KR102254330B1 - Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same - Google Patents

Abstract

The present invention relates to a primer set for discriminating between Miichthys miiuy and Sciaenops ocellatus, capable of being rapidly and conveniently used in, being analyzed in not only raw materials but also processed foods, and enabling discrimination even when multiple species are mixed, to a method for discriminating between Miichthys miiuy and Sciaenops ocellatus using the same and to a kit comprising the same.

Description

Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same}

The present invention relates to a set of primers for discriminating seaweed and redfish, a method for discriminating seaweed and redfish using the same, and a kit including the same, and in more detail, it can be used quickly and conveniently in the field, and not only in raw materials but also in processed foods. It is possible to analyze, even when several species are mixed, to a set of primers for discriminating seafish and redfish, a method for discriminating seafish and redfish using the same, and a kit including the same.

DNA is generally more stable against processing such as heating and grinding than protein, and can be analyzed with only a small amount of tissue. In addition, unlike proteins that vary depending on age, condition, and tissue type, they exist identically in all cells, and have the advantage of being highly specific and capable of more sensitive detection.

Polymerase chain reaction (PCR) is a method of amplifying a specific nucleotide sequence of a specific gene, and there are various species discrimination methods based on this.

Random amplified polymorphic DNA (RAPD) is a method of amplifying a gene using a short fragment of a primer and then separating the species through the pattern shown by electrophoresis. The advantage of being able to analyze species without genetic information However, it has a disadvantage that reproducibility is poor, and analysis is possible only when high-quality DNA is used.

Restriction fragment length polymorphism (RFLP) is a method of classifying genes according to patterns that appear after treatment with restriction enzymes, and it is possible to classify related species, but it has poor reproducibility and is difficult to analyze in mixed or processed samples.

Forensically informative nucleotide sequencing (FINS) targets sites such as the cytochrome c oxidase subunit I (COI) gene, which shows good variation between species, and uses primers that amplify a wide section. It is a method to determine the species by analyzing the base sequence of the amplified product obtained by using it and comparing it with databases such as National Center for biotechnology information (NCBI) or Barcode of life data system (BOLD). As a result, it is difficult to analyze a sample that is damaged by a gene or a mixture of several species, and it has a disadvantage that it cannot be analyzed if there is no genetic information in the database.

The method of using a species-specific primer is a method of accurately amplifying only the species by designing a primer in the nucleotide sequence section specific to the species. The experimental method and result determination method are simple, fast and accurate, and can be applied to processed products in which several species are mixed. It is a possible method (non-patent document 1 to non-patent document 3).

The analysis method using species-specific PCR is to check the presence of pig genes in order to determine the authenticity of halal products (Non-Patent Document 4), or check the raw materials of cattle and poultry products sold because the raw materials are incorrectly marked (non-patented Document 5) It is applied in various ways, and the amplification result can be checked in real time during the reaction, and after the reaction, ostrich meat is detected in commercially distributed meat processed products using real-time PCR that does not require electrophoresis, or It is applied to various fields such as determining the authenticity (non-patent document 6 and non-patent document 7).

On the other hand, since it is difficult to distinguish between blackfish and redfish, some companies in the fishery market exploit this point to cheat and sell redfish as afish, or sell them under the name of aquaculture to take advantage of money. Therefore, there is an urgent need to develop an analysis method capable of quickly and accurately distinguishing between fish and redfish in the field.

Trends in Food Science & Technology, 21(8), 408-421 (2010) Comprehensive reviews in food science and food safety, 7(3), 280-295 (2008) Trends in biotechnology, 23(7), 359-366 (2005) Food Control, 18(7), 885-889 (2007) Food Control, 62, 157-164 (2016) Food Control, 22(3-4), 523-531 (2011) Food Control, 50, 9-17 (2015)

The present invention is to solve the problems of the prior art described above, and an object of the present invention is to distinguish the species that can be distinguished using species-specific PCR and ultra-high-speed real-time PCR methods when redfish (astrologers), which are relatively inexpensive, are sold as croaker. It is to provide a set of specific primers.

Another object of the present invention is to provide a method for quickly and easily discriminating between croaker and redfish using the primer set.

Another object of the present invention is to provide a kit capable of quickly and simply discriminating between a whitefish and a redfish by including the primer set.

The present invention, in order to achieve the above object, a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; And it provides a primer set for discrimination of seaweed and redfish comprising a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4.

In addition, the present invention comprises the steps of (a) extracting genomic DNA from a sample, (b) amplifying the extracted genomic DNA by using the primer set by ultra-fast real time PCR, And (c) analyzing the Cq value and the Tm value of the amplified product generated by the above step to determine the species of whitefish and redfish.

In addition, the present invention provides a kit for discrimination of seaweed and redfish including the primer set.

The primer set for discrimination of flounder and redfish of the present invention, a method for discriminating flounder and redfish using the same, and a kit for discriminating flounder and redfish including the primer set, can be used quickly and conveniently in the field, and processed as well as the original product. It has the advantage of being able to analyze even foods that have been prepared, and that it is possible to discriminate even when several species are mixed.

In addition, according to the present invention, it is possible to specifically discriminate between redfish and redfish, and it is possible to prevent the sale of redfish as expensive seaweed, thereby contributing to the activation of the consumption market.

Figure 1 shows the location of the primers specific for sea bass and redfish, redfish for redfish and blue for redfish.
Figure 2 shows the general PCR results using a specific primer for Mineo (Lane M: size marker; lane 1: Mineo; lane 2: Hongmineo; lane 3: negative control).
Figure 3 shows the general PCR results using the redminfish specific primers (Lane M: size marker; lane 1: Hongminfish; lane 2: Mineo; lane 3: negative control).
Figure 4 shows the results of the specific PCR (Lane M: size marker; lane 1: 5 ng; lane 2: 0.5 ng; lane 3: 0.05 ng; lane 4: 0.005 ng; lane 5: 0.0005 ng).
Figure 5 shows the redfish specific PCR results (Lane M: size marker; lane 1: 5 ng; lane 2: 0.5 ng; lane 3: 0.05 ng; lane 4: 0.005 ng; lane 5: 0.0005 ng).
6 shows the configuration information of Rapi:Chip ^™.
Figure 7 shows an ultra-fast real-time PCR amplification curve using a Miner specific primer.
Figure 8 shows an ultra-fast real-time PCR amplification curve using the Hongminfish-specific primer.
Figure 9 shows the ultra-fast real-time PCR melting curve using the Mineo specific primer.
Figure 10 shows the ultra-fast real-time PCR melting curves using the Hongminfish-specific primers.

The present invention is a primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; And a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, and a primer set for discriminating seaweed and redfish.

Said croaker ( Miichthys miiuy ) is a representative fish species of the perch family, and is also called Gaeuchi, Hongchi, Buldeunggeori, and Bogulchi, and is mainly distributed in the Northwest Pacific Ocean. The redfish (Sciaenops ocellatus ), a fish of the flounder family, also known as astrocytes, has excellent environmental adaptability and meets the requirements as a fish for aquaculture, such as growing rapidly at high water temperatures. It is inexpensive at about a quarter of the fish.

The redfish has a black spot on its tail and has an external difference from that of the redfish, but there is a problem that it is difficult to distinguish it through a simple processing process such as removing the shell and floating sashimi.

In the present invention, a "primer" is a single-stranded oligonucleotide, under suitable conditions (the presence of four different nucleoside triphosphates and a polymerase such as DNA or RNA polymerase), a template- It is meant to serve as an initiation point from which the synthesis of directive DNA can be initiated.

In the present invention, the suitable length of the primer is determined by the characteristics of the primer to be used, but is usually used as a length of 15 to 30 bp. The primer does not have to be exactly complementary to the sequence of the template, but must be complementary enough to form a hybrid-complex with the template.

The present inventors tried to solve this by designing a primer so that the size of the amplification product is around 200 bp so that the damage degree of DNA due to heat treatment may be a problem in the analysis in processed foods (Trends in biotechnology, 22 ( 5), 222-226 (2004)).

In the primer set of the present invention, a primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 may be used for discriminating fish.

In the primer set of the present invention, a pair of primers consisting of the forward primer of SEQ ID NO: 3 and the reverse direction of SEQ ID NO: 4 may be used for discriminating redfish.

In the primer set of the present invention, the primer set may be a species-specific primer set for ultra-fast real time PCR.

In addition, the present invention relates to a method of discriminating between a whitefish and a redfish using the primer set.

The method of discriminating the flounder and redfish of the present invention has a high copy number and a fast evolution rate, so that a species-specific primer is designed as a target to amplify a short fragment of a mitochondrial gene useful for species identification, and uses DNA. It is a discrimination method that can distinguish between and hongmineo.

In order to optimize the experimental method, experiments by temperature and number of repetitions were performed, and the primer set developed under the optimized conditions specifically amplified only seafish and redfish and exhibited sensitivity to detect 0.005 ng/μL of DNA. .

Also. Because ultra-high-speed real-time PCR is used, the process from gene extraction to result determination can be performed within 30 minutes, and the analysis equipment is small and simple, so it can be used in the field. It was confirmed that the method of discriminating between seafish and redfish according to the present invention was applied to 12 types of processed products to specifically detect only the target species even in processed foods.

The method of discriminating the flounder and redfish according to the present invention comprises the steps of: (a) extracting genomic DNA from a sample, (b) using the primer set to apply the extracted genomic DNA to ultra-fast real-time polymerase chain reaction (ultra-fast real-time polymerase chain reaction). time PCR) to amplify, and (c) analyzing the Cq value and Tm value of the amplified product generated by the above step to determine the species of seaweed and redfish.

Extracting DNA from the sample in step (a) can extract DNA from each tissue or various processed products of the sample by various methods, and this is to determine the species by analyzing the extracted DNA. There is no particular limitation on where the DNA is extracted or how it is extracted.

In step (b), since the amplification of the extracted DNA by ultra-fast real time PCR is also for increasing test samples, the method of obtaining the amplified product is not particularly limited. It is clear that the amplification method known to those skilled in the art to which the present invention pertains also falls within the scope of the present invention.

For example, the high-speed real-time polymerase chain reaction increases the reaction time compared to conventional PCR (Conventional PCR) through methods such as increasing the temperature control rate, improving the thermal conductivity of the reaction tube or chip, and reducing the volume of reaction reagents. It is a method that can amplify genes quickly and easily not only in the laboratory but also in the field by shortening and miniaturizing the device. For this, see Nucleic acids research, 34(11), e77-e77 (2006) and Analytical Chemistry, 72(13). , 2995-3000 (2000)).

In the polymerase chain reaction, various DNA polymerases can be used in the amplification reaction, including, for example, "Klenow fragment" of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. do.

Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which is Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Includes.

In the present invention, the amplification reaction solution may include a dNTP mixture (dATP, dCTP, dGTP, dTTP), a PCR buffer, and a DNA polymerase cofactor.

On the other hand, when carrying out the amplification reaction by the polymerase chain reaction, it is preferable to provide an excess of the components necessary for the reaction in the reaction vessel. The excessive amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the component.

So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach optimal reaction conditions.

Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

In the present invention, the ultrafast real-time polymerase chain reaction can be performed under the conditions of Table 3 below.

[Table 3]

In addition, in the Cq value of step (c), the Cq is a cycle in which a statistically significant fluorescence signal appears (the number of repetitions), the number of repetitions in which the fluorescence value is higher than the minimum detection level, and the T _M In value The T _M denotes the melting temperature of the primers.

In the step (c), when the Cq value is less than 30 and the T _M value is 79.6±1.5° C., it may be determined as a miner. In addition, when the Cq value is less than 30 and the T _M value is 75.7±1.5° C., it may be determined as Hongmin.

In addition, the present invention relates to a kit for discrimination of seaweed and redfish comprising a primer set.

The kit for discriminating seafish and redfish of the present invention may be manufactured in a plurality of separate packaging or compartments including reagent components commonly used in polymerase chain reactions.

According to the DNA microarray technology, the present invention can simultaneously determine the species of croaker and redfish on a single slide, or use each primer independently for each tube to determine the authenticity of raw fish or redfish in food. have.

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

<Example 1> (Securing of samples)

Flounder and redfish are available in local markets, as well as purpura, semi-dried croaker Processed products such as, etc. were purchased through online shopping malls, and DNA was extracted and used to optimize PCR conditions.

<Example 2> (gene extraction from sample)

Gene extraction was performed using a DNA Blood & Tissue kit (Qiagen, Germany), and was performed as follows according to the manual provided by the manufacturer.

To about 100 mg of the sample, 180 μL of ATL buffer and 20 μL of Proteinase K were added and mixed, and then sufficiently reacted at 56° C. until all the samples were dissolved by the buffer. Then, 200 μL of AL buffer was added to the reaction solution, followed by mixing, and then 200 μL of ethanol (96-100%) was added and mixed.

The mixed mixture was placed on a DNeasy Mini spin column and centrifuged at 6,000 × g for 1 minute. After removing the lower layer after centrifugation, 500 μL of AW1 buffer was added to the column and centrifuged at 6,000 × g for 1 minute to wash the DNeasy Mini spin column.

After removing the lower layer, 500 μL of AW2 buffer was added to the column and centrifuged at 20,000 × g for 3 minutes to wash the DNeasy Mini spin column again. 100 μL of AE buffer was added to the column and allowed to stand for 5 minutes, followed by centrifugation at 6,000 × g for 1 minute to elute.

The concentration and purity of the extracted DNA were confirmed using NanoDrop 2000 (Thermo Scientific, USA), and used as template DNA for amplification of marker genes using species-specific primers.

<Example 3> (Selection of target gene and primer design)

Both nuclear and mitochondrial genes are widely used for species identification, but mitochondrial genes have higher amplification efficiency due to the presence of more DNA per cell, and have an advantage in classifying related species due to their rapid evolution. It is often used (Non-Patent Document 1, Non-Patent Document 2).

Therefore, in this study, the mitochondrial genetic information of seafish and astrocytes registered in the National Center for Biological Information (NCBI) gene bank was confirmed, and the nucleotide sequence was secured, and then primers were designed.

For primer design, the BioEdit Sequence Alignment Editor (ver. 7.2.2) program was used, the length was 20-30 bp, and the G+C content was 45-60%, so that a pair of primers were not complementary to each other. , The formation of hair pins (hair pin loops) was avoided by not having three or more G or C consecutively located at the 3'end of the primer.

In addition, the melting temperature (T _M ) value of the primer was set to be the same. In order to be applicable to processed foods, the size of the amplified product was set to be 200 bp or less, and the NCBI Primer-BLAST search function was used to check whether cross-reactions other than the target species were found.

FIG. 1 shows the locations of specific primers for seaweed and redfish (red for seaweed and blue for redfish), and Table 1 below shows the nucleotide sequence and information of the designed primers.

<Example 4> (gene amplification and nucleotide sequence analysis using general PCR)

The PCR reaction was performed in a total of 20.0 μL including 5 ng of template DNA and 1 uM of each primer, 1 × PCR buffer, 0.2 mM dNTPs, 1.0 mM MgCl ₂ , 1 U recombinant Taq polymerase (Takara, Japan) and sterile distilled water. Was done.

Experiments by temperature to find the optimum binding temperature, experiments by number of repetitions to determine the optimal PCR repetition number, and detection limit experiments to confirm the minimum detection limit were conducted to establish the optimal PCR conditions for species-specific primers as shown in Table 2 below. .

The short-length fragments amplified using the developed species-specific primers in Table 1 and the optimal PCR conditions in Table 2 were purely isolated on an agarose gel and cloned into pGEM-T easy vector (Promega, USA), After the base sequence was analyzed by requesting Bioneer (South Korea), the species was identified using the BLAST search function of NCBI.

As a result of confirming the specificity of each of the whitefish and redfish using the specific primers of the above Table 1, each primer was specifically amplified only for the species as shown in FIGS. 2 and 3, and PCR amplification of the expected size It was confirmed that the product was generated (124 bp, respectively, specific primers for seafish and redfish).

After purification and cloning of the specifically obtained amplification product, the nucleotide sequence was analyzed and compared with the nucleotide sequence information in the NCBI database. As a result, accession numbers obtained through the BLAST search function for both seafish and redfish were obtained (minfish: MK560626.1, redminfish: KP641522. It was found that 1) coincided with the accession number of the genetic information used when designing the primer, and the nucleotide sequence of the amplified portion was also 100% identical.

In addition, by using NCBI's primer-BLAST search function, the presence or absence of species with the same nucleotide sequence and amplification product size at the primer site was further identified to identify species that can be analyzed with the corresponding primers.

In the case of croquet-specific primers, in addition to crockery, Eopsetta It was shown that grigorjewi (flounder), Engraulis japonicus (anchovy), and redfish specific primers were able to additionally analyze the genus Actinopterygii.

The detection limit was analyzed in the range of 5 to 0.0005 ng/μL by diluting the template DNA (5 ng) stepwise by 10 times, and the detection limits of the specific primers for seaweed and redfish were as shown in FIGS. 4 and 5 (minfish specific primer: 0.005 ng /μL, redfish specific primer: 0.005 ng/μL).

<Example 5> (gene amplification using ultra-fast real-time PCR)

40 mg of tissue samples were taken from the raw fish and redfish raw and processed products, mixed with 200 μL of a solution (Direct lysis buffer, DLB, Genesystem, South Korea), mixed at 1 minute intervals, and reacted at room temperature for 5 minutes.

10 μL of the supernatant of the reaction solution was taken, mixed with 90 μL of sterile distilled water, diluted 10 times, and diluted to 100 times the initial concentration by repeating this. 2 μL of the final diluted solution diluted 100 times the initial concentration was used as a template DNA, and the plasmid DNA cloned in Example 4 was used as a positive control.

The reaction was carried out with a total of 10.0 μL including 1 × Rapi qPCR premix buffer (Genesystem, South Korea) and sterile distilled water.

Experiments by temperature to find the optimum temperature and detection limit experiments to confirm the minimum detection limit were conducted to establish the optimal conditions for ultra-high-speed real-time PCR as shown in Table 3 below.

As a result of ultra-high-speed real-time PCR using species-specific primers, C _q Value and T _M It was confirmed that the values were used to specifically amplify the whitefish and redfish (FIGS. 6 to 10 ).

_{6 to 10, C q} amplified with a Flour-specific primer Value is less than 30 and T _M When the value is 79.6±1.5℃, it was judged as a fish, and C _{q amplified with a redfish specific primer} When the value is less than 30 and the T _M value is 75.7±1.5°C, it was determined as redfish, and it was confirmed that the established method specifically amplifies the redfish and redfish through the monitoring of 12 fish and redfish products.

As described above, the present invention has been described with reference to preferred embodiments of the present invention, but those skilled in the art can variously modify and modify the present invention within the scope not departing from the spirit and scope of the present invention described in the following claims. You will understand that you can change it.

According to the present invention, since it is possible to specifically discriminate between redfish and redfish, and it is possible to prevent the sale of redfish as expensive seaweed, thereby contributing to the activation of the consumption market, it is useful in the technical field to which the present invention belongs. Can be applied.

<110> Republic of Korea (Ministry of Food and Drug Safety) <120> Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same <130> 11408 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Miichthys miiuy <400> 1 aggtgtttcc tcaattctag gt 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Miichthys miiuy <400> 2 gaggactgct gtgatcagg 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Sciaenops ocellatus <400> 3 tttcgggaac tgactcgtac c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Sciaenops ocellatus <400> 4 tacacctgag gaggtaagga ga 22

Claims (9)

A primer pair consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; And In the primer set composition for discriminating seafish and redfish, comprising a primer pair consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4,
The primer set composition is characterized in that a species-specific primer set for ultra-fast real time PCR (ultra-fast real time PCR), a primer set composition for discriminating fish and redfish. The primer set composition of claim 1, wherein the primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 is used for discriminating fishfish. According to claim 1, wherein the primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse direction of SEQ ID NO: 4 is characterized in that for the identification of redfish, a primer set composition for identification of redfish and redfish. delete (a) extracting genomic DNA from the sample;
(b) amplifying the extracted genomic DNA by using the primer set composition of any one of claims 1 to 3 by ultra-fast real time PCR; And
_{(c) Analyzing the Cq value and T M} value of the amplified product generated by the above step to determine the species of croaker and redfish. The method of claim 5, wherein the ultra-fast real-time polymerase chain reaction is performed under the conditions of Table 3 below.
[Table 3]

[6] The method of claim 5, wherein the Cq value is less than 30 and the T _M value is 79.6±1.5°C. [6] The method of claim 5, wherein the Cq value is less than 30 and the T _M value is 75.7±1.5°C. Claims 1 to 3, comprising the primer set composition of any one of claims, a kit for discrimination of seaweed and redfish.

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