KR102017236B1 - The primer set for analysis of cephalopods and methods for their qualitativeand quantitative analysis - Google Patents

Abstract

The present invention is a composition for producing a gene library for cephalopod analysis or analysis comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2, a kit for analysis of cephalopods comprising the composition for analyzing cephalopods, SEQ ID NO: 1 and 2 It relates to a cephalopod analysis method using a primer set consisting of the nucleotide sequence represented by and a method for producing a gene library for cephalopod analysis. Unlike conventional morphological analysis, the CPD universal primer set of the present invention can classify cephalopods with high specificity in mixed samples without separating cephalopod plankton. In addition, when using the CPD universal primer set of the present invention, there is an effect that can be economically and quickly mass analysis of qualitative and quantitative analysis of cephalopods compared to the existing sequencing system. Therefore, the primer set of the present invention can be used for cephalopods, cephalopods spawning and breeding and marine ecosystem investigation, food bioanalysis, ecosystem disturbance and analysis of foreign invasive species and raw material analysis of processed foods, It can also be used for origin and identification of distribution. In addition, the existing cephalopod analysis gene library can be used, as well as a new cephalopod analysis gene library can be used to identify more precise cephalopod species.

Description

The primer set for analysis of cephalopods and methods for their qualitative and quantitative analysis}

The present invention is a composition for producing a gene library for cephalopod analysis or analysis comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2, a kit for analysis of cephalopods comprising the composition for analyzing cephalopods, SEQ ID NO: 1 and 2 It relates to a cephalopod analysis method using a primer set consisting of the nucleotide sequence represented by and a method for producing a gene library for cephalopod analysis.

In Korea, three sides are surrounded by the sea, and various types of reservoirs and rivers have been developed, and a total of 1,186 native fish are inhabited (National Institute of Biological Resources, 2011). Cephalopods are one of the main food sources for fish and are widely distributed in various habitats of the ocean and include many commercially available species such as squid, octopus and octopus. In particular, according to the report on fish consumption in major countries of the world (2016) surveyed by the United Nations Food and Agriculture Organization (FAO), Korean consumers consume 58.4 kg of fish per capita per year, compared to Norway (53.3 kg) or Japan (50.2 kg). It was found to be at a higher level. In addition, it is reported that by 2025, Korean consumers' seafood consumption will increase by about 10%. In Korea, the highest preference among various aquatic products was mackerel, and cephalopods such as squid and octopus were also the third most preferred.

In order to manage resources of commercial species such as cephalopods, it is important to understand the larval distribution in addition to the environmental conditions of the spawning ground and the hatching rate of eggs. In addition, an overall understanding of larval distribution studies not only helps to understand the bioecological characteristics of species, but can also provide basic data on fisheries by predicting changes in resources. However, studies on the distribution of larvae in cephalopods in the surrounding waters of Korea are lacking due to difficulties in sampling and morphological identification.

In the past, species analysis using morphological classification features has been mainly performed, but in recent years, species testing using DNA markers has been in full swing. Although the analysis using Sanger sequencing has been carried out with the development of molecular techniques, the analysis method is limited to a specific cephalopod group, which makes it difficult to analyze all the cephalopod group.

In order to classify cephalopods, species identification based on past forms, especially in the case of fish eggs and larvae, was difficult because the core of species identification was unclear. However, the recent development of molecular identification techniques such as next generation sequencing (NGS) has significantly solved these difficulties.

The development of biology has led to changes in the biological classification system, and the field of studying biological systems using molecular traits centered on DNA sequences is called molecular systematics. Has brought great changes to the law of sympathy. Among them, DNA-based species analysis is a useful and promising tool when compared to morphological analysis due to less labor or time consumption. Cephalopods have well-documented mitochondrial genome sequences, of which Ribosomal DNA gene regions are frequently used for species analysis.

Conventionally, all sequencers were based on Sanger's dideoxyribonucleotide analog. Recently, new sequencing technology has been rapidly spread. Among them, next-generation or second-generation sequencers capable of producing massively parallel data unlike the Sanger method. (Next Generation Sequencer, 2nd Generation Sequencer) was developed. Next generation sequencing (NGS) is an analysis method that obtains the sequence of the entire genome by assembling the obtained sequencing fragment after reading the genome, which is a collection of all genes, into a myriad of fragments. Since the technology has been commercialized, as the related technology has developed rapidly, a large amount of genome information has been obtained in a short time, and it can be used for various research fields. In particular, as the cost and time of genome analysis decreases, the demand for genome analysis of various organisms is rapidly increasing.

In particular, with the announcement of the Nagoya Protocol in October 2014, the importance and value of biological resources in their own countries has emerged. Accordingly, the systematic integrated management at the national level on securing and preservation of marine life resources is necessary. In addition, Korea's Act on the Conservation and Management of Marine Ecosystems (2007) stipulates the basic plan for investigating, observing and managing marine protected areas by designating areas that maintain primitiveness or are rich in marine biodiversity. I've done it. For the management of aquatic resources, it is necessary to investigate the composition of cephalopod species and the spawning season and spawning season for the entire coastal waters of Korea. In addition, as part of establishing policies such as marine protected areas, it is necessary to identify spawning and breeding grounds.

Therefore, many universal primers used for DNA meta-barcoding can be used to classify and analyze commercial cephalopods using NGS, but there are some problems in detecting cephalopod plankton in samples containing various taxonomic species. First, cephalopods are very low in plankton samples, making them difficult to detect by other relatively high species. Most general primers target non-targeted cross-reactivity because they target a well-conserved COI region. There may be detection of species. Therefore, there is a need for a cephalopod-specific (CPD-specific) universal primer set for detecting cephalopods in planktonic samples with high specificity.

Therefore, the present inventors studied to classify only cephalopods from a zooplankton sample containing taxonomically diverse marine organisms using a next-generation sequencer (NGS), and thus optimized length (465-471bp) that can be analyzed by Illumina Miseq sequencer. We have designed a new set of universal primers for the classification of cephalopods, and analyzed the amplified DNA sequences with Illumina Miseq sequencer, resulting in economical, rapid, mass-classification and qualitative analysis of cephalopods compared to conventional sequencing systems. As well as confirming that quantitative analysis is possible, the present invention has been completed.

Accordingly, an object of the present invention is a composition for preparing a gene library for cephalopod analysis or analysis comprising a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2, kit for analysis of cephalopods comprising the composition for analyzing cephalopods, sequence The present invention provides a method for analyzing cephalopods using a primer set consisting of base sequences represented by numbers 1 and 2 and a method for preparing a gene library for cephalopods.

In order to achieve the above object, there is provided a cephalopod analysis composition comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2.

In another aspect, the present invention provides a kit for cephalopod analysis comprising the composition.

The present invention also comprises the steps of (a) extracting the DNA of the sample; (b) performing amplification using a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 as a template of the DNA of (a); And (c) analyzing the sequence for the amplification product of (b); Provides a cephalopod analysis method comprising a.

In another aspect, the present invention provides a composition for preparing a gene library for cephalopod analysis, comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2.

The present invention also comprises the steps of (a) extracting the DNA of the sample; (b) performing amplification using a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 as a template of the DNA of (a); And (c) analyzing the sequence for the amplification product of (b); Provided, a method for producing a gene library for cephalopod analysis.

Unlike conventional morphological analysis, the CPD universal primer set of the present invention can classify cephalopods with high specificity in mixed samples without separating cephalopod plankton. In addition, when using the CPD universal primer set of the present invention, there is an effect that can be economically and quickly mass analysis of qualitative and quantitative analysis of cephalopods compared to the existing sequencing system. Therefore, the primer set of the present invention can be used for cephalopods, cephalopods spawning and breeding and marine ecosystem investigation, food bioanalysis, ecosystem disturbance and analysis of foreign invasive species and raw material analysis of processed foods, It can also be used for origin and identification of distribution. In addition, the existing cephalopod analysis gene library can be used, as well as a new cephalopod analysis gene library can be used to identify more precise cephalopod species.

1 is a diagram showing 258 vertices of a Korean sea area from which samples for analysis are extracted.
2 is a diagram showing the results of electrophoresis confirming whether only cephalopods can be specifically detected in a mixed sample of marine organisms using the universal primer set of the present invention.
Figure 3 is a graph showing the cephalopod composition confirmed using the primer set of the present invention in 2016 monthly egg fry samples.
Figure 4 is a diagram showing the results of systematic analysis of cephalopods in the Korean sea area using the CPD universal primer set.
Figure 5 is a diagram showing the results of the analysis of the ratio between the single phase for analyzing the monthly standard haplotype for three cephalopod dominant species.

The present invention provides a cephalopod analysis composition comprising a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2.

Hereinafter, the present invention will be described in more detail.

As used herein, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow to start the synthesis of extension products. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

In the present invention, the primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 targets cytochrome b and ND6 regions of cephalopod mitochondrial DNA, and various taxonomic species such as cephalopods and crustaceans are mixed. Only cephalopods present in trace amounts in a zooplankton sample present can be specifically detected.

The primer set of the present invention may be characterized by amplifying a sequence so that the amplified PCR product can obtain a comparative size of an optimal size for classifying cephalopods using a next generation sequencer (NGS), preferably from 300bp to The 600 bp PCR product can be amplified, and more preferably, 465 to 471 bp amplified PCR product can be optimized for NGS. When amplifying the gene of 300bp to 600bp, it is possible to analyze the longer sequence than the conventional amplification method can increase the accuracy of species analysis.

The primer set of the present invention may be characterized by amplifying common genes of cephalopods of various species present in the sample. By using the primer set of the present invention, it is possible to amplify 1 to 100 kinds of cephalopod genes, preferably 1 to 70 kinds of cephalopod genes. In the present invention, the cephalopod to be amplified is not limited thereto, but may be at least one selected from Table 1 below.

TABLE 1

The primer set of the present invention can be used for Next Generation Sequencer (NGS) analysis.

The primer set of the present invention does not amplify the sequences of any other taxa except Cephalopoda. Thus, with the primer set of the present invention, except for unidentified due to lack of database, more than 87% of the entire sequence is identified as species level, 0.8% as genus level, and enough species for species identification are identified. Cephalopods can be diagnosed, including variations. In addition, the primer set of the present invention can be used to analyze standard haplotypes, and one or more types of analysis selected from the group consisting of the types of cephalopods, the ratio of cephalopods, and the standard haplotype analysis of cephalopods are possible. Do.

The "analysis" can be divided into "quantitative analysis" and "quantitative analysis," "quantitative analysis" refers to finding out what proportion of a particular taxon on a mixed sample. "Qualitative analysis" means that you can identify what species are on the mixed sample.

In another aspect, the present invention provides a cephalopod analysis kit comprising a cephalopod analysis composition comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2.

The kit of the present invention may include a reagent for performing an amplification reaction, and may include heat resistant DNA polymerase, dNTPs, buffers, and the like. In addition, the kits of the present invention may further comprise a user guide describing the optimal reaction performance conditions. The guide is a printed document that explains how to use the kit, such as how to prepare a PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

In another aspect, the present invention (a) extracting the DNA of the sample; (b) performing amplification using a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 as a template of the DNA of (a); And (c) analyzing the sequence for the amplification product of (b); Provides a cephalopod analysis method comprising a.

The amplification of step (b) includes polymerase chain reaction, multiplex polymerase chain reaction, multiplex PCR, competitive polymerase chain reaction, and real time polymerase chain reaction. any one selected from the group consisting of real-time polymerase chain reaction, real-time quantitative polymer chain reaction, DNA chip, loop-mediated isothermal amplification, and combinations thereof Can be used.

The amplification of step (b) may be repeated 15 to 30 times using PCR, and preferably, a total of 28 times may be repeated. Initial denaturation at 94 ° C. for 30 seconds; The denaturation was repeated 30 times at 94 ° C., 30 seconds at 51 ° C., and 30 seconds at 72 ° C., and the final extension may be performed at 72 ° C. for 5 minutes, but is not limited thereto.

In the step (c), the analysis of the sequence for the amplification product may use a next-generation sequencer (NGS), and the analysis equipment of the NGS may have unique characteristics and advantages and disadvantages according to specific techniques such as chemical reaction and sequencing principle. Various platforms have been released, such as Illumina MiSeq sequencer, Illumina HiSeq sequencer, Roche 454 GS FLX + sequencer, Ion Torrent PGM sequencer from Thermo Fisher, or Illumina Genome Analyser IIx sequencer.

For the purposes of the present invention, Illumina MiSeq sequencer can be used, but is not limited thereto.

In the present invention, the "Illumina Miseq sequencer" is one of using a next generation or second generation sequencer technique capable of producing a large amount of parallel data, unlike the conventional Sanger method. The NGS amplifies the DNA sequence, and then reads the base through a process of taking an image of a fluorescent marker or the like image, and the Illumina Miseq sequencer of the present invention uses solid-phase amplification. . Illumina Miseq sequencer of the present invention is economical because it has a low price and various applications while maintaining the chemistry of HiSeq, which is a large-capacity sequencer. If the cost of reading one cyt.b-ND6 sequence is about 1 cent using the existing 454 sequencer, the Illumina Miseq sequencer applied in the present invention is 2 x 300bp. At 0.02 cents, it is about 50 times more competitive.

In another aspect, the present invention provides a composition for preparing a gene library for cephalopod analysis, comprising a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 1 and 2.

The primer set can be used to generate a cephalopod DNA gene pool, ie, a gene library, for cephalopod analysis. In the present invention, the "gene pool" refers to a gene population composed of a plurality of genes, and may be used interchangeably with a gene library. At this time, the presence ratio of the mutant gene in the gene to be searched is not asked. For example, 100% may be a wild type gene, 50% may be a wild type gene and the remaining 50% may be a variant gene. By primer amplification of 1 to 100 kinds of cephalopod genes in common, the primer set of the present invention can effectively form a cephalopod DNA gene library for cephalopod analysis and then use it for analysis using next-generation sequencers.

The present invention also comprises the steps of (a) extracting the DNA of the sample; (b) performing amplification using a primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2 as a template of the DNA of (a); And (c) analyzing the sequence for the amplification product of (b); Provided, a method for producing a gene library for cephalopod analysis.

Hereinafter, the present invention will be described in detail through examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example  One. CPD  General purpose primer  Fabrication of Set and Analysis of Cephalopod Species

1.1 Sample Collection and DNA Extraction

In 2016, a total of 739 zooplankton samples collected using bongo nets (333 μm) were used at 258 vertices in Korea, and the vertices from which the samples were collected are shown in FIG. 1. The collected sample fixed with alcohol was sufficiently dehydrated in running water, homogenized with 6x 1X Lysis buffer with respect to 1/2 of the total wet weight and homogenized to extract genomic DNA of the sample.

1.2 CPD  General purpose primer  Design of sets

Primer sets were designed to analyze cephalopods. The longer the sequence is compared, the more species or populations can be compared, so it is important to secure the maximum length using the Illumina Miseq sequencer. Therefore, the maximum reading sequence of the Miseq analysis system was 600 bp (300 + 300), and thus, a region capable of specifically classifying and detecting only cephalopods was found for a primer set amplifying at about 300 to 600 bp. Specifically, cytochrome b and ND6 regions of highly variable mitochondrial DNA sufficient to classify the taxonomic location and species of cephalopods capable of specifically detecting cephalopods using sequence data to design cephalopod specific universal primers. A CPD universal primer set was designed to amplify. The designed primer sets are shown in SEQ ID NOs: 1 and 2.

1.3 CPD  General purpose primer  Diagnosis of Cephalopods Using Sets

In addition, in order to confirm whether the primer set designed in Example 1.2 can diagnose a target cephalopod species at a higher taxonomic level, the existing primer sets 16Sar and 16Sbr (Simon et al., 1994) Experiment was performed to measure the diagnostic level of cephalopods in the sample between the primer set of the present invention and the CPD universal primer set. Genetic distances between species of the two pairs of primer sets were calculated and are shown in Table 2.

TABLE 2

As shown in Table 2, the 16Sar and 16Sbr primer sets have an average level of 0.084 ± 0.012, 0.081 ± 0.012, and 0.083 ± 0.011, which can be analyzed for cephalopod order, family, and genus, respectively. It was confirmed that the primer set region of the present invention appears in order of 0.131 ± 0.015, 0.127 ± 0.015, 0.116 ± 0.014. As a result of measuring the average genetic distance value, it was confirmed that the species variation of the primer set of the present invention was higher than the existing 16S rRNA region. Therefore, it was confirmed that the primer set of the present invention, which amplifies the cytocrome b region as a target, enables an excellent level of identification between species in cephalopods.

Example  2. CPD  General purpose primer  Specificity evaluation of sets

PCR was performed to evaluate the specificity and efficiency of the cephalopods of the primer set designed in Example 1.2. PCR conditions were repeated 28 times in a cycle of 3 minutes at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 51 ° C, and 30 seconds at 72 ° C, and finally 5 minutes at 72 ° C. The template used 200ng of each genomic DNA of cephalopod plankton individuals and eggs samples collected from Korean waters in 2016. 1 μl CPD universal primer set, 2 μl dNTPs (10 mM), 0.2 μl Ex Taq Hot Start Version (Takara Bio Inc. Japan), 2 μl 10X Ex Taq buffer (TakaraBio Inc. Japan), 1 μl DMSO PCR was performed by mixing 20 μl of the final volume with distilled water. AccuPrep PCR product  After purification using Gel PurificationKit (Bioneer. Korea), the sequence was obtained and subjected to sequencing. The PCR amplified using the primer set of the present invention on four cephalopod plankton individuals and four mixed zooplankton samples. The product was placed on a 1.5% agarose gel and subjected to electrophoresis, which is shown in FIG. 2.

As shown in Figure 2, using the CPD universal primer set of the present invention it was confirmed that one specific amplicon of constant length in all four cephalopod plankton individuals and four mixed zooplankton samples. In addition, sequencing analysis indicated that lanes 1 and 2 were Todarodes. pacificus ), Nos. 3 and 4 were Sepiola birostrata , and in the zooplankton samples of lanes 5 to 6, 69% of squid, 24% of squid, and squid ( Watasenia) scintillans ) accounted for 1%. That is, using the CPD universal primer set of the present invention, it is possible to specifically detect only cephalopods in a sample mixed with marine organisms, and also to analyze the species of cephalopods constituting the sample so that only cephalopods in the mixed sample can be detected. It was confirmed that qualitative and quantitative analysis is possible.

Example  3. Large sequencing NGS Species Diversity of Cephalopods in the Korean Sea

3.1 CPD  General purpose primer  Diagnosis of Species Monthly Diversity of Cephalopods in the Korean Sea Area

Experiments were carried out to identify species diversity of cephalopods inhabiting Korean waters using the CPD universal primer set of the present invention. The first PCR conditions were repeated 28 times in a cycle of 3 minutes at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 51 ° C, and 30 seconds at 72 ° C, and finally 5 minutes at 72 ° C. In addition, PCR was carried out in 2016, 200 ng of genomic DNA and 1 μl each of the larval larvae samples, 1 μl of CPD universal primer set, 2 μl of dNTPs (10 mM), 0.2 μl of Ex Taq Hot Start Version (Takara Bio Inc. Japan), 2 μl of 10 × Ex Taq buffer (TakaraBio Inc. Japan), 1 μl of DMSO, were mixed with tertiary distilled water to a final volume of 20 μl. AccuPrep PCR product  Purification was performed using Gel PurificationKit (Bioneer. Korea) and used for secondary PCR. The PCR was performed under the same conditions as the first PCR except the annealing temperature of 55 ° C., and the amplification was repeated 15 times. Thereafter, the PCR product obtained by amplification was purified by AccuPrep Gel PurificationKit (Bioneer, Korea) after cutting the gel after 1.5% agarose gel electrophoresis. Libraries were prepared using the Nextera XT index Kit (lllumina, USA) for sequencing using the MiSeq platform (lllumina. USA) and sequencing was performed using lllumina MiSeq (2 X 300 bp pair-ends). It was. The BLASTn search was performed using the NCBI nt database, and in the case of OTUs having more than 99% sequence identity, the species was designated as a species, and the sequences between 90% and 98% were designated as the genus. OTUs with less than 90% sequence identity were designated "unidentified". The results of the sequencing analysis are shown in Table 3 and FIG. 3.

TABLE 3

As shown in Table 3 and FIG. 3, sequencing was performed using NGS. As a result, the cephalopods could be analyzed at six oval levels, ten genus levels, and six species levels in the monthly egg fry samples. In general, the percentage of squid was high in 65% of the whole, followed by 16% of the squid and 9% of the cuttlefish. Small locust uyii ) appeared only in August, confirming that August is the month in which various cephalopod species live, with the highest Shannon-Wiener index index of 1.271. As a result, it was confirmed that species diversity analysis of cephalopods inhabiting Korean waters without cross-reactivity was possible using the CPD universal primer set of the present invention in samples mixed with various taxonomic organisms.

3.2 CPD  General purpose primer  Phylogenetic Analysis of Cephalopods in the Korean Waters Using the Set

 Cephalopods were systematically analyzed to build a database of cephalopods existing in Korean waters based on the species diversity analyzed in Example 3.1. Genotypes occupying more than 10% of the total contig of the samples analyzed monthly were selected as the standard OTU to prepare a phylogenetic tree, which is shown in FIG. 4.

As shown in Figure 4, using the CPD universal primer set of the present invention, a total of 50 standard OTU can be analyzed by dividing the cephalopods into a total of seven families and sub-genus and species of the family according to each taxonomy It was.

3.3 CPD  General purpose primer  Single Phases of Three Cephalopod Dominant Species in the Korean Sea haplotype ) Diagnosis

To determine which haplotype is the standard haplotype for each of the three major cephalopod dominant species in Korea, the ratio was analyzed using the CPD primer set of the present invention. As a standard haplotype, genotypes that occupy more than 10% of the total contig of monthly samples of three dominant species were selected as standard haplotypes, and the results of confirming the monthly ratio are shown in FIG. 5.

As shown in Figure 5, cuttlefish ( Todarodes pacificus ) showed only one standard haplotype in November, and the genotypes of November were distinct from other months, and the population of November was clearly different from that of other months. In the case of squid ( Watasenia scintillans ), five standard haplotypes were identified as the most diverse standard haplotypes. Little Squid ( Sepiola) In case of birostrata ), the standard haplotype 2 accounted for 70% in June and the standard haplotype 3 occupied in November. Therefore, based on the above results, using the CPD universal primer set of the present invention, it was confirmed that cephalopods can be analyzed from the cephalopods of a specific period to the standard haplotype.

From the above experiments, the CPD universal primer set of the present invention can be analyzed up to the neck, family, genus, and species level of cephalopods, and specifically detects only cephalopods from a mixture of multiple species, up to the amount contained in the sample. It was confirmed that it could be detected. In addition, the CPD universal primer set of the present invention can be used as a composition for preparing a genetic library for the analysis of cephalopods, and at the same time it was confirmed that it can be used for the study of genetic diversity with respect to cephalopods.

Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the spirit of the invention.

<110> Pukyong National University Industry-University Cooperation Foundation <120> The primer set for analysis of Cephalopoda and methods for their          qualitative and quantitative analysis <130> 1-49P <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of cephalopod-specific universal primer          set <400> 1 gayatytgnc cycadgg 17 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer sequence of cephalopod-specific universal primer          set <400> 2 atttgytayt aytgtgangg 20

Claims (12)

A primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2, wherein the primer set is for analysis of Next Generation Sequencer (NGS) and includes the types of cephalopods, the ratio of cephalopods, and the standard single-phase type of cephalopods ( haplotype) to analyze at least one selected from the group consisting of, cephalopod analysis composition. The cephalopod analysis composition according to claim 1, wherein the primer set amplifies the cytochrome b and ND6 (NADH dehydrogenase subunit6) regions of the cephalopod mitochondrial DNA. According to claim 1, wherein the primer set is characterized in that for amplifying the PCR product of 300 to 600bp, cephalopod analysis composition. The cephalopod analysis composition of claim 1, wherein the primer set amplifies a gene of 1 to 100 cephalopods. According to claim 1, wherein the primer set is characterized in that for amplifying one or more cephalopod genes selected from Table 1, cephalopod analysis composition.
TABLE 1

delete delete For analysis of cephalopods comprising the composition of any one of claims 1 to 5 and analyzing one or more selected from the group consisting of cephalopod types, proportions of cephalopods, and standard haplotype analysis of cephalopods. Kit.
(a) extracting the DNA of the sample;
(b) performing amplification using a primer set consisting of a nucleotide sequence represented by SEQ ID NOs: 1 and 2 as a template using the DNA of step (a); And
(c) analyzing the sequence for the amplification product of step (b) using a Next Generation Sequencer (NGS); Cephalopod analysis method comprising the analysis of one or more selected from the group consisting of the type of cephalopods, cephalopods, and the standard haplotype analysis of cephalopods.
delete A primer set consisting of the nucleotide sequences represented by SEQ ID NOs: 1 and 2, wherein the primer set is for analysis of Next Generation Sequencer (NGS) and includes the types of cephalopods, the ratio of cephalopods, and the standard single-phase type of cephalopods ( Haplotype) A composition for preparing a genetic library for cephalopod analysis that analyzes one or more selected from the group consisting of.
(a) extracting the DNA of the sample;
(b) performing amplification using a primer set consisting of a nucleotide sequence represented by SEQ ID NOs: 1 and 2 as a template using the DNA of step (a); And
(c) analyzing the sequence for the amplification product of step (b) using a Next Generation Sequencer (NGS); Method for producing a gene library for cephalopod analysis comprising a, a type of cephalopod, the ratio of cephalopods, and one or more selected from the group consisting of the standard haplotype analysis of cephalopods.

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