KR101817107B1 - Method For Identification Of Beringraja pulchra Using Real-Time PCR assay - Google Patents
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Abstract
The present invention provides a primer set comprising a true-horn primer consisting of the base sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 and a probe composed of the base sequence shown in SEQ ID NO: 3, And to a method for identifying a true hare.
The real-time polymerase chain reaction using the true-pinhole-specific sequence according to the present invention does not require a lot of time-consuming and costly sequencing analysis, and the DNA that can quickly and accurately identify the true- As a technique, it is possible to prevent fraud or ambiguous notation of true hongo.
Description
More particularly, the present invention relates to a method for identification of a true-pheromone using a real-time PCR, and more particularly to a method for identifying a true-pheromone-primer- And a real time PCR identification method using the same.
Chamaele is a cartilaginous fish belonging to the stingray family of hippocampus. It is a cold-water fish species with a habitat layer of 30-200m and a water temperature of 5-15 ℃. It mainly feeds on shrimp and small fish. Due to these characteristics, the true hongo are mainly found in the Southwest Sea of Korea, the East China Sea, and the south and south of Japan, mainly in the Black Sea coast and Daechung Island. Rajiforms are composed of 245 species in 3 families and 30 genera. B. pulchra was classified as Raja pulchra until a new genera Beringraja was established by Ishihara. In the Yellow Sea around the island of Heuksan and Daechungdo Island in Korea, it was very common up to 20 years ago, but the number of serious hunters has been steadily declining and is currently weakened by the International Union for Conservation of Nature (IUCN) Species. Until recently, 11 species of Hongkong belonging to five genera have been identified in Korea, and among them, Hongkong is widely known as one of the most economically valuable fishes in Korea. It is mainly consumed in the form of fermented flesh pieces. As the value of B. pulchra species increases day by day, food-related crimes are increasing, and police are currently cracking down on these food-related crimes in Korea. However, a rapid and accurate identification method has not yet been developed for the foods that are not morphologically distinguishable.
The commercially available food samples are the only way to identify species using molecular markers because their morphological characteristics have disappeared. Protein and DNA (deoxyribonucleic acid) are used as molecular markers. While protein analysis techniques are based on physicochemical differences, DNA technology consists of detecting nucleotide variation in a sequence. Nonetheless, the suitability of the marker is very important because the sample has already undergone various processes such as temperature, chemical addition, humidity, and processing. Electrophoretic analysis of proteins extracted from samples is not suitable for samples with irreversible changes due to thermal treatment. However, the use of DNA as a molecular marker is the most powerful tool for identification of species, since DNA molecules are much more stable to heat treatment than others.
DNA-based methods widely used for the identification of various food species include polymerase chain reaction (PCR), restriction enzyme-polymerase chain reaction (PCR-RFLP), and polymerase chain reaction-restriction Fragment Length Polymerism, Forensically Informative Nucleotide Sequencing (FINS), and Real-time Fluorescence Chain Reaction (PCR). The RT-PCR technique is based on the detection and quantification of a fluorescence reporter containing a specific fluorogenic probe using a high-quality optical detection instrument. And this is better suited for sample handling which meets standard lab conditions more carefully and clearly and jeopardizes DNA integrity.
The present invention uses RT-PCR analysis based on the use of a MGB (Minor Groove Binding) TaqMan probe that binds to the diagnostic region of the target DNA and amplifies during amplification. In addition, the mitochondrial cytochrome oxidase subunit 1 (COI) gene was applied to commercial food and B. pulchra as a genetic marker for species identification. The mitochondrial (mt) gene has a higher copy number than the nuclear DNA and therefore has a higher recovery rate in the trace sample. In addition, the mitochondrial gene generally lacks recombination promoting the loss or immobilization of mitochondrial DNA haplotypes, which makes it possible to identify interspecies due to its low diversity in species.
The present invention provides a TaqMan real-time PCR technique based on the amplification of mitochondrial COI gene base sequence fragments for the identification of B. pulchra species. This system is based on the genomic sequence of the true-to-the-spider, which was found by comparing and analyzing the COI gene sequence of 157 species of sponges and stingrays reported in the GenBank DNA database of the National Institute of Health. And applied to 27 commercially available food samples, 29 of which were undefined. Therefore, the inventors of the present invention confirmed that the real-time PCR assay can distinguish only true hongbong species from species similar to true hongbong species, and that the test method is also simple and completed the present invention.
Korean Priority Patent Application No. 10-0806208 (a method for determining the classification system of fishes belonging to the sponges and the stigma and related polynucleotide probes, DNA chips and kits) and Korea Patent No. 10-1232878 (mitochondria A primer for a polymerase chain reaction for amplifying a COI gene DNA bar coding region).
SUMMARY OF THE INVENTION An object of the present invention is to provide a primer probe set for identification of true hairs that can accurately detect and identify true hairs.
Another object of the present invention is to provide a kit for identification of a true hippocampus including a primer probe set for identification of true hippocampus capable of accurately detecting and identifying a true hippocampus.
It is still another object of the present invention to provide a method for identification of true hairs using a real-time PCR method using a kit for identification of true hairs including a primer probe set for identification of true hairs, which can accurately detect and identify true hairs have.
In order to achieve the above object, the present invention provides a chromosome identification set comprising a primer for identification of a true hermoid consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a probe comprising the nucleotide sequence shown in SEQ ID NO: 3 to provide.
The primers and probes are characterized by specificity to the codon of mitochondrial COI gene.
The end of the probe 5 'is labeled with a reporter FAM and the end of the probe 3' is labeled with a non-fluorescence quenching group MGB.
The present invention is characterized in that real-time polymerase chain reaction (RT-PCR) is performed using a primer set of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, The method of the present invention provides a method for identifying a true-to-the-right person.
The Real-Time Polymerase Chain Reaction (RT-PCR) is a method of obtaining DNA from a separated sample. Performing a real-time PCR using the primers of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 and the probe of the nucleotide sequence shown in SEQ ID NO: 3; And detecting fluorescence emitted from the probe.
The present invention provides a kit for identification of a true hippocampus comprising a primer probe set consisting of a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
The present invention provides a primer probe set for identification of true hippocampus capable of accurately detecting and identifying a true hippocampus, a kit for identification of a true hippocampus including the same, and a real hippocampus identification method using a real-time polymerase chain reaction method using the same. According to the above method, it is possible to provide a means by which the inspection can be simple, accurate, quick, and cost efficient and can be identified efficiently.
Brief Description of the Drawings Fig. 1 is a diagram showing a QIAxcel gel image of a product obtained by amplifying a part of a COI gene by amplifying a sample of 27 commercially available food samples with three primer sets shown in Table 1; Fig.
Fig. 2 is a diagram showing the 22 nucleotide sequences most similar to the primer and probe base sequences which can be identified only to the true hippocampus. The box shows the location of the primer-probe set of the present invention.
FIG. 3 is a view showing a real-time PCR amplification pattern of the true horseshoe and other species.
(A) true horny amplification pattern
(B) (a) true-iris amplification pattern
(b) Sample No. 3 amplification pattern
(C) No. 11 sample amplification pattern
(D) The remaining 25 samples except for the true-skewer Amplification pattern
Hereinafter, the present invention will be described in detail.
In one aspect, the present invention relates to a primer probe set comprising a primer for identification of a true hoe having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 and a probe comprising the nucleotide sequence shown in SEQ ID NO: 3.
The nucleotide sequence of the primer probe set of the present invention is characterized by a specific ability to analyze nucleotide sequences of mitochondrial COIs of the true hermaphrodite species, including specific mutations that do not differ between nucleotides in the species of true hippocampus and differ from other species I have.
In another aspect of the present invention, there is provided a method for detecting the presence or absence of a mutation in a nucleic acid sequence comprising a primer having a nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a real-time polymerase chain reaction (RT) using a probe comprising the nucleotide sequence shown in SEQ ID NO: -PCR). ≪ / RTI >
In another aspect, the present invention relates to a kit for identification of a true hippocampus comprising a primer having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 and a probe having the nucleotide sequence shown in SEQ ID NO: 3.
More particularly, the present invention provides a method for identifying DNA comprising the steps of: obtaining DNA from a separated sample; real-time polymerase chain reaction (RT) using a primer set capable of amplifying a mitochondrial COI site -PCR), and detecting fluorescent light emitted from the probe.
The real-time PCR reaction (RT-PCR) used in the identification method of the present invention is not particularly limited and may be one of several known modification methods. For example, RT-PCR solution is prepared by mixing reagents such as Tris-HCl, KCl, MgCl 2 , dNTP, and Taq DNA polymerase in addition to primer set and DNA template.
Specifically, the reagents included in the kit include dNTP, MgCl 2 , Taq polymerase, Tris-HCl, glycerol, DMSO, DNA for positive control, DNA for negative control, and distilled water. The reagents may be packaged independently in a kit or may be provided in a mixed form of two or more kinds of reagents. There is no particular limitation on the concentration of each reagent contained in the kit, but preferably, the real time polymerase And can be as far as possible for performing a real-time polymerase chain reaction (RT-PCR). In addition, the kit of the present invention may include only information on the preferable RT-PCR conditions, or may include only a primer set.
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
In this case, unless otherwise defined, technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the following description and the accompanying drawings, A description of known functions and configurations that may unnecessarily obscure the description of the present invention will be omitted.
Sample collection and DNA extraction
The tail tissue samples of the true hippocampus were isolated from 29 wild - caught hippocampus species captured in Korea. Commercially available food samples, mostly composed of fermented fish, of unknown species were obtained in supermarkets in Seoul and Jeolla provinces. These tissues were preserved in 100% ethanol and transported to the laboratory for DNA extraction. Approximately 0.2 g of tissue was used for DNA extraction, respectively. DNA extraction was performed using QIAamp DNA micro kit (Qiagen, Hilden, Germany) and separated from each sample according to instructions. The extracted DNA was quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Barrington, IL, USA) and stored at -20 ° C.
PCR analysis and DNA sequencing
1. Experimental Method
DNA fragments of the mitochondrial COI gene of all samples were amplified using the three pairs of primers shown in Table 1. Polymerase chain reaction (PCR) amplification was performed using 10 ng template DNA, 2.5 μl Gold ST * R 10x buffer (Promega, Madison, Wis. USA), 2.5 U AmpliTaq Gold® DNA polymerase (Applied Biosystems, Foster City, ) And 1.0 [mu] M of each primer. Thermal cycling was performed on a GeneAmp PCR System 9700 (Applied Biosystems). The reaction conditions were denaturation at 95 ° C for 11 min and repeated for 30 cycles at 94 ° C for 1 min, followed by 1 min at 58, 54 and 52 ° C for three pairs of primers, Lt; / RTI > for 7 minutes. The amplified PCR products were QIAxcel Advanced using QX cartridges composed of a QX Size 50-800 base pair (bp), a 15bp / 1000bp alignment Marker (Qiagen), and an array of 12 capillaries filled with gel polymers system (Qiagen).
Sequencing reactions were performed according to instructions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing was performed by capillary electrophoresis using a 3500xL genetic analyzer (Applied Biosystems).
Raw data were analyzed by Sequence Analysis software v.5.4 (Applied Biosystems). The nucleotide sequences were analyzed by Molecular Evolutionary Genetics Analysis (Mega) Version 5.1 software and the results were analyzed using the BLAST (Basic Local Alignment Search Tool) of the GenBank Databse database (National Center for Biotechnology Information, Bethesda, Maryland) Species were identified.
2. Experimental results
In order to quickly and accurately distinguish the true horseshoe between the hippocampus and the stingray species, the present invention has investigated the nucleotide sequences of mitochondrial COI and found a specific nucleotide sequence of the true hippocampus COI, and developed a real-time PCR reaction method based on the nucleotide sequence. Until the method proposed in the present invention was developed, identification of the species was carried out by analyzing the nucleotide sequence of the PCR amplification product after the general PCR. According to the previous method, DNA of 29 indi- vidual species and 27 unclear food samples were extracted and successfully amplified by the three primer sets described in Table 1.
size (bp)
The primers were designed based on a partial sequence of the COI gene of GenBank accession number EU327184. The amplified product was analyzed by QIAxcel system. As a result, as shown in FIG. 1, about 600 bp (base pair), 507 bp and 439 bp size were observed, respectively. Sequencing analysis using primers used for amplification Respectively. Sequence similarity searches were performed using GenBank BLAST on fragments of COI genes obtained from all samples to identify the species. As shown in Table 2, the results of the GenBank database of 27 species of unknown food samples were Dipturus chilensis (N = 14) (N = number, number of samples), Bathyraja brachyurops (N = 7), Dipturus laevis 2), Beringraja pulchra (N = 2), Rajiforms sp. (N = 1) and Bathyraja albomaculata (N = 1) were 99% -100% identical. As a result, only two food samples (No. 3 and No. 11) were identified as true rubella species and 7.4% of the food samples were examined proportionally. Among the 27 food samples, D. chilensis (52%) and B. brachyurops (26%) showed a relatively high ratio. According to the red list of the International Union for Conservation of Nature (IUCN) (www.iucnredlist.org.), The two species are classified as Atlantic-southwest, Pacific -southwest area).
Sample
EU074403.1, EU074404.1
Example 1: Design of primers and probes
Identification of the species in the unclear food collected from the true hermit crab and mart provided by the National Fisheries Research and Development Institute (NFRDI) is shown in Table 1 as GenBank accession number EU327184.1, Of the mitochondrial COI gene.
Primer and probe sequences used in real-time PCR should be free of nucleotide differences in the same species and should contain specific mutations that differ from other species. The nucleotide sequence regions meeting these two conditions were compared with the nucleotide sequences of 12S rRNA, cytochrome b and COI genes in 29 mitochondria of the true hippocampus species and the 157 hippocampus and stingray species reported in NCBI. . The nucleotide sequence of mitochondrial COI from all samples was able to search and compare the data registered with GenBank using NCBI BLAST. The COI sequence of the 157 spores and stingray species reported in GenBank was downloaded and compared with the COI sequence of the true hippocampus using the Clustal W function of the MEGA program, and then a specific MGB TaqMan primer-probe system A suitable fragment for design was selected. The two most similar base sequences to the primer and probe base sequences which can be identified only by the true haremongi are shown in Fig.
This system, which is capable of identifying true hermaphrodites, is composed of 59 bp nucleotide sequences. Primer Express software (Applied Biosystems) is used to design the primers and probe sets for compatibility and error potential. . Using this program, it was confirmed that annealing does not occur when the sequences are not 100% identical due to differences in SNP positions on probes and primers, I was able to increase the degree.
The sequence of the primer-probe set prepared in this study is as follows.
BPCOIRT-F (forward; SEQ ID NO: 1): 5 'GGGCCGGAACAGGTTGA-3'
BPCOIRT-R (reverse; SEQ ID NO: 2): 5 'CCCAGCGTGGGCTATATTTC-3'
BPCORT-P (probe; SEQ ID NO: 3): 5 'CTGTGTACCCCCCCTT-3'
The probe was labeled with the fluorescent dye FAM at the 5 'end and Minor Groove Binding (MGB) at the 3' end.
Example 2: Real-time PCR system setup
1. Experimental Method
The real-time PCR assays were covered with a MicroAmp optical adhesive film (Applied Biosystems) and transferred to a MicroAmp high-speed optical micro-amp 96-well reaction plate (Applied Biosystems) Lt; / RTI > The experiment detects the VIC fluorescence emitted from the probe against the internal positive control (IPC) (Applied Biosystems), a synthetic sequence that does not exist in nature, and the FAM fluorescence emitted from the probe to the COI gene It was investigated by doing. Each reaction contained 10 ng of DNA, 12.5 μl of TaqMan Fast Universal PCR Master Mix ™ UNG Amperase (2X) and a final 900 nM primer and a probe concentration of 225 nM. The TaqMan reaction was run on a 7500 PCR System (Applied Biosystems). The thermal cycling conditions were 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 15 seconds and 60 ° C for 15 seconds. Fluorescence data emitted from the plate was analyzed by 7500 system SDS software v1.3.0. Negative controls and positive controls were included to confirm inhibition of amplification of DNA samples. Each Ct value was obtained by performing an average of three values. The average Ct values obtained from the samples of true horseshoe were compared with those of unclear commercial food samples.
2. Experimental results
Preliminary tests were performed using various concentrations of primers (50, 300 and 900 nM) and probes (25-225 nM) to determine the optimal concentration of primers and TaqMan probes. Identification of true horses was performed using a 900 nM primer and a final probe concentration of 225 nM with the lowest Cycle threshold (Ct value) and the highest fluorescence value.
The efficiency of the developed method was calculated based on the slope of the standard curve obtained by using various amounts of DNA (0.1 pg - 100 ng) as a template for RT-PCR, and the mean value of the obtained slope was - 3.32 Respectively. From this slope, the amplification efficiency was calculated using E = [10 (-1 / -slope) -1]
The average Ct values obtained from the true hermit crab species and food samples Nos. 3 and 11 were 19.1 ± 0.1, 19.1 ± 0.0 and 26.7 ± 0.1, respectively, and the rest were 37.5 ± 0.3 (Ct values of unidentified samples were given as 40) The mean Ct value was shown (Fig. 3A). As shown in FIG. 3B, in the case of the commercial food sample No. 11, Ct values were higher than those of the 29 real sample samples and the commercial food sample sample No. 3, even though they were identified as the true species by sequencing. The IPC (Internal Positive Control) test was performed with all the DNA samples, and no RT-PCR reaction problems or RT-PCR inhibitors were observed in the extracted DNA samples. As shown in the RT-PCR result, the
Example 3: Real-time PCR analysis to identify true hippocampal species
As shown in Table 2, twenty-seven species-labeled commercial food samples and 29 true-irrigation samples identified by Forensic Informative Nucleotide Sequencing (FINS) were analyzed by RT-PCR analysis . RT-PCR results were consistent with those obtained by FINS. The average Ct value obtained from the 29 true humpback samples was 19.1 ± 0.1, and the average Ct values of the two samples (Sample No. 3, 11) identified as the true humpback species in FINS among the 27 commercial samples were 19.1 ± 0.1 0.0 and 26.7 ± 0.1. The higher Ct value of the
The present invention first provides a method for quickly and reliably identifying a Beringraja pulchra species using a real-time PCR method, which specifically analyzes the mitochondrial COI nucleotide sequence of the true hippocampal species. This method is an economically useful technique that can effectively deal with food crimes such as rising signs and unclear signs of an increasing number of true hermit crab related foods. In particular, the real-time PCR method based on TaqMan technology is one of the most valuable and preferred food species for food, and it has genetic discrimination ability to distinguish true hongo species, which are one of the most valuable fish species, to provide. In the future, if this method is mainly used for identification of true hermit crab species, it will prevent food fraud, such as replacing true hermit crab species with other hermit crabs and stingray species, contribute to adherence to food label regulations, and ultimately, It is believed that it will contribute to protection.
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION)
<120> Method For Identification Of Beringraja pulchra Using Real-Time
PCR assay
<130> 1
<160> 3
<170> KoPatentin 3.0
<210> 1
<211> 17
<212> DNA
<213> Unknown
<220>
<223> Beringraja pulchra
<400> 1
Claims (6)
The primers and probes are specific for the < RTI ID = 0.0 > codon < / RTI >
Wherein the probe is labeled with a reporter FAM at the 5 'end and MGB labeled at the 3' end with a non-fluorescent quenching group (Real-Time Polymerase Chain Reaction, RT-PCR A primer-probe set for identification of a true < RTI ID = 0.0 >
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| CN108801989A (en) * | 2018-04-16 | 2018-11-13 | 番禺出入境检验检疫局综合技术服务中心 | It is a kind of to be used to detect aptamer-molecular engram fluorescent optical sensor of cytochrome c and preparation method thereof |
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| KR101940977B1 (en) | 2017-09-13 | 2019-01-23 | 대한민국 | Primer set for identifying skate ray, imported skate ray and stingray, analysis kit using its primers, and identifying method using thereof |
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| KR101395344B1 (en) | 2013-10-21 | 2014-05-16 | 대한민국 | Peptide nucleic acids set for identifying raja kenojei mueller et henle species and identifying method of raja kenojei mueller et henle species using the same |
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| KR101395344B1 (en) | 2013-10-21 | 2014-05-16 | 대한민국 | Peptide nucleic acids set for identifying raja kenojei mueller et henle species and identifying method of raja kenojei mueller et henle species using the same |
Non-Patent Citations (2)
| Title |
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| Molecular Phylogenetics and Evolution, Vol. 75, pp. 245-251 (2014.01.31.)* |
| NCBI, GenBank Accession No. KF317730.1 (2014.08.14.) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108801989A (en) * | 2018-04-16 | 2018-11-13 | 番禺出入境检验检疫局综合技术服务中心 | It is a kind of to be used to detect aptamer-molecular engram fluorescent optical sensor of cytochrome c and preparation method thereof |
| CN108801989B (en) * | 2018-04-16 | 2020-10-02 | 番禺出入境检验检疫局综合技术服务中心 | Aptamer-molecularly imprinted fluorescent sensor for detecting cytochrome c and preparation method thereof |
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