KR20140024672A - Probes and dna chip for identifying place of origin of hairtails, and method for identifying place of origin of hairtails using the same - Google Patents

Abstract

The present invention provides a probe composition and a DNA chip to simply, rapidly, and specifically identify the place of origin of hairtails, and a method for identifying the place of origin of hairtails using the same. According to the present invention, the present invention specifically identifies the place of origin of the hairtails, domestic hairtails and Chinese hairtails, and additionally identifies hairtails maid in Pakistan, India, Senegal, Iran, and/or Morocco by the place of origin. Therefore, the present invention can simply, rapidly, and accurately identify the place of origin of the hairtails and specifically identify the place of origin of the hairtails in order to prepare a measure which rapidly prevents imported hairtails, especially Chinese hairtails from being changed into domestic hairtails during import, quarantine, and distribution processes so that transparency of the distribution process of the hairtails can be enhanced, and profit and health of the domestic consumers can be protected.

Description

TECHNICAL FIELD [0001] The present invention relates to a probe composition, a gene chip, and a method for discriminating the origin of a hairtail using the same, and a method for identifying the origin of hairtail using the same. }

The present invention relates to a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the same, and more particularly, to a method for discriminating between domestic and domestic hairbrushes The present invention relates to a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the same.

In addition, the present invention further relates to a probe composition, a gene chip and a method for determining the origin of a hairbrush using the probe composition, which can discriminate, by country of origin, brackish from Pakistan, India, Senegal, Iran and / or Morocco.

In particular, the present invention provides a method of quickly, precisely and specifically discriminating the origin of the hairbrush from the origin of the imported hairbrush, in particular the Chinese domestic brassiere, to quickly cut off the importation, quarantine and distribution of the domestic brassiere To improve the transparency of the process of distributing haircuts and to protect the interests and health of domestic consumers.

The brassicae are seahorse fishes of the perennials, and they are about 1m in length and thin and long and flat. The hairbrush is shaped like a string with a long tail at the end of the tail. It has a long dorsal fin covering the entire surface of the back, and the body color is silver-white. Ecologically, the warts live on the sandy clay bottom of the continental shelf, mainly at night, and are known to eat plankton, sardines, bulgogi, and squids as carnivorous.

The seaweed caught in the waters of the Korean peninsula, especially in the sea of Jeju Island, is cooked as a variety of dishes that are well suited to the taste of Koreans and are sold at a relatively high price compared to other fish. However, it is very difficult to distinguish between domestic and imported ones, unless they are experts. Domestic horses are distributed at high prices compared to imported horses. Some of the importers, distributors, and restaurants use the price difference to import domestic horses, especially Chinese horses, into domestic horses. Is invisible.

From this point of view, it is necessary to confirm the origin in the importation and quarantine process of imported haircatcher and to require the indication of origin in the distribution process. However, it is necessary to discriminate the origin of haircrumbs from the cost of genetic testing There has been a problem that it is not possible to promptly identify the origin and to improve the transparency of the process of distributing the hairbrush in response to the demands of suppliers and consumers who demand freshness maintenance and rapid distribution as well as inefficiency.

Korean Patent Laid-Open Publication No. 10-2011-0077174 discloses a polynucleotide probe, a DNA chip, and a kit for determining the species of the hairy species or determining the origin of the hairy breed, Patent Publication No. 10-2011-0077174 discloses that the hairy brackishes from Korea / Chinese / Japanese are distinguished from each other by the same species (Trichiurus japonicus) and distinguish them from the Indonesian brackishes (Trichiurus lepturus) and the Indian brackishes (Trichiurus sp.) , We are introducing a probe and a DNA chip that discriminate between species other than Korean, Chinese, and Japanese.

However, the above-mentioned prior art does not pay attention to the problem of the reckless imports of Chinese agricultural products and the problem that Chinese agricultural and marine products turn into domestic ones, and it is not effective to distinguish Chinese braids from domestic warts, There was a problem.

Therefore, in the technical field to which the present invention belongs, considering the unreasonable import of Chinese agricultural and marine products and the problems that the Chinese agricultural and marine products are turned into domestic products, it is possible to simply, quickly, accurately and specifically It is necessary to provide a technology that can enhance the transparency of the distribution process and protect the interests and health of domestic consumers.

Korean Patent Publication No. 10-2011-0077174 (July 7, 2011)

It is an object of the present invention to provide a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the probe composition for specifically identifying the origin of the hairbrush.

Specifically, the present invention provides a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the probe composition, which can specifically discriminate between domestic and overseas hairbirds.

It is another object of the present invention to provide a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the probe composition, which can discriminate, by country of origin, brackish from Pakistan, India, Senegal, Iran and / or Morocco.

Further, the present invention can easily, quickly, accurately, and specifically discriminate the origin of the hairbrush from the imported hairbrushes, especially the Chinese domestic hairbrushes, to quickly cut off the importation, quarantine and distribution processes The purpose of this study is to provide a technology that can enhance the transparency of the hair-cutting distribution process and protect the interests and health of domestic consumers.

The present invention provides a probe composition, a gene chip, and a method for determining the origin of a hairbrush using the probe composition, which can easily, accurately, and specifically discriminate the origin of the hairbrush.

The probe composition of the present invention for the identification of the source of the glutinous origin is a probe for the identification of Trichiurus japonicus in Korea, which comprises oligonucleotides of SEQ ID NOS: 3 to 5 and oligonucleotides complementary to these oligonucleotides And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 6 to SEQ ID NO: 7 and oligonucleotides complementary to these oligonucleotides as probes for discrimination from Chinese / Pakistani / Indian goat ( Trichiurus lepturus ) And at least one probe selected from the group.

In one embodiment of the present invention, the probe composition for determining the origin of an artificial wolf is selected from the group consisting of Chinese, Pakistani / Indian, and Iranian hairbirds, A probe for distinguishing a Senegal horseshoe crab further comprises at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 8 to SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides.

In addition, the probe composition for determining the origin of the hair of the present invention of the present invention can be used as a hairy product of Chinese, Pakistani / Indian, and Senegal, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 10 to SEQ ID NO: 11 and oligonucleotides complementary to these oligonucleotides .

In addition, the probe composition for identifying an origin of the horseshoe crab according to an embodiment of the present invention is a probe for the identification of a Moroccan hare ( Lepidopus caudatus ), and oligonucleotides of SEQ ID NOs: 12 to 13 and oligonucleotides complementary to these oligonucleotides And at least one probe selected from the group consisting of nucleotides.

The gene chip for discriminating the origin of the horseshoe crab according to an embodiment of the present invention is a probe for the identification of a domestic horseshoe crab ( Trichiurus japonicus ) comprising oligonucleotides of SEQ ID NOS: 3 to 5 and oligonucleotides complementary to these oligonucleotides And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 6 to SEQ ID NO: 7 and oligonucleotides complementary to these oligonucleotides as probes for discrimination from Chinese / Pakistani / Indian goat ( Trichiurus lepturus ) And at least one probe selected from the group.

In one embodiment of the present invention, the genetic chip for determining the origin of the hair of the origin of the warts is a genetic chip of Senegal, which is the same species as Chinese, Pakistani / Indian, and Iranian, such as Chinese, Pakistani / A probe for distinguishing a Senegal horseshoe crab further comprises at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 8 to SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides.

In addition, the gene chip for the identification of the origin of the horses in one embodiment of the present invention is a genetic chip for discriminating the origin of horses from the Chinese, Pakistani / Indian, and Senegal horses, the Chinese horses, the Chinese horses, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 10 to SEQ ID NO: 11 and oligonucleotides complementary to these oligonucleotides .

In addition, the genetic chip for the identification of the origin of the warts in one embodiment of the present invention is a probe for discriminating Moruso L. ( Lepidopus caudatus ), and oligonucleotides of SEQ ID NOs: 12 to 13 and oligonucleotides complementary to these oligonucleotides And at least one probe selected from the group consisting of nucleotides.

A gene chip for discriminating the origin of an artificial wisdom of an embodiment of the present invention is a gene chip for discriminating domestic worms ( Trichiurus japonicus ), a probe for discriminating Chinese / Pakistani / Indian worms, a probe for discriminating worms from Senegal, The probe for discriminating the horseshoe crab and / or the probe for discriminating the Lepidopus caudatus may be microarrayed on the same substrate such as a glass slide. It is preferable that the substrate is a glass substrate or a plastic substrate generally used for producing a gene chip. In addition, it is preferable to additionally fix a position marker indicated by a specific nucleotide sequence in the gene chip in which the probe is microarrayed, in order to enhance the hybridization and detection accuracy with the probe.

The method for determining the origin of the hair of the hair of the present invention comprises the steps of amplifying the DNA of the hairy sample of each country of origin by performing PCR, and hybridizing the PCR amplification product to the gene chip for discriminating the origin of the hair of origin as described above And discriminating the origin of the horses according to whether hybridization has occurred or not.

In at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 3 to SEQ ID NO: 5 and oligonucleotides complementary to these oligonucleotides, ( Trichiurus japonicus ) when hybridized with an amplification product, and at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 6 to SEQ ID NO: 7 and oligonucleotides complementary to these oligonucleotides When hybridized with the PCR amplification product, it is distinguished as Chinese / Pakistani / Indian ( Trichiurus lepturus ).

In addition, in the method for determining the origin of the hair of the hair of the present invention, at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 8 to SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides When hybridizing with the PCR amplification product, the oligosaccharides of Senegal are discriminated, and at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 10 to SEQ ID NO: 11 and oligonucleotides complementary to these oligonucleotides is identified When hybridized with the PCR amplification product, it is distinguished as a silkworm.

In addition, in the method of determining the origin of the hair of the hair of an embodiment of the present invention, at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 12 to SEQ ID NO: 13 and oligonucleotides complementary to these oligonucleotides Is distinguished from Trichiurus japonicus and Trichiurus lepturus when it is hybridized with the PCR amplification product, and is distinguished as a Moroccan outburst ( Lepidopus caudatus ).

The hairpin origin identification kit according to one embodiment of the present invention comprises a genetic chip for identifying the origin of the hair extinction as described above and a pair of primers of SEQ ID NOS: 1 and 2 for amplifying the mitochondrial COI gene of the hair- And a labeling substance for detecting an amplified gene product hybridized with the gene chip.

On the other hand, in the present invention, the labeling material is not limited to a specific fluorescent material, as well as a variety of fluorescent materials that can be identified as a fluorescence detector can be used, enzymatic coloration reaction and isotope labeling is also possible. For example, the label may be Cy5, Cy3, biotin-binding material, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethyltamine (TMR), tetramethyltamine isocyanate (TMRITC ), x-rhodamine or Texas red and the like, and it is preferable that the phosphor is Cy3 or Cy5. However, the present invention is not limited thereto, and various labeling materials such as various fluorescent materials, radioactive materials, or luminescent materials known in the art may be used.

According to the present invention, it is possible to specifically discriminate the origin of the hairbrush, and to specifically discriminate domestic hairbrush and Chinese hairbrush.

In addition, according to the present invention, it is further possible to discriminate, by country of origin, brackish from Pakistan, India, Senegal, Iran and / or Morocco.

Accordingly, the present invention provides a method for quickly, accurately, and specifically discriminating the origin of the hairbrush from the origin of the imported hairbrush, particularly, the Chinese domestic brassiere, so as to promptly block the importation, quarantine and distribution of the domestic brassiere It is possible to enhance the transparency of the process of distributing haircuts and to protect the interests and health of domestic consumers.

FIG. 1 shows a layout of a microarray gene chip on which probes for the identification of the origin of hairbrushes are integrated according to an embodiment of the present invention.
FIGS. 2A to 2E are fluorescence images of a gene chip obtained by hybridizing a PCR amplification product of a sample of each genus of the hairbrush of Table 2 to the microarray gene chip of FIG. 1.
FIGS. 3A to 3E are graphs showing the results of hybridization of the PCR amplification products of the insect hairy sample of Table 2 to the gene chip of FIG. 1 in which the probes for discriminating origin of origin (Table 4) (Fluorescence intensity) obtained by scanning and analyzing the fluorescence of the post-fluorescence.

Hereinafter, the present invention will be described in more detail based on examples. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The references cited in the present invention are incorporated herein by reference.

Example 1: Preparation of primer

Using genetic information of mitochondrial COIs obtained from GenBank and previous studies, it is possible to identify Korean domestic warts ( Trichiurus japonicus ), Chinese / Pakistani / Indian warts ( Trichiurus lepturus ), Senegalic warts ( Trichiurus lepturu s) A primer capable of amplifying the DNA extracted from the sample of the wild goose ( Trichiurus lepturus ) and the Moroccan hairtail ( Lepidopus caudatus ) was synthesized. On the other hand, according to NCBI BLAST results, Trichiurus lepturus is classified as the same species in the catch , but the nucleotide sequence of each species by origin (China / Pakistan / India, Senegal, Iran) Respectively.

Table 1:

In the present embodiment described below, the following primers are used. These primers were synthesized by a request from Korea Bioneer (Daejeon Metropolitan City).

Primers for PCR amplification for discrimination by hairy origin

Forward primer

VF2_t1_M13: 5'-CAACCAACCACAAAGACATTGGCAC-3 '(SEQ ID NO: 1)

Reverse primer

FishR2_t1_M13: 5'-ACTTCAGGGTGACCGAAGAATCAGAA-3 '(SEQ ID NO: 2)

The forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 used in the symmetric or asymmetric polymerase chain reaction (PCR) were hybridized with rhodamine, cy3, Or cy5, or attached with biotin. When biotin was attached, it was combined with streptavidin-cyanine after hybridization. A preferred example is to attach cy3 to the primer terminus.

Example 2: Performing PCR amplification reaction on DNA extracted from a hairy sample

Examples of domestic wrist samples, Pakistan wrist samples, Senegal wart samples, Iran wart samples, Chinese wart samples, Japanese wart samples, Indian wart samples and Moroccan wart samples (Table 2 DNA extracted from Qingdao's Qiagen's DNeasy tissue kit.

Table 2: Samples of warts by origin (index number and origin mark)

Then, DNAs extracted from the hairy sample of Table 2 were subjected to PCR using the methods known in the art, for example, a DNA engine (Takara Shuzo Co., Ltd.) under PCR conditions as shown in Table 3 below. PCR amplification reaction was carried out using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2. For reference, the PCR amplification premix shown in Table 3 below is provided by the above-mentioned DNA engine manufacturer, and includes, for example, PCR buffer, dNTP, and TaKaRa Ex Taq ^TM .

Table 3: PCR reaction composition and reaction conditions

2 μl of gel loading buffer (0.2% orange G, 0.25% xylene cyanol FF, 60% glycerol) was added to 3 μl of the PCR amplification product under the above conditions and 1 μg / ml of ethidium bromide bromide in a 2% agarose gel, and the band of the PCR amplification product was confirmed in an image analyzer (HITACHI, Japan) equipped with a UV transilluminator.

Example 3: Fabrication of a probe for discriminating the origin of the hairbrush

In this example, probes capable of specifically discriminating domestic goose hair (discriminated from the same species as Japanese goose) and Chinese goat hair were synthesized, and furthermore, probes of Pakistan, India, Senegal, Iran and / or Moroccan Probes were also synthesized to identify the species by country of origin. Sequence information of the probe for the hybridization reaction is shown in Table 4 below. These probes were prepared according to a method for synthesizing oligonucleotide probes generally known in the art or were commissioned by an institute specialized in oligonucleotide synthesis (for example, Bioni Company, Daejeon, Korea). On the substrate coated with aldehyde In order to covalently bind the oligonucleotides, an amino link was attached to the 5 'end of all probes, and 10 to 20 oligos (dT) were added to minimize spatial interference between the probes integrated on the slide glass during the hybridization reaction The probes used in the embodiments of the invention were completed.

Table 4: Probe nucleotide sequence for the identification of the origin of the warts

As described in the following hybridization reaction examples, it is possible to specifically identify the origin of the haircut according to whether the PCR amplification product of the hairy originating sample of the origin of Table 2 and the hybridization of the probes of Table 4 are listed, It is possible to discriminate between domestic domestic and overseas Chinese army worms.

For example, when the PCR amplification product of the hairless sample to be examined is hybridized with at least one of the probes of SEQ ID NOS: 3 to 5 (domestic probes for distinguishing hairy species) and hybridized with the probes of SEQ ID NOS: 6 to 7 , It can be judged that the sample of the burrows to be inspected is not a Chinese hairbrush and is a domestic hairbrush ( Trichiurus japonicus ).

On the other hand, when the PCR amplification product of the insect hairy sample is not hybridized with the probes of SEQ ID NOS: 3 to 5 (domestic hairy species discrimination probes), at least one of the probes of SEQ ID NOS: 6 to 7 In case of hybridization with one, it is judged that the sample of the burrows to be inspected is Trichurus lepturus from Chinese / Pakistani / Indian.

In addition, the probe composition for determining the origin of the burdock of the present invention can be judged by discriminating accurately from Chinese / Pakistan / Indian salmon, Senegal salmon, and Iranian salmon from the same species of Tridenturus lepturus . For example, when the PCR amplification product of the hairy sample to be tested is hybridized with at least one of the probes of SEQ ID NO: 6 to SEQ ID NO: 7, the hairy sample to be inspected is selected from the group consisting of Chinese braids / Pakistan braids / Trichiurus lepturus ), and when the PCR amplification product of the hairless sample to be tested is hybridized with at least one of the probes of SEQ ID NO: 8 to SEQ ID NO: 9, it is judged that the hairy sample to be examined is a Senegalate hair- When the PCR amplification product of the hairless sample to be examined is hybridized with at least one of the probes of SEQ ID NO: 10 to SEQ ID NO: 11, it is judged that the hairy sample to be inspected is the Iranian hair-killer, so that it can be determined accurately for each origin.

In addition, the probe composition for determining the origin of the burdock of the present invention can discriminate a Moroccan hairtail ( Lepidopus caudatus ) unlike the prior art, wherein the PCR amplification product of the burdock sample to be tested comprises SEQ ID NO: 12 to SEQ ID NO: It is judged that the hair sample to be examined is a Moroccan hairbrush ( Lepidopus caudatus ).

Therefore, the inventive probe composition for distinguishing the origin of the hair of the present invention can be used for the purpose of easily, quickly, accurately and specifically discriminating the origin of the hairbrush from the imported hairbrush, especially the Chinese hairbrush, And provides the advantage of being able to quickly block the distribution process.

Example 4 Fabrication of Microarray Chips

Fabrication of microarrayed gene chips is performed using methods well known to those skilled in the art.

First, an aminolink was modified at the 5'-end of an oligonucleotide having a probe base sequence of Example 3 on a glass having an aldehyde functional group, and then the spatial interference between the probes integrated on the slide glass during the hybridization reaction 10 to 20 oligo (dT) were added to minimize probes to complete the probes used in the examples of the present invention.

In order to prepare the microarray chip in which the oligonucleotide probes were immobilized, the amino link was immobilized on a CSS-100 silylated slide (Cel Associate, USA) at a concentration of 50 μM. The same amount of 3X SSC solution as that of the probe was mixed and accumulated and reacted at room temperature for 16 hours. After the reaction was completed, the slides were washed once with 0.1% SDS for 5 minutes and twice with distilled water for 5 minutes. The prepared chips were reacted with 250 ml of sodium borohydride solution (0.625 g NaBH ₄ , 187.5 ml of PBS, 62.5 ml of 100% EtOH) for 5 minutes, then washed twice with distilled water for 5 minutes and then at 800 rpm for 5 minutes And centrifuged.

The fabricated microarray chips were divided into four sub-arrays and covered with a CoverWell perfusion chamber (Grace-Bio Labs, USA) so that the microarray chips prepared in the hybridization experiment could be independently reacted. The layout (layout) of the microarray chip in which the probes for the identification of the origin of the stocks of glutinous rice (SEQ ID NO: 3 to SEQ ID NO: 13) in Table 4 are integrated is shown in FIG. In the structure of the microarray chip of Fig. 1, the probe name PM is a position marker and is composed of the non-homologous sequence to the mitochondrial COI gene for the identification of the origin of the warts.

Example 5: Hybridization Reaction

PCR amplification products amplified according to Examples 1 and 2 and bound with a label (eg cy3) were mixed with a hybridization solution (3X SSC, 0.3% Sarcosyl) in a ratio of 1: 9. The thus prepared hybridization solution was reacted with the microarray chip of Fig.

That is, the hybridization solution prepared as described above was applied to a chamber containing the microarray chip of FIG. 1 prepared in Example 4, and reacted at 60 ° C for 1 hour. It was then first washed for 5 minutes in 1X SSC, 0.1% SDS solution and again for 5 minutes in 1X SSC solution. It was then further washed in 0.1X SSC solution for 5 minutes. The washed slides were dried for 5 minutes at a speed of 800 rpm in a centrifuge.

In order to confirm the hybridization result of the microarray chip of FIG. 1, the hybridization signal (fluorescence signal) of the microarray chip on which the reaction has been completed was performed using a Photomultiplier Tube (PMT) 550 using GenePix 4000B (Axon Instrument, , Laser power of 100% and resolution of 5 mu m, fluorescence images were stored, and positive and negative of each probe were judged as fluorescence values (see FIGS. 2A to 2E). Fluorescence intensity data was analyzed using GenePix Pro 4.1 software (Axon instrument, USA), and the local background value was subtracted from the mean value of each spot. All probes were spotted double and the signal intensities of the target probes were calculated from the average of the two spots (see Figures 3A-3E).

FIGS. 2A to 2E are photographs showing the result of combining the probes for the identification of the origin of the shoots (see Table 4) according to the present invention and the PCR amplification products of the hairless hairy sample of Table 2. FIGS. 3A to 3E are photographs (Table 4) in which the probes for the identification of the origin of the glutinous rice of Example 1 were arrayed was subjected to hybridization reaction of the PCR amplification product of the glutamate samples of Table 2 to the gene chip of FIG. 1, (Fluorescence intensity).

As shown in Figs. 2A and 3A, in the case of a domestic hair extrudate sample (index number 1-13 samples in Table 2; Korea-1 to Korea-13 samples), fluorescence of the gene chip was analyzed and analyzed. 3 to SEQ ID NO: 5 (domestic probes for discrimination), fluorescence spots were observed at positions where the fluorescent signals of the probes of SEQ ID NOS: 6 to 7 The discrimination probe of the present invention has a relatively stronger analysis than the fluorescent signal of the Chinese / Pakistani / Indian haircut discrimination probes). Thus, the discriminating probes according to the present invention can be applied to imported hairbrushes, especially Chinese brazes, It can be confirmed that it can be discriminated accurately.

On the other hand, as shown in Figs. 2B and 3B, in the case of Chinese goat hair samples (index number 40-44 samples in Table 2; China-1 to China -5 samples), fluorescence of gene chips was analyzed and analyzed Results Fluorescent spots were strongly identified at the locations where the probes of SEQ ID NOS: 6 to 7 (probes for discrimination from Chinese / Pakistan / Indian stocks) were arrayed and the fluorescence signals of other probes other than these fluorescence signals, 3 to SEQ ID NO: 5 (probes for identification of domestic hair extrudate) were hardly observed.

Also, as shown in Figs. 2C and 3C, a Senegalian hairbrush sample classified as Chinese / Pakistani / Indian haircatcher and Iranian haircatcher and the same type of haircatcher Trichiurus lepturus (index numbers 19-28 in Table 2 (Senegal-1 to Senegal-10 samples), the fluorescence of the gene chip was analyzed and analyzed. As a result, it was found that when the probes of SEQ ID NO: 8 to SEQ ID NO: 9 Spots were strongly confirmed and fluorescence signals of other probes other than these fluorescence signals, particularly the probes of SEQ ID NOS: 6 to 7 (probes for discriminating Chinese / Pakistani / Indian goat species) and the probes of SEQ ID NOS: 10 to 11 The fluorescence signals of the probes (the probes for discriminating the hatchlings of an egg) were rarely observed.

On the other hand, as shown in Figs. 2 (d) and 3 (d), the samples of Chinese brackish / brackish and brackish silver and the brackish , Trichiurus lepturus , 10) to SEQ ID NO: 11 (probes for discriminating the anchovy horses) from the fluorescence of the gene chip, it was found that the fluorescence The spots were strongly confirmed and the fluorescence signals of other probes other than these fluorescence signals, particularly the probes of SEQ ID NOS: 6 to 7 (probes for discrimination of Chinese / Pakistan / Indian salmon) and the probes of SEQ ID NOS: 8 to 9 Fluorescence signals of the probes (probes for discrimination from the Senegal hairbrush) were scarcely observed.

Therefore, the probe composition for the identification of the origin of the burdock of the present invention can discriminate the Trichiurus lepturus , which is the same type of burrow , from the Chinese / Pakistan / Indian salmon, Senegal salmon and Iranian salmon I could confirm.

In addition, as shown in Figs. 2E and 3E, in the case of the Moroccan hair extrudate sample (index number 56-60 sample in Table 2; Morocco-1 to Morocco-5 sample), fluorescence of the gene chip was scanned As a result, fluorescence spots were strongly confirmed at the positions where the probes of SEQ ID NO: 12 to SEQ ID NO: 13 (probes for discriminating Moroccan anchovy) were arrayed, and fluorescence signals of other probes other than these fluorescence signals were hardly observed. Therefore, it was confirmed that the probe composition for the identification of the hairy origin of the present invention can accurately discriminate the Moroccan hairtail ( Lepidopus caudatus ) against Trichiurus japonicus and Trichiurus lepturus , unlike the prior art.

As a result, the probe composition for the identification of the origin of the burdock of the present invention can distinguish the origin of the burdock from the origin of the imported burdock, especially Chinese burdock, into domestic domestic burdock by simple, fast, accurate and specific discrimination. It is advantageous to make it possible to prevent the profit and health of the domestic consumers and to establish the measures to protect the domestic consumers.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

<110> GENOCHECK CO., LTD.          REPUBLIC OF KOREA <120> Probes and DNA chip for identifying place of origin of hairtails,          and method for identifying place of origin of hairtails using the          same <130> P12-0294KR <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VF2_t1_M13 Forward Primer <400> 1 caaccaacca caaagacatt ggcac 25 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> FishR2_t1_M13 Reverse Primer <400> 2 acttcagggt gaccgaagaa tcagaa 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Kor / Jap-4_AS Probe <400> 3 ggggtagaag tcagaagctc atatta 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Kor / Jap-6 Probe <400> 4 acaatactcc taactgaccg aaatct 26 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kor / Jap-9 Probe <400> 5 cccactagct gggaatctag cac 23 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Pak / Ind / chi-3 Probe <400> 6 taactatctt ctccctccat ctggca 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Pak / Ind / chi-6_AS Probe <400> 7 tccaattatt agggggacga gtcagt 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> sen-3 Probe <400> 8 cttcttcttc tagcttcttc tggagt 26 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> sen-4_AS Probe <400> 9 ctgtaacgat aacattgtaa atttgg 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ira-1 Probe <400> 10 gccggaatta caatgcttct aactga 26 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Ira-3 Probe <400> 11 tcttttccct ccatttagca ggaatt 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Mor-2 Probe <400> 12 tacagccatc cttttacttc tgtccc 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Mor-3 Probe <400> 13 cccttattcg tgtggtccgt attaat 26

Claims (14)

A probe for discriminating Trichiurus japonicus in Korea, at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 3 to SEQ ID NO: 5 and oligonucleotides complementary to these oligonucleotides,
6 to 9 and oligonucleotides complementary to these oligonucleotides as a probe for discriminating Trichurus lepturus from Chinese / Pakistani / Indian ( Trichiurus lepturus ), and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides A probe composition for discriminating the origin of hair. The method of claim 1,
As a probe for distinguishing Senegal hairbirds from Chinese, Pakistani and Indian hairbirds and Iranian hairbirds distinguished from Chinese, Pakistani / Indian and Brazilian hairbirds, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides. The method of claim 1,
A probe for the identification of an antelope from the Chinese, Pakistani, and Indian braids and Senegal from the Chinese, Pakistani, and Indian braids and Senegal, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 11 and oligonucleotides complementary to these oligonucleotides. 4. The method according to any one of claims 1 to 3,
A Lepidopus caudatus discriminating probe comprising at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 12 to SEQ ID NO: 13 and oligonucleotides complementary to these oligonucleotides, A probe composition for discriminating origin. A probe for discriminating Trichiurus japonicus in Korea, at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 3 to SEQ ID NO: 5 and oligonucleotides complementary to these oligonucleotides,
6 to 9 and oligonucleotides complementary to these oligonucleotides as a probe for discriminating Trichurus lepturus from Chinese / Pakistani / Indian ( Trichiurus lepturus ), and at least one probe selected from the group consisting of oligonucleotides complementary to these oligonucleotides Genetic chip for the identification of the origin of the burdock. 6. The method of claim 5,
As a probe for distinguishing Senegal hairbirds from Chinese, Pakistani and Indian hairbirds and Iranian hairbirds distinguished from Chinese, Pakistani / Indian and Brazilian hairbirds, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides. 6. The method of claim 5,
A probe for the identification of an antelope from the Chinese, Pakistani, and Indian braids and Senegal from the Chinese, Pakistani, and Indian braids and Senegal, And at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 11 and oligonucleotides complementary to these oligonucleotides. 6. The method of claim 5,
A Lepidopus caudatus discriminating probe comprising at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 12 to SEQ ID NO: 13 and oligonucleotides complementary to these oligonucleotides, Genetic chip for discrimination of origin. Amplifying the DNA of the cutlass sample by place of origin by PCR;
Hybridizing the PCR amplification product to a gene chip for identification of an origin of warts as defined in any one of claims 5 to 8,
And determining the origin of the hairbrush according to whether hybridization has occurred or not. 10. The method of claim 9,
When at least one probe selected from the group consisting of oligonucleotides of SEQ ID NOS: 3 to 5 and oligonucleotides complementary to these oligonucleotides is hybridized with the PCR amplification product, it is judged to be a domestic Trichurus japonicus and,
When at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 6 to SEQ ID NO: 7 and oligonucleotides complementary to these oligonucleotides is hybridized with the PCR amplification products, Trichiurus lepturus ) to determine the origin of cutlass, characterized in that. 10. The method of claim 9,
Characterized in that when at least one probe selected from the group consisting of oligonucleotides of SEQ ID NO: 8 to SEQ ID NO: 9 and oligonucleotides complementary to these oligonucleotides is hybridized with the PCR amplification product, To determine the origin of the hairbrush. 10. The method of claim 9,
Characterized in that when at least one probe selected from the group consisting of oligonucleotides of SEQ ID NOS: 10 to 11 and oligonucleotides complementary to these oligonucleotides is hybridized with the PCR amplification product, To determine the origin of the hairbrush. 10. The method of claim 9,
When at least one probe selected from the group consisting of oligonucleotides of SEQ ID NOS: 12 to 13 and oligonucleotides complementary to these oligonucleotides is hybridized with the PCR amplification product, it is distinguished from Trichiurus japonicus and Trichiurus lepturus , ( Lepidopus caudatus ). &Lt; / RTI &gt; A gene chip for discriminating the origin of the warts as defined in any one of claims 5 to 8,
1 and 2 for amplifying the mitochondrial COI gene of the wild-type hairy sample,
And a labeling substance for detecting an amplified gene product hybridized with the gene chip.

Images