KR101918124B1 - Primer set for determining identity of crap material in food, method of determining identity of crap material in food using the same and kit comprising the same - Google Patents

Abstract

The present invention relates to a primer set for discriminating authenticity of a species used as a food material, a method for discriminating authenticity of a food material using the primer set, and a kit comprising the primer set, DNA sequences can be arbitrarily created, so that classification of raw materials in food can be simplified and facilitated.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for discriminating authenticity of a raw material in a food, a method for determining authenticity of a raw material in a food using the same, and a kit including the same food using the same and kit comprising the same}

The present invention relates to a method for discriminating four varieties to be used as a food ingredient, and particularly, a genotype for a crab used as a food ingredient is analyzed based on an arbitrary DNA sequence distinguished among species of four crabs, A primer set for identifying a raw material, a method for easily and quickly identifying a raw material using the primer set, and a kit including the same.

Crabs are aquatic products belonging to crustaceans among arthropods, and it is known that there are 4,500 species in the world and about 183 species in Korea. It is also one of the major marine resources used in our diet. It is commonly used as a raw material for processed foods such as smoked fish and sausage, in addition to boiling or boiling.

According to the Ministry of Maritime Affairs and Fisheries, about 29% of total crustacean production is reported in 2016, according to the Ministry of Maritime Affairs and Fisheries.

In general, crustaceans, including crabs, have distinguished species by analyzing the appearance such as color, nipple, and attachment. However, it is difficult to apply the existing distinction method because it is similar to the interspecific phenotype and eliminates nippers and appendages during processing (Food Control, 25 (1), 239-244 (2012)).

Due to these characteristics, there has been a case in Korea where crab cakes are sold in relatively expensive markets, crabs are not included in other countries,

In using crabs as food materials, it is possible to intentionally replace expensive materials with similar low-cost materials, or to make errors in the representation of raw materials. Therefore, there is a need for a method that can quickly and accurately determine the presence or absence of crab and species in both raw and non-processed foods that have external features (Oceana. Retrieved from (2015) http://usa.oceana.org / publications / reports / oceana-reveals-mislabeling-iconic-chesapeake-bluecrab ).

In addition to morphological species discrimination, predominantly protein-based methods have been used to identify species of crustaceans. Protein-based methods such as isoelectric focusing (IEF), capillary electrophoresis (CE), and immunoassay system are mainly used to analyze fresh or rapidly frozen tissues, Or if it is dried, the biochemical properties or structure of the protein is altered and can not be analyzed.

In addition, the expression of a protein differs depending on the tissue, even in the same organism, and since commercially available methods detect most plasma proteins, products that are mixed with other species are likely to be contaminated, making analysis difficult.

Methods such as enzyme-linked immunosorbent assay (ELISA) can also be used to analyze thermally processed products, but antibodies can cross-react when in close proximity. In addition, it is necessary to develop a specific antibody according to a specific protein, and the commercially available antibody has a disadvantage that it is difficult to apply to the species discrimination (Trends in Food Science & Technology, 11 (2), 67-77 7 (3), 280-295 (2008); Journal of Agricultural and Food Chemistry, 47 (4), 1350-1355 (1999)).

DNA is generally more stable than proteins and less likely to degrade during thermal processing. It is also present in all biological tissues and has the advantage of being able to analyze even a small amount of samples.

In the case of mitochondrial DNA, it is generally more advantageous to analyze the myofilaments than the nuclear DNA, and since there are many DNAs per cell, amplification is easy and is often used as a genetic marker for species discrimination.

Polymerase chain reaction (PCR) is a method of amplifying a desired part of a specific gene using a pair of primers.

In addition, when used in conjunction with species-specific primers capable of amplifying species-specific genetic markers, it is easy to identify the presence of a desired species in a variety of processed food products (Erlich, H. (2015). PLS One, 5 (9), e12620 (2010); Gene, 311 129-135 (2003).

The marine processed food market is growing, and interest in processed marine products is increasing. However, the fact that the economic value of aquatic products differs depending on the species, the case that Honggega is popularly sold in Korea, the case that overseas craze is different from the one specified in the crab cake, or the case of replacing crabs with low cost fish It has been reported.

Accordingly, there is a growing need for a method for accurately and accurately determining the content and species of crabs in both raw and non-processed foods that have external characteristics.

The species identification of crabs should be fast and easy to use, and because species must be distinguishable in samples and processed foods that are mixed with other species as well as other species, DNAs that are relatively more stable than proteins, It is urgently required to develop a discrimination method which can identify species of crabs easily by designing species specific primers to be amplified.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a method and apparatus for distinguishing crabs according to genotypes of crabs existing in a food raw material, Lt; / RTI > primer set.

Another object of the present invention is to provide a method for distinguishing species of crabs according to genotypes of crabs existing in a food raw material, and identifying the crab species simply and quickly.

It is still another object of the present invention to provide a kit that can distinguish species of crabs according to the genotype of crabs existing in a food raw material and can easily and quickly identify the crab species.

In order to achieve the above object, the present invention provides a primer set for discriminating authenticity of a food ingredient in a food, comprising a pair of primers consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2.

The present invention also provides a primer set comprising a primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, a primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, A primer pair consisting of a forward primer of SEQ ID NO: 6 and a reverse primer of SEQ ID NO: 6, and a primer pair consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: Lt; / RTI >

(B) amplifying the extracted genomic DNA by a polymerase chain reaction using the primer set; and (c) amplifying the genomic DNA obtained by the step (a) And a step of fractionating the amplified product and confirming authenticity of the crab varieties in the food.

Further, the present invention provides a kit for discriminating authenticity of a crab raw material in a food including the primer set.

The present invention is capable of distinguishing species with a simple, rapid, accurate and low detection limit according to the genotype of a target crab based on an arbitrary DNA sequence which is distinguished among species of various crabs mainly used as food raw materials .

The present invention is also applicable to a seasoned workpiece or a powdered powder which is made of a DNA chip or a kit containing the primer set and is hard to be visually distinguished, and even if a sample is put in one kit, . ≪ / RTI >

In addition, by using the microarray method using the primer according to the present invention, the time required for analyzing a sample can be greatly shortened compared with the conventional method, and a large amount of the sample can be inspected within a short time.

Fig. 1 (a) is a comparison of nucleotide sequence and primer position, Fig. 1 (b) is a comparison of nucleotide sequence and primer position, Fig. 1 (c) is a nucleotide sequence comparison and primer position, .
Figure 2 shows the gene of mostly cytochrome C oxidase subunit I (COI) (SEQ ID NO: 9).
FIGS. 3A and 3B show the genes of internal transcribed spacer (ITS) in Hong Kong (SEQ ID NO: 10).
4A and 4B show the gene of the King Krab's cytochrome B (CYTB) (SEQ ID NO: 11).
5 shows the gene of cytochrome C oxidase subunit I (COI) of crab (SEQ ID NO: 12).
6a shows a PCR result using a specific primer (lane 1, lane 2, lane 3, kinkaku, lane 4, crab, lane N, negative control).
6B shows the result of PCR using a red spot specific primer (Lane 1; Hongge, Lane 2; Daegyeo, Lane 3; King Crab, Lane 4; Crab, Lane N; Negative control).
6C shows the results of PCR using a specific primer of the Kungkrap (lane 1, king crab, lane 2, mungbean, lane 3, red mug, lane 4, crab, lane N, negative control).
6D shows PCR results using a crab-specific primer (lane 1, crab, lane 2, sand crab, lane 3, red crab, lane 4, king crab, lane N, negative control).
Figure 7a shows the results of specific PCR (lane 1; 5 ng, lane 2; 0.5 ng, lane 3; 0.05 ng, lane 4; 0.005 ng, lane 5; 0.0005 ng, lane N; negative control).
Figure 7b shows the results of specific PCR for lupus erythematosus (lane 1: 5 ng, lane 2: 0.5 ng, lane 3: 0.05 ng, lane 4: 0.005 ng, lane 5: 0.0005 ng, lane N; negative control).
Fig. 7c shows the results of the specific PCR for the Kungkap (lane 1: 5 ng, lane 2: 0.5 ng, lane 3: 0.05 ng, lane 4: 0.005 ng, lane 5: 0.0005 ng, lane N; negative control).
Figure 7d shows the crab specific PCR results (lane 1; 5 ng, lane 2; 0.5 ng, lane 3; 0.05 ng, lane 4; 0.005 ng, lane 5; 0.0005 ng, lane N; negative control).
Figure 8a shows the results of a typical PCR (lane P: positive control (usually), lanes 1 to 5, crab processed food 1 to 5, lane 6, red lane 7, kinkaku, lane 8, crab, lane N; Negative control).
Fig. 8B shows the result of specific PCR for lupus erythematosus (lane P: positive control (red ginseng), lanes 1 to 5, crab processed food 1 to 5, lane 6, lane 7, king crab, lane 8, crab, lane N; Negative control).
Figure 8c shows the results of the specific PCR of the Kungkap (lane P: positive control (kink), lane 1 to 5, crab processed food 1 to 5, lane 6, crab 7, lane 7, lane 8, crab, lane N; Negative control).
Figure 8d shows the results of the crab-specific PCR (lane P: positive control (crab), lane 1 to 5, crab meat 1 to 5, lane 6, crab 7, lane 7, lane 8, Negative control).

The present invention relates to a primer set for discriminating authenticity of a crab raw material in a food, which comprises a pair of primers consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2.

The present invention also provides a primer pair comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a primer pair of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4, a forward primer of SEQ ID NO: A reverse primer of SEQ. ID. NO. 7 and a reverse primer of SEQ. ID. NO. 8, and a pair of primers consisting of a reverse primer of SEQ ID NO: 8 and a primer pair of reverse primer of SEQ ID NO.

In the present invention, a "primer" is a single strand of oligonucleotides, which is hybridized under suitable conditions (presence of four different nucleoside triphosphates and a polymerase such as DNA or RNA polymerase), a template- Quot; means acting as a starting point at which to initiate directed DNA synthesis.

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

The present inventors tried to solve the problem by designing the primers so that the size of the amplified product is about 200 bp (Trends in Biotechnology, 22 (5) ), 222-226 (2004)).

In the primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 can be generally used for discrimination.

In the primer set of the present invention, the pair of primers consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 may be used for red flag determination.

In the above primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 may be used for discriminating the kinks.

In the primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 can be used for crab identification.

The present inventors chose the group of genes most suitable for discrimination as crab genotypes, that is, genes encoding CI (Cytochrome C oxidase subunit I) (mungbean, crab), ITS gene (Hongge) and cytochrome b gene , And to identify species specific sites. Based on this, DNA sequences which are distinguished according to species in various crabs were arbitrarily created to facilitate species discrimination.

In particular, the inventors of the present invention have found that DNA sequences corresponding to SEQ ID NO: 1 to SEQ ID NO: 8 in COI, ITS or cytochrome b gene among four mitochondrial DNAs, which are widely used as food materials, And it is found that each species can be distinguished from other conventional methods by species specificity and sensitivity.

FIGS. 1A to 1D respectively show the nucleotide sequences and positions of the cytochrome oxidase subunit I (COI) gene, the red spot ITS gene and the cytochrome b gene region of the king crab in the mitochondrial DNA of the main foodstuff, will be.

Based on this, the present inventors have derived an arbitrary DNA sequence which can be distinguished from each other among four species (SEQ ID NO: 1 to SEQ ID NO: 8 in Table 1 below) It can be used as a marker.

The marker can be used as a forward primer or a reverse primer for determining the species of crabs, and FIGS. 1A to 1D show the position of a gene on which the marker can be joined.

After many studies and efforts, the inventors of the present invention have found a region that is differentiated among COI gene, ITS gene, or cytochrome b gene of crab DNA, and based on this, it is possible to identify any DNA sequence most suitable for distinguishing crab species And it was confirmed that species of crab can be distinguished by using a probe including such a DNA sequence.

Accordingly, the present invention uses the same or a complementary base sequence as each DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 8, which will be described later, as a probe.

Since these probes are based on different DNA sequences depending on the species to which they belong, it is desirable that they bind with PCR amplification products belonging to the intended crab, but not with those of other crabs belonging to other species.

Further, the present invention relates to a method for determining authenticity of a crab raw material in a food using the primer set.

A method for determining authenticity of a raw material in the food of the present invention comprises the steps of (a) extracting genomic DNA from a sample, (b) amplifying the extracted genomic DNA by a polymerase chain reaction using the primer set , And (c) isolating the amplification product produced by the above step and confirming authenticity of the crab variety in the food.

Extraction of DNA from the sample can extract DNA from various tissues or various workpieces of the sample by various methods. Since DNA is extracted for analyzing the extracted DNA to identify the species, DNA is extracted from any part of the sample The extraction method and the method of extraction are not particularly limited.

Since the amplification of the extracted DNA by polymerase chain reaction (PCR) is also intended to increase the number of test specimens, the method of obtaining the amplification product is not particularly limited. Amplification methods known to those of ordinary skill in the art are also within the scope of the present invention.

For example, the polymerase chain reaction is a method for selectively amplifying a specific DNA fragment, which is described in Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822).

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

Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) .

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

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

It is required to provide the reaction mixture with such a partner as Mg ^{2 +} , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions.

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

In the present invention, the amplification reaction with a polymerase can be carried out by repeating a cycle consisting of (i) a pre-denaturation process, (ii) denaturation, annealing, and elongation several to several tens times (Iii) a final heat treatment process or a thermal cycle program suitably modified for these processes.

The present invention can prepare a plurality of probes each containing the same or a complementary base sequence as the respective DNA sequences of SEQ ID NO: 1 to SEQ ID NO: 8, and by binding at least one of these probes to the PCR product , It is possible to distinguish the species of crab according to the combination.

In addition, the probe according to the present invention binds each DNA sequence of SEQ ID NOS: 1 to 8 present in COI, ITS, CYTB gene or mitochondrial gene region of crab mitochondrial DNA, .

As described above, the present invention is characterized in that a probe comprising the same or a complementary base sequence as each DNA sequence of at least one of SEQ ID NO: 1 to SEQ ID NO: 8 is used, A DNA chip and a kit containing one probe. It is also possible that the chips and kits are provided with additional position markers indicated by a specific nucleotide sequence in order to improve the accuracy of binding and detection with the probes.

The kit for determining the authenticity of crab raw materials in the food of the present invention can be produced from a number of separate packages or compartments containing reagent ingredients conventionally used for polymerase chain reaction.

In accordance with the DNA microarray technology, the present invention can identify multiple species simultaneously on one slide, and independently determine the authenticity of raw materials in the food by using each primer independently in each tube.

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

≪ Example 1 > (securing sample)

In order to extract the DNA used to optimize species specific PCR conditions, weeds, crabs, king crabs and crabs were purchased in local markets.

≪ Example 2 > (Extraction of DNA from crab samples)

DNA extraction was performed using DNA Blood and Tissue Kit (Qiagen, Germany) according to the manual provided by the manufacturer.

Approximately 100 mg of the sample was mixed with 360 μL of ATL buffer and 40 μL of Proteinase K, followed by sufficient reaction at 56 ° C. until the sample was completely dissolved by the buffer. Subsequently, 400 μL of AL buffer was added to the reaction solution, and 400 μL of ethanol (96-100%) was added thereto and mixed.

The mixture was placed in a DNeasy Mini spin column and centrifuged at 6,000 x g for 1 minute. After centrifugation, the supernatant was removed, 500 μL of AW1 buffer was added to the column, and the DNeasy Mini spin column was washed by centrifugation at 6,000 × g for 1 minute.

After removing the supernatant, 500 μL of AW2 buffer was added to the column, and the DNeasy Mini spin column was washed once again by centrifugation at 20,000 × g for 3 minutes. 100 μL of AE buffer was added to the column and left for 5 minutes, followed by centrifugation at 6,000 x g for 1 minute.

The concentration and purity of the extracted DNA was confirmed using NANODROP 2000 (Thermo Scientific) and used as template DNA for marker gene amplification using species specific primers.

≪ Example 3 > (Selection of target genes and primer design)

In order to amplify species-specific genes of a particular species, consideration should be given to the number of copies of the gene, the size of the amplified portion, and the specificity with other species.

Mitochondria are the organelles present in the cytoplasm, and mitochondrial DNA is a molecular marker that can be efficiently used to identify closely related species with higher copy number per cell and faster evolution rate (Meat Science, 83 (2 ), 165-174 (2009)).

In the present invention, for primer design, GenBank Accession No. HQ322669.1, GenBank Accession No. AB501212.1, GenBank Accession No. JX944381. 1) and crab (GenBank Accession No. FJ919808.1), and the primer was designed after securing the base sequence. When the base sequence of the comparative species could not be obtained, the specificity was confirmed through experiments .

When designing the primer, the length is 20 to 30 bp and the position of G + C% is 45 to 60% so that the pair of primers are not partially complementary to each other. The formation of a hair pin loop was avoided so that G + C did not come in succession more than three times.

In addition, the melting temperatures (Tm) of the two primers were made equal. In order to be specific for other species, the species should be as specific as possible and the above conditions should be satisfied as much as possible. The size of the amplified products should be 200 bp or less so that they can be applied to processed foods. Using the NCBI Primer-BLAST search function (Trends in Biotechnology, 22 (5), 222-226 (2004)).

Fig. 1 (a) is a comparison of nucleotide sequence and primer position, Fig. 1 (b) is a comparison of nucleotide sequence and primer position, Fig. 1 (c) is a nucleotide sequence comparison and primer position, .

The nucleotide sequence of the designed primer set and the information thereof are shown in Table 1 below.

≪ Example 4 > (Gene amplification and nucleotide sequence analysis)

PCR reactions were performed with a total of 20.0 μL of 10 ng template DNA and 1 μM primer, 1 × PCR buffer, 0.2 mM dNTPs, 2.0 mM MgCl ₂ , 1 U rTaq polymerase and sterile distilled water, respectively.

In order to find the optimal annealing temperature, we conducted temperature experiments, cycle experiments to determine optimal PCR cycles, and detection limit experiments to confirm the minimum detection limits. 2.

PCR was performed under the optimized conditions shown in Table 2 above using DNA extracted from four kinds of standard samples. As a result, as shown in FIGS. 6A to 6D, four kinds of crabs (crab, crab, king crab, crab) It can be seen that no nonspecific bands due to cross-reactivity were generated.

As shown in FIGS. 6A to 6D, each primer set produced a PCR product of the expected size for the corresponding DNA. Generally, the size of the specific primer was 105 bp, the size of the primer specific for red spot was 140 bp, The size of the specific primer was 137 bp and the size of the crab specific primer was 174 bp.

In order to confirm the nucleotide sequence of the short-length fragments amplified using the developed species-specific primer, the amplification product was purified from the agarose gel and cloned into pGEM-T easy vector.

The plasmid DNA was purified and sent to Bioneer Corp. (Seoul, South Korea) for analysis of the nucleotide sequence. The analyzed nucleotide sequence was compared with nucleotide sequence information in the NCBI database using the BLAST search function.

In all four species, the accession number obtained from the BLAST search coincided with the number of the genetic information used in designing the primer, and the nucleotide sequence of the amplified region was 100% identical.

In addition, using the primer-BLAST function of NCBI, the presence or absence of species having the same base sequence and amplification product size at the primer site was searched and further species capable of being analyzed by the primer were further confirmed.

In case of crab specific primer, it was possible to further analyze Charybdis japonica and Sophrops striata in addition to crab.

Example 5 (Analysis of detection limit of each species specific primer)

The detection limits of each species are shown in FIGS. 7A to 7D, where the template DNA extracted from the standard sample was diluted 10-fold in steps, and the experiment was performed in the range of 5 to 0.00005 ng / uL.

As shown in FIGS. 7A to 7D, the detection limit of the specific primer was 0.005 ng / μL, the detection limit of the red spot specific primer was 0.05 ng / μL, the detection limit of the Kingpact specific primer was 0.05 ng / The detection limit of the specific primer was 0.5 ng / μL.

Example 6 (Classification of crab processed food using species-specific primers)

Identification of the authenticity of the food ingredients contained in the product by using species-specific primers developed for five processed foods such as fish paste, fish paste, sauce, sausage, The results are shown in Figs. 8A to 8D.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, many variations and modifications may be made without departing from the scope of the present invention as defined by the following claims. It will be understood that the present invention can be changed.

According to the present invention, it is possible to confirm the authenticity of a raw material using a species-specific primer not only in a sample raw material but also in a processed food which is hard to be visually confirmed, Therefore, the present invention can be applied to a technical field to which the present invention belongs.

<110> Republic of Korea (Ministry of Food and Drug Safety) <120> Primer set for determining identity of crap material in food,          method of determining identity of crap material in food using the          same and kit comprising the same <130> 10728 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Chionoecetes opilio <400> 1 gtataagcct agatcaaata cca 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Chionoecetes opilio <400> 2 aaagtatggt aattgctcca gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Chionoecetes japonicus pacificus <400> 3 acgaaggtgt gcccttaaga 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Chionoecetes japonicus pacificus <400> 4 cacaactagt aacgcgtcaa c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Paralithodes camtschaticus <400> 5 cctgggtatt tctagacaag taga 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Paralithodes camtschaticus <400> 6 ctggatctat tagagcgtat ggga 24 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Portunus trituberculatus <400> 7 ttacaatgtt gtagtcacag ctcat 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Portunus trituberculatus <400> 8 agagttaatg aaggaggaag aagtc 25 <210> 9 <211> 748 <212> DNA <213> Chionoecetes opilio <400> 9 ggtatcatta agattaatta ttcgagctga acttggacaa cccggaactc ttatcggaaa 60 tgaccaaatt tataatgtag ttgttacagc ccacgctttt gttataatct tcttcatagt 120 tatacctatt ataattggag ggtttggaaa ttgactaatt cccttaatac tgggagcccc 180 tgatatagcc tttcctcgaa taaataacat aagattttgg ttattgcctc cttctttaac 240 actactatta ataagaggta tagtagaaag aggagtagga actggatgga ctgtttaccc 300 tcctttagca gctgcaatcg ctcacgctgg agcctccgtt gatatgggga ttttttctct 360 acatttagct ggagtttcct ctattttagg agctgtaaat tttataacta ccgtaattaa 420 tatacgatcc ttcggtataa gcctagatca aataccactt tttgtttgag ctgtatttat 480 tacagctatt ttacttctat tatctctacc tgttttagct ggagcaatta ccatactttt 540 aactgatcga aacttaaata catctttttt tgaccctgca ggagggggag accctgtttt 600 ataccaacac ctcttttgat tctttggaca cccagaagtt tatattctta ttctaccagc 660 atttggtata gtttcacaca ttgttagtca agagtccggt aaaaaagaat ctttcgggac 720 attaggaata atttatgcta tattagca 748 <210> 10 <211> 1527 <212> DNA <213> Unknown <220> <223> ITS gene of Chionoecetes japonicus <400> 10 aaaaaaaaag tacgtgagta cgtgtgtgtg ggtgtgtgtt ggcattgcat acatactacc 60 actaccatct ttgactaggc ttcagaccaa gctctctacg ttgtcccaca caccgctaat 120 attttgcctc gatctctgcg ctctcatacg ggataagtcc accggaaacg gggcattcgg 180 caggaagcag agatgagttg ggcggtgtag cgctgttaga gggtttgtgt ctgtgcctag 240 ctgctgattc gttcattcaa gtggctcccc cggccagaac acctaaccat cagcagtaga 300 ctgttgcgac agactgctga tgacccatgt tattaatact acttctccct cccgtcaagt 360 gacggctacg agggtagact ataacacgct tacacactgg atggagtgtg taagcgtatt 420 gtcggcattg tcacccacca caggtggtgg ttgtctggtg actttgaccg ccttggggcg 480 tcgctactgt cttgtcttac aacgacgaag gtgtgccctt aagaactgtc tgcgagtcgc 540 ggctctaaaa cggggtcgcg ggctcgggat aaggtggtgg tacgttgcca ttattactaa 600 acaacctctc ccgggcagag catccgggag cttggttgga aaagtaaggt aacgagagtc 660 cagagccgta ctacgttctg gtctcggcca cagccgccca gtgagcgttg agcgctcgac 720 tgaatctttt ttaaacctta acactacctg tcactactac gacaaacctt caaacccata 780 gtactaagtc ttcccacatg gtctcttgag gcagagattg tggaagtgat aaaagtcggc 840 acagcccgac gtcaatataa aaatacaact cttaacggtg gatcactcgg ctcgtgggtc 900 gatgaagacc gcagcaagct gcgcgtcgtt atgtgaatcg caagaaaacc agatacatcg 960 acaagtcgaa cgcacattgc ggcggcggca ctctcatgtg ctgccgtcac tcctacacga 1020 gggtcggaca tcatataacc atacatcggc attgtcaccc accacaggtg gtggttgtct 1080 ggtgactttg accgccttgg ggcgtcgcta ctgtcttgtc ttacaacaac gacgaaggtg 1140 tgcccttaag aactgtctgc gagtcgcggc tctaaaacgg ggtcgcgggc tcgggataag 1200 gaccactgca ttccagccag aatacgtaaa tcacggcagc gacttgcgcc gttgacgcgt 1260 tactagttgt gtttgtcaga gggccgtcca tacatacctg ctttgctgct tgtcaaccct 1320 tgtcagcagg ctcgtcagac ggtgaccggg tcggcgctac ccgatctgct cggatcacca 1380 gagcgagctt gccgcccaga gtattattat atccccatgc tgatataata tgggtgtgaa 1440 aaagacgaga gggagacttg cagcagccaa acgcgagcta gtctccagtc tgactaactg 1500 agtgactgct ccaacccatg tcgacct 1527 <210> 11 <211> 1137 <212> DNA <213> Unknown <220> <223> CYTB gene of Paralithodes camtschaticus <400> 11 atgaaaatac cagttcgtaa aactcaccct ttaattagaa ttattaatgg agcattagtt 60 gatatgccac tacctagtaa tatttcaaca ttttgaaatt ttggatcttt acttggattg 120 tgtttaatta ttcaaattat aacaggatta tttttatcta tacattatac agcacatgta 180 gatttagctt tttcttctgt agctcatatt caccgagatg taaattatgg ctgattaatt 240 cggtcgctcc atgctaatgg ggcttccttc ttttttgtat gtttatatct tcatattggg 300 cgtggagttt attatggatc ttatataaat attccagcgt gaattgttgg agttattatt 360 ttatttttag ttatagcaac agcattttta ggttacgttc taccttgggg gcagatgtct 420 ttttgaggtg ctacagtaat tacaaattta ttctcagcaa ttccatatgt aggaacttat 480 ttagtaaatt gaatttgagg tggatttgca gttgaaaatg ctactttaac acgattttat 540 tctttacact ttttatttcc ctttgtagtg atagctctat ctgtagtaca tattatattt 600 ttacaccaaa caggggctaa taaccccctg ggtatttcta gacaagtaga taaagttccc 660 tttcacccct attttacata taaggatatt gtaggattcg tagttatatt atgagcttta 720 ttaatattat cccttttatt cccatacgct ctaatagatc cagataattt tattccggct 780 aacccaatag taacaccaaa acatattcaa cctgagtgat attttttatt tgcctatgca 840 attttgcgat caattcctaa taaagcattg ggagttattg cgttagtttt atcagttgca 900 gccctaataa ctttcccttt cactcacaaa tcaaaatttc gaggattggc cttttatcct 960 gtaaatcaat gattattctg attttttatc tcaatagtta ttcaactaac atgaattgga 1020 gctcgacccg tagaagaacc ttacgtttta acaggacaaa ttttaacttt tttatatttt 1080 tcatattata ttttaaatcc attaattctt cgatgatgag atagaaaaat agtttaa 1137 <210> 12 <211> 658 <212> DNA <213> Unknown <220> <223> COI gene of Portunus trituberculatus <400> 12 tacattatat tttatttttg gagcatgatc aggaatagta ggaacttcac ttagtctaat 60 tattcgtgct gaattaggac aacccggtac tcttattggc aacgaccaaa tttacaatgt 120 tgtagtcaca gctcatgctt tcgtcatgat tttctttata gttataccaa tcataattgg 180 aggattcggt aattgattag tacccctaat attaggagct cctgatatag ccttcccccg 240 tataaataac ataagattct gacttcttcc tccttcatta actcttcttc ttataagagg 300 tatagtagaa agaggtgtag gtactggatg aactgtatat cctcctcttt ctgccgcaat 360 tgcccatgcc ggtgcatcag tagacttagg aattttttct cttcatttag caggagtttc 420 atctatttta ggtgcagtaa attttataac cactgttatt aatatacgat cttttggtat 480 aagaatagac caaataccac tatttgtatg atcggtattt attactgcaa ttcttcttct 540 tttatctctc cctgttctgg caggagctat tactatactt ctcacagatc gtaatttaaa 600 tacttcattc ttcgaccctg ccgggggtgg agaccccgtt ctttaccaac atctcttc 658

Claims (8)

A primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the primer pair of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: And a pair of primers consisting of a pair of primers consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8, wherein the pair of primers comprises:
The primer pair of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 are usually discriminated and the detection limit is 0.005 ng /
The primer pairs of SEQ ID NO: 3 and reverse sequence of SEQ ID NO: 4 are for red spot discrimination, the detection limit is 0.05 ng /
The primer pair of SEQ ID NO: 5 and the reverse sequence of SEQ ID NO: 6 is for the determination of the Kokapu, the detection limit is 0.05 ng /
A primer set of SEQ ID NO: 7 and a primer pair in the reverse direction of SEQ ID NO: 8 are for crab identification and a detection limit of 0.5 ng / μL. delete delete delete delete delete (a) extracting genomic DNA from a sample;
(b) amplifying the extracted genomic DNA by a polymerase chain reaction using the primer set of claim 1; And
(c) fractionating the amplification product produced by the step (a) and confirming authenticity of a crab variety in a food product,
Wherein the crab consists of generally crab, red crab, king crab and crab. A kit for determining authenticity of a crab raw material in a food, comprising the primer set of claim 1,
The kit for determining the authenticity of a crab raw material in a food, comprising the crab, crab, king crab, and crab crab.

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