 5 below shows the candidate groups of the designed IPC primer set.

In the case of the existing IPC (primary primer set 1), the amplification efficiency is very low due to competition with other primers, a new IPC DNA target was collected through the literature information and verified. The final new IPC primer set was selected through electrophoresis analysis after PCR reaction was performed for 8 fish IPC primer set candidate groups under 10 ng condition which is the minimum amount of gDNA extracted during general gDNA extraction.

3 is a view showing the results of electrophoresis and IPC primer set finally selected through the IPC primer candidate set.

Referring to FIG. 3, electrophoresis results were analyzed in terms of band intensity and sensitivity, and finally, primer number 3 was selected as an IPC primer set.

As a result, the primer set included in the primer composition for genetically modified fluorescent ornamental fish according to one embodiment of the present invention was determined. Confirmed primer sets are shown in Table 6 below. In addition, sequence list numbers were assigned to each primer set as shown in Table 4.

Establish Multiplex PCR Conditions

1. Primer mixing concentration adjustment

Experiments were conducted to determine the primer input amount which is advantageous in terms of effect. The primer composition for genetically engineered fluorescent ornamental fish according to one embodiment of the present invention is a composition applied to a qualitative test, and the amplification of the fluorescent gene from the sample is made in about 1 or 2 and not all bands are amplified together. Since it is not necessary to adjust the balance of the target primer, the experiment was conducted on two concentrations of 5 pmol and 10 pmol.

4 is a view showing the results of electrophoresis according to the primer mixture concentration. Referring to FIG. 4, as a result of verifying the dosage of primers using 1 ng of plasmid and 10 ng of genomic DNA as a template, 5 pmol of mixed concentration test group showed no specific amplification, so the mixed concentration was about 5 pmol, for example. For example, it is effective to adjust to 4 to 6 pmol.

2. Annealing reaction time experiment

As described above, annealing was carried out at the time conditions of 40 seconds, 1 minute, and 1 minute 30 seconds, respectively, at a temperature of 62 ° C. which was found to be an optimum annealing temperature.

5 is a view showing the results of electrophoresis according to the annealing time at 62 ℃ temperature. Referring to FIG. 5, in the case where annealing was performed for 1 minute, non-specific amplification was not confirmed and sensitivity performance up to 100 pg gDNA was confirmed.

Thus, the annealing temperature and time of PCR were found to exhibit the most effective non-specific amplification inhibition and excellent sensitivity between approximately 61-63 ° C. and 50 seconds to 1 minute 10 seconds, respectively.

3. Extension reaction time experiment

The optimal extension time to improve detection specificity was determined. As described above, PCR was performed at a temperature of 72 ° C. for 1 minute and 50 seconds, respectively, and the results of electrophoresis were confirmed.

6 is a view showing the results of electrophoresis according to extension time under a temperature of 72 ℃. Referring to FIG. 6, since no singularity was found according to the extension time, a relatively short 50 seconds in terms of process efficiency was determined as the extension time.

IPC Primer Concentration Experiment

The effectiveness of the four compositions constituted by adjusting the concentration ratio of the selected target primer and IPC primer was confirmed.

FIG. 7 is a diagram showing electrophoresis results of four test group mixtures having different concentration ratios of a target primer and an IPC primer. Referring to FIG. 7, the specific bands for all the samples were confirmed in the mixture D corresponding to TEST 4 (MIX D), and the IPC amplification signals of all the samples were confirmed.

Stability evaluation

1. Performance test by drying

Specificity performance evaluation was performed on the premix before and after drying the prototype.

8 is an electrophoresis result showing the performance change before and after drying for the premix prototype. Referring to Figure 8, it was confirmed that there is no change in the performance of the product even after drying, even if the composition according to an embodiment of the present invention in the form of a dry product was confirmed that no performance degradation occurs.

2. Stability Test

Verification experiments were conducted to confirm the performance change according to three different batches. Experiments were performed using 10 ng of each fluorescent tuber shark gDNA, and as shown in FIG. 9, it was confirmed that all three batches had no abnormality in performance. 9 is a diagram showing the results of electrophoresis according to three batches for the prototype.

Positive marker performance evaluation

1. Amplification Product Location Comparison Experiment

After PCR cloning for each fluorescent gene, it was confirmed by electrophoresis by PCR reaction in an amount of 1 ng.

10 shows electrophoresis results for PC products and positive markers. Referring to FIG. 10, five fluorescent genes and an IPC PCR product could be identified, but the amount of amplification products was large, and as a result of the test, the size of the band was confirmed below the normal position. Therefore, each fluorescent PCR product was purified and mixed to prepare a marker. As a result of 2% agarose gel electrophoresis, there was no problem in the size of the positive marker, and for the detection of fluorescence, six multiple amplification kits showed that the positive marker was more stable and useful than the conventional positive control. Verified.

2. Positive marker manufacturing stability experiment

Comparative experiments were performed on positive markers of three different batches and the stability was evaluated. FIG. 11 shows electrophoresis results for positive markers in three different configurations. Referring to FIG. 11, 2% agarose gel electrophoresis confirmed that there was no abnormality in the positive marker size in all the batches.

3. Positive Marker Validation Experiment

By comparing the target size of the positive marker and the genomic DNA PCR product of the fluorescent ornamental fish by electrophoresis, it was confirmed that the performance of the positive marker was effective.

Each gDNA performed a PCR reaction using 10 ng as a template. Positive markers were confirmed by electrophoresis using 5 μl each. 12 is a diagram showing electrophoresis results for validating positive markers. Referring to FIG. 12, it was confirmed by 2% agarose gel electrophoresis that the band sizes of the target PCR products of the positive marker and the genomic DNA were identical. Experimental results confirmed that the positive marker of the present invention is effective for the analysis criteria.

<110> NATIONAL MARINE BIODIVERSITY INSTITUTE OF KOREA <120> PRIMER COMPOSITION FOR DETECTING LIVING MODIFIED FLUORESCENT          AQUARIUM FISH AND METHOD OF DETECTING LIVING MODIFIED FLUORESCENT          AQUARIUM FISH BY USING THE SAME <130> DP-18-263 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ORANGE-F <400> 1 accaactggg aggccagct 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ORANGE-R <400> 2 gatggcgtgc tcggtcag 18 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GREEN-F <400> 3 actgcatgta ccacgagtcc aag 23 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GREEN-R <400> 4 gatggcgtgc tcggtcag 18 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PURPLE-F <400> 5 ccggtcgcca ccatgag 17 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PURPLE-R <400> 6 ggagcagtgc aggtagccc 19 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BLUE-F <400> 7 atgtgcatca agatgaccat gga 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BLUE-R <400> 8 cctccataca tgcaacaagg tgc 23 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RED-F <400> 9 ggaacgttcc agtacggctc caagg 25 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RED-R <400> 10 gcagctgcac gggcttcttg gc 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IPC-F <400> 11 tggcctcttc cttggccgtc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IPC-R <400> 12 ggcgaaaagc cagacgagtt 20

Claims (6)

A composition for detecting a fluorescently labeled gene of a genetically modified fluorescent ornamental fish,
A first primer set for detecting a fluorescently labeled gene comprising a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 2 and expressing an orange series of fluorescent colors;
A second primer set for detecting a fluorescently labeled gene comprising a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4 and expressing a green series of fluorescent colors;
A third primer set for detecting a fluorescently labeled gene comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 6 and expressing a purple or pink series fluorescent color;
A fourth primer set for detecting a fluorescently labeled gene comprising a primer of SEQ ID NO: 7 and a primer of SEQ ID NO: 8 and expressing a blue series of fluorescent colors;
A fifth primer set for detecting a fluorescently labeled gene comprising a primer of SEQ ID NO: 9 and a primer of SEQ ID NO: 10 and expressing a red series of fluorescent colors; And
A primer of SEQ ID NO: 11 and a primer of SEQ ID NO: 12, and a sixth primer set (IPC) for detecting IPC (internal positive control),
A primer composition for determining a genetically modified fluorescent ornamental fish that can determine whether or not genetically modified fluorescent ornamental fish by multiplexing PCR.
The method of claim 1,
The primer composition is a primer composition for genetically modified fluorescent ornamental fish, characterized in that the dried form.
delete delete Taking a DNA sample from the fluorescent ornamental fish;
Mixing the DNA sample and a primer composition for determining a genetically modified fluorescent ornamental fish of claim 1; And
Performing and analyzing the multiplexing PCR,
Method for discriminating genetically modified fluorescent ornamental fish.
The method of claim 5,
The annealing temperature of the PCR is 61 to 63 ℃ characterized in that the genetically modified fluorescent ornamental fish.

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