KR101536177B1 - Method and kit for identifying Larimichthys polyactis and Larimichthys crocea using real-time PCR - Google Patents

Abstract

The present invention provides a technique to specifically, rapidly, and accurately discriminate Larimichthys polyactis and Larimichthys crocea by performing real-time PCR using a real-time PCR primer pair and a probe respectively specific to Larimichthys polyactis and Larimichthys crocea. In the present invention, Larimichthys polyactis and Larimichthys crocea are simply, rapidly, accurately, and specifically discriminated by using real-time PCR, so that a countermeasure for rapidly blocking that Chinese Larimichthys crocea is disguised as Larimichthys polyactis is ensured during import, quarantine, and distribution processes. Accordingly, transparency is promoted in a distribution process of Larimichthys polyactis and Larimichthys crocea, and benefits and health of domestic consumers may be protected.

Description

[0001] The present invention relates to a method and kit for discriminating reference and taxation using real-time PCR,

The present invention relates to a method and kit for discriminating between reference and reference using real-time PCR (real-time polymerase chain reaction), and more particularly, to a method and kit capable of discriminating reference and homing specifically and rapidly And a kit.

Specifically, the present invention relates to a method and a kit for identifying a reference and a tax base during import, quarantine and distribution using real-time PCR, with high specificity and sensitivity based on quick, accurate and quantified data.

In addition, the present invention provides a method for quickly and accurately discriminating reference strains and reference strains using real-time PCR, thereby promptly preventing the importation, quarantine, and distribution of strains, To improve the transparency of reference and subscription distribution processes and to protect the interests and health of domestic consumers.

The reference fish (Larimichthys polyactis) is a sea cucumber fish with a body length of about 30 cm. It lives in the southern part of Korea, the west sea and the south sea, the western part of Japan, and the East China Sea. It is caught from summer to early spring. It is used for dried fish, salt grilled, steamed, hot water.

The larvae (Larimichthys crocea) are distributed in the Pacific Northwest of Korea, Japan, and the East China Sea. Spawning takes place twice in spring and autumn. Spawning occurs in the East China Sea in spring and in the South China Sea in autumn. Busan is widely used as a bath or bath, and its price is low, similar to early taste.

The Chinese reference and the domestic reference are all the same species, and there is no way to distinguish them at this time, and the taxation is very similar to the reference system. It is a fish that is traditionally favored by our people because of its taste. In the past, the catches of reference cucumbers were abundant, but now the catches are significantly reduced, which is more expensive than taxation. However, it is very difficult to distinguish between reference devices and subsidies currently in circulation unless they are experts.

Compared to taxation, referrals are distributed at high prices. In some importers, distributors and restaurants, abuse of such price differences makes it difficult to take profits from consumers by converting the value-based taxation system, especially the Chinese taxation system, into domestic reference taxpayers.

In this respect, the necessity of discrimination between reference and subsistence in the process of importation, quarantine and distribution of reference and subsidies is very large. However, compared to the cost of genetic testing, In addition to this inefficiency, there was a problem that it was not enough to promptly identify the reference and taxation in response to the demands of suppliers and consumers who demand freshness maintenance and rapid distribution, and to enhance the transparency of reference and tax distribution.

In this regard, Korean Patent Registration No. 10-0382172 discloses a method for quickly determining whether or not an artificial coloration of a reference airflow is caused by coloring with peracetic acid to prevent the coloring of the pigment to a reference fish, Thereby releasing the dye.

However, the above-mentioned prior art focuses only on the problem of reference guide and coloring matters due to coloring of reference-like fishes such as taxation, and there is a limit that can not distinguish between taxation and reference, There was a low effectiveness in the quarantine and distribution process. In addition, in the case of the conventional method for sequencing the species, there is a disadvantage in that it takes a long time to test and it is difficult to distinguish the sample from the mixed sample. In the case of the general polymerase chain reaction method, one base mismatching It is true that there are many error results in the following.

Therefore, in the technical field to which the present invention belongs, in order to fundamentally solve the problem of reckless importation of foreign agricultural and marine products and the problem of importing and distributing tax, especially cheap Chinese import tax, It is necessary to provide the technology that can enhance the transparency of reference and subscription distribution process and protect the interests and health of domestic consumers by simple, prompt, accurate and specific identification of taxation.

Korean Patent Registration No. 10-0382172 (2003.05.01)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method and kit for specifically and rapidly and precisely discriminating reference vectors and bases using real- It has its purpose.

Specifically, the present invention provides a method and a kit that can discriminate between a reference and a tax base during import, quarantine and distribution using real-time PCR with a high degree of specificity and sensitivity based on quick, accurate and quantified data. .

Furthermore, the present invention provides a method for quickly, accurately, and specifically discriminating reference and miscarriage using real-time PCR, thereby promptly preventing the importation, quarantine, and distribution of taxable goods, The purpose of this study is to provide the technology that can enhance the transparency of the circulation process of reference and subscription and protect the interests and health of domestic consumers.

As a result of intensive researches in order to solve the technical problem of the invention described above, the present inventors analyzed the mtDNA sequence of the reference gene and the subtilis mRNA and analyzed the sequence of the 16S rRNA gene, , A probe specific to reference and bias, respectively. The present invention provides a technology capable of specifically and quickly and accurately discriminating reference vectors and bases by performing real-time PCR using a pair of primers for real-time PCR and a probe specific for reference and base, respectively.

As used herein, the term "real-time PCR" refers to the application of a fluorescent material to a PCR technique, in which the amplification of a target gene existing in a specimen during the reaction, It is a method that can quickly and accurately analyze the presence or absence of amplification of a target gene and its pattern by real-time detection and quantitative analysis. This real-time PCR is divided into two methods, one using SYBR Green and the other using double-labeled probes.

The SYBR green technique combines the SYBR green dye into the DNA amplified during real-time PCR amplification and binds to the double-stranded DNA synthesized by the PCR reaction to generate a fluorescent signal. The fluorescent signal is detected, It is a method that can measure the presence and the amount of amplification product from it. The real-time PCR technique using a dual labeled probe is a method using a double-labeled probe in which a fluorescent substance is labeled at the 5 'end and a quencher is labeled at the 3' end, Through the fluorescence signal from the labeled probe, it is possible to confirm whether or not the double-labeled probe is annealed with the PCR amplification product of the target gene, and the presence or absence of the target gene and the amount of the amplification product generated therefrom can be measured. It goes without saying that real-time PCR known in the art such as a TaqMan probe method and the like are applicable in the present invention.

On the other hand, the extraction of DNA from the reference and auxiliary samples can be carried out from various tissues of the sample by various methods, since DNA is extracted and analyzed to determine reference and homology, DNA is extracted from any part of the sample Or how it is extracted is also not particularly limited. It is clear that other DNA extraction methods known to those skilled in the art are also within the scope of the present invention.

The present invention relates to a primer pair for real-time PCR amplification, which is used in a method of discriminating between reference and non-primer pairs using real-time PCR, and which comprises a primer pair of SEQ ID NOS: 3 and 4 for amplifying a 16S rRNA target gene, And a set of primers for amplifying the 16S rRNA target gene of BSE.

The present invention also relates to a probe specific for a 16S rRNA target gene of the reference gene, comprising at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 5 and an oligonucleotide complementary to the oligonucleotide and at least one probe selected from the group consisting of a 16S rRNA target gene A probe and at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 6 and an oligonucleotide complementary to the oligonucleotide as a specific probe.

In addition, the present invention is a kit for discriminating between a reference vector and a tax base using real-time PCR, and is characterized in that a primer pair of SEQ ID NOS: 3 and 4 for amplifying a 16S rRNA target gene of a reference vector and a primer pair of a 16S rRNA target gene And at least one probe selected from the group consisting of an oligonucleotide complementary to such an oligonucleotide and a probe specifically binding to the amplification product of a 16S rRNA target gene There is provided a kit for discriminating between a reference and a reference using real-time PCR, which comprises at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 6 and an oligonucleotide complementary to such oligonucleotide.

The reference kit and the biosensor determination kit using real-time PCR in one embodiment of the present invention further include DNA polymerase, dNTPs, PCR buffer solution, and labeling substance for PCR amplification product.

In a real-time PCR amplification kit for discriminating between a reference vector and a reference vector according to an embodiment of the present invention, the labeling substance comprises one end of a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference vector, For example, at the 5 ' end of the probe that specifically binds to the amplification product of the 16S rRNA target gene.

A real-time PCR amplification kit for identifying a reference vector and an oligonucleotide according to an embodiment of the present invention, comprising: a probe specifically binding to an amplification product of a 16S rRNA target gene of the reference vector; and an amplification product of the 16S rRNA target gene The specifically binding probes are preferably labeled with a labeling substance that can be detected at different wavelengths. As such, a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference vector and a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference gene can be detected as markers capable of detecting at different wavelengths It is possible to detect the 16S rRNA target gene of the reference and the 16S rRNA target gene of the reference at a time in one test tube when labeled.

The reference substance is a fluorescent substance, and the intensity of fluorescence from the labeled substance is measured to determine the amplification amount of the 16S rRNA target gene of the reference gene and the fluorescence intensity Of 16S rRNA target gene amplification can be calculated.

The reference method and the method of determining bias using real-time PCR according to an embodiment of the present invention,

Preparing a gene sample from a specimen,

Using the prepared gene sample as a template, the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene in the gene sample of the specimen were amplified in real time using a reference kit and a discrimination kit as described above -time) amplification by PCR;

After amplification of the 16S rRNA target gene of the reference strain and the 16S rRNA target gene of the reference strain, the real-time PCR result is checked to determine whether the sample is a reference-based substance or a tax-derived substance or whether they are mixed .

The reference method and the method of determining bias using real-time PCR according to an embodiment of the present invention,

Before amplification, a positive standard, which is a gene sample that knows the copy number of the 16S rRNA target gene of the reference gene and the copy number of the 16S rRNA target gene of the reference gene, was amplified using a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4 amplifying the 16S rRNA target gene Amplifying the 16S rRNA target gene amplification amount of the reference gene and the labeled 16S rRNA target gene amplification amount of the reference gene by real-time PCR;

Measuring a change in fluorescence intensity of the labeling substance with respect to the entire PCR amplification period of the positive standard and analyzing the number of PCR reactions (Cp value or Ct value) reaching a constant fluorescence intensity;

Correlating the number of copies of the positive standard with the value of Cp or Ct from a standard curve obtained by measuring a change in fluorescence intensity of the labeling substance;

The change in fluorescence intensity of the labeling substance with respect to the entire PCR amplification period of each of the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene existing in the gene sample of the specimen was measured and PCR Analyzing the number of reactions (Cp value or Ct value)

A Cp value or a Ct value obtained by PCR amplification of each of the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene existing in the gene sample of the specimen is made to correspond to the copy number of the positive standard, Lt; RTI ID = 0.0 > 16S < / RTI > rRNA target gene or subgeneric 16S rRNA target gene.

In a method of identifying a reference vector and a reference vector using real-time PCR according to an embodiment of the present invention, the labeling substance may include one end of a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference vector, Can be labeled at one end of the probe specifically binding to the amplification product of the 16S rRNA target gene, for example, at the 5 'end.

In this regard, a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference vector and a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference gene are capable of detecting a labeling substance . ≪ / RTI > As such, a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference vector and a probe specifically binding to the amplification product of the 16S rRNA target gene of the reference gene can be detected as markers capable of detecting at different wavelengths It is possible to detect the 16S rRNA target gene of the reference and the 16S rRNA target gene of the reference at a time in one test tube when labeled.

In a method of discriminating between a reference and a reference using real-time PCR according to an embodiment of the present invention, the labeling substance is a fluorescent substance, and the intensity of fluorescence from the labeling substance is measured to determine the 16S rRNA target gene amplification amount of the reference gene, Of 16S rRNA target gene amplification can be calculated.

In the present invention, one end of the probe, for example, the 5 'terminus may be selected from Cy5, Cy3, x-Rhodamine, Texas Red, SYBR green, FAM, VIC, JOE, NED, HEX and TET The other end of the probe, for example, the 3 'terminus, may be labeled with a small molecule such as IABkFQ, DABCYL, TAMRA, ECLIPSE, BHQ (BHQ-1, BHQ-2, BHQ- . However, it should be understood that the present invention is not limited thereto, and a variety of fluorescent materials and minerals known in the art can be used.

According to the present invention, real-time PCR can be used to specifically and rapidly and accurately determine the reference and bias, and in particular, the reference and bias during the import, quarantine and distribution process can be quickly, accurately and quantitatively determined based on the quantified data And the sensitivity can be determined to be high.

In addition, the present invention provides a method for quickly and accurately discriminating reference strains and reference strains using real-time PCR, thereby promptly preventing the importation, quarantine, and distribution of strains, It is possible to enhance the transparency of the distribution process of reference and taxation and to protect the interests and health of domestic consumers.

FIG. 1 is a photograph of the results of electrophoresis of PCR amplification products of 16S rRNA gene contained in a DNA sample extracted from domestic reference, Chinese reference, and Chinese origin, on an agarose gel.
Fig. 2 is a diagram showing homology and mutation analysis results of the 16S rRNA target gene sequence of the reference gene and the 16S rRNA target gene sequence of the subgenus.
FIG. 3A is a real-time PCR result graph of a negative control without reference and / or specific specific 16S rRNA target gene sequence in a sample, and FIG. 3B is a graph showing real- This is a real-time PCR result graph.
FIG. 4A is a graph of real-time PCR results of a negative control without reference and / or specific specific 16S rRNA target gene sequence in the sample, and FIG. 4B is a graph showing real- This is a real-time PCR result graph. In the graphs of FIGS. 4A and 4B, the threshold value was set to 0.3, and FAM fluorescence values less than 0.3 were regarded as noise and the false negative results were excluded.
FIG. 5 is a graph of the detection limit obtained by performing real-time PCR after stepwise dilution of the DNA extracted directly from the reference strain with the sterilized distilled water. FIG. 5A is a graph of the detection limit result of the real-time PCR obtained in the case of the reference instrument, and FIG. 5B is a graph of the detection limit result of the real-time PCR obtained in the case of the bias.

Hereinafter, the present invention will be described in more detail based on the embodiments of the present invention. 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

Example 1: Obtaining 16S rRNA gene sequence information for specific discrimination between reference and non-reference and performing pre-PCR amplification

For specific identification of the reference strain (Larimichthys polyactis) and the taxane (Larimichthys crocea), reference was made to the prior art and the GenBank gene sequence, and as a result, the mitochondrial 16S rRNA gene was selected as the species identification molecule marker. Specifically, the gene sequence of FJ618559.1 registered in NCBI was selected (see SEQ ID NO: 7) as the target gene sequence for the specific discrimination of the reference gene, and the target gene sequence for the specific discrimination of the subgenus was registered in NCBI The gene sequence of FJ595214.1 was selected (see SEQ ID NO: 8). Based on these 16S rRNA target gene sequences, primer pairs and probes are designed to perform real-time PCR.

To analyze the homology and variation between the target gene sequences for various reference and subsampling samples and to select the optimal primer pairs and probes for performing real-time PCR, pre-PCR amplification from reference and sub- PCR amplification was performed.

Domestic reference crops were purchased at the Gunsan Fisheries Market, Noryangjin Market, and Busan Fisheries Cooperative Fisheries Market. The Chinese reference fisheries were purchased at the Incheon Fish Market, and the Chinese fisheries were purchased at the Agriculture, Forestry and Fisheries Quarantine Inspection Headquarters and the Incheon Fish Market. DNA was extracted from DNase Blood & Tissue Kit (Qiagen, Cat. 69506) of Qiagen, Germany, from the reference and the subsamples thus purchased. Although the reference portion and the body portion of the trunk are used as the extraction region, the present invention is not limited thereto, and it is easily understood by those skilled in the art that various tissues can be used.

Then, pre-PCR amplification was performed using the DNA extracted from the reference strain and the bases as a template and using primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 as a universal primer pair for mitochondrial 16S rRNA gene amplification.

16S L (forward primer): 5'-CGC CTG TTT AAC AAA AAC AT-3 '(SEQ ID NO: 1)

16S H (reverse primer): 5'-CCG GTC TGA ACT CAG ATC ACG T-3 '(SEQ ID NO: 2)

The PCR amplification composition and amplification conditions for the pre-PCR are shown in Table 1 below, and the master mix for PCR amplification in the PCR amplification composition is composed of, for example, PCR buffer, dNTP, DNA polymerase and the like. In the example, Premix Taq (Ex Taq Version 2.0) (TaKaRa, Cat. RR003A) was used.

After completion of the pre-PCR amplification under the above conditions, the amplified product was confirmed by electrophoresis using agarose gel (3%). A band of the PCR amplification product was confirmed in an image analyzer (HITACHI, Japan) equipped with a UV transilluminator. As a result of confirmation of the PCR amplification products, formation of nonspecific bands could not be confirmed, and the amplification products of the 16S rRNA genes of Korean reference, Chinese reference and Chinese origin were confirmed (FIG. 1).

Example 2: Design of primers and probes for real-time PCR for specific discrimination of reference and homology

Sequencing methods and sequence analysis methods widely used and known in the art were carried out on 16S rRNA gene samples of various reference and secondary reference primers amplified in Example 1. From this, the homology and mutation to the 16S rRNA target gene sequence for specifically discriminating between the reference gene and the taxa were analyzed (Fig. 2). (SEQ ID NO: 3 and SEQ ID NO: 4) capable of amplifying both the reference gene and the 16S rRNA target gene sequence of the bases based on the analyzed nucleotide sequences, and a pair of fluorescent-labeled probes (SEQ ID NO: 5 and SEQ ID NO: 6) were designed (see Table 2 below). These primers and probes were synthesized by requesting Bioneer (Daejeon, Republic of Korea).

Primers and probes for 16S rRNA gene amplification for discrimination between reference and primers SEQ ID NO: designation The base sequence (5 '- > 3') Remarks SEQ ID NO: 3 16S rRNA Forward Primer ACC AAC AAG ATC CGG CAA SEQ ID NO: 4 16S rRNA Reverse Primer GGA TTG CGC TGT TAT CCC TA SEQ ID NO: 5 CO-KOR probe CGC CGA TCA ACG AAC CCA GTT ACC Reference (CO-KOR) SEQ ID NO: 6 CO-CHN probe CGC CGA TCA ACG AAC CGA GTT ACT (CO-CHN)

The probe of SEQ ID NO: 5 specifically binds to the amplification product of the 16S rRNA target gene sequence of the reference gene (the gene sequence of FJ618559.1), and the probe of SEQ ID NO: 6 is the 16S rRNA target gene sequence of FJ595214. 1 < / RTI > gene sequence). Therefore, the specific discrimination between the reference vector and the bases can be made through the real-time PCR using the primer pair and the probe of Table 2 above.

Example 3: Evaluation of primer and probe performance for specific discrimination between reference and non-reference

The 16S rRNA gene contained in the DNA extracted directly from the reference strain and the bases, as described in Example 1, was used as a template or the amplified product of the 16S rRNA target gene sequence of the pre-amplified reference gene and the 16S rRNA target gene sequence in Example 1 was used as a template (SEQ ID NO: 3 and SEQ ID NO: 4) and specific probes (SEQ ID NO: 5 and SEQ ID NO: 6) for the reference and secondary 16S rRNA target genes.

To identify and quantitatively analyze the results of real-time PCR amplification reactions, probes are double-labeled with fluorescent and small-molecule materials. In this example, the probe of SEQ ID NO: 5 was labeled with FAM (maximum absorption wavelength: 495 nm, maximum emission wavelength: 520 nm) at the 5 'end of the probe, and TAMRA, which is a mineralsubstance, was labeled at the 3' end. In addition, a fluorescence substance HEX (maximum absorption wavelength: 535 nm, maximum emission wavelength: 556 nm) was labeled at the 5 'end of the probe of SEQ ID NO: 6, and TAMRA was detected at the 3' end.

Thus, the presence of a reference-specific 16S rRNA target gene sequence in one test tube by reference-specific and bespecific probes labeled with different fluorescent materials (with different emission wavelengths) The presence or absence of the 16S rRNA target gene sequence can be simultaneously detected. Meanwhile, the fluorescent substance and the small-molecule substance for labeling the probe are not limited to the above-mentioned examples, and it goes without saying that various fluorescent substances and small-molecule substances known in the art may be used.

The real-time PCR method is applied to the PCR technique using such a fluorescent substance and a small-scale substance. It amplifies the target gene sequence present in the sample during the reaction and detects the emission level of the fluorescent substance in real time, Analysis of the amplification of the target gene sequence and its aspects can be quickly and accurately analyzed. The basic principle of real-time PCR lies in the detection and quantification of fluorescence, which is performed in real time every PCR cycle by the principle of polymerase and fluorescence resonance energy transfer (FRET).

Quantification in real-time PCR is a method of measuring the amplification product in real time within the exponential phase by monitoring the progress of each cycle during PCR. In this case, important concept is Ct (threshold cycle) or Cp point. This value is the number of cycles at which the intensity of fluorescence produced by the double-labeled probe described above increases markedly beyond the baseline level, indicating that the initial template quantity of the target gene sequence The number of copies of the reference and / or tax specific 16S rRNA target gene sequence). Therefore, PCR amplification of a sample containing reference and / or bispecific specific 16S rRNA target gene sequence was performed to measure the change in fluorescence intensity of the fluorescent substance over the entire PCR amplification period, and the number of PCR reactions (Cp value Or Ct value) and a standard curve of fluorescence intensity is obtained, and then the presence or absence of reference and / or specific specific 16S rRNA target gene sequence and the initial amount of these target gene sequences contained in the sample are quantitatively analyzed .

Real-time PCR was performed in this example using the 7900HT Fast Real Time PCR System (Life Technologies, USA). (Primer pair of SEQ ID NO: 3 and SEQ ID NO: 4) for the real-time PCR amplification of the reference and the secondary 16S rRNA target gene, and the reference and secondary 16S rRNA target genes, respectively, Specific probe of SEQ. ID. NO. 5 and the specific probe of SEQ ID NO. 6 that were fluorescently labeled with different fluorescent substances (having different emission wavelengths) and a reference specimen to be amplified and / or a secondary 16S rRNA Real-time PCR compositions such as a target gene template and a real-time PCR master mixture were performed as shown in Table 3 below, and real-time PCR was performed under the real-time PCR amplification conditions shown in Table 3 below . Meanwhile, TaqMan Universal Master Mix II, no UNG (Life Technologies, Cat. 4440040) was used as a real-time PCR master mix.

In this example, real-time PCR was repeated under the real-time PCR conditions of Table 3 under the real-time PCR amplification composition of Table 3 above for reference and / or bispecific specific 16S rRNA target gene sequences. Each time the real-time PCR amplification was repeated, the fluorescence values were measured at the FAM and HEX wavelengths, and the fluorescence intensity for the reaction cycle was analyzed using SDS software. The results are shown in Figs. 3A and 3B.

3A is a real-time PCR result graph of a negative control without a reference and / or specific specific 16S rRNA target gene sequence in a real-time PCR amplification sample, and FIG. 3B is a graph showing real- Lt; RTI ID = 0.0 > 1 < / RTI >

As shown in FIG. 3B, in the confirmation of the two real-time PCR amplification reaction products produced in one test tube, the reference-specific probe of SEQ ID NO: 5 labeled 5 'end with FAM was used as the 16S rRNA target The presence of the 16S rRNA target gene of the reference gene can be confirmed by the fluorescence value coming from the 520 nm wavelength since it specifically binds to the amplification product of the gene sequence. On the other hand, the presence of the 16S rRNA target gene of the subunit was found to be 556 nm, because the specific probe of SEQ ID NO: 6 labeled with 5 'end with HEX specifically binds to the amplification product of the 16S rRNA target gene sequence of subtilis It can be confirmed by the fluorescence value coming from the wavelength. Therefore, the method of discriminating between reference and non-reference using the real-time PCR of the present invention can be carried out by using a probe specific for reference and sub-assay, and a labeled substance having different emission wavelengths labeled for each of these specific probes, The discrimination can be clarified.

4A and 4B are graphs showing the results of real-time PCR of the second-order real-time PCR under the real-time PCR composition and conditions shown in Table 3 above. In the results of FIGS. 4A and 4B, the FAM fluorescence noise value from the probe-specific probe of SEQ ID NO: 5 labeled with 5'-terminal was relatively large and the threshold value was set to 0.3 during the analysis. . In other words, FAM fluorescence values less than 0.3 were regarded as noise and the false negative results were excluded.

As a result, it was confirmed that the real-time PCR reaction clearly distinguished the two reactants in one tube, that is, the discrimination between the reference reagent and the virulent reagent, and the false positives or false negatives of the two reactants were not found.

As a result, as shown in FIG. 3A to FIG. 4B, the 16S rRNA target gene of the reference and the 16S rRNA target gene of the reference were respectively amplified by real-time PCR and the primers of Table 2 Pair and specific probes effectively amplify the corresponding reference and subsequence 16S rRNA target gene sequences and the curve of fluorescence intensity for the amplification cycle is also used for specific discrimination of reference and subspecies (reference 16S rRNA target gene sequence and / Or the presence of the 16S rRNA target gene sequence of virulence) and quantitative analysis.

Example 4 Analysis of Detection Limit Concentration of 16S rRNA Target Gene of Reference and Subtilis Using Real Time PCR Method of the Present Invention

In this example, the detection limit concentration of the reference gene and the 16S rRNA target gene of the reference gene was analyzed using the real-time PCR method of the present invention.

As described in Example 1, the DNA extracted directly from the reference strain and each of the subtiles was diluted with sterilized distilled water in seven steps ranging from 10 ng / ul to 0.00001 ng / ul, followed by real-time PCR Respectively. The detection limit results of the real-time PCR method of the present invention are as shown in Figs. 5A and 5B.

As a result, it was possible to effectively amplify the target gene sequence of the 16S rRNA of the reference strain to a concentration of 0.001 ng / ul in the case of the reference strain (see FIG. 5A), and to the concentration of 0.01 ng / Effective amplification of the target gene sequence was possible (see Fig. 5b). Therefore, in the case of the real-time PCR method of the present invention at a concentration of 0.01 ng / ul, which is a concentration that can not be detected by conventional PCR in terms of detection sensitivity (detection limit concentration), the 16S rRNA target gene sequence of the reference gene and the 16S rRNA target gene sequence could be detected. As a result of this quantitative analysis, the method of discriminating between the reference and the tax using the real-time PCR method of the present invention not only discriminates between the reference and the tax, but also discriminates the reference and the tax with the detection sensitivity much superior to the conventional PCR I can confirm that I can do it.

Therefore, the method of discriminating between the reference and the tax using the real-time PCR of the present invention can discriminate the reference and the tax base quickly, accurately and quantitatively based on the data during the import, quarantine and distribution process, In particular, it has the advantage of enhancing the transparency of the circulation process of referrals and subsidies and protecting the interests and health of domestic consumers by providing measures to promptly block the importation, quarantine, .

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. <120> Method and kit for identifying Larimichthys polyactis and          Larimichthys crocea using real-time PCR <130> P14-0009KR <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S L universal forward primer <400> 1 cgcctgttta acaaaaacat 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 16S H universal reverse primer <400> 2 ccggtctgaa ctcagatcac gt 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA Forward Primer <400> 3 accaacaaga tccggcaa 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA Reverse Primer <400> 4 ggattgcgct gttatcccta 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CO-KOR probe <400> 5 cgccgatcaa cgaacccagt tacc 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CO-CHN probe <400> 6 cgccgatcaa cgaaccgagt tact 24 <210> 7 <211> 564 <212> DNA <213> Larimichthys polyactis <400> 7 aggtcctgcc tgccctgtga ccatgagttc aacggccgcg gtattttgac cgtgcaaagg 60 tagcgcaatc acttgtcttt taaataaaga cccgtatgaa tggcaagacg agggcttagc 120 tgtctccttt ttcaggtcaa tgaaattgat ctttccgtgc agaagcggga atgtcaacat 180 aagacgagaa gaccctatgg agctttagac accaagacag atcacgtcaa agccccctaa 240 ttaaggatta aactaactga gccctgtcct aatgtctttg gttggggcga ccacggggaa 300 ctacaaaacc cccgcgtgga atgaaagcac caccctgctt ttacaactaa gagcctcccg 360 ctctaataaa cagaatatct gaccaacaag atccggcaac gccgatcaac gaacccagtt 420 accctaggga taacagcgca atcctctttt agagtccata tcgacaagag ggtttacgac 480 ctcgatgttg gatcaggaca tcctaatggt gcagccgcta ttaagggttc gtttgttcaa 540 cgattaaagt cctacgtgat ctga 564 <210> 8 <211> 563 <212> DNA <213> Larimichthys crocea <400> 8 aggtcctgcc tgccctgtga ccatgagttc aacggccgcg gtattttgac cgtgcaaagg 60 tagcgcaatc acttgtcttt taaataaaga cccgtatgaa tggcaagacg agggcttagc 120 tgtctccttt ttcaggtcaa tgaaattgat ctttccgtgc agaagcggga atgtcaacat 180 aagacgagaa gaccctatgg agctttagac accaagacag atcacgtcaa agccccctaa 240 ttaaggatta aactaactga gccctgtcct aatgtctttg gttggggcga ccacggggaa 300 ctacaaaacc cccgcgtgga atgaaagcac caccctgctt ttacaactaa gagcctccgc 360 tctaataaac agaatatctg accaacaaga tccggcaacg ccgatcaacg aacccagtta 420 ccctagggat aacagcgcaa tcctctttta gagtccatat cgacaagagg gtttacgacc 480 tcgatgttgg atcaggacat cctaatggtg cagccgctat taagggttcg tttgttcaac 540 gattaaagtc ctacgtgatc tga 563

Claims (12)

As reference kit and subscription discrimination kit using real-time PCR,
The primer pairs of SEQ ID NOS: 3 and 4, which amplify the 16S rRNA target gene of the reference and secondary,
A probe specifically binding to the amplification product of the 16S rRNA target gene of the reference gene, wherein the probe comprises at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 5 and an oligonucleotide complementary to the oligonucleotide,
A real-time PCR, comprising at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 6 and an oligonucleotide complementary to the oligonucleotide, as a probe specifically binding to the amplification product of a secondary 16S rRNA target gene A reference kit and a tax assessment kit used. The method according to claim 1,
A reference kit and a discrimination kit using real-time PCR, further comprising DNA polymerase, dNTPs, PCR buffer solution and a marker substance of the PCR amplification product. 3. The method of claim 2,
The labeling substance may be,
One end of a probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene,
Wherein the primer is labeled at one end of a probe that specifically binds to the amplification product of the subsequence 16S rRNA target gene. The method of claim 3,
A probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene and a probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene are each labeled with a detectable labeling substance at different wavelengths A reference kit and a tax determination kit using real-time PCR. 5. The method of claim 4,
The 16S rRNA target gene of the reference vector and the 16S rRNA target gene of the reference vector can be detected at a time in one test tube by simultaneously using two probes labeled with a labeling substance detectable at different wavelengths A reference kit and a tax determination kit using real-time PCR. In a reference method and a method of determining bias using real-time PCR,
Preparing a gene sample from a specimen,
The 16S rRNA target gene of the reference gene and the 16S rRNA target gene in the gene sample of the specimen and the 16S rRNA target gene in the gene sample of the specimen are measured using the prepared gene sample as a template and the reference kit defined in any one of claims 1 to 5, Amplifying the 16S rRNA target gene of the present invention by real-time PCR,
Determining the real-time PCR result after amplification of the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene to determine whether the sample is a reference-based substance, a tax-derived substance, or a mixture thereof A method of distinguishing between reference and bias using real - time PCR. The method according to claim 6,
Before amplification, a positive standard, which is a gene sample that knows the copy number of the 16S rRNA target gene of the reference gene and the copy number of the 16S rRNA target gene of the reference gene, was amplified using a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4 amplifying the 16S rRNA target gene Amplifying the 16S rRNA target gene amplification amount of the reference gene and the labeled 16S rRNA target gene amplification amount of the reference gene by real-time PCR;
Measuring a change in fluorescence intensity of the labeling substance with respect to the entire PCR amplification period of the positive standard and analyzing the number of PCR reactions (Cp value or Ct value) reaching a constant fluorescence intensity;
Correlating the number of copies of the positive standard with the value of Cp or Ct from a standard curve obtained by measuring a change in fluorescence intensity of the labeling substance;
The change in fluorescence intensity of the labeling substance with respect to the entire PCR amplification period of each of the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene existing in the gene sample of the specimen was measured and PCR Analyzing the number of reactions (Cp value or Ct value)
A Cp value or a Ct value obtained by PCR amplification of each of the 16S rRNA target gene of the reference gene and the 16S rRNA target gene of the reference gene existing in the gene sample of the specimen is made to correspond to the copy number of the positive standard, Quantifying the 16S rRNA target gene or the virulent 16S rRNA target gene using the real-time PCR. 8. The method of claim 7,
The labeling substance may be,
One end of a probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene,
Wherein the primer is labeled at one end of a probe that specifically binds to the amplification product of the subtilis 16S rRNA target gene. 9. The method of claim 8,
A probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene and a probe that specifically binds to the amplification product of the 16S rRNA target gene of the reference gene are each labeled with a detectable labeling substance at different wavelengths Wherein the real time PCR is used to determine the reference and bias. 10. The method of claim 9,
The 16S rRNA target gene of the reference vector and the 16S rRNA target gene of the reference vector can be detected at a time in one test tube by simultaneously using two probes labeled with a labeling substance detectable at different wavelengths How to Identify Reference and Taxes Using Real Time PCR. A pair of primers for real-time PCR amplification used in a reference and a method of discrimination of taxa using real-time PCR, which comprises a primer pair of SEQ ID NOS: 3 and 4 amplifying a reference gene and a 16S rRNA target gene, Primer set for 16S rRNA target gene amplification. At least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 5 and an oligonucleotide complementary to the oligonucleotide as a probe specific to the 16S rRNA target gene of the reference gene,
A probe and at least one probe selected from the group consisting of an oligonucleotide of SEQ ID NO: 6 and an oligonucleotide complementary to the oligonucleotide as a probe specific to a sub-16S rRNA target gene.

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